WorldWideScience

Sample records for human adipocytes secrete

  1. Human adipocytes stimulate invasion of breast cancer MCF-7 cells by secreting IGFBP-2.

    Directory of Open Access Journals (Sweden)

    Chen Wang

    Full Text Available A better understanding of the effects of human adipocytes on breast cancer cells may lead to the development of new treatment strategies. We explored the effects of adipocytes on the migration and invasion of breast cancer cells both in vitro and in vivo.To study the reciprocal effects of adipocytes and cancer cells, we co-cultured human mature adipocytes and breast cancer cells in a system devoid of heterogeneous cell-cell contact. To analyze the factors that were secreted from adipocytes and that affected the invasive abilities of breast cancer cells, we detected different cytokines in various co-culture media. To study the communication of mature adipocytes and breast cancer cells in vivo, we chose 10 metastatic pathologic samples and 10 non-metastatic pathologic samples to do immunostaining.The co-culture media of human MCF-7 breast cancer cells and human mature adipocytes increased motility of MCF-7 cells. In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells. Based on our co-culture medium chip results, we chose four candidate cytokines and tested their influence on metastasis individually. We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors. In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.IGFBP-2 secreted by mature adipocytes plays a key role in promoting the metastatic ability of MCF-7 breast cancer cells.

  2. Differentiation of human adipocytes at physiological oxygen levels results in increased adiponectin secretion and isoproterenol-stimulated lipolysis.

    Science.gov (United States)

    Famulla, Susanne; Schlich, Raphaela; Sell, Henrike; Eckel, Jürgen

    2012-07-01

    Adipose tissue (AT) hypoxia occurs in obese humans and mice. Acute hypoxia in adipocytes causes dysregulation of adipokine secretion with an increase in inflammatory factors and diminished adiponectin release. O2 levels in humans range between 3 and 11% revealing that conventional in vitro culturing at ambient air and acute hypoxia treatment (1% O2) are performed under non-physiological conditions. In this study, we mimicked physiological conditions by differentiating human primary adipocytes under 10% or 5% O2 in comparison to 21% O2. Induction of differentiation markers was comparable between all three conditions. Adipokine release by adipocytes differentiated at lower oxygen levels was altered, with a marked upregulation of adiponectin, IL-6 and DPP4 secretion, and reduced leptin levels compared with adipocytes differentiated at 21% O2. Isoproterenol-induced lipolysis was significantly elevated in adipocytes differentiated at 10% and 5% compared with 21% O2. This effect was accompanied by increased protein expression of β-1 and -2 adrenergic receptor, HSL and perilipin. Conditioned medium (CM) of adipocytes differentiated at the three different conditions was generated for stimulation of human skeletal muscle cells (SkMC) or smooth muscle cells (SMC). CM-induced insulin resistance in SkMC was comparable for the different CMs. However, the SMC proliferative effect of CM from adipocytes differentiated at 10% O2 was significantly reduced compared with 21% O2. This study demonstrates that oxygen levels during adipogenesis are important factors altering adipocyte functionality such as adipokine release, in particular adiponectin secretion, as well as the hormone-induced lipolytic pathway.

  3. Insulin: pancreatic secretion and adipocyte regulation.

    Science.gov (United States)

    Baumgard, L H; Hausman, G J; Sanz Fernandez, M V

    2016-01-01

    Insulin is the primary acute anabolic coordinator of nutrient partitioning. Hyperglycemia is the main stimulant of insulin secretion, but other nutrients such as specific amino acids, fatty acids, and ketoacids can potentiate pancreatic insulin release. Incretins are intestinal hormones with insulinotropic activity and are secreted in response to food ingestion, thus integrating diet chemical composition with the regulation of insulin release. In addition, prolactin is required for proper islet development, and it stimulates β-cell proliferation. Counterintuitively, bacterial components appear to signal insulin secretion. In vivo lipopolysaccharide infusion acutely increases circulating insulin, which is paradoxical as endotoxemia is a potent catabolic condition. Insulin is a potent anabolic orchestrator of nutrient partitioning, and this is particularly true in adipocytes. Insulin dictates lipid accretion in a dose-dependent manner during preadipocyte development in adipose tissue-derived stromal vascular cell culture. However, in vivo studies focused on insulin's role in regulating adipose tissue metabolism from growing, and market weight pigs are sometimes inconsistent, and this variability appears to be animal, age and depot dependent. Additionally, porcine adipose tissue synthesizes and secretes a number of adipokines (leptin, adiponectin, and so forth) that directly or indirectly influence insulin action. Therefore, because insulin has an enormous impact on agriculturally important phenotypes, it is critical to have a better understanding of how insulin homeostasis is governed.

  4. Short-term regulation of adiponectin secretion in rat adipocytes.

    Science.gov (United States)

    Szkudelski, T; Nogowski, L; Szkudelska, K

    2011-01-01

    Adiponectin belongs to the group of biologically active substances secreted by adipocytes and referred to as adipokines. Disturbances in its secretion and/or action are thought to be involved in the pathogenesis of some metabolic diseases. However, regulation of adiponectin secretion is poorly elucidated. In the present study, short-term regulation of adiponectin secretion in primary rat adipocytes was investigated. Isolated rat adipocytes were incubated in Krebs-Ringer buffer containing 5 mM glucose and insulin alone or in the combination with epinephrine, dibutyryl-cAMP, adenosine A(1) receptor antagonist (DPCPX), palmitate, 2-bromopalmitate or inhibitor of mitochondrial electron transport (rotenone). Adipocyte exposure for 2 h to insulin (1-100 nM) significantly increased secretion of adiponectin compared with secretion observed without insulin. Furthermore, secretion of adiponectin from adipocytes incubated with glucose and insulin was reduced by 1 and 2 microM epinephrine, but not by 0.25 and 0.5 microM epinephrine. Under similar conditions, 1 and 2 mM dibutyryl-cAMP substantially diminished secretion of adiponectin, whereas 0.5 mM dibutyryl-cAMP was ineffective. Secretion of adiponectin was found to be effectively decreased by DPCPX. Moreover, adipocyte exposure to rotenone also resulted in a substantial diminution of secretory response of adipocytes incubated for 2 h with glucose and insulin. It was also demonstrated that palmitate and 2-bromopalmitate (0.06-0.5 mM) failed to affect secretion of leptin. The obtained results indicated that in short-term regulation of adiponectin secretion, insulin and epinephrine exert the opposite effects. These effects appeared as early as after 2 h of exposure. Moreover, deprivation of energy or blockade of adenosine action substantially decreased secretion of adiponectin.

  5. Hypoxia induces apelin expression in human adipocytes.

    Science.gov (United States)

    Geiger, K; Muendlein, A; Stark, N; Saely, C H; Wabitsch, M; Fraunberger, P; Drexel, H

    2011-06-01

    Adipokines play a central role in the development of diseases associated with insulin resistance and obesity. Hypoxia in adipose tissue leads to a dysregulation of the expression of adipokines. The effect of hypoxia on the more recently identified adipokine apelin in human adipocytes is unclear. Therefore, we aimed at investigating the role of hypoxia on the expression of the adipokine apelin. Differentiated human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were cultured under hypoxic conditions for varying time periods. A modular incubator chamber was used to create a hypoxic tissue culture environment (defined as 1% O(2), 94% N, and 5% CO(2)). In addition, hypoxic conditions were mimicked by using CoCl(2). The effect of hypoxia on the expression of the investigated adipokines was measured by real-time PCR and the secretion of apelin was quantified by ELISA. Induction of hypoxia significantly induced mRNA expression of leptin and apelin in differentiated SGBS adipocytes compared with the normoxic control condition. Expression of adiponectin was significantly decreased by hypoxia. In addition, the amount of secreted apelin protein in response to hypoxia was elevated compared to untreated cells. Furthermore, we could demonstrate that the observed hypoxia-induced induction of apelin mRNA expression is in the first phase dependent on HIF-1α. In our study, we could demonstrate for the first time that apelin expression and secretion by human adipocytes are strongly induced under hypoxic conditions and that the early response on hypoxia with apelin induction is dependent on HIF-1α. © Georg Thieme Verlag KG Stuttgart · New York.

  6. Fibroblast growth factor 21 is elevated in metabolically unhealthy obesity and affects lipid deposition, adipogenesis, and adipokine secretion of human abdominal subcutaneous adipocytes

    Directory of Open Access Journals (Sweden)

    Lucia Berti

    2015-07-01

    Conclusions: The hepatokine FGF21 exerts weak lipogenic and anti-adipogenic actions and marked adiponectin-suppressive and leptin and interleukin-6 release-promoting effects in human differentiating preadipocytes. Together with the higher serum concentrations in MUHO subjects, our findings reveal FGF21 as a circulating factor promoting the development of metabolically unhealthy adipocytes.

  7. Adipocyte secreted factors enhance aggressiveness of prostate carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Ângela Moreira

    Full Text Available Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1 and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01. Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01. Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05. Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

  8. Human adipocyte function is impacted by mechanical cues.

    Science.gov (United States)

    Pellegrinelli, V; Heuvingh, J; du Roure, O; Rouault, C; Devulder, A; Klein, C; Lacasa, M; Clément, E; Lacasa, D; Clément, K

    2014-06-01

    Fibrosis is a hallmark of human white adipose tissue (WAT) during obesity-induced chronic inflammation. The functional impact of increased interstitial fibrosis (peri-adipocyte fibrosis) on adjacent adipocytes remains unknown. Here we developed a novel in vitro 3D culture system in which human adipocytes and decellularized material of adipose tissue (dMAT) from obese subjects are embedded in a peptide hydrogel. When cultured with dMAT, adipocytes showed decreased lipolysis and adipokine secretion and increased expression/production of cytokines (IL-6, G-CSF) and fibrotic mediators (LOXL2 and the matricellular proteins THSB2 and CTGF). Moreover, some alterations including lipolytic activity and fibro-inflammation also occurred when the adipocyte/hydrogel culture was mechanically compressed. Notably, CTGF expression levels correlated with the amount of peri-adipocyte fibrosis in WAT from obese individuals. Moreover, dMAT-dependent CTGF promoter activity, which depends on β1-integrin/cytoskeleton pathways, was enhanced in the presence of YAP, a mechanosensitive co-activator of TEAD transcription factors. Mutation of TEAD binding sites abolished the dMAT-induced promoter activity. In conclusion, fibrosis may negatively affect human adipocyte function via mechanosensitive molecules, in part stimulated by cell deformation. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  9. Effects of glucocorticoids on human brown adipocytes.

    Science.gov (United States)

    Barclay, Johanna L; Agada, Hadiya; Jang, Christina; Ward, Micheal; Wetzig, Neil; Ho, Ken K Y

    2015-02-01

    Clinical cases of glucocorticoid (GC) excess are characterized by increased fat mass and obesity through the accumulation of white adipocytes. The effects of GCs on growth and function of brown adipose tissue are unknown and may contribute to the negative energy balance observed clinically. This study aims to evaluate the effect of GCs on proliferation, differentiation, and metabolic function of brown adipocytes. Human brown adipocytes sourced from supraclavicular fat biopsies were grown in culture and differentiated to mature adipocytes. Human white adipocytes sourced from subcutaneous abdominal fat biopsies were cultured as controls. Effects of dexamethasone on growth, differentiation (UCP1, CIDEA, and PPARGC1A expression), and function (oxygen consumption rate (OCR)) of brown adipocytes were quantified. Dexamethasone (1 μM) significantly stimulated the proliferation of brown preadipocytes and reduced that of white preadipocytes. During differentiation, dexamethasone (at 0.1, 1, and 10 μM) stimulated the expression of UCP1, CIDEA, and PPARGC1A in a concentration-dependent manner and enhanced by fourfold to sixfold the OCR of brown adipocytes. Isoprenaline (100 nM) significantly increased (POCR of brown adipocytes. These effects were significantly reduced (Pbiology of human brown adipose tissue (BAT) and for the involvement of the BAT system in the metabolic manifestation of Cushing's syndrome. © 2015 Society for Endocrinology.

  10. Maraviroc shows differential effects on glucose uptake and lipolysis in human subcutaneous cultured adipocytes in comparison with omental adipocytes

    Directory of Open Access Journals (Sweden)

    P Perez-Matute

    2012-11-01

    Full Text Available Maraviroc (MVC, the first approved CC-chemokine receptor 5 (CCR5 antagonist, is used for treating HIV-1-infected patients with CCR5 tropism. MVC has been proved safe in all respects and showed beneficial effects on lipid profile of HIV patients with dislipidemia. Adipocyte dysfunction seems to be responsible for many metabolic alterations such as insulin resistance and dislipidemia. Subcutaneous and visceral fat depots are not only physiologically but also metabolically different and metabolic disturbances are more closely associated with visceral than subcutaneous fat accumulation. It has been suggested that antiretrovirals affect both fat depots in a different extent. Thus, whether isolated human adipocytes display regio-specific sensitivity to the metabolic effects of MVC have been tested in this study. Human subcutaneous and omental preadipocytes were used as the source of human adipocytes. These cells were treated with therapeutic concentrations of MVC (0.5–25 µM at day 14 post-differentiation (4 and 24 hours of treatment. Glucose utilization, lactate production and glycerol released into the media were measured using an autoanalyzer. Adiponectin secretion was determined by an ELISA array. A dose-dependent increase in glucose uptake was observed in subcutaneous adipocytes treated with MVC (+72% of stimulation for MVC 25 µM, p < 0.01. This stimulatory effect was tissue specific, as no effects were observed in omental adipocytes. MVC did not exert any significant effect on adiponectin secretion. No significant effects were observed on lactate production neither in subcutaneous nor omental adipocytes. Interestingly, 4 hours of treatment with MVC induced a significant increase in the amount of glycerol released into the media by subcutaneous adipocytes (p < 0.001, but this effect disappeared with longer exposure of adipocytes to MVC (24 h. No effects were observed on lipolysis in omental adipocytes although a slight tendency to

  11. Interleukin-17A inhibits adipocyte differentiation in human mesenchymal stem cells and regulates pro-inflammatory responses in adipocytes.

    Science.gov (United States)

    Shin, Jennifer H; Shin, Dong Wook; Noh, Minsoo

    2009-06-15

    The immune system is closely linked to human metabolic diseases. Serum levels of IL-6 increase with obesity and insulin resistance. Not only does IL-6 decrease the insulin sensitivity of human cells such as adipocytes, but it also regulates the lineage commitment of naïve T cells into interleukin (IL)-17A-producing CD4(+) T (Th17) cells. Although IL-17A exerts a variety of effects on somatic tissues, its functional role in human adipocytes has not been identified. In this work, we show that IL-17A inhibits adipocyte differentiation in human bone marrow mesenchymal stem cells (hBM-MSCs), while promoting lipolysis of differentiated adipocytes. We find that IL-17A increases both mRNA and protein secretion of IL-6 and IL-8 during adipocyte differentiation in hBM-MSCs. IL-17A up-regulates cyclooxygenase (COX)-2 gene expression and thereby increases the level of prostaglandin (PG) E(2) in differentiated adipocyes. The suppression of anti-adipogenic PGE(2) by COX inhibitors such as aspirin and NS-398 partially blocked the effect of IL-17A on adipocyte differentiation in hBM-MSCs. Therefore, IL-17A exhibits its inhibitory effect in part via the COX-2 induction in differentiated adipocytes. In addition, treatment with anti-IL-17A antibody neutralizes IL-17A-mediated effects on adipocyte differentiation and function. These results suggest that IL-17A plays a regulatory role in both the metabolic and inflammatory processes of human adipocytes, similar to other pro-inflammatory cytokines such as IL-1, IFNgamma, and TNFalpha.

  12. Dynamics of Adipocyte Turnover in Humans

    Energy Technology Data Exchange (ETDEWEB)

    Spalding, K; Arner, E; Westermark, P; Bernard, S; Buchholz, B; Bergmann, O; Blomqvist, L; Hoffstedt, J; Naslund, E; Britton, T; Concha, H; Hassan, M; Ryden, M; Frisen, J; Arner, P

    2007-07-16

    Obesity is increasing in an epidemic fashion in most countries and constitutes a public health problem by enhancing the risk for cardiovascular disease and metabolic disorders such as type 2 diabetes. Owing to the increase in obesity, life expectancy may start to decrease in developed countries for the first time in recent history. The factors determining fat mass in adult humans are not fully understood, but increased lipid storage in already developed fat cells is thought to be most important. We show that adipocyte number is a major determinant for the fat mass in adults. However, the number of fat cells stays constant in adulthood in lean and obese and even under extreme conditions, indicating that the number of adipocytes is set during childhood and adolescence. To establish the dynamics within the stable population of adipocytes in adults, we have measured adipocyte turnover by analyzing the integration of {sup 14}C derived from nuclear bomb tests in genomic DNA. Approximately 10% of fat cells are renewed annually at all adult ages and levels of body mass index. Neither adipocyte death nor generation rate is altered in obesity, suggesting a tight regulation of fat cell number that is independent of metabolic profile in adulthood. The high turnover of adipocytes establishes a new therapeutic target for pharmacological intervention in obesity.

  13. Apolipoprotein E promotes lipid accumulation and differentiation in human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lasrich, Dorothee; Bartelt, Alexander [Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany); Grewal, Thomas, E-mail: thomas.grewal@sydney.edu.au [Faculty of Pharmacy A15, The University of Sydney, Sydney, NSW 2006 (Australia); Heeren, Joerg, E-mail: heeren@uke.de [Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany)

    2015-09-10

    Several studies in mice indicate a role for apolipoprotein E (APOE) in lipid accumulation and adipogenic differentiation in adipose tissue. However, little is yet known if APOE functions in a similar manner in human adipocytes. This prompted us to compare lipid loading and expression of adipocyte differentiation markers in APOE-deficient and control adipocytes using the differentiated human mesenchymal stem cell line hMSC-Tert as well as primary human and mouse adipocytes as model systems. Differentiated hMSC-Tert were stably transduced with or without siRNA targeting APOE while murine adipocytes were isolated from wild type and Apoe knockout mice. Human APOE knockdown hMSC-Tert adipocytes accumulated markedly less triglycerides compared to control cells. This correlated with strongly decreased gene expression levels of adipocyte markers such as adiponectin (ADIPOQ) and fatty acid binding protein 4 (FABP4) as well as the key transcription factor driving adipocyte differentiation, peroxisome proliferator activator receptor gamma (PPARG), in particular the PPARG2 isoform. Similarly, differentiation of murine Apoe-deficient adipocytes was characterized by reduced gene expression of Adipoq, Fabp4 and Pparg. Interestingly, incubation of APOE-deficient hMSC-Tert adipocytes with conditioned media from APOE3-overexpressing adipocytes or APOE-containing Very Low Density Lipoprotein (VLDL) partially restored triglyceride accumulation, but were unable to induce adipocyte differentiation, as judged by expression of adipocyte markers. Taken together, depletion of endogenous APOE in human adipocytes severely impairs lipid accumulation, which is associated with an inability to initiate differentiation. - Highlights: • Immortalized human mesenchymal stem cells were used to study adipocyte development. • Knockdown of endogenous APOE lead to impaired lipid accumulation and adipogenesis. • APOE supplementation partially restored lipid accumulation but not differentiation.

  14. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, Cormac T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland); Ryan, Silke, E-mail: silke.ryan@ucd.ie [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland)

    2014-05-16

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  15. LS14: a novel human adipocyte cell line that produces prolactin.

    Science.gov (United States)

    Hugo, Eric R; Brandebourg, Terry D; Comstock, Clay E S; Gersin, Keith S; Sussman, Jeffrey J; Ben-Jonathan, Nira

    2006-01-01

    Adipose tissue is an integral component within the endocrine system. Adipocytes produce numerous bioactive substances, and their dysregulation has serious pathophysiological consequences. We previously reported that human adipose tissue from several depots produces significant amounts of prolactin (PRL). To study locally produced PRL, we sought an acceptable in vitro model. Consequently, we developed an adipocyte cell line derived from a metastatic liposarcoma. The cell line, designated LS14, has been in continuous culture for 2 yr. These cells exhibit many properties of primary preadipocytes, including the ability to undergo terminal differentiation, as judged by morphological alterations, lipid accumulation, and increase in glycerol-3-phosphate dehydrogenase. LS14 cells express many adipose-associated genes, such as adipocyte fatty acid-binding protein (aP(2)), hormone-sensitive lipase, lipoprotein lipase, preadipocyte factor 1, adiponectin, leptin, and IL-6. Similar to primary adipocytes, LS14 cells also produce and respond to PRL, thus making them an attractive model to study adipose PRL production and function. The expression of PRL was confirmed at the transcriptional level by RT-PCR, and PRL secretion was determined by the Nb2 bioassay. Addition of exogenous PRL to LS14 cells resulted in a dose-dependent inhibition of IL-6 release. In summary, we have established a novel human adipocyte cell line with many characteristics of primary adipocytes. The LS14 cells open up new avenues for research on human adipocyte biology and add to the repertoire of nonpituitary, PRL-producing cell lines.

  16. Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes.

    Science.gov (United States)

    Xie, Linglin; O'Reilly, Cormac P; Chapes, Stephen K; Mora, Silvia

    2008-02-01

    Adiponectin and leptin are two adipokines secreted by white adipose tissue that regulate insulin sensitivity. Previously we reported that adiponectin but not leptin release depends on GGA-coated vesicle formation, suggesting that leptin and adiponectin may follow different secretory routes. Here we have examined the intracellular trafficking pathways that lead to the secretion of these two hormones. While adiponectin and leptin displayed distinct localization in the steady-state, treatment of adipocytes with brefeldin A inhibited both adiponectin and leptin secretion to a similar level, indicating a common requirement for class III ADP-ribosylating factors and an intact Golgi apparatus. Adiponectin secretion was significantly reduced by endosomal inactivation in both 3T3L1 and rat isolated adipocytes, whereas this treatment had no effect on leptin secretion. Importantly, endosomal inactivation completely abolished the insulin stimulatory effect on adiponectin release in rat adipocytes. Confocal microscopy studies revealed colocalization of adiponectin with endogenous rab11 a marker for the recycling endosome, and with expressed rab5-GFP mutant (rab5Q75L) a marker for the early endosome compartment. Colocalization of adiponectin and rab5Q75L was increased in endosome inactivated cells. Consistent with these findings adiponectin secretion was reduced in cells expressing mutants of Rab11 and Rab5 proteins. In contrast, expression of an inactive (kinase dead) mutant of Protein Kinase D1 moderately but significantly inhibited leptin secretion without altering adiponectin secretion. Taken together, these results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and that endosomal compartments are required for adiponectin but not for leptin secretion.

  17. Resveratrol metabolites modify adipokine expression and secretion in 3T3-L1 pre-adipocytes and mature adipocytes.

    Directory of Open Access Journals (Sweden)

    Itziar Eseberri

    Full Text Available OBJECTIVE: Due to the low bioavailability of resveratrol, determining whether its metabolites exert any beneficial effect is an interesting issue. METHODS: 3T3-L1 maturing pre-adipocytes were treated during differentiation with 25 µM of resveratrol or with its metabolites and 3T3-L1 mature adipocytes were treated for 24 hours with 10 µM resveratrol or its metabolites. The gene expression of adiponectin, leptin, visfatin and apelin was assessed by Real Time RT-PCR and their concentration in the incubation medium was quantified by ELISA. RESULTS: Resveratrol reduced mRNA levels of leptin and increased those of adiponectin. It induced the same changes in leptin secretion. Trans-resveratrol-3-O-glucuronide and trans-resveratrol-4'-O-glucuronide increased apelin and visfatin mRNA levels. Trans-resveratrol-3-O-sulfate reduced leptin mRNA levels and increased those of apelin and visfatin. CONCLUSIONS: The present study shows for the first time that resveratrol metabolites have a regulatory effect on adipokine expression and secretion. Since resveratrol has been reported to reduce body-fat accumulation and to improve insulin sensitivity, and considering that these effects are mediated in part by changes in the analyzed adipokines, it may be proposed that resveratrol metabolites play a part in these beneficial effects of resveratrol.

  18. Genistein inhibits differentiation of primary human adipocytes.

    Science.gov (United States)

    Park, Hea Jin; Della-Fera, Mary Anne; Hausman, Dorothy B; Rayalam, Srujana; Ambati, Suresh; Baile, Clifton A

    2009-02-01

    Genistein, a major soy isoflavone, has been reported to exhibit antiadipogenic and proapoptotic potential in vivo and in vitro. It is also a phytoestrogen which has high affinity to estrogen receptor beta. In this study, we determined the effect of genistein on adipogenesis and estrogen receptor (ER) alpha and beta expression during differentiation in primary human preadipocytes. Genistein inhibited lipid accumulation in a dose-dependent manner at concentrations of 6.25 microM and higher, with 50 microM genistein inhibiting lipid accumulation almost completely. Low concentrations of genistein (3.25 microM) increased cell viability and higher concentrations (25 and 50 microM) decreased it by 16.48+/-1.35% (P<.0001) and 50.68+/-1.34% (P<.0001). Oil Red O staining was used to confirm the effects on lipid accumulation. The inhibition of lipid accumulation was associated with inhibition of glycerol-3-phosphate dehydrogenase activity and down-regulation of expression of adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, glycerol-3-phosphate dehydrogenase, adipocyte fatty acid binding protein, fatty acid synthase, sterol regulatory element-binding protein 1, perilipin, leptin, lipoprotein lipase and hormone-sensitive lipase. These effects of genistein during the differentiation period were associated with down-regulation of ERalpha and ERbeta expression. This study adds to the elucidation of the molecular pathways involved in the inhibition of adipogenesis by phytoestrogens.

  19. The control of insulin secretion by adipokines: current evidence for adipocyte-beta cell endocrine signalling in metabolic homeostasis.

    Science.gov (United States)

    Cantley, James

    2014-10-01

    Metabolic homeostasis is maintained by the coordinated action of multiple organ systems. Insulin secretion is often enhanced during obesity or insulin resistance to maintain glucose and lipid homeostasis, whereas a loss of insulin secretion is associated with type 2 diabetes. Adipocytes secrete hormones known as adipokines which act on multiple cell types to regulate metabolism. Many adipokines have been shown to influence beta cell function by enhancing or inhibiting insulin release or by influencing beta cell survival. Insulin, in turn, regulates lipolysis and promotes glucose uptake and lipid storage in adipocytes. As adipokine secretion and action is strongly influenced by obesity, this provides a potential route by which beta cell function is coordinated with adiposity, independently of alterations in blood glucose or lipid levels. In this review, I assess the evidence for the direct regulation of beta cell function by the adipokines leptin, adiponectin, extracellular nicotinamide phosphoribosyltransferase, apelin, resistin, retinol binding protein 4, fibroblast growth factor 21, nesfatin-1 and fatty acid binding protein 4. I summarise in vitro and in vivo data and discuss the influence of obesity and diabetes on circulating adipokine concentrations, along with the potential for influencing beta cell function in human physiology. Finally, I highlight future research questions that are likely to yield new insights into the exciting field of insulinotropic adipokines.

  20. Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines.

    Directory of Open Access Journals (Sweden)

    Bao G Vu

    Full Text Available BACKGROUND: Human adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin. METHODOLOGY/PRINCIPAL FINDINGS: Primary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8. CONCLUSIONS/SIGNIFICANCE: Endotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to

  1. Gallic Acid, the Active Ingredient of Terminalia bellirica, Enhances Adipocyte Differentiation and Adiponectin Secretion.

    Science.gov (United States)

    Makihara, Hiroko; Koike, Yuka; Ohta, Masatomi; Horiguchi-Babamoto, Emi; Tsubata, Masahito; Kinoshita, Kaoru; Akase, Tomoko; Goshima, Yoshio; Aburada, Masaki; Shimada, Tsutomu

    2016-01-01

    Visceral obesity induces the onset of metabolic disorders such as insulin resistance and diabetes mellitus. Adipose tissue is considered as a potential pharmacological target for treating metabolic disorders. The fruit of Terminalia bellirica is extensively used in Ayurvedic medicine to treat patients with diseases such as diabetes mellitus. We previously investigated the effects of a hot water extract of T. bellirica fruit (TB) on obesity and insulin resistance in spontaneously obese type 2 diabetic mice. To determine the active ingredients of TB and their molecular mechanisms, we focused on adipocyte differentiation using mouse 3T3-L1 cells, which are widely used to study adipocyte physiology. We show here that TB enhanced the differentiation of 3T3-L1 cells to mature adipocytes and that one of the active main components was identified as gallic acid. Gallic acid (10-30 µM) enhanced the expression and secretion of adiponectin via adipocyte differentiation and also that of fatty acid binding protein-4, which is the target of peroxisome proliferator-activated receptor gamma (PPARγ), although it does not alter the expression of the upstream genes PPARγ and CCAAT enhancer binding protein alpha. In the PPARγ ligand assay, the binding of gallic acid to PPARγ was undetectable. These findings indicate that gallic acid mediates the therapeutic effects of TB on metabolic disorders by regulating adipocyte differentiation. Therefore, TB shows promise as a candidate for preventing and treating patients with metabolic syndrome.

  2. Hypoxia-mimetic effects in the secretome of human preadipocytes and adipocytes.

    Science.gov (United States)

    Rosenow, Anja; Noben, Jean-Paul; Bouwman, Freek G; Mariman, Edwin C M; Renes, Johan

    2013-12-01

    White adipose tissue (WAT) regulates energy metabolism by secretion of proteins with endocrine and paracrine effects. Dysregulation of the secretome of obesity-associated enlarged WAT may lead to obesity-related disorders. This can be caused by hypoxia as a result of poorly vascularized WAT. The effect of hypoxia on the secretome of human (pre)adipocytes is largely unknown. Therefore, we investigated the effect of CoCl2, a hypoxia mimetic, on the secretome of human SGBS (pre)adipocytes by a proteomics approach combined with bioinformatic analysis. In addition, regulation of protein secretion was examined by protein turnover experiments. As such, secretome changes were particularly associated with protein down-regulation and extracellular matrix protein dysregulation. The observed up-regulation of collagens in adipocytes may be essential for cell survival while down-regulation of collagens in preadipocytes may indicate a disturbed differentiation process. These CoCl2-induced changes reflect WAT dysfunction that ultimately may lead to obesity-associated complications. In addition, 9 novel adipocyte secreted proteins were identified from which 6 were regulated by CoCl2. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000162. © 2013.

  3. A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

    DEFF Research Database (Denmark)

    Kratchmarova, Irina; Kalume, Dario E; Blagoev, Blagoy;

    2002-01-01

    We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secre...

  4. Glucose-dependent insulinotropic polypeptide induces cytokine expression, lipolysis, and insulin resistance in human adipocytes.

    Science.gov (United States)

    Timper, Katharina; Grisouard, Jean; Sauter, Nadine S; Herzog-Radimerski, Tanja; Dembinski, Kaethi; Peterli, Ralph; Frey, Daniel M; Zulewski, Henryk; Keller, Ulrich; Müller, Beat; Christ-Crain, Mirjam

    2013-01-01

    Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1β, and the IL-1 receptor antagonist IL-1Ra, whereas TNFα, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-κB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1β, and TNFα. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-κB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-κB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes.

  5. Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Wang, Zai [Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Li, Jian; Cai, Jian-Ping [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [School of Biomedical Sciences and Shenzhen Institute of Research and Innovation, The University of Hong Kong, Pokfulam (Hong Kong); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China); Zhang, Tie-Mei, E-mail: tmzhang126@126.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China)

    2016-08-05

    Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

  6. Signaling pathways involved in LPS induced TNFalpha production in human adipocytes

    Directory of Open Access Journals (Sweden)

    Festy Franck

    2010-01-01

    Full Text Available Abstract Background The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation. Methods Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays. Results We show for the first time that the production of TNFalpha in mature human adipocytes is mainly dependent upon two pathways: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages. Conclusion This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

  7. Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Sárvári, Anitta K., E-mail: anittasarvari@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Veréb, Zoltán, E-mail: jzvereb@gmail.com [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); Uray, Iván P., E-mail: ipuray@mdanderson.org [Clinical Cancer Prevention Department, The University of Texas, MD Anderson Cancer Center, Houston, TX (United States); Fésüs, László, E-mail: fesus@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary); MTA DE Apoptosis, Genomics and Stem Cell Research Group of the Hungarian Academy of Sciences (Hungary); Balajthy, Zoltán, E-mail: balajthy@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Debrecen (Hungary)

    2014-08-08

    Highlights: • Antipsychotics modulate the expression of adipogenic genes in human adipocytes. • Secretion of proinflammatory cytokine IL8 and MCP-1 is induced by antipsychotics. • Adipocyte-dependent inflammatory abnormality could develop during chronic treatment. • Infiltrated macrophages would further enhance proinflammatory cytokine production. - Abstract: Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin

  8. Wogonin enhances intracellular adiponectin levels and suppresses adiponectin secretion in 3T3-L1 adipocytes.

    Science.gov (United States)

    Yang, Tan; Liu, Hua; Zhao, Bo; Xia, Zhongyuan; Zhang, Yemin; Zhang, Deling; Li, Mingxin; Cao, Yingkang; Zhang, Zhijiang; Bi, Yongyi; Wang, Changhua

    2017-01-30

    As an insulin sensitizer and modulator of inflammatory responses, adiponectin has become a therapeutic target for insulin resistance, diabetes, and diabetes-related complications. Wogonin possesses anti-oxidative, anti-inflammatory, and anti-diabetic abilities. However, its effect on generation and secretion of adiponectin is ill-defined in adipocytes. Here, we demonstrated that wogonin administration augmented intracellular adiponectin levels and attenuated adiponectin release in a dose- and time-dependent manner in mature 3T3-L1 adipocytes, along with a suppression of PKCδ phosphorylation. Wogonin treatment also prevented PKCδ overexpression-induced reduction of intracellular adiponectin levels and enhancement of adiponectin release. In addition, wogonin supplementation dramatically increased AMPK phosphorylation and SirT1 expression. Inhibition of either AMPK or SirT1 mitigated wogonin action on adiponectin production and release. Furthermore, inhibition of AMPK by its specific inhibitor markedly reduced wogonin-enhanced mRNA and protein expressions of SirT1. These results suggested that wogonin regulated expression and secretion of adiponectin via PKCδ/AMPK/SirT1 signaling pathway in mature 3T3-L1 adipocytes.

  9. Vitamin C inhibits leptin secretion and some glucose/lipid metabolic pathways in primary rat adipocytes.

    Science.gov (United States)

    Garcia-Diaz, D F; Campion, J; Milagro, F I; Boque, N; Moreno-Aliaga, M J; Martinez, J A

    2010-07-01

    Antioxidant-based treatments are emerging as an interesting approach to possibly counteract obesity fat accumulation complications, since this is accompanied by an increased systemic oxidative stress. The aim of this study was to analyze specific metabolic effects of vitamin C (VC) on epididymal primary rat adipocytes. Cells were isolated and incubated for 72 h in culture medium, in the absence or presence of 1.6 nM insulin, within a range of VC concentrations (5-1000 microM). Glucose- and lipid-related variables as well as the secretion/expression patterns of several obesity-related genes were assessed. It was observed that VC dose dependently inhibited glucose uptake and lactate production, and also reduced glycerol release in both control and insulin-treated cells. Also, VC caused a dramatic concentration-dependent fall in leptin secretion especially in insulin-stimulated cells. In addition, VC (200 microM) induced Cdkn1a and Casp8, partially inhibited Irs3, and together with insulin drastically reduced Gpdh (listed as Gpd1 in the MGI database) gene expressions. Finally, VC and insulin down-regulatory effects were observed on extracellular and intracellular reactive oxygen species production respectively. In summary, this experimental assay describes a specific effect of VC in isolated rat adipocytes on glucose and fat metabolism, and on the secretion/expression of important obesity-related proteins.

  10. Effect of the Cannabinoid Receptor-1 antagonist SR141716A on human adipocyte inflammatory profile and differentiation

    Directory of Open Access Journals (Sweden)

    Murumalla Ravi

    2011-11-01

    Full Text Available Abstract Background Obesity is characterized by inflammation, caused by increase in proinflammatory cytokines, a key factor for the development of insulin resistance. SR141716A, a cannabinoid receptor 1 (CB1 antagonist, shows significant improvement in clinical status of obese/diabetic patients. Therefore, we studied the effect of SR141716A on human adipocyte inflammatory profile and differentiation. Methods Adipocytes were obtained from liposuction. Stromal vascular cells were extracted and differentiated into adipocytes. Media and cells were collected for secretory (ELISA and expression analysis (qPCR. Triglyceride accumulation was observed using oil red-O staining. Cholesterol was assayed by a fluorometric method. 2-AG and anandamide were quantified using isotope dilution LC-MS. TLR-binding experiments have been conducted in HEK-Blue cells. Results In LPS-treated mature adipocytes, SR141716A was able to decrease the expression and secretion of TNF-a. This molecule has the same effect in LPS-induced IL-6 secretion, while IL-6 expression is not changed. Concerning MCP-1, the basal level is down-regulated by SR141716A, but not the LPS-induced level. This effect is not caused by a binding of the molecule to TLR4 (LPS receptor. Moreover, SR141716A restored adiponectin secretion to normal levels after LPS treatment. Lastly, no effect of SR141716A was detected on human pre-adipocyte differentiation, although the compound enhanced adiponectin gene expression, but not secretion, in differentiated pre-adipocytes. Conclusion We show for the first time that some clinical effects of SR141716A are probably directly related to its anti-inflammatory effect on mature adipocytes. This fact reinforces that adipose tissue is an important target in the development of tools to treat the metabolic syndrome.

  11. Human adipocytes from the subcutaneous superficial layer have greater adipogenic potential and lower PPAR-γ DNA methylation levels than deep layer adipocytes.

    Science.gov (United States)

    Kosaka, Kentaro; Kubota, Yoshitaka; Adachi, Naoki; Akita, Shinsuke; Sasahara, Yoshitaro; Kira, Tomoe; Kuroda, Masayuki; Mitsukawa, Nobuyuki; Bujo, Hideaki; Satoh, Kaneshige

    2016-08-01

    Human subcutaneous fat tissue consists of two layers, superficial adipose tissue (SAT) and deep adipose tissue (DAT). Some recent reports suggest that a disproportionate accumulation of DAT is related to obesity-associated metabolic complications. However, the differences in adipocyte function between SAT and DAT are unclear. To clarify the differences in human adipocyte characteristics between SAT and DAT, human ceiling culture-derived proliferative adipocytes (ccdPAs) were primary cultured from SAT and DAT of three lean female patients. Differences in adipogenic differentiation potential and sensitivity to exogenous adipogenic factors were examined. Epigenetic modification of the CpG island DNA methylation levels of genes related to adipogenesis was measured. In histological analyses, the mean adipocyte size in SAT was significantly larger than that in DAT (8,741 ± 416 vs. 7,732 ± 213 μm(2), P < 0.05). Primary cultured adipocytes from SAT showed significantly greater adipogenesis than did those of DAT. Sensitivity to partial adipogenic stimulation was significantly different between ccdPAs of SAT and DAT. Peroxisome proliferator-activated receptor-γ (PPAR-γ) protein expression and leptin protein secretion from ccdPAs were significantly higher in SAT than DAT. DNA methylation levels of PPAR-γ were significantly lower in ccdPAs of SAT than DAT. Adipocyte size was larger in SAT than DAT in vivo. This is consistent with the findings of an in vitro study that, compared with ccdPAs in DAT, ccdPAs in SAT have higher adipogenic potential and lower DNA methylation levels of PPAR-γ.

  12. Persistent organic pollutants alter DNA methylation during human adipocyte differentiation

    NARCIS (Netherlands)

    Dungen, van den Myrthe; Murk, Tinka; Steegenga, Wilma; Gils-Kok, van Dieuwertje

    2016-01-01

    Genome-wide DNA methylation profiling was performed in human mesenchymal stem cells (hMSCs) differentiated into adipocytes (day 10) while being continuously exposed to either one of three different persistent organic pollutants (POPs), namely TCDD, PFOS, and TBT. The Illumina Infinium 450K Human DNA

  13. Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Alessi Marie-Christine

    2008-02-01

    Full Text Available Abstract Background It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS at an early step of commitment to adipocytes and osteoblasts. Results A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice. Conclusion Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.

  14. Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

    Science.gov (United States)

    Chiellini, Chiara; Cochet, Olivia; Negroni, Luc; Samson, Michel; Poggi, Marjorie; Ailhaud, Gérard; Alessi, Marie-Christine; Dani, Christian; Amri, Ez-Zoubir

    2008-01-01

    Background It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts. Results A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice. Conclusion Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis. PMID:18302751

  15. TLR4-dependant pro-inflammatory effects of HMGB1 on human adipocyte.

    Science.gov (United States)

    Gunasekaran, Manoj Kumar; Virama-Latchoumy, Anne-Laurence; Girard, Anne-Claire; Planesse, Cynthia; Guérin-Dubourg, Alexis; Ottosson, Lars; Andersson, Ulf; Césari, Maya; Roche, Régis; Hoareau, Laurence

    2016-01-01

    Chronic low grade inflammation is one of the major metabolic disorders in case of obesity and associated pathologies. By its important secretion function, the role of adipose tissue in this metabolic low grade inflammation is well known. Recently, it was demonstrated that the alarmin high mobility group box protein 1 (HMGB1) is involved in obesity-related pathologies by its increased serum levels in obese compared to normal weight individuals, and by its pro-inflammatory effects. However, the role of HMGB1 on adipocytes inflammation is poorly documented and we propose to investigate this point. Primary culture of human subcutaneous adipocytes were performed from human adipose tissue samples. Cells were treated with recombinant HMGB1 with/without anti-TLR4 antibody and inhibitors of NF-κB and P38 MAPK. Supernatants were collected for IL-6 and MCP-1 ELISA. HMGB1 initiates Toll-like receptor 4 (TLR4)-dependent activation of inflammation through the downstream NF-κB and P38 MAPK signaling pathway to upregulate the secretion of the pro-inflammatory cytokine IL-6. HMGB1 has pro-inflammatory effects on adipocytes. This reinforces the role of TLR4 in adipose tissue inflammation and antagonizing the HMGB1 inflammatory pathway could bring on new therapeutic targets to counteract obesity-associated pathologies.

  16. Silibinin regulates lipid metabolism and differentiation in functional human adipocytes

    Directory of Open Access Journals (Sweden)

    Ignazio eBarbagallo

    2016-01-01

    Full Text Available Silibinin, a natural plant flavonoid, is the main active constituent found in milk thistle (Silybum marianum. It is known to have hepatoprotective, anti-neoplastic effect and suppresses lipid accumulation in adipocytes. Objective of this study was to investigate the effect of silibinin on adipogenic differentiation and thermogenic capacity of human adipose tissue derived mesenchymal stem cells. Silibinin (10 μM treatment, either at the beginning or at the end of adipogenic differentiation, resulted in an increase of SIRT-1, PPARα, Pgc-1α and UCPs gene expression. Moreover, silibinin administration resulted in a decrease of PPARγ, FABP4, FAS and MEST/PEG1 gene expression during the differentiation, confirming that this compound is able to reduce fatty acid accumulation and adipocyte size. Our data showed that silibinin regulated adipocyte lipid metabolism, inducing thermogenesis and promoting a brown remodelling in adipocyte. Taken together, our findings suggest that silibinin increases UCPs expression by stimulation of SIRT1, PPARα and Pgc-1α, improved metabolic parameters, decreased lipid mass leading to the formation of functional adipocytes.

  17. Disruption of Lipid Raft Function Increases Expression and Secretion of Monocyte Chemoattractant Protein-1 in 3T3-L1 Adipocytes.

    Science.gov (United States)

    Lu, Juu-Chin; Chiang, Yu-Ting; Lin, Yu-Chun; Chang, Yu-Tzu; Lu, Chia-Yun; Chen, Tzu-Yu; Yeh, Chia-Shan

    2016-01-01

    The adipocyte is unique in its capacity to store lipids. In addition to triglycerides, the adipocyte stores a significant amount of cholesterol. Moreover, obese adipocytes are characterized by a redistribution of cholesterol with depleted cholesterol in the plasma membrane, suggesting that cholesterol perturbation may play a role in adipocyte dysfunction. We used methyl-β-cyclodextrin (MβCD), a molecule with high affinity for cholesterol, to rapidly deplete cholesterol level in differentiated 3T3-L1 adipocytes. We tested whether this perturbation altered adipocyte secretion of monocyte chemoattractant protein-1 (MCP-1), a chemokine that is elevated in obesity and is linked to obesity-associated chronic diseases. Depletion of cholesterol by MβCD increased MCP-1 secretion as well as the mRNA and protein levels, suggesting perturbation at biosynthesis and secretion. Pharmacological inhibition revealed that NF-κB, but not MEK, p38 and JNK, was involved in MβCD-stimulated MCP-1 biosynthesis and secretion in adipocytes. Finally, another cholesterol-binding drug, filipin, also induced MCP-1 secretion without altering membrane cholesterol level. Interestingly, both MβCD and filipin disturbed the integrity of lipid rafts, the membrane microdomains enriched in cholesterol. Thus, the depletion of membrane cholesterol in obese adipocytes may result in dysfunction of lipid rafts, leading to the elevation of proinflammatory signaling and MCP-1 secretion in adipocytes.

  18. Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

    Science.gov (United States)

    van den Dungen, Myrthe W; Murk, Albertinka J; Kok, Dieuwertje E; Steegenga, Wilma T

    2017-04-01

    Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes. Copyright © 2017 Wageningen University. Published by Elsevier Ltd.. All rights reserved.

  19. The glucocorticoid receptor, not the mineralocorticoid receptor, plays the dominant role in adipogenesis and adipokine production in human adipocytes.

    Science.gov (United States)

    Lee, M-J; Fried, S K

    2014-09-01

    Both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) are expressed in adipose tissue and assumed to mediate cortisol actions on adipose tissue. The relative significance of the two receptors in mediating glucocorticoid regulation of adipogenesis and adipokine expression in human adipocytes has not been addressed. We investigated the differential roles of the GR and MR in mediating glucocorticoid actions on adipogenesis and adipokine production using RNA interference in primary cultures of human preadipocytes and adipocytes. Both types of receptors are expressed, but levels of GR were several hundred fold higher than MR in both human preadipocytes and adipocytes. As expected, cortisol added during adipogenesis increased the differentiation of human preadipocytes. Silencing of GR, but not MR, blocked these proadipogenic actions of cortisol. In differentiated human adipocytes, addition of cortisol increased leptin and adiponectin, while suppressing interleukin-6 (IL-6), messenger RNA levels and protein secretion. Knockdown of GR by 65% decreased leptin and adiponectin while increasing IL-6 production. In addition, GR silencing blocked the effects of cortisol on adipokine expression. In contrast, although MR knockdown increased leptin, it did not affect adiponectin and IL-6 expression. Our data demonstrate that although both GR and MR have roles in regulating leptin expression, GR plays more important roles in mediating the actions of cortisol to regulate adipogenesis and adipokine production in human adipocytes.

  20. Effects of selected bioactive food compounds on human white adipocyte function

    DEFF Research Database (Denmark)

    Björk, Christel; Wilhelm, Uta; Mandrup, Susanne

    2016-01-01

    BACKGROUND: Previous studies suggest that intake of specific bioactive compounds may have beneficial clinical effects on adipose tissue partly due to their anti-inflammatory and insulin-sensitizing properties. With the overall aim to contribute to better understanding of the mechanisms of selecte...... uptake albeit only with the combination of DHA and AC. Taken together, our results may link the reported health benefits of the selected bioactives on metabolic disorders such as insulin resistance, hypertension and dyslipidemia to effects on white adipocytes....... (PI) on adipokine secretion, fatty acid metabolism (lipolysis/lipogenesis) and adipocyte differentiation (lipid accumulation) was studied in human fat cells differentiated in vitro. To investigate possible synergistic, additive or antagonistic effects, DHA was also combined with AC or PI. RESULTS......: Each compound, alone or together with DHA, suppressed basal adipocyte lipolysis compared to control treated cells. DHA alone attenuated the secretion of pro-inflammatory adipokines such as chemerin, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1/CCL2), whereas AC suppressed only...

  1. Interacting Effects of TSH and Insulin on Human Differentiated Adipocytes.

    Science.gov (United States)

    Felske, D; Gagnon, A; Sorisky, A

    2015-08-01

    Subclinical hypothyroidism, characterized by an isolated rise in TSH serum levels with normal thyroid function, is a pro-inflammatory state associated with insulin resistance. Adipocytes express TSH receptors, but it is not known if TSH can directly inhibit insulin signaling. Using primary human differentiated adipocytes, we examined the effects of TSH on insulin-stimulated Akt phosphorylation, and whether conventional PKC (cPKC) were involved. The effect of insulin on TSH-stimulated lipolysis was also investigated. TSH inhibited insulin-stimulated Akt phosphorylation in adipocytes by 54%. TSH activated cPKC, and Gö6976, a PKCα and -β1 inhibitor, prevented the inhibitory effect of TSH on the insulin response. Insulin reduced the ability of TSH to activate cPKC and to stimulate lipolysis.Our data reveal novel interactions between TSH and insulin. TSH inhibits insulin-stimulated Akt signaling in a cPKC-dependent fashion, whereas insulin blocks TSH-stimulated cPKC activity and lipolysis. TSH and insulin act on differentiated human adipocytes to modulate their respective intracellular signals. © Georg Thieme Verlag KG Stuttgart · New York.

  2. Fatty acids do not pay the toll: effect of SFA and PUFA on human adipose tissue and mature adipocytes inflammation

    Directory of Open Access Journals (Sweden)

    Murumalla Ravi Kumar

    2012-12-01

    Full Text Available Abstract Background On the basis that high fat diet induces inflammation in adipose tissue, we wanted to test the effect of dietary saturated and polysunsaturated fatty acids on human adipose tissue and adipocytes inflammation. Moreover we wanted to determine if TLR2 and TLR4 are involved in this pathway. Methods Human adipose tissue and adipocytes primary cultures were treated with endotoxin-free BSA conjugated with SFA (lauric acid and palmitic acid - LA and PA and PUFA (eicosapentaeneic acid, docosahexaenoic acid and oleic acid - EPA, DHA and OA with or without LPS. Cytokines were then assayed by ELISA (TNF-alpha, IL-6 and MCP-1. In order to determine if TLR2 and TLR4 are activated by fatty acid (FA, we used HEK-Blue cells transfected by genes from TLR2 or TLR4 pathways associated with secreted alkaline phosphatase reporter gene. Results None of the FA tested in HEK-Blue cells were able to activate TLR2 or TLR4, which is concordant with the fact that after FA treatment, adipose tissue and adipocytes cytokines levels remain the same as controls. However, all the PUFA tested: DHA, EPA and to a lesser extent OA down-regulated TNF-alpha, IL-6 and MCP-1 secretion in human adipose tissue and adipocytes cultures. Conclusions This study first confirms that FA do not activate TLR2 and TLR4. Moreover by using endotoxin-free BSA, both SFA and PUFA tested were not proinflammatory in human adipose tissue and adipocytes model. More interestingly we showed that some PUFA exert an anti-inflammatory action in human adipose tissue and adipocytes model. These results are important since they clarify the relationship between dietary fatty acids and inflammation linked to obesity.

  3. OXIDATIVE STRESS: ITS ROLE IN INSULIN SECRETION, HORMONE RECEPTION BY ADIPOCYTES AND LIPOLYSIS IN ADIPOSE TISSUE

    Directory of Open Access Journals (Sweden)

    V. V. Ivanov

    2014-01-01

    Full Text Available Oxidative stress is one of the pathogenetic components of many diseases during which generation of reactive oxigen species increases and the capacity of the antioxidant protection system diminishes. In the research of the last decades special attention has been given to adipose tissue, production of adipokines by it and their role in development of immunoresistance associated with formation of the metabolic syndrome and diabetes.Search for methods of therapeutic correction of adipokine secretion disorders, their influence on metabolism of separate cells and the organism on the whole as well as development of new approaches to correction of disorders in cell sensitivity to insulin are extremely topical nowadays. Systematization and consolidation of accumulated data allow to determine the strategies of further research more accurately; as a result, we have attempted to summarize and analyze the accumulated data on the role of adipose tissue in oxidative stress development.On the basis of literature data and the results of the personal investigations, the role of adipose tissue in forming oxidative stress in diabetes has been analyzed in the article. Brief description of adipose tissue was given as a secretory organ regulating metabolic processes in adipocytes and influencing functions of various organs and systems of the body. Mechanisms of disorder in insulin secretion as well as development of insulin sesistance in type I diabetes were described along with the contribution of lipolysis in adipose tissue to these processes.

  4. Uraemic sera stimulate lipolysis in human adipocytes: role of perilipin.

    Science.gov (United States)

    Axelsson, Jonas; Aström, Gaby; Sjölin, Eva; Qureshi, Abdul Rashid; Lorente-Cebrián, Silvia; Stenvinkel, Peter; Rydén, Mikael

    2011-08-01

    Although chronic kidney disease (CKD) is associated with dyslipidaemia and insulin resistance, the exact cause(s) are unknown. Since adipose tissue plays an important role in the development of these complications, we investigated the effect of uraemic sera on human adipocytes in vitro. Cultured human adipocytes were incubated for 48 h with media containing sera from eight CKD Stage 5 patients or four (matched for age, sex and body mass index) healthy controls. Glycerol release (an index of lipolysis) was determined in conditioned media. RNA was isolated from the cells and quantitative polymerase chain reaction of genes involved in lipolysis was performed. In vivo lipolysis was determined by the plasma glycerol/total fat mass (from dual energy X-ray absorptiometry) ratio in 28 CKD patients and 28 matched controls. Incubation with uraemic, but not control, sera resulted in a significant ∼30% increase in spontaneous (basal) lipolysis (P lipolysis in human adipocytes in vitro, probably by attenuating the expression of the lipolytic regulator PLIN. Since in vivo lipolysis is a well-established risk factor for insulin resistance and cardiovascular disease, these effects may promote increased morbidity and mortality in CKD.

  5. Effects of clozapine on adipokine secretions/productions and lipid droplets in 3T3-L1 adipocytes

    Directory of Open Access Journals (Sweden)

    Tomomi Tsubai

    2017-02-01

    Full Text Available Clozapine, a second-generation antipsychotic (SGA, is a cause of side effects related to metabolic syndrome. The participation of serotonin 5-HT2C and histamine H1 receptors in the central nervous system has been reported as a mechanism of the weight gain caused by clozapine. In the present study, we investigated the direct pharmacological action of clozapine on the 3T3-L1 adipocytes and compared it to that of blonanserin, an SGA with low affinity for both receptors. Short-term exposure to clozapine decreased secretion and mRNA expression of leptin. Long-term exposure decreased leptin as well as adiponectin secretion, and further increased lipid droplets accumulation. However, short- and long-term exposures to blonanserin did not affect these parameters. A selective serotonin 5-HT2C, but not a histamine H1, receptor antagonist enhanced the decreased secretion of leptin induced by short-term exposure to clozapine, but did not affect the increased accumulation of lipid droplets. Our findings indicate that clozapine, but not blonanserin, strongly and directly affected the secretion of adipokines, such as leptin, in adipocytes and caused adipocyte enlargement.

  6. Effects of clozapine on adipokine secretions/productions and lipid droplets in 3T3-L1 adipocytes.

    Science.gov (United States)

    Tsubai, Tomomi; Yoshimi, Akira; Hamada, Yoji; Nakao, Makoto; Arima, Hiroshi; Oiso, Yutaka; Noda, Yukihiro

    2017-02-01

    Clozapine, a second-generation antipsychotic (SGA), is a cause of side effects related to metabolic syndrome. The participation of serotonin 5-HT2C and histamine H1 receptors in the central nervous system has been reported as a mechanism of the weight gain caused by clozapine. In the present study, we investigated the direct pharmacological action of clozapine on the 3T3-L1 adipocytes and compared it to that of blonanserin, an SGA with low affinity for both receptors. Short-term exposure to clozapine decreased secretion and mRNA expression of leptin. Long-term exposure decreased leptin as well as adiponectin secretion, and further increased lipid droplets accumulation. However, short- and long-term exposures to blonanserin did not affect these parameters. A selective serotonin 5-HT2C, but not a histamine H1, receptor antagonist enhanced the decreased secretion of leptin induced by short-term exposure to clozapine, but did not affect the increased accumulation of lipid droplets. Our findings indicate that clozapine, but not blonanserin, strongly and directly affected the secretion of adipokines, such as leptin, in adipocytes and caused adipocyte enlargement. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  7. Adipocyte lipases and defect of lipolysis in human obesity.

    Science.gov (United States)

    Langin, Dominique; Dicker, Andrea; Tavernier, Geneviève; Hoffstedt, Johan; Mairal, Aline; Rydén, Mikael; Arner, Erik; Sicard, Audrey; Jenkins, Christopher M; Viguerie, Nathalie; van Harmelen, Vanessa; Gross, Richard W; Holm, Cecilia; Arner, Peter

    2005-11-01

    The mobilization of fat stored in adipose tissue is mediated by hormone-sensitive lipase (HSL) and the recently characterized adipose triglyceride lipase (ATGL), yet their relative importance in lipolysis is unknown. We show that a novel potent inhibitor of HSL does not inhibit other lipases. The compound counteracted catecholamine-stimulated lipolysis in mouse adipocytes and had no effect on residual triglyceride hydrolysis and lipolysis in HSL-null mice. In human adipocytes, catecholamine- and natriuretic peptide-induced lipolysis were completely blunted by the HSL inhibitor. When fat cells were not stimulated, glycerol but not fatty acid release was inhibited. HSL and ATGL mRNA levels increased concomitantly during adipocyte differentiation. Abundance of the two transcripts in human adipose tissue was highly correlated in habitual dietary conditions and during a hypocaloric diet, suggesting common regulatory mechanisms for the two genes. Comparison of obese and nonobese subjects showed that obesity was associated with a decrease in catecholamine-induced lipolysis and HSL expression in mature fat cells and in differentiated preadipocytes. In conclusion, HSL is the major lipase for catecholamine- and natriuretic peptide-stimulated lipolysis, whereas ATGL mediates the hydrolysis of triglycerides during basal lipolysis. Decreased catecholamine-induced lipolysis and low HSL expression constitute a possibly primary defect in obesity.

  8. Identification and validation of novel adipokines released from primary human adipocytes.

    Science.gov (United States)

    Lehr, Stefan; Hartwig, Sonja; Lamers, Daniela; Famulla, Susanne; Müller, Stefan; Hanisch, Franz-Georg; Cuvelier, Claude; Ruige, Johannes; Eckardt, Kristin; Ouwens, D Margriet; Sell, Henrike; Eckel, Juergen

    2012-01-01

    Adipose tissue is a major endocrine organ, releasing signaling and mediator proteins, termed adipokines, via which adipose tissue communicates with other organs. Expansion of adipose tissue in obesity alters adipokine secretion, which may contribute to the development of metabolic diseases. Although recent profiling studies have identified numerous adipokines, the amount of overlap from these studies indicates that the adipokinome is still incompletely characterized. Therefore, we conducted a complementary protein profiling on concentrated conditioned medium derived from primary human adipocytes. SDS-PAGE/liquid chromatography-electrospray ionization tandem MS and two-dimensional SDS-PAGE/matrix-assisted laser desorption ionization/time of flight MS identified 347 proteins, 263 of which were predicted to be secreted. Fourty-four proteins were identified as novel adipokines. Furthermore, we validated the regulation and release of selected adipokines in primary human adipocytes and in serum and adipose tissue biopsies from morbidly obese patients and normal-weight controls. Validation experiments conducted for complement factor H, αB-crystallin, cartilage intermediate-layer protein, and heme oxygenase-1 show that the release and expression of these factors in adipocytes is regulated by differentiation and stimuli, which affect insulin sensitivity, as well as by obesity. Heme oxygenase-1 especially reveals to be a novel adipokine of interest. In vivo, circulating levels and adipose tissue expression of heme oxygenase-1 are significantly increased in obese subjects compared with lean controls. Collectively, our profiling study of the human adipokinome expands the list of adipokines and further highlights the pivotal role of adipokines in the regulation of multiple biological processes within adipose tissue and their potential dysregulation in obesity.

  9. n3 and n6 polyunsaturated fatty acids differentially modulate prostaglandin E secretion but not markers of lipogenesis in adipocytes

    Directory of Open Access Journals (Sweden)

    Saxton Arnold M

    2009-01-01

    Full Text Available Abstract A dramatic rise in the incidence of obesity in the U.S. has accelerated the search for interventions that may impact this epidemic. One recently recognized target for such intervention is adipose tissue, which secretes a variety of bioactive substances including prostaglandins. Prostaglandin E2 (PGE2 has been shown to decrease lipolysis in adipocytes, but limited studies have explored alternative mechanisms by which PGE2 might impact obesity, such as adipogenesis or lipogenesis. Studies conducted on ApcMin/+ mice indicated that selective inhibition of the cyclooxygenase (COX-2 enzyme led to significant reductions in fatty acid synthase (FAS activity in adipose tissue suggesting lipogenic effects of PGE2. To further investigate whether these lipid mediators directly regulate lipogenesis, we used 3T3-L1 adipocytes to determine the impact of eicosapentaenoic acid (EPA and celecoxib on PGE2 formation and FAS used as a lipogenic marker. Both arachidonic acid (AA and EPA dose-dependently increased PGE secretion from adipocytes. AA was expectedly more potent and exhibiting at 150 uM dose a 5-fold increase in PGE2 secretion over EPA. Despite higher secretion of PGE by EPA and AA compared to control, neither PUFA significantly altered FAS activity. By contrast both AA and EPA significantly decreased FAS mRNA levels. Addition of celecoxib, a selective COX-2 inhibitor, significantly decreased PGE2 secretion (p 2 and celecoxib further decreased the FAS activity compared to PGE2 alone or untreated controls. In conclusion, EPA-mediated inhibition of AA metabolism did not significantly alter FAS activity while both AA and EPA significantly decreased FAS mRNA expression. COX-2 inhibition significantly decreased PGE2 production resulting in a decrease in FAS activity and expression that was not reversed with the addition of exogenous PGE2, suggesting an additional mechanism that is independent of COX-2.

  10. Effects of Monoclonal Antibody Against Porcine 40-kDa Adipocyte-Specific Membrane Protein on Endocrine Secretion in Pigs

    Institute of Scientific and Technical Information of China (English)

    LIU Ling-yun; HU Hong-mei; ZHAO Su-mei; ZHANG Xi; DUAN Gang; GAO Shi-zheng

    2009-01-01

    The present study was to investigate the effect of monocional antibody against porcine 40-kDa adipocyte-specific membrane protein on endocrine secretion in pigs, in order to provide the evidence for application of this antibody to reduce excessive fat deposition in pig production. 40 Landrace × Saba pigs were randomly divided into 8 groups: 2 control groups were given saline with 10 mL, respectively, and the 6 treatment groups were given monoclonal antibody against porcine 40-kDa adipocyte-specific membrane protein with 0.1,0.5, and 1.0 mg kg-1 body weight at 15 or 60 kg body weight,respectively, all treatments were performed by intraperitoneal injection. The results showed that this monoclonal antibody could significantly reduce serum insulin level and increase levels of serum growth hormone (GH), insulin-like growth factor-1 (IGF-1), triiodothyronine (T3), and tetraiodothyronine (T4) either at 15 or 60 kg body weight injection. However,more marked effect was observed at 15 kg body weight treatment. Moreover, the dose-dependent effect of this monoclonal antibody on endocrine secretion was also observed. This result revealed that this monoclonal antibody increased secretion of hormones regulating fat lysis and reduced secretion of hormones regulating fat synthesis, suggests the reduction of porcine excessive fat deposition by this monoclonal antibody was carried out through affecting hormones regulating fat metabolism.

  11. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha

    2008-01-01

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged...

  12. Natriuretic peptides: a new lipolytic pathway in human adipocytes.

    Science.gov (United States)

    Sengenès, C; Berlan, M; De Glisezinski, I; Lafontan, M; Galitzky, J

    2000-07-01

    Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.

  13. Characterization of adipocyte differentiation from human mesenchymal stem cells in bone marrow

    Directory of Open Access Journals (Sweden)

    Huang Hai-Yan

    2010-05-01

    Full Text Available Abstract Background Adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. The mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models (i.e. 3T3-L1 and 3T3-F442A, revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors (C/EBPβ, C/EBPα and PPARγ. However, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells (hBMSCs, remain poorly understood. This study investigated cell cycle regulation and the roles of C/EBPβ, C/EBPα and PPARγ during adipocyte differentiation from hBMSCs. Results Utilising a BrdU incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3T3-L1 pre-adipocytes but not hBMSCs undergoing mitotic clonal expansion. Knock-down and over-expression assays revealed that C/EBPβ, C/EBPα and PPARγ were required for adipocyte differentiation from hBMSCs. C/EBPβ and C/EBPα individually induced adipocyte differentiation in the presence of inducers; PPARγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. Therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse. Conclusions The characteristics of hBMSCs during adipogenic differentiation are different from those of murine cells. These findings could be important in elucidating the mechanisms underlying human obesity further.

  14. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  15. ER stress in adipocytes inhibits insulin signaling, represses lipolysis, and alters the secretion of adipokines without inhibiting glucose transport.

    Science.gov (United States)

    Xu, L; Spinas, G A; Niessen, M

    2010-08-01

    The endoplasmic reticulum (ER) is the intra-cellular site, where secreted and membrane proteins are synthesized. ER stress and activation of the unfolded protein response (UPR) contribute to insulin resistance and the development of diabetes in obesity. It was shown previously in hepatocytes that the UPR activates c-jun N-terminal kinase (JNK), which phosphorylates insulin receptor substrate (IRS) proteins on serine residues thereby inhibiting insulin signal transduction. Here we describe how ER stress affects insulin signaling and the biological function of adipocytes. In addition to inhibition of IRS we found that ER stress downregulates the expression of the insulin receptor. Concomitantly, insulin-induced activation of Akt/PKB and of ERK1/2 was strongly inhibited. Ectopic expression of IRS1 or IRS2 strongly counteracted the inhibitory effect of ER stress on insulin signaling while pharmacological inhibition of JNK with SP600125 resulted only in a mild improvement. ER stress decreased the secretion of the adipokines adiponectin and leptin, but strongly increased secretion of IL-6. ER stress inhibited expression and insulin-induced phosphorylation of AS160, reduced lipolysis but did not inhibit glucose transport. Finally, supernatants collected from 3T3-L1 adipocytes undergoing ER stress improved or impaired proliferation when used to condition the culture medium of INS-1E beta-cells dependent on the degree of ER stress. It appears that ER stress in adipocytes might initially lead to changes resembling early prediabetic stages, which at least in part support the regulation of systemic energy homeostasis. Copyright Georg Thieme Verlag KG Stuttgart New York.

  16. Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression.

    Science.gov (United States)

    Gesta, S; Lolmède, K; Daviaud, D; Berlan, M; Bouloumié, A; Lafontan, M; Valet, P; Saulnier-Blache, J S

    2003-03-01

    Primary culture of adipose tissue has often been used to investigate pharmacological and nutritional regulation of adipocyte gene expression. Possible alteration of adipocyte gene expression by primary culture on its own has not been explored in detail. In order to address this issue, explants were prepared from human subcutaneous adipose tissue recovered from plastic surgery and maintained for 0 to 48 h in DMEM supplemented with 10 % serum. At different time points, adipocytes were isolated from the explants by collagenase digestion, and mRNA expression and lipolysis were studied. Culture was associated with an accumulation of tumor necrosis factor-alpha (TNFalpha) in the culture medium, an increase in anaerobic glycolysis, and an increase in the basal lipolysis. In parallel, a rapid and dramatic decrease in the level of mRNA encoding for several adipocyte-specific proteins such as adipocyte lipid-binding protein, hormone-sensitive lipase, lipoprotein lipase, and peroxisome proliferation activating receptor-gamma2 was observed in isolated adipocytes. These downregulations were reminiscent of a dedifferentiation process. In parallel, primary culture was associated with an increase in adipocyte beta-actin, TNFalpha, glucose transporter-1 and hypoxia-induced factor-1alpha mRNAs. Treatment of explants with agents that increase cAMP (isobutylmethylxanthine and forskolin) prevented TNFalpha production and expression and culture-induced alterations of adipocyte gene expression. These data show that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression.

  17. Effect of Sfrp5 on cytokine release and insulin action in primary human adipocytes and skeletal muscle cells.

    Directory of Open Access Journals (Sweden)

    Maren Carstensen

    Full Text Available Secreted frizzled-related protein 5 (Sfrp5 is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC. Sfrp5 neither affected interleukin (IL-6, monocyte chemoattractant protein-1 (MCP-1 and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05, but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01, 31% (p<0.05, 37% (p<0.05 and 34% (p<0.01, respectively, and the stimulation of glucose uptake by 25% (p<0.05. Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state.

  18. Effect of Sfrp5 on Cytokine Release and Insulin Action in Primary Human Adipocytes and Skeletal Muscle Cells

    Science.gov (United States)

    Röhrig, Karin; Fahlbusch, Pia; Roden, Michael; Herder, Christian; Ouwens, D. Margriet

    2014-01-01

    Secreted frizzled-related protein 5 (Sfrp5) is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC). Sfrp5 neither affected interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF) α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05), but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01), 31% (p<0.05), 37% (p<0.05) and 34% (p<0.01), respectively, and the stimulation of glucose uptake by 25% (p<0.05). Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state. PMID:24465779

  19. Ubc9 Impairs Activation of the Brown Fat Energy Metabolism Program in Human White Adipocytes.

    Science.gov (United States)

    Hartig, Sean M; Bader, David A; Abadie, Kathleen V; Motamed, Massoud; Hamilton, Mark P; Long, Weiwen; York, Brian; Mueller, Michaela; Wagner, Martin; Trauner, Michael; Chan, Lawrence; Bajaj, Mandeep; Moore, David D; Mancini, Michael A; McGuire, Sean E

    2015-09-01

    Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis.

  20. Zinc-transporter genes in human visceral and subcutaneous adipocytes: lean versus obese.

    Science.gov (United States)

    Smidt, Kamille; Pedersen, Steen B; Brock, Birgitte; Schmitz, Ole; Fisker, Sanne; Bendix, Jørgen; Wogensen, Lise; Rungby, Jørgen

    2007-01-29

    Zinc ions influence adipose tissue metabolism by regulating leptin secretion and by promoting free fatty acid release and glucose uptake. The mechanisms controlling zinc metabolism in adipose tissue are unknown. We therefore examined the gene-expression levels of a number of zinc-transporting proteins in adipose tissue, comparing subcutaneous fat with visceral fat from lean and obese humans. Both ZnT-proteins responsible for zinc transport from cytosol to extracellular compartments and intracellular vesicles and Zip-proteins responsible for zinc transport to the cytoplasm were expressed in all samples. This suggests that zinc metabolism in adipocytes is actively controlled by zinc-transporters. The expression levels were different in lean and obese subjects suggesting a role for these proteins in obesity. Furthermore, the expression levels were different from subcutaneous fat to intra-abdominal fat suggesting that the metabolic activity in adipocytes is to some extent dependent upon zinc and the activity of zinc-transporting proteins or vice versa.

  1. Differentiation of human adipose-derived stem cells into brite (brown-in-white adipocytes

    Directory of Open Access Journals (Sweden)

    Didier F Pisani

    2011-11-01

    Full Text Available It is well established now that adult humans possess active brown adipose tissue which represents a potential pharmacological target to combat obesity and associated diseases. We had shown previously that human multipotent adipose-derived stem (hMADS cells are able to differentiate into cells which exhibit the key properties of human white adipocytes, and to convert into functional brown adipocytes upon PPARγ activation that could explain UCP1-expressing cells within islets surrounded by white adipocytes. Herein we further characterize hMADS cells differentiation into brown adipocytes that behave like mouse brite adipocytes previously described. We analyzed the expression of gene markers known to be associated with mouse white and brown adipocytes. When shifting from a white to a brown fat cell phenotype, the striking enhancement of uncoupling activity appears mainly due, if not all, to an increase in UCP1 expression whereas induction of UCP2 is weak and UCP3 expression is unchanged. Conversion of white hMADS adipocytes is dependent on PPARγ activation with rosiglitazone as the most potent agonist and is inhibited by a PPARγ antagonist. Furthermore our data show that, in contrast to mouse cellular models, hMADS cells conversion into brown adipocytes is not induced by BMP7 treatment and not modulated by activation of the Hedgehog pathway. No primary or clonal precursor cells of human brown adipocytes have been obtained so far that can be used as a tool to develop therapeutic drugs and to gain further insights into the molecular mechanisms of brown adipogenesis in humans. Thus hMADS cells represent a suitable cell model to delineate the formation and/or the uncoupling capacity of human brown/brite adipocytes that could help to dissipate caloric excess intake among individuals.

  2. Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes.

    Science.gov (United States)

    Roche, Régis; Hoareau, Laurence; Bes-Houtmann, Sandrine; Gonthier, Marie-Paule; Laborde, Christine; Baron, Jean-François; Haffaf, Yacine; Cesari, Maya; Festy, Franck

    2006-08-01

    To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.

  3. Aquaporin-10 represents an alternative pathway for glycerol efflux from human adipocytes.

    Directory of Open Access Journals (Sweden)

    Umberto Laforenza

    Full Text Available BACKGROUND: Glycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1 the exact localization of aquaporin-7 in human white adipose tissue; 2 the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes. METHODOLOGY/PRINCIPAL FINDINGS: Aquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability. CONCLUSIONS/SIGNIFICANCE: The results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is

  4. MicroRNAs regulate human adipocyte lipolysis: effects of miR-145 are linked to TNF-α.

    Directory of Open Access Journals (Sweden)

    Silvia Lorente-Cebrián

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that regulate gene expression and have multiple effects in various tissues including adipose inflammation, a condition characterized by increased local release of the pro-lipolytic cytokine tumor necrosis factor-alpha (TNF-α. Whether miRNAs regulate adipocyte lipolysis is unknown. We set out to determine whether miRNAs affect adipocyte lipolysis in human fat cells. To this end, eleven miRNAs known to be present in human adipose tissue were over-expressed in human in vitro differentiated adipocytes followed by assessments of TNF-α and glycerol levels in conditioned media after 48 h. Three miRNAs (miR-145, -26a and let-7d modulated both parameters in parallel. However, while miR-26a and let-7d decreased, miR-145 increased both glycerol release and TNF-α secretion. Further studies were focused therefore on miR-145 since this was the only stimulator of lipolysis and TNF-α secretion. Time-course analysis demonstrated that miR-145 over-expression up-regulated TNF-α expression/secretion followed by increased glycerol release. Increase in TNF-α production by miR-145 was mediated via activation of p65, a member of the NF-κB complex. In addition, miR-145 down-regulated the expression of the protease ADAM17, resulting in an increased fraction of membrane bound TNF-α, which is the more biologically active form of TNF-α. MiR-145 overexpression also increased the phosphorylation of activating serine residues in hormone sensitive lipase and decreased the mRNA expression of phosphodiesterase 3B, effects which are also observed upon TNF-α treatment in human adipocytes. We conclude that miR-145 regulates adipocyte lipolysis via multiple mechanisms involving increased production and processing of TNF-α in fat cells.

  5. Additive Regulation of Adiponectin Expression by the Mediterranean Diet Olive Oil Components Oleic Acid and Hydroxytyrosol in Human Adipocytes

    Science.gov (United States)

    Scoditti, Egeria; Massaro, Marika; Carluccio, Maria Annunziata; Pellegrino, Mariangela; Wabitsch, Martin; Calabriso, Nadia; Storelli, Carlo; De Caterina, Raffaele

    2015-01-01

    Adiponectin, an adipocyte-derived insulin-sensitizing and anti-inflammatory hormone, is suppressed in obesity through mechanisms involving chronic inflammation and oxidative stress. Olive oil consumption is associated with beneficial cardiometabolic actions, with possible contributions from the antioxidant phenol hydroxytyrosol (HT) and the monounsaturated fatty acid oleic acid (OA, 18:1n-9 cis), both possessing anti-inflammatory and vasculo-protective properties. We determined the effects of HT and OA, alone and in combination, on adiponectin expression in human and murine adipocytes under pro-inflammatory conditions induced by the cytokine tumor necrosis factor(TNF)-α. We used human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes and murine 3T3-L1 adipocytes as cell model systems, and pretreated them with 1-100 μmol/L OA, 0.1-20 μmol/L HT or OA plus HT combination before stimulation with 10 ng/mL TNF-α. OA or HT significantly (Padiponectin secretion (by 42% compared with TNF-α alone) as well as mRNA levels (by 30% compared with TNF-α alone). HT and OA also prevented—by 35%—TNF-α-induced downregulation of peroxisome proliferator-activated receptor PPARγ. Co-treatment with HT and OA restored adiponectin and PPARγ expression in an additive manner compared with single treatments. Exploring the activation of JNK, which is crucial for both adiponectin and PPARγ suppression by TNF-α, we found that HT and OA additively attenuated TNF-α-stimulated JNK phosphorylation (up to 55% inhibition). In conclusion, the virgin olive oil components OA and HT, at nutritionally relevant concentrations, have additive effects in preventing adiponectin downregulation in inflamed adipocytes through an attenuation of JNK-mediated PPARγ suppression. PMID:26030149

  6. Low-dose radiation pretreatment improves survival of human ceiling culture-derived proliferative adipocytes (ccdPAs) under hypoxia via HIF-1 alpha and MMP-2 induction

    Energy Technology Data Exchange (ETDEWEB)

    Adachi, Naoki [Department of Plastic Surgery, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba, #260-8677 (Japan); Kubota, Yoshitaka, E-mail: kubota-cbu@umin.ac.jp [Department of Plastic Surgery, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba, #260-8677 (Japan); Kosaka, Kentarou; Akita, Shinsuke; Sasahara, Yoshitarou; Kira, Tomoe [Department of Plastic Surgery, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba, #260-8677 (Japan); Kuroda, Masayuki [Center for Advanced Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba, #260-8677 (Japan); Mitsukawa, Nobuyuki [Department of Plastic Surgery, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba, #260-8677 (Japan); Bujo, Hideaki [Department of Clinical-Laboratory and Experimental-Research Medicine, Toho University, Sakura Medical Center, 564-1 Shimoshizu, Sakura-shi, Chiba, #285-8741 (Japan); Satoh, Kaneshige [Department of Plastic Surgery, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-city, Chiba, #260-8677 (Japan)

    2015-08-07

    Poor survival is a major problem of adipocyte transplantation. We previously reported that VEGF and MMPs secreted from transplanted adipocytes are essential for angiogenesis and adipogenesis. Pretreatment with low-dose (5 Gy) radiation (LDR) increased VEGF, MMP-2, and HIF-1 alpha mRNA expression in human ceiling culture-derived proliferative adipocytes (hccdPAs). Gene expression after LDR differed between adipose-derived stem cells (hASCs) and hccdPAs. Pretreatment with LDR improved the survival of hccdPAs under hypoxia, which is inevitable in the early stages after transplantation. Upregulation of VEGF and MMP-2 after LDR in hccdPAs is mediated by HIF-1 alpha expression. Our results suggest that pretreatment with LDR may improve adipocyte graft survival in a clinical setting through upregulation of VEGF and MMP-2 via HIF-1 alpha. - Highlights: • Ceiling culture-derived proliferative adipocytes (ccdPAs) react to radiation. • Low-dose radiation (LDR) pretreatment improves survival of ccdPAs under hypoxia. • Gene expression after LDR differs between ccdPAs and adipose-derived stem cells. • LDR-induced increase in MMP-2 and VEGF is dependent on HIF-1 alpha induction. • LDR pretreatment may improve the adipocyte graft survival rate in clinical settings.

  7. Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle

    Science.gov (United States)

    Arrighi, N; Moratal, C; Clément, N; Giorgetti-Peraldi, S; Peraldi, P; Loubat, A; Kurzenne, J-Y; Dani, C; Chopard, A; Dechesne, C A

    2015-01-01

    A population of fibro/adipogenic but non-myogenic progenitors located between skeletal muscle fibers was recently discovered. The aim of this study was to determine the extent to which these progenitors differentiate into fully functional adipocytes. The characterization of muscle progenitor-derived adipocytes is a central issue in understanding muscle homeostasis. They are considered as being the cellular origin of intermuscular adipose tissue that develops in several pathophysiological situations. Here fibro/adipogenic progenitors were isolated from a panel of 15 human muscle biopsies on the basis of the specific cell-surface immunophenotype CD15+/PDGFRα+CD56−. This allowed investigations of their differentiation into adipocytes and the cellular functions of terminally differentiated adipocytes. Adipogenic differentiation was found to be regulated by the same effectors as those regulating differentiation of progenitors derived from white subcutaneous adipose tissue. Similarly, basic adipocyte functions, such as triglyceride synthesis and lipolysis occurred at levels similar to those observed with subcutaneous adipose tissue progenitor-derived adipocytes. However, muscle progenitor-derived adipocytes were found to be insensitive to insulin-induced glucose uptake, in association with the impairment of phosphorylation of key insulin-signaling effectors. Our findings indicate that muscle adipogenic progenitors give rise to bona fide white adipocytes that have the unexpected feature of being insulin-resistant. PMID:25906156

  8. On the origin of human adipocytes and the contribution of bone marrow-derived cells.

    Science.gov (United States)

    Rydén, Mikael

    2016-01-01

    In the last decade, results in both animal models and humans have demonstrated that white adipocytes are generated over the entire life-span. This adds to the plasticity of adipose tissue and alterations in adipocyte turnover are linked to metabolic dysfunction. Adipocytes are derived from precursors present primarily in the perivascular areas of adipose tissue but their precise origin remains unclear. The multipotent differentiation capacity of bone marrow-derived cells (BMDC) has prompted the suggestion that BMDC may contribute to different cell tissue pools, including adipocytes. However, data in murine transplantation models have been conflicting and it has been a matter of debate whether BMDC actually differentiate into adipocytes or just fuse with resident fat cells. To resolve this controversy in humans, we recently performed a study in 65 subjects that had undergone bone marrow transplantation. Using a set of newly developed assays including single cell genome-wide analyses of mature adipocytes, we demonstrated that bone marrow contributes with approximately 10 % to the adipocyte pool. This proportion was more than doubled in obesity, suggesting that BMDC may constitute a reserve pool for adipogenesis, particularly upon weight gain. This commentary discusses the possible relevance of these and other recent findings for human pathophysiology.

  9. Maraviroc reduces cytokine expression and secretion in human adipose cells without altering adipogenic differentiation.

    Science.gov (United States)

    Díaz-Delfín, Julieta; Domingo, Pere; Giralt, Marta; Villarroya, Francesc

    2013-03-01

    Maraviroc (MVC) is a drug approved for use as part of HAART in treatment-experienced HIV-1 patients with CCR5-tropic virus. Despite the current concerns on the alterations in adipose tissue that frequently appear in HIV-infected patients under HAART, there is no information available on the effects of MVC on adipose tissue. Here we studied the effects of MVC during and after the differentiation of human adipocytes in culture, and compared the results with the effects of efavirenz (EFV). We measured the acquisition of adipocyte morphology; the gene expression levels of markers for mitochondrial toxicity, adipogenesis and inflammation; and the release of adipokines and cytokines to the medium. Additionally, we determined the effects of MVC on lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in adipocytes. Unlike EFV-treated pre-adipocytes, MVC-treated pre-adipocytes showed no alterations in the capacity to differentiate into adipocytes and accumulated lipids normally. Consistent with this, there were no changes in the mRNA levels of PPARγ or SREBP-1c, two master regulators of adipogenesis. In addition, MVC caused a significant decrease in the gene expression and release of pro-inflammatory cytokines, whereas EFV had the opposite effect. Moreover, MVC lowered inflammation-related gene expression and inhibited the LPS-induced expression of pro-inflammatory genes in differentiated adipocytes. We conclude that MVC does not alter adipocyte differentiation but rather shows anti-inflammatory properties by inhibiting the expression and secretion of pro-inflammatory cytokines. Collectively, our results suggest that MVC may minimize adverse effects on adipose tissue development, metabolism, and inflammation, and thus could be a potentially beneficial component of antiretroviral therapy.

  10. Fish oil prevents changes induced by a high-fat diet on metabolism and adipokine secretion in mice subcutaneous and visceral adipocytes.

    Science.gov (United States)

    de Sá, Roberta D C da Cunha; Crisma, Amanda R; Cruz, Maysa M; Martins, Amanda R; Masi, Laureane N; do Amaral, Catia L; Curi, R; Alonso-Vale, Maria I C

    2016-11-01

    Fish oil (FO), rich in omega-3 polyunsaturated fatty acids, has beneficial effects on changes induced by obesity and partially prevents associated comorbidities. The effects of FO on adipocytes from different adipose tissue depots in high-fat (HF) diet induced obese mice have not been uninvestigated. This is the first study to examine the effects of FO on changes in metabolism and adipokine production in adipocytes from s.c. (inguinal; ING) or visceral (retroperitoneal; RP) white adipose depots in a HF diet-induced obese mice. Unlike most studies performed previously, FO supplementation was initiated 4 weeks before the induction of obesity. HF diet caused marked changes in ING (glucose uptake and secretion of adiponectin, tumour necrosis factor-α and interleukin-6 in ING) and RP (lipolysis, de novo lipogenesis and secretion of pro-inflammatory cytokines) adipose depots. Previous and concomitant FO administration prevented the changes in ING and RP adipocytes induced by the HF diet. In the present study, we investigated the effect of fish oil (FO) on metabolism and adipokine production by adipocytes from s.c. (inguinal; ING) and visceral (retroperitoneal; RP) white adipose depots in high-fat (HF) diet-induced obese mice. Mice were divided into CO (control diet), CO+FO, HF and HF+FO groups. The HF group presented higher body weight, glucose intolerance, insulin resistance, higher plasma total and low-density lipoprotein cholesterol levels, and greater weights of ING and RP adipose depots accompanied by hypertrophy of the adipocytes. FO exerted anti-obesogenic effects associated with beneficial effects on dyslipidaemia and insulin resistance in mice fed a HF diet (HF+FO group). HF raised RP adipocyte lipolysis and the production of pro-inflammatory cytokines and reduced de novo synthesis of fatty acids, whereas, in ING adipocytes, it decreased glucose uptake and adiponectin secretion but did not change lipolysis. Therefore, the adipose depots play different roles in

  11. Protective Effects of Diallyl Sulfide Against Ethanol-Induced Injury in Rat Adipose Tissue and Primary Human Adipocytes.

    Science.gov (United States)

    Kema, Venkata Harini; Khan, Imran; Jamal, Reshma; Vishwakarma, Sandeep Kumar; Lakki Reddy, Chandrakala; Parwani, Kirti; Patel, Farhin; Patel, Dhara; Khan, Aleem A; Mandal, Palash

    2017-06-01

    Alcohol consumption is the fourth leading cause of death and disability worldwide. Several cellular pathways contribute to alcohol-mediated tissue injury. Adipose tissue apart from functioning as an endocrine organ secretes several hormones and cytokines known as adipokines that are known to play a significant role in alcohol-induced tissue damage. This study was designed to test the efficacy of diallyl sulfide (DAS) in regulating the alcohol-induced outcomes on adipose tissue. Male Wistar rats were fed with 36% Lieber-DeCarli liquid diet containing ethanol (EtOH) for 4 weeks. Control rats were pair-fed with isocaloric diet containing maltodextrin instead of EtOH. During the last week of feeding protocol, the EtOH-fed rat group was given 200 mg/kg body weight of DAS through diet. We also studied DAS effect on isolated human primary adipocytes. Viability of human primary adipocytes on DAS treatment was assessed by MTT assay. Malondialdehyde (MDA), a marker of oxidative stress, was measured by HPLC and the thiobarbituric acid method. Expression of inflammatory genes and lipogenic genes was studied by qRT-PCR and Western blotting. Serum inflammatory gene expression was studied by ELISA. Our study results showed that DAS could alleviate EtOH-induced expression levels of proinflammatory and endoplasmic reticulum (ER) stress genes and improve adipose tissue mass and adipocyte morphology in male Wistar rats fed Lieber-DeCarli diet containing 6% EtOH. Further, we showed that DAS reduced the expression of lipogenic genes and improved lipid accumulation and adipocyte mass in human primary adipocytes treated with EtOH. Subsequently, we also showed that oxidative stress, as measured by the changes in MDA levels, was reduced in both male Wistar rats and human primary adipocytes treated with EtOH plus DAS. Our study results prove that DAS is effective in ameliorating EtOH-induced damage to adipose tissue as evidenced by the reduction brought about by DAS in oxidative stress

  12. Yohimbine increases human salivary secretion.

    Science.gov (United States)

    Chatelut, E; Rispail, Y; Berlan, M; Montastruc, J L

    1989-01-01

    The effect of oral yohimbine (14 mg) on salivary secretion was evaluated in healthy volunteers. Yohimbine significantly increased salivary secretion when compared with placebo. This effect was significant from 60 min until 180 min after administration under our experimental conditions. Yohimbine (or alpha 2-adrenoceptor blocking agents) could have a potential interest in the treatment of dry mouths. PMID:2789932

  13. Reduced DPP4 activity improves insulin signaling in primary human adipocytes.

    Science.gov (United States)

    Röhrborn, Diana; Brückner, Julia; Sell, Henrike; Eckel, Jürgen

    2016-03-11

    DPP4 is a ubiquitously expressed cell surface protease which is also released to the circulation as soluble DPP4 (sDPP4). Recently, we identified DPP4 as a novel adipokine oversecreted in obesity and thus potentially linking obesity to the metabolic syndrome. Furthermore, sDPP4 impairs insulin signaling in an autocrine and paracrine fashion in different cell types. However, it is still unknown which functional role DPP4 might play in adipocytes. Therefore, primary human adipocytes were treated with a specific DPP4 siRNA. Adipocyte differentiation was not affected by DPP4 silencing. Interestingly, DPP4 reduction improved insulin responsiveness of adipocytes at the level of insulin receptor, proteinkinase B (Akt) and Akt substrate of 160 kDa. To investigate whether the observed effects could be attributed to the enzymatic activity of DPP4, human adipocytes were treated with the DPP4 inhibitors sitagliptin and saxagliptin. Our data show that insulin-stimulated activation of Akt is augmented by DPP4 inhibitor treatment. Based on our previous observation that sDPP4 induces insulin resistance in adipocytes, and that adipose DPP4 levels are higher in obese insulin-resistant patients, we now suggest that the abundance of DPP4 might be a regulator of adipocyte insulin signaling.

  14. Momordica charantia (bitter melon inhibits primary human adipocyte differentiation by modulating adipogenic genes

    Directory of Open Access Journals (Sweden)

    Nerurkar Vivek R

    2010-06-01

    Full Text Available Abstract Background Escalating trends of obesity and associated type 2 diabetes (T2D has prompted an increase in the use of alternative and complementary functional foods. Momordica charantia or bitter melon (BM that is traditionally used to treat diabetes and complications has been demonstrated to alleviate hyperglycemia as well as reduce adiposity in rodents. However, its effects on human adipocytes remain unknown. The objective of our study was to investigate the effects of BM juice (BMJ on lipid accumulation and adipocyte differentiation transcription factors in primary human differentiating preadipocytes and adipocytes. Methods Commercially available cryopreserved primary human preadipocytes were treated with and without BMJ during and after differentiation. Cytotoxicity, lipid accumulation, and adipogenic genes mRNA expression was measured by commercial enzymatic assay kits and semi-quantitative RT-PCR (RT-PCR. Results Preadipocytes treated with varying concentrations of BMJ during differentiation demonstrated significant reduction in lipid content with a concomitant reduction in mRNA expression of adipocyte transcription factors such as, peroxisome proliferator-associated receptor γ (PPARγ and sterol regulatory element-binding protein 1c (SREBP-1c and adipocytokine, resistin. Similarly, adipocytes treated with BMJ for 48 h demonstrated reduced lipid content, perilipin mRNA expression, and increased lipolysis as measured by the release of glycerol. Conclusion Our data suggests that BMJ is a potent inhibitor of lipogenesis and stimulator of lipolysis activity in human adipocytes. BMJ may therefore prove to be an effective complementary or alternative therapy to reduce adipogenesis in humans.

  15. Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

    Science.gov (United States)

    Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

    2009-01-01

    Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

  16. Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers

    DEFF Research Database (Denmark)

    Loft, Anne; Forss, Isabel; Siersbæk, Majken Storm

    2015-01-01

    Long-term exposure to peroxisome proliferator-activated receptor γ (PPARγ) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone...... reprogramming of PPARγ binding, leading to the formation of PPARγ "superenhancers" that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel......-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARγ and appears to cooperate with PPARγ in a feed-forward manner to activate...

  17. Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate.

    Science.gov (United States)

    Ždychová, J; Čejková, S; Králová Lesná, I; Králová, A; Malušková, J; Janoušek, L; Kazdová, L

    2014-01-01

    The abnormal proliferation of vascular smooth muscle cells (VSMC) is thought to play a role in the pathogenesis of atherosclerosis. Adipocytes produce several bioactive paracrine substances that can affect the growth and migration of VSMCs. Our study focuses on the direct effect of the bioactive substances in conditioned media (CM) that was obtained by incubation with primary adipocyte-derived cell lines, including cell lines derived from both preadipocytes and from more mature cells, on the proliferation rate of human aortic smooth muscle cells (HAoSMCs). We used a Luminex assay to measure the adipokine content of the CM and showed that there was a higher concentration of monocyte chemoattractant protein-1 in renal preadipocyte-CM compared with the HAoSMC control (p<0.5). The addition of both renal preadipocyte- and epicardial adipocyte- CM resulted in the elevated production of vascular endothelial growth factor compared with the control HASoSMC CM (p<0.001). The adiponectin content in renal adipocyte-CM was increased compared to all the remaining adipocyte-CM (p<0.01). Moreover, the results showed a higher proliferation rate of HAoSMCs after co-culture with epicardial adipocyte-CM compared to the HAoSMC control (p<0.05). These results suggest that bioactive substances produced by adipocytes have a stimulatory effect on the proliferation of VSMCs.

  18. Adeno-Associated Viral Vectors Transduce Mature Human Adipocytes in Three-Dimensional Slice Cultures.

    Science.gov (United States)

    Kallendrusch, Sonja; Schopow, Nikolas; Stadler, Sonja C; Büning, Hildegard; Hacker, Ulrich T

    2016-10-01

    Adipose tissue plays a pivotal role, both in the regulation of energy homeostasis and as an endocrine organ. Consequently, adipose tissue dysfunction is closely related to insulin resistance, morbid obesity, and metabolic syndrome. To study molecular mechanisms and to develop novel therapeutic strategies, techniques are required to genetically modify mature adipocytes. Here, we report on adeno-associated viral (AAV) vectors as a versatile tool to transduce human mature adipocytes in organotypic three-dimensional tissue cultures.

  19. Interleukin-1β mediates macrophage-induced impairment of insulin signaling in human primary adipocytes.

    Science.gov (United States)

    Gao, Dan; Madi, Mohamed; Ding, Cherlyn; Fok, Matthew; Steele, Thomas; Ford, Christopher; Hunter, Leif; Bing, Chen

    2014-08-01

    Adipose tissue expansion during obesity is associated with increased macrophage infiltration. Macrophage-derived factors significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. Identification of the major factors that mediate detrimental effects of macrophages on adipocytes may offer potential therapeutic targets. IL-1β, a proinflammatory cytokine, is suggested to be involved in the development of insulin resistance. This study investigated the role of IL-1β in macrophage-adipocyte cross-talk, which affects insulin signaling in human adipocytes. Using macrophage-conditioned (MC) medium and human primary adipocytes, we examined the effect of IL-1β antagonism on the insulin signaling pathway. Gene expression profile and protein abundance of insulin signaling molecules were determined, as was the production of proinflammatory cytokine/chemokines. We also examined whether IL-1β mediates MC medium-induced alteration in adipocyte lipid storage. MC medium and IL-1β significantly reduced gene expression and protein abundance of insulin signaling molecules, including insulin receptor substrate-1, phosphoinositide 3-kinase p85α, and glucose transporter 4 and phosphorylation of Akt. In contrast, the expression and release of the proinflammatory markers, including IL-6, IL-8, monocyte chemotactic protein-1, and chemokine (C-C motif) ligand 5 by adipocytes were markedly increased. These changes were significantly reduced by blocking IL-1β activity, its receptor binding, or its production by macrophages. MC medium-inhibited expression of the adipogenic factors and -stimulated lipolysis was also blunted with IL-1β neutralization. We conclude that IL-1β mediates, at least in part, the effect of macrophages on insulin signaling and proinflammatory response in human adipocytes. Blocking IL-1β could be beneficial for preventing obesity-associated insulin resistance and inflammation in human adipose tissue. Copyright

  20. Random Secretion of Growth Hormone in Humans

    Science.gov (United States)

    Prank, Klaus; Kloppstech, Mirko; Nowlan, Steven J.; Sejnowski, Terrence J.; Brabant, Georg

    1996-08-01

    In normal humans, growth hormone (GH) is secreted from a gland located adjacent to the brain (pituitary) into the blood in distinct pulses, but in patients bearing a tumor within the pituitary (acromegaly) GH is excessively secreted in an irregular manner. It has been hypothesized that GH secretion in the diseased state becomes random. This hypothesis is supported by demonstrating that GH secretion in patients with acromegaly cannot be distinguished from a variety of linear stochastic processes based on the predictability of the fluctuations of GH concentration in the bloodstream.

  1. Human aquaporin-11 is a water and glycerol channel and localizes in the vicinity of lipid droplets in human adipocytes.

    Science.gov (United States)

    Madeira, Ana; Fernández-Veledo, Sonia; Camps, Marta; Zorzano, Antonio; Moura, Teresa F; Ceperuelo-Mallafré, Victoria; Vendrell, Joan; Soveral, Graça

    2014-09-01

    For a long time Aquaporin-7 has been the only aquaporin associated with the adipose tissue, and its dysregulation has been linked to the underlying mechanisms of obesity. However, the presence of alternative glycerol channels within the adipose tissue has been postulated, which has prompted us to the search of alternate glycerol transport routes in adipocytes. In view of this, it is hypothesized that Aquaporin-11 (AQP11) would have a role in adipocyte cell biology. The expression, the localization and the function of human AQP11 (hAQP11) in cultured differentiated adipocytes were investigated. Gene expression analysis revealed the presence of AQP11 in both subcutaneous and visceral human mature adipocytes. It is found that hAQP11 is primarily located intracellularly in human adipocytes and partially colocalizes with perilipin, pointing towards AQP11 preferential location in the vicinity of lipid droplets. Overexpression of hAQP11 in 3T3-L1 adipocytes enabled to validate its function as a water channel and reveal its glycerol permeation activity. hAQP11 permeates both water and glycerol, localizing in the vicinity of lipid droplets in human adipocytes. © 2014 The Obesity Society.

  2. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells.

    Science.gov (United States)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

    2008-02-15

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor gamma (PPARgamma) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPARgamma agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPARgamma-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.

  3. Pluripotent stem cells derived from mouse and human white mature adipocytes.

    Science.gov (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R; Shahmirian, Laurine J; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A; Yao, Yucheng; Boström, Kristina I

    2014-02-01

    White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5-7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies.

  4. Effects of Two Different Rhodiola rosea Extracts on Primary Human Visceral Adipocytes

    Directory of Open Access Journals (Sweden)

    Elena Pomari

    2015-05-01

    Full Text Available Rhodiola rosea (Rro has been reported to have various pharmacological properties, including anti-fatigue, anti-stress and anti-inflammatory activity. It is also known to improve glucose and lipid metabolism, but the effects of Rhodiola rosea on adipocyte differentiation and metabolism are not still elucidated. In this study the anti-adipogenic and lipolytic activity of two extracts of Rhodiola rosea, containing 3% salidroside (RS or 1% salidroside and 3% rosavines (RR on primary human visceral adipocytes was investigated. Pre-adipocytes were analyzed after 10 and 20 days of treatment during differentiation and after 7 days of treatment when they reached mature shape. The RS extract significantly induced higher apoptosis and lipolysis in comparison to control cells and to RR extract. In contrast, RR extract significantly reduced triglyceride incorporation during maturation. Differentiation of pre-adipocytes in the presence of RS and RR extracts showed a significant decrease in expression of genes involved in adipocyte function such as SLC2A4 and the adipogenic factor FGF2 and significant increase in expression of genes involved in inhibition of adipogenesis, such as GATA3, WNT3A, WNT10B. Furthermore RR extract, in contrast to RS, significantly down-regulates PPARG, the master regulator of adipogenesis and FABP4. These data support the lipolytic and anti-adipogenetic activity of two different commercial extracts of Rhodiola rosea in primary human visceral pre-adipocytes during differentiation.

  5. Adipocyte property evaluation with photoacoustic spectrum analysis: a feasibility study on human tissues

    Science.gov (United States)

    Cao, Meng; Zhu, Yunhao; O'Rourke, Robert; Wang, Huaideng; Yuan, Jie; Cheng, Qian; Xu, Guan; Wang, Xueding; Carson, Paul

    2017-03-01

    Photoacoustic spectrum analysis (PASA) offers potential advantages in identifying optically absorbing microstructures in biological tissues. Working at high ultrasound frequency, PASA is capable of identifying the morphological features of cells based on their intrinsic optical absorption. Adipocyte size is correlated with metabolic disease risk in the form of diabetes mellitus, thus it can be adopted as a pathology predictor to evaluate the condition of obese patient, and can be helpful for assessing the patient response to bariatric surgery. In order to acquire adipocyte size, usually adipose tissue biopsy is performed and histopathology analysis is conducted. The whole procedure is not well tolerated by patients, and is also labor and cost intensive. An unmet need is to quantify and predict adipocyte size in a mild and more efficient way. This work aims at studying the feasibility to analyze the adipocyte size of human fat tissue using the method of PASA. PA measurements were performed at the optical wavelength of 1210 nm where lipid has strong optical absorption, enabling the study of adipocyte without need of staining. Both simulation and ex vivo experiments have been completed. Good correlation between the quantified photoacoustic spectral parameter slope and the average adipocyte size obtained by the gold-standard histology has been established. This initial study suggests the potential opportunity of applying PASA to future clinical management of obesity.

  6. The eicosapentaenoic acid metabolite 15-deoxy-δ(12,14-prostaglandin J3 increases adiponectin secretion by adipocytes partly via a PPARγ-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Jennifer Lefils-Lacourtablaise

    Full Text Available The intake of ω-3 polyunsaturated fatty acids (PUFAs, which are abundant in marine fish meat and oil, has been shown to exert many beneficial effects. The mechanisms behind those effects are numerous, including interference with the arachidonic acid cascade that produces pro-inflammatory eicosanoids, formation of novel bioactive lipid mediators, and change in the pattern of secreted adipocytokines. In our study, we show that eicosapentaenoic acid (EPA increases secreted adiponectin from 3T3-L1 adipocytes and in plasma of mice as early as 4 days after initiation of an EPA-rich diet. Using 3T3-L1 adipocytes, we report for the first time that 15-deoxy-δ(12,14-PGJ3 (15d-PGJ3, a product of EPA, also increases the secretion of adiponectin. We demonstrate that the increased adiponectin secretion induced by 15d-PGJ3 is partially peroxisome proliferator-activated receptor-gamma (PPAR-γ-mediated. Finally, we show that 3T3-L1 adipocytes can synthesize 15d-PGJ3 from EPA. 15d-PGJ3 was also detected in adipose tissue from EPA-fed mice. Thus, these studies provide a novel mechanism(s for the therapeutic benefits of ω-3 polyunsaturated fatty acids dietary supplementation.

  7. Impact of metabolic regulators on the expression of the obesity associated genes FTO and NAMPT in human preadipocytes and adipocytes.

    Directory of Open Access Journals (Sweden)

    Daniela Friebe

    Full Text Available BACKGROUND: FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators. METHODOLOGY AND PRINCIPAL FINDINGS: We assessed FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively. CONCLUSION: FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes.

  8. Impact of Metabolic Regulators on the Expression of the Obesity Associated Genes FTO and NAMPT in Human Preadipocytes and Adipocytes

    Science.gov (United States)

    Schönberg, Maria; Bernhard, Falk; Büttner, Petra; Landgraf, Kathrin; Kiess, Wieland; Körner, Antje

    2011-01-01

    Background FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators. Methodology and Principal Findings We assessed FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively. Conclusion FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes. PMID:21687707

  9. Role of calcitriol and cortisol on human adipocyte proliferation and oxidative and inflammatory stress: a microarray study.

    Science.gov (United States)

    Sun, Xiaocun; Morris, Kristin L; Zemel, Michael B

    2008-01-01

    Dietary calcium inhibits adiposity, and a key underlying mechanism is suppression of calcitriol, which modulates Ca(2+) signaling and mitochondrial uncoupling in adipocytes. We demonstrated that calcitriol directly regulates adipocyte 11beta-HSD-1 expression and cortisol production in human adipocytes in vitro and dietary calcium inhibits visceral adipose tissue 11beta-HSD-1 expression in mice, indicating an interaction of calcitriol and cortisol in obesity. Consequently, we have evaluated the gene expression profile of human subcutaneous adipocytes treated with calcitriol and/or cortisone. Data analysis demonstrated significant calcitriol modulation of gene expression toward inhibition of the adipocyte apoptosis (e.g., VEGF and STC-2) and promotion of adipocyte proliferation (e.g., IGF-1 and IGF-1R). Calcitriol also up-regulated oxidative stress and inflammatory genes such as NOX-4 and TLR-3. The calcitriol/cortisone combination resulted in significant additional up-regulation of 11beta-HSD-1 and down-regulation of adiponectin expression, while cortisone exerted little independent effect in the absence of calcitriol. Overall, calcitriol stimulated a pattern of adipocyte gene expression which favored adipocyte proliferation, oxidative and inflammatory stress and visceral adiposity, and these effects were amplified in the presence of cortisone; however, this conclusion must be tempered by the adipocyte source (subcutaneous) and requires confirmation in visceral adipocytes. Copyright 2007 S. Karger AG, Basel.

  10. Endothelin-1 stimulates human adipocyte lipolysis through the ET A receptor.

    Science.gov (United States)

    Eriksson, A K S; van Harmelen, V; Stenson, B M; Aström, G; Wåhlén, K; Laurencikiene, J; Rydén, M

    2009-01-01

    Levels of the vascular peptide endothelin-1 (ET-1) are significantly elevated in obesity. Adipose tissue-derived ET-1 attenuates insulin-mediated antilipolysis in human visceral adipocytes through the activation of the ET receptor B (ET(B)R), thereby linking ET-1 to insulin resistance. Whether ET-1 has direct effects on lipolysis in human adipocytes is not known. Endothelin-1 receptor (ETR) mRNA expression was determined by quantitative PCR in 130 non-obese and obese subjects. ET-1 mRNA in different adipose tissue regions was also assessed. ETR protein expression was analyzed by western blotting in 37 subjects. The effect of ET-1 on lipolysis was assessed in freshly isolated adipocytes and in vitro differentiated adipocytes from human donors. Freshly isolated human adipocytes incubated with different concentrations of ET-1 showed no acute effect on lipolysis. In contrast, a 24 h incubation in primary cultures of human adipocytes resulted in a significant 50% increase in lipolysis. This effect was concentration dependent and could be mimicked by an agonist of the ET(A) receptor but not with a selective ET(B)R agonist. Adipocyte differentiation was not affected by any of the agonists. In subcutaneous (s.c.) adipose tissue from 19 non-obese and 18 obese subjects, the protein expression of ET(A)R was significantly higher in obese subjects whereas there was no difference in ET(B)R expression. Interestingly, the differences in protein expression were not observed at the mRNA level as ET(A)R expression was similar between lean and obese subjects. Long-term but not acute incubation of human adipocytes with ET-1 results in a significant increase in lipolysis. This appears to be mediated through the activation of ET(A)R, demonstrating a yet another receptor-specific effect of ET-1. In addition, the protein expression of ET(A)R is increased in s.c. adipose tissue in obesity, possibly through post-transcriptional mechanisms. An increased effect of ET-1 could be a mechanism

  11. Differentiation of human adipose stromal cells in vitro into insulin-sensitive adipocytes.

    Science.gov (United States)

    Huttala, Outi; Mysore, R; Sarkanen, J R; Heinonen, T; Olkkonen, V M; Ylikomi, T

    2016-10-01

    Adipose tissue-related diseases such as obesity and type 2 diabetes are worldwide epidemics. In order to develop adipose tissue cultures in vitro that mimic more faithfully the in vivo physiology, new well-characterized and publicly accepted differentiation methods of human adipose stem cells are needed. The aims of this study are (1) to improve the existing natural adipose tissue extract (ATE)-based induction method and (2) to study the effects of a differentiation method on insulin responsiveness of the resulting adipocytes. Different induction media were applied on human adipose stromal cell (hASC) monocultures to study the differentiation capacity of the induction media and the functionality of the differentiated adipocytes. Cells were differentiated for 14 days to assess triglyceride accumulation per cell and adipocyte-specific gene expression (PPARγ, adiponectin, AP2, leptin, Glut4, Prdm16, CIDEA, PGC1-α, RIP140, UCP and ADCY5). Insulin response was studied by measuring glucose uptake and inhibition of lipolysis after incubation with 100 or 500 nM insulin. The selected differentiation method included a 3-day induction with ATE, 6 days in serum-free medium supplemented with 1.15 μM insulin and 9.06 μM Troglitazone, followed by 4 days in a defined serum- and insulin-free stimulation medium. This protocol induced prominent general adipocyte gene expression, including markers for both brown and white adipocytes and triglyceride accumulation. Moreover, the cells were sensitive to insulin as observed from increased glucose uptake and inhibition of lipolysis. This differentiation protocol provides a promising approach for the induction of hASC adipogenesis to obtain functional and mature human adipocytes.

  12. Creating capillary networks within human engineered tissues: impact of adipocytes and their secretory products.

    Science.gov (United States)

    Aubin, Kim; Vincent, Caroline; Proulx, Maryse; Mayrand, Dominique; Fradette, Julie

    2015-01-01

    The development of tissue-engineered substitutes of substantial volume is closely associated with the need to ensure rapid vascularization upon grafting. Strategies promoting angiogenesis include the in vitro formation of capillary-like networks within engineered substitutes. We generated both connective and adipose tissues based on a cell sheet technology using human adipose-derived stromal cells. This study evaluates the morphology and extent of the capillary networks that developed upon seeding of human microvascular endothelial cells during tissue production. We posited that adipocyte presence/secretory products could modulate the resulting capillary network when compared to connective substitutes. Analyses including confocal imaging of CD31-labeled capillary-like networks indicated slight differences in their morphological appearance. However, the total volume occupied by the networks as well as the frequency distribution of the structure's volumes were similar between connective and adipose tissues. The average diameter of the capillary structures tended to be 20% higher in reconstructed adipose tissues. Quantification of pro-angiogenic molecules in conditioned media showed greater amounts of leptin (15×), angiopoietin-1 (3.4×) and HGF (1.7×) secreted from adipose than connective tissues at the time of endothelial cell seeding. However, this difference was attenuated during the following coculture period in endothelial cell-containing media, correlating with the minor differences noted between the networks. Taken together, we developed a protocol allowing reconstruction of both connective and adipose tissues featuring well-developed capillary networks in vitro. We performed a detailed characterization of the network architecture within engineered tissues that is relevant for graft assessment before implantation as well as for in vitro screening of angiogenic modulators using three-dimensional models. Copyright © 2014 Acta Materialia Inc. Published by

  13. Bisphenol-A impairs insulin action and up-regulates inflammatory pathways in human subcutaneous adipocytes and 3T3-L1 cells.

    Science.gov (United States)

    Valentino, Rossella; D'Esposito, Vittoria; Passaretti, Federica; Liotti, Antonietta; Cabaro, Serena; Longo, Michele; Perruolo, Giuseppe; Oriente, Francesco; Beguinot, Francesco; Formisano, Pietro

    2013-01-01

    Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1 nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

  14. Bisphenol-A impairs insulin action and up-regulates inflammatory pathways in human subcutaneous adipocytes and 3T3-L1 cells.

    Directory of Open Access Journals (Sweden)

    Rossella Valentino

    Full Text Available Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1 nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

  15. Thermogenic activity of UCP1 in human white fat-derived beige adipocytes.

    Science.gov (United States)

    Bartesaghi, Stefano; Hallen, Stefan; Huang, Li; Svensson, Per-Arne; Momo, Remi A; Wallin, Simonetta; Carlsson, Eva K; Forslöw, Anna; Seale, Patrick; Peng, Xiao-Rong

    2015-01-01

    Heat-producing beige/brite (brown-in-white) adipocytes in white adipose tissue have the potential to suppress metabolic disease in mice and hold great promise for the treatment of obesity and type 2 diabetes in humans. Here, we demonstrate that human adipose-derived stromal/progenitor cells (hASCs) from subcutaneous white adipose tissue can be efficiently converted into beige adipocytes. Upon pharmacological activation of peroxisome proliferator-activated receptor-γ, hASC-derived adipocytes activated beige fat-selective genes and a brown/beige fat-selective electron transport chain gene program. Importantly, hASC-derived beige fat cells displayed the bioenergetic characteristics of genuine brown fat cells, including a capacity for increased respiratory uncoupling in response to β-adrenergic agonists. Furthermore, knock-down experiments reveal that the thermogenic capacity of human beige fat cells was entirely dependent on the presence of Uncoupling protein 1. In summary, this study reveals that hASCs can be readily differentiated into beige adipocytes that, upon activation, undergo uncoupling protein 1-dependent thermogenesis.

  16. Inhibition of fatty acid biosynthesis prevents adipocyte lipotoxicity on human osteoblasts in vitro.

    Science.gov (United States)

    Elbaz, Alexandre; Wu, Xiying; Rivas, Daniel; Gimble, Jeffrey M; Duque, Gustavo

    2010-04-01

    Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell-cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase.

  17. Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes.

    Science.gov (United States)

    Bour, Sandy; Daviaud, Danièle; Gres, Sandra; Lefort, Corinne; Prévot, Danielle; Zorzano, Antonio; Wabitsch, Martin; Saulnier-Blache, Jean-Sébastien; Valet, Philippe; Carpéné, Christian

    2007-08-01

    A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.

  18. Ursodeoxycholic acid but not tauroursodeoxycholic acid inhibits proliferation and differentiation of human subcutaneous adipocytes.

    Directory of Open Access Journals (Sweden)

    Lucia Mališová

    Full Text Available Stress of endoplasmic reticulum (ERS is one of the molecular triggers of adipocyte dysfunction and chronic low inflammation accompanying obesity. ERS can be alleviated by chemical chaperones from the family of bile acids (BAs. Thus, two BAs currently used to treat cholestasis, ursodeoxycholic and tauroursodeoxycholic acid (UDCA and TUDCA, could potentially lessen adverse metabolic effects of obesity. Nevertheless, BAs effects on human adipose cells are mostly unknown. They could regulate gene expression through pathways different from their chaperone function, namely through activation of farnesoid X receptor (FXR and TGR5, G-coupled receptor. Therefore, this study aimed to analyze effects of UDCA and TUDCA on human preadipocytes and differentiated adipocytes derived from paired samples of two distinct subcutaneous adipose tissue depots, abdominal and gluteal. While TUDCA did not alter proliferation of cells from either depot, UDCA exerted strong anti-proliferative effect. In differentiated adipocytes, acute exposition to neither TUDCA nor UDCA was able to reduce effect of ERS stressor tunicamycin. However, exposure of cells to UDCA during whole differentiation process decreased expression of ERS markers. At the same time however, UDCA profoundly inhibited adipogenic conversion of cells. UDCA abolished expression of PPARγ and lipogenic enzymes already in the early phases of adipogenesis. This anti-adipogenic effect of UDCA was not dependent on FXR or TGR5 activation, but could be related to ability of UDCA to sustain the activation of ERK1/2 previously linked with PPARγ inactivation. Finally, neither BAs did lower expression of chemokines inducible by TLR4 pathway, when UDCA enhanced their expression in gluteal adipocytes. Therefore while TUDCA has neutral effect on human preadipocytes and adipocytes, the therapeutic use of UDCA different from treating cholestatic diseases should be considered with caution because UDCA alters functions of

  19. Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α.

    Science.gov (United States)

    Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

    2015-01-01

    Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells

  20. Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α

    Science.gov (United States)

    Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

    2015-01-01

    Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells

  1. Derivation of Adipocytes from Human Endometrial Stem Cells (EnSCs)

    Science.gov (United States)

    Ai, Jafar; Shahverdi, Ahmad Reza; Barough, Somayeh Ebrahimi; Kouchesfehani, Homa Mohseni; Heidari, Saeed; Roozafzoon, Reza; Verdi, Javad; Khoshzaban, Ahad

    2012-01-01

    Background Due to increasing clinical demand for adipose tissue, a suitable cell for reconstructive adipose tissue constructs is needed. In this study, we investigated the ability of Human Endometrial-derived stem cells (EnSCs) as a new source of mesenchymal stem cells to differentiate into adipocytes. EnSCs are the abundant and easy available source with no immunological response, for cell replacement therapy. Methods Single-cell suspensions of EnSCs were obtained from endometrial tissues from 10 women experiencing normal menstrual cycles, and were cultured at clonal density (10 cells/cm 2) or limiting dilution. Endometrial mesenchymal stem cell markers were examined flow cytometry. These cells were treated with adipogenic-inducing medium for 28 days. The adipogenic differentiation of the EnSC was assessed by cellular morphology and further confirmed by Oil Red O staining and RT-PCR. The BM-MSC differentiated into adipocytes in the presence of adipogenic stimuli for 3 weeks. Results The flow cytometric analysis showed that the cells were positive for CD90, CD105, CD146 and were negative for CD31, CD34.We showed that the key adipocytes marker PPARa was expressed in mRNA level after 28 days post treatment (PT). Conclusion According to our finding, it can be concluded that EnSCs represent a useful in vitro model for human adipogenesis, and provide opportunities to study the stages prior to commitment to the adipocyte lineage. PMID:23926540

  2. Comparative expression analysis of isolated human adipocytes and the human adipose cell lines LiSa-2 and PAZ6

    NARCIS (Netherlands)

    Beek, van E.A.; Bakker, A.H.; Kruyt, P.M.; Vink, C.; Saris, W.H.; Keijer, J.

    2008-01-01

    Objective: To obtain insight in the extent to which the human cell lines LiSa-2 and PAZ6 resemble isolated primary human adipocytes. Design: A combination of cDNA subtraction (representative difference analysis; RDA) and cDNA microarray analysis was used to select adipose specific genes to compare i

  3. Human multipotent adipose-derived stem cells differentiate into functional brown adipocytes

    DEFF Research Database (Denmark)

    Elabd, Christian; Chiellini, Chiara; Carmona, Mamen

    2009-01-01

    adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells that exhibit the key properties of human white adipocytes, that is, uncoupling protein two expression, insulin-stimulated glucose uptake, lipolysis in response to beta......In contrast to the earlier contention, adult humans have been shown recently to possess active brown adipose tissue with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent......-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein, we show that, upon chronic exposure to a specific PPARgamma but not to a PPARbeta/delta or a PPARalpha agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both uncoupling protein one...

  4. St. John's Wort inhibits insulin signaling in murine and human adipocytes.

    Science.gov (United States)

    Richard, Allison J; Amini, Zhaleh J; Ribnicky, David M; Stephens, Jacqueline M

    2012-04-01

    Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.

  5. B Lymphocyte Stimulator (BLyS) is expressed in human adipocytes in vivo and is related to obesity but not to insulin resistance.

    Science.gov (United States)

    Müller, Nike; Schulte, Dominik M; Hillebrand, Susann; Türk, Kathrin; Hampe, Jochen; Schafmayer, Clemens; Brosch, Mario; von Schönfels, Witigo; Ahrens, Markus; Zeuner, Rainald; Schröder, Johann O; Blüher, Matthias; Gutschow, Christian; Freitag-Wolf, Sandra; Stelmach-Mardas, Marta; Saggau, Carina; Schreiber, Stefan; Laudes, Matthias

    2014-01-01

    Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (pr = 0.43, panti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.

  6. Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Sandra C. [Translational Sciences - Translational Medicine, Novartis Institutes for Biomedical Research, Inc., 220 Massachusetts Avenue, Cambridge, MA 02139 (United States); Chau, Mary D.L.; Yang, Qing [Cardiovascular and Metabolism Disease Area, Novartis Institutes for Biomedical Research, Inc., 100 Technology Square, Cambridge, MA 02139 (United States); Gauthier, Marie-Soleil [Department of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, MA 02140 (United States); Clairmont, Kevin B.; Wu, Zhidan; Gromada, Jesper [Cardiovascular and Metabolism Disease Area, Novartis Institutes for Biomedical Research, Inc., 100 Technology Square, Cambridge, MA 02139 (United States); Dole, William P., E-mail: bill.dole@novartis.com [Translational Sciences - Translational Medicine, Novartis Institutes for Biomedical Research, Inc., 220 Massachusetts Avenue, Cambridge, MA 02139 (United States)

    2011-07-08

    Highlights: {yields} Treatment of differentiated human adipocytes with atrial natriuretic peptide (ANP) increased lipolysis and oxygen consumption by activating AMP-activated protein kinase (AMPK). {yields} ANP stimulated lipid mobilization by selective activation of the alpha2 subunit of AMPK and increased energy utilization through activation of both the alpha1 and alpha2 subunits of AMPK. {yields} ANP enhanced adipocyte mitochondrial oxidative capacity as evidenced by induction of oxidative mitochondrial genes and increase in oxygen consumption. {yields} Exposure of human adipocytes to fatty acids and (TNF{alpha}) induced insulin resistance and decreased expression of mitochondrial genes which was restored to normal by ANP. -- Abstract: Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation. Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and

  7. Metabolic interplay between white, beige, brown adipocytes and the liver.

    Science.gov (United States)

    Scheja, Ludger; Heeren, Joerg

    2016-05-01

    In mammalian evolution, three types of adipocytes have developed, white, brown and beige adipocytes. White adipocytes are the major constituents of white adipose tissue (WAT), the predominant store for energy-dense triglycerides in the body that are released as fatty acids during catabolic conditions. The less abundant brown adipocytes, the defining parenchymal cells of brown adipose tissue (BAT), internalize triglycerides that are stored intracellularly in multilocular lipid droplets. Beige adipocytes (also known as brite or inducible brown adipocytes) are functionally very similar to brown adipocytes and emerge in specific WAT depots in response to various stimuli including sustained cold exposure. The activation of brown and beige adipocytes (together referred to as thermogenic adipocytes) causes both the hydrolysis of stored triglycerides as well as the uptake of lipids and glucose from the circulation. Together, these fuels are combusted for heat production to maintain body temperature in mammals including adult humans. Given that heating by brown and beige adipocytes is a very-well controlled and energy-demanding process which entails pronounced shifts in energy fluxes, it is not surprising that an intensive interplay exists between the various adipocyte types and parenchymal liver cells, and that this influences systemic metabolic fluxes and endocrine networks. In this review we will emphasize the role of hepatic factors that regulate the metabolic activity of white and thermogenic adipocytes. In addition, we will discuss the relevance of lipids and hormones that are secreted by white, brown and beige adipocytes regulating liver metabolism in order to maintain systemic energy metabolism in health and disease.

  8. The acute phase protein Serum Amyloid A induces lipolysis and inflammation in human adipocytes through distinct pathways.

    Directory of Open Access Journals (Sweden)

    Aurélie Faty

    Full Text Available BACKGROUND: The acute phase response (APR is characterized by alterations in lipid and glucose metabolism leading to an increased delivery of energy substrates. In adipocytes, there is a coordinated decrease in Free Fatty acids (FFAs and glucose storage, in addition to an increase in FFAs mobilization. Serum Amyloid A (SAA is an acute phase protein mainly associated with High Density Lipoproteins (HDL. We hypothesized that enrichment of HDL with SAA, during the APR, could be implicated in the metabolic changes occurring in adipocytes. METHODOLOGY/PRINCIPAL FINDINGS: In vitro differentiated human adipocytes (hMADS were treated with SAA enriched HDL or recombinant SAA and the metabolic phenotype of the cells analyzed. In hMADS, SAA induces an increased lipolysis through an ERK dependent pathway. At the molecular level, SAA represses PPARγ2, C/EBPα and SREBP-1c gene expression, three transcription factors involved in adipocyte differentiation or lipid synthesis. In addition, the activation of the NF-κB pathway by SAA leads to the induction of pro-inflammatory cytokines and chemokines, as in the case of immune cells. These latter findings were replicated in freshly isolated mature human adipocytes. CONCLUSIONS/SIGNIFICANCE: Besides its well-characterized role in cholesterol metabolism, SAA has direct metabolic effects on human adipocytes. These metabolic changes could be at least partly responsible for alterations of adipocyte metabolism observed during the APR as well as during pathophysiological conditions such as obesity and conditions leading to insulin resistant states.

  9. Combined effects of genistein, quercetin, and resveratrol in human and 3T3-L1 adipocytes.

    Science.gov (United States)

    Park, Hea Jin; Yang, Jeong-Yeh; Ambati, Suresh; Della-Fera, Mary Anne; Hausman, Dorothy B; Rayalam, Srujana; Baile, Clifton A

    2008-12-01

    The natural compounds genistein (G), quercetin (Q), and resveratrol (R) have been reported to each exhibit anti-adipogenic activities in adipocytes and antiproliferative and pro-apoptotic activities in several cell types. We studied the combined effects of G, Q, and R on adipogenesis and apoptosis in primary human adipocytes (HAs) and 3T3-L1 murine adipocyte (MAs). Combined treatment with 6.25 microM G, 12.5 microM Q, and 12.5 microM R during the 14-day differentiation period caused an enhanced inhibition of lipid accumulation in maturing HAs that was greater than the responses to individual compounds and to the calculated additive response. Glycerol 3-phosphate dehydrogenase activity, a marker of late adipocyte differentiation, was decreased markedly in HAs treated with the combination of G+Q+R. In addition, combined treatment with 50 microM G, 100 microM Q, and 100 microM R for 3 days decreased cell viability and induced apoptosis in early- and mid- phase maturing and lipid-filled mature HAs. In contrast, no compound alone induced apoptosis. Oil Red O stain and Hoechst 33342 stain were performed to confirm the effects on lipid accumulation and apoptosis, respectively. We also determined whether MAs responded to the combination treatment similarly to HAs. As in HAs, G+Q+R treatment decreased lipid accumulation in maturing MAs and increased apoptosis in pre- and lipid-filled mature MAs more than the responses to G, Q, and R when used separately. These results show that lower concentrations of combined treatments with several natural compounds may be useful for treatments for obesity through the suppression of adipogenesis and enhanced adipocyte apoptosis.

  10. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    Science.gov (United States)

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction.

  11. THP-1 macrophages and SGBS adipocytes - a new human in vitro model system of inflamed adipose tissue

    Directory of Open Access Journals (Sweden)

    Michaela eKeuper

    2011-12-01

    Full Text Available Obesity is associated with an accumulation of macrophages in adipose tissue. This inflammation of adipose tissue is a key event in the pathogenesis of several obesity-related disorders, particularly insulin resistance.Here, we summarized existing model systems that mimic the situation of inflamed adipose tissue in vitro, most of them being murine. Importantly, we introduce our newly established human model system which combines the THP-1 monocytic cell line and the preadipocyte cell strain SGBS. THP-1 cells, which originate from an acute monocytic leukemia, differentiate easily into macrophages in vitro. The human preadipocyte cell strain SGBS (Simpson-Golabi-Behmel syndrome was recently introduced as a unique to tool to study human fat cell functions. SGBS cells are characterized by a high capacity for adipogenic differentiation. SGBS adipocytes are capable of fat cell-specific metabolic functions such as insulin-stimulated glucose uptake, insulin-stimulated de novo lipogenesis and beta-adrenergic-stimulated lipolysis and they secrete typical adipokines including leptin, adiponectin, and RBP4. Applying either macrophage-conditioned medium or a direct co-culture of macrophages and fat cells, our model system can be used to distinguish between paracrine and cell-contact dependent effects.In conclusion, we propose this model as a useful tool to study adipose inflammation in vitro. It represents an inexpensive, highly reproducible human system. The methods described here can be easily extended for usage of primary human macrophages and fat cells.

  12. C-peptide modifies leptin and visfatin secretion in human adipose tissue.

    Science.gov (United States)

    Garcia-Serrano, Sara; Gutiérrez-Repiso, Carolina; Gonzalo, Montserrat; Garcia-Arnes, Juan; Valdes, Sergio; Soriguer, Federico; Perez-Valero, Vidal; Alaminos-Castillo, Miguel A; Francisco Cobos-Bravo, Juan; Moreno-Ruiz, Francisco J; Rodriguez-Cañete, Alberto; Rodríguez-Pacheco, Francisca; Garcia-Escobar, Eva; García-Fuentes, Eduardo

    2015-08-01

    The effects of C-peptide on adipose tissue, an organ involved in the development of obesity and insulin resistance, are not yet well known. The aim of this study was to determine whether C-peptide could be involved in the regulation of the adipocytokine synthesis in human visceral adipose tissue. The association between C-peptide and different serum adipocytokines, with an intravenous glucose tolerance test (IVGTT), and in an in vitro study in subjects without obesity and in subjects with morbid obesity were analyzed. In different multiple regression analysis models, C-peptide and C-peptide increase above basal levels during total IVGTT and between 0 and 10 min were associated positively with leptin and negatively with visfatin. Rhodamine-labeled C-peptide binds to human adipocytes, and this binding was blocked with excess of unlabeled C-peptide. Exposure of human visceral explants and adipocytes from subjects with morbid obesity to C-peptide at 1 and 10 nM induced a significant increase in leptin and a decrease in visfatin secretion. In subjects without obesity, these C-peptide effects were found mainly at 10 nM. These effects can be inhibited by phosphatidylinositol 3-kinase (PI3K) or protein kinase B (PKB) inhibitors. C-peptide may be involved in the regulation of leptin and visfatin secretion, molecules intimately involved in energy homeostasis processes, through PI3K or PKB pathways. © 2015 The Obesity Society.

  13. Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell-derived adipocytes.

    Science.gov (United States)

    Clabaut, Aline; Grare, Céline; Léger, Thibaut; Hardouin, Pierre; Broux, Odile

    2015-10-01

    One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.

  14. White Tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre-adipocytes

    Directory of Open Access Journals (Sweden)

    Wenck Horst

    2009-05-01

    Full Text Available Abstract Background The dramatic increase in obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. To investigate how natural substances influence lipolysis and adipogenesis, we determined the effects of White Tea extract on cultured human subcutaneous preadipocytes and adipocytes. Methods For our in vitro studies we used a White Tea extract solution that contained polyphenols and methylxanthines. Utilizing cultured human preadipocytes we investigated White Tea extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. In vitro studies on human adipocytes were performed aiming to elucidate the efficacy of White Tea extract solution to stimulate lipolytic activity. To characterize White Tea extract solution-mediated effects on a molecular level, we analyzed gene expression of essential adipogenesis-related transcription factors by qRT-PCR and determined the expression of the transcription factor ADD1/SREBP-1c on the protein level utilizing immunofluorescence analysis. Results Our data show that incubation of preadipocytes with White Tea extract solution significantly decreased triglyceride incorporation during adipogenesis in a dose-dependent manner (n = 10 without affecting cell viability (n = 10. These effects were, at least in part, mediated by EGCG (n = 10, 50 μM. In addition, White Tea extract solution also stimulated lipolytic activity in adipocytes (n = 7. Differentiating preadipocytes cultivated in the presence of 0.5% White Tea extract solution showed a decrease in PPARγ, ADD1/SREBP-1c, C/EBPα and C/EBPδ mRNA levels. Moreover, the expression of the transcription factor ADD1/SREBP-1c was not only decreased on the mRNA but also on the protein level. Conclusion White Tea extract is a natural source that effectively inhibits adipogenesis and stimulates lipolysis-activity. Therefore, it can be utilized to

  15. Tumor necrosis factor alpha and interleukin 11 secreted by malignant breast epithelial cells inhibit adipocyte differentiation by selectively down-regulating CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma: mechanism of desmoplastic reaction.

    Science.gov (United States)

    Meng, L; Zhou, J; Sasano, H; Suzuki, T; Zeitoun, K M; Bulun, S E

    2001-03-01

    /EBPbeta and C/EBPdelta transcripts. In these 3T3-L1 cells, inhibition of differentiation was also confirmed by markedly suppressed levels of aP2 mRNA, which is an adipocyte-specific gene. These in vitro observations were confirmed in sections of human malignant breast tumors, where immunoreactive C/EBPalpha was readily detectable in adipose flbroblasts distant to the tumor but not in intratumoral fibroblasts. Treatment of 3T3-L1 cells with T47D cell conditioned medium or TNF-alpha changed neither the numbers of cells in G0-G1, S, and G2 phases nor the rate of [3H]thymidine incorporation, thus ruling out a proliferative effect of malignant cells on the surrounding fibroblasts. In summary, desmoplastic reaction primarily occurs via the action of cytokines (TNF-alpha and IL-11) secreted by the malignant epithelial cells to inhibit differentiation of adipose fibroblasts to mature adipocytes. This tumor-induced block in adipocyte differentiation is mediated by the selective inhibition of expression of the essential adipogenic transcription factors, i.e., PPARgamma and C/EBPalpha.

  16. Human induced pluripotent stem cells: A new source for brown and white adipocytes

    Institute of Scientific and Technical Information of China (English)

    Anne-Laure; Hafner; Christian; Dani

    2014-01-01

    Mesenchymal stem cells(MSCs) derived from human induced pluripotent stem cells(hiPSCs) provide a novel source for generating adipocytes, thus opening new avenues for fundamental research and clinical medicine. We present the adipogenic potential of hiPSCs and the various methods to derive hiPSC-MSCs. We discuss the main characteristic of hiPSC-MSCs, which is their low adipogenic capacity as compared to adult-MSCs. Finally, we propose several hypotheses to explanation this feature, underlying a potential critical role of the micro-environment. We favour the hypothesis that the range of factors or culture conditions required to induce adipocyte differentiation of MSCs derived from adult tissues and from embryonic-like cells could differ.

  17. Dopaminergic Differentiation of Human Embryonic Stem Cells on PA6-Derived Adipocytes.

    Science.gov (United States)

    Guloglu, M Oktar; Larsen, Anna

    2016-01-01

    Human embryonic stem cells (hESCs) are a promising source for cell replacement therapies. Parkinson's disease is one of the candidate diseases for the cell replacement therapy since the motor manifestations of the disease are associated with the loss of dopaminergic neurons in the substantia nigra pars compacta. Stromal cell-derived inducing activity (SDIA) is the most commonly used method for the dopaminergic differentiation of hESCs. This chapter describes a simple, reliable, and scalable dopaminergic induction method of hESCs using PA6-derived adipocytes. Coculturing hESCs with PA6-derived adipocytes markedly reduces the variable outcomes among experiments. Moreover, the colony differentiation step of this method can also be used for the dopaminergic induction of mouse embryonic stem cells and NTERA2 cells as well.

  18. Anti-diabetic effects of Caulerpa lentillifera:stimulation of insulin secretion in pancreatic β-cells and enhancement of glucose uptake in adipocytes

    Institute of Scientific and Technical Information of China (English)

    Bhesh; Raj; Sharma; Dong; Young; Rhyu

    2014-01-01

    Objective:To evaluate anti-diabetic effect of Caulrpa kntillifera(C.lentillifera).Methods:The inhibitory effect of C.lentillifera extract on dipeptidyl peptidase-IV and a-glucosidase enzyme was measured in a cell free system.Then,interleukin-1βand interferon-γinduced cell death and insulin secretion were measured in rat insulinoma(RIN)cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA kit,respectively.Glucose uptake and glucose transporter expression were measured by fluorometry and western blotting,using 3T3-Ll adipocytes.Results:C.lentillifera extract significantly decreased dipeptidyl peptidase-IV and a-glucosidase enzyme activities,and effectively inhibited cell death and iNOS expression in interleukin-1βand interfcron-γinduced RIK cells.Furthermore,C.lntillifera extract significantly enhanced insulin secretion in RTN cells and glucose transporter expression and glucose uptake in 3T3-L1adipocytes.Conclusions:Thus,our results suggest that C.lentillifera could be used as a potential antidiabetic agenl.

  19. Anti-diabetic effects of Caulerpa lentillifera:stimulation of insulin secretion in pancreatic β-cells and enhancement of glucose uptake in adipocytes

    Institute of Scientific and Technical Information of China (English)

    Bhesh Raj Sharma; Dong Young Rhyu

    2014-01-01

    Objective: To evaluate anti-diabetic effect of Caulerpa lentillifera (C. lentillifera).Methods:The inhibitory effect of C. lentillifera extract on dipeptidyl peptidase-IV andα-glucosidase enzyme was measured in a cell free system. Then, interleukin-1β and interferon-γinduced cell death and insulin secretion were measured in rat insulinoma (RIN) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA kit, respectively. Glucose uptake and glucose transporter expression were measured by fluorometry and western blotting, using 3T3-L1 adipocytes.Results: C. lentillifera extract significantly decreased dipeptidyl peptidase-IV and α-glucosidase enzyme activities, and effectively inhibited cell death and iNOS expression in interleukin-1βand interferon-γ induced RIN cells. Furthermore, C. lentillifera extract significantly enhanced insulin secretion in RIN cells and glucose transporter expression and glucose uptake in 3T3-L1 adipocytes.Conclusions:Thus, our results suggest that C. lentillifera could be used as a potential anti-diabetic agent.

  20. Anti-diabetic effects of Caulerpa lentillifera: stimulation of insulin secretion in pancreatic β-cells and enhancement of glucose uptake in adipocytes.

    Science.gov (United States)

    Sharma, Bhesh Raj; Rhyu, Dong Young

    2014-07-01

    To evaluate anti-diabetic effect of Caulerpa lentillifera (C. lentillifera). The inhibitory effect of C. lentillifera extract on dipeptidyl peptidase-IV and α-glucosidase enzyme was measured in a cell free system. Then, interleukin-1β and interferon-γ induced cell death and insulin secretion were measured in rat insulinoma (RIN) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA kit, respectively. Glucose uptake and glucose transporter expression were measured by fluorometry and western blotting, using 3T3-L1 adipocytes. C. lentillifera extract significantly decreased dipeptidyl peptidase-IV and α-glucosidase enzyme activities, and effectively inhibited cell death and iNOS expression in interleukin-1β and interferon-γ induced RIN cells. Furthermore, C. lentillifera extract significantly enhanced insulin secretion in RIN cells and glucose transporter expression and glucose uptake in 3T3-L1 adipocytes. Thus, our results suggest that C. lentillifera could be used as a potential anti-diabetic agent.

  1. Endothelial differentiation in multipotent cells derived from mouse and human white mature adipocytes.

    Science.gov (United States)

    Jumabay, Medet; Abdmaulen, Raushan; Urs, Sumithra; Heydarkhan-Hagvall, Sepideh; Chazenbalk, Gregorio D; Jordan, Maria C; Roos, Kenneth P; Yao, Yucheng; Boström, Kristina I

    2012-12-01

    White mature adipocytes give rise to multipotent cells, so-called de-differentiated fat (DFAT) cells, when losing their fat in culture. The objective of this study was to examine the ability of DFAT cells to give rise to endothelial cells (ECs) in vitro and vivo. We demonstrate that mouse and human DFAT cells, derived from adipose tissue and lipospirate, respectively, initially lack expression of CD34, CD31, CD146, CD45 and pericyte markers, distinguishing them from progenitor cells previously identified in adipose stroma. The DFAT cells spontaneously differentiate into vascular ECs in vitro, as determined by real-time PCR, fluorescence activated cell sorting, immunostaining, and formation of tube structures. Treatment with bone morphogenetic protein (BMP)4 and BMP9, important in regulating angiogenesis, significantly enhances the EC differentiation. Furthermore, adipocyte-derived cells from Green Fluorescent Protein-transgenic mice were detected in the vasculature of infarcted myocardium up to 6 weeks after ligation of the left anterior descending artery in mice. We conclude that adipocyte-derived multipotent cells are able to spontaneously give rise to ECs, a process that is promoted by BMPs and may be important in cardiovascular regeneration and in physiological and pathological changes in fat and other tissues.

  2. LDL but not HDL increases adiponectin release of primary human adipocytes.

    Science.gov (United States)

    Krautbauer, Sabrina; Neumeier, Markus; Eisinger, Kristina; Hader, Yvonne; Dada, Ashraf; Schmitz, Gerd; Aslanidis, Charalampos; Buechler, Christa

    2013-12-01

    Adipocytes in obesity have inappropriately low cholesterol while adiponectin release is reduced. Cholesterol shortage may contribute to low adiponectin and 3T3-L1 cells treated with lovastatin have diminished adiponectin in cell supernatants. LDL and HDL deliver cholesterol to adipocytes. LDL but not HDL increases adiponectin in cell supernatants of primary human adipocytes. The effect of LDL is not blocked by receptor associated protein suggesting that members of the LDL-receptor family are not involved. To evaluate whether these in vitro observations translate into changes in systemic adiponectin, adiponectin was measured in serum of three patients before, immediately after and 3d after LDL-apheresis. Whereas circulating lipoproteins are reduced immediately after apheresis adiponectin is not changed. Therefore, acute lowering of lipoproteins does not affect systemic adiponectin also excluding that plenty of adiponectin is bound to lipoprotein particles. Accordingly, levels of adiponectin in purified lipoproteins are quite low. Familial hypobetalipoproteinemia (FHBL) is a rare disorder associated with low plasma LDL. Serum adiponectin is, however, similar compared to healthy controls. Thus, neither LDL nor HDL directly contributes to circulating adiponectin concentrations.

  3. B Lymphocyte Stimulator (BLyS is expressed in human adipocytes in vivo and is related to obesity but not to insulin resistance.

    Directory of Open Access Journals (Sweden)

    Nike Müller

    Full Text Available Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001. Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001 and are positively correlated to the BMI (r = 0.43, p<0.0002. In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.

  4. Differential roles of CIDEA and CIDEC in insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes.

    Science.gov (United States)

    Ito, Minoru; Nagasawa, Michiaki; Hara, Tomoko; Ide, Tomohiro; Murakami, Koji

    2010-07-01

    Both insulin and the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. However, regulation of the CIDE family by insulin and the contribution of the CIDE family to insulin actions remain unclear. Here, we investigated whether insulin regulates expression of the CIDE family and which subtypes contribute to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. Insulin decreased CIDEA and increased CIDEC but not CIDEB mRNA expression. Starvation-induced apoptosis in adipocytes was significantly inhibited when insulin decreased the CIDEA mRNA level. Small interfering RNA-mediated depletion of CIDEA inhibited starvation-induced apoptosis similarly to insulin and restored insulin deprivation-reduced adipocyte number, whereas CIDEC depletion did not. Lipid droplet size of adipocytes was increased when insulin increased the CIDEC mRNA level. In contrast, insulin-induced enlargement of lipid droplets was markedly abrogated by depletion of CIDEC but not CIDEA. Furthermore, depletion of CIDEC, but not CIDEA, significantly increased glycerol release from adipocytes. These results suggest that CIDEA and CIDEC are novel genes regulated by insulin in human adipocytes and may play key roles in the effects of insulin, such as anti-apoptosis and lipid droplet formation.

  5. PRDM16 sustains white fat gene expression profile in human adipocytes in direct relation with insulin action.

    Science.gov (United States)

    Moreno-Navarrete, José María; Ortega, Francisco; Moreno, María; Xifra, Gemma; Ricart, Wifredo; Fernández-Real, José Manuel

    2015-04-15

    In the present study, we aimed to evaluate the possible role of PRDM16 in human adipocytes and in whole adipose tissue according to obesity and insulin sensitivity. PRDM16 knockdown (KD) had a dual behavior. While KD in preadipocytes led to enhanced gene expression markers of adipocyte differentiation, PRDM16 KD in fully differentiated adipocytes resulted in decreased adipogenic gene expression and insulin action. In line with KD in adipocytes, PRDM16 was positively associated with the expression of several genes involved in adipogenesis, insulin signaling, mitochondrial function and brown adipocyte-related markers in whole adipose tissue from two independent cohorts. PRDM16 was decreased in obese subjects in relation with the decrease of insulin sensitivity [HOM(AIR) (cohort 1) and M clamp value (cohort 2)]. Rosiglitazone (5 µmol/l) and metformin (5 mmol/l) led to increased PRDM16 mRNA and protein levels in isolated human adipocytes and in whole adipose tissue. In conclusion, PRDM16 might contribute to maintain adipose tissue "white fat" gene expression profile and systemic metabolic homeostasis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Effects of glucose and insulin on secretion of amyloid-β by human adipose tissue cells.

    Science.gov (United States)

    Tharp, William G; Gupta, Dhananjay; Smith, Joshua; Jones, Karen P; Jones, Amanda M; Pratley, Richard E

    2016-07-01

    Obesity and type 2 diabetes mellitus are risk factors for developing Alzheimer disease. Overlapping patterns of metabolic dysfunction may be common molecular links between these complex diseases. Amyloid-β (Aβ) precursor protein and associated β- and γ-secretases are expressed in adipose tissue. Aβ precursor protein is up-regulated with obesity and correlated to insulin resistance. Aβ may be secreted by adipose tissue, its production may be regulated through metabolic pathways, and Aβ may exert effects on adipose tissue insulin receptor signaling. Human stromal-vascular cells and differentiated adipocytes were cultured with different combinations of glucose and insulin and then assayed for Aβ in conditioned media. Aβ was measured in vivo using adipose tissue microdialysis. Aβ secretion was increased by glucose and insulin in vitro. Adipose tissue microdialysates contained Aβ. Adipocytes treated with Aβ had decreased expression of insulin receptor substrate-2 and reduced Akt-1 phosphorylation. Aβ was made by adipose tissue cells in vitro at concentrations similar to in vivo measurements. Regulation of Aβ production by glucose and insulin and effects of Aβ on the insulin receptor pathway suggest similar cellular mechanisms may exist between neuronal dysfunction in Alzheimer disease and adipose dysfunction in type 2 diabetes. © 2016 The Authors Obesity published by Wiley Periodicals, Inc. on behalf of The Obesity Society (TOS).

  7. AMP-Activated Kinase (AMPK Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype.

    Directory of Open Access Journals (Sweden)

    Omar Abdul-Rahman

    Full Text Available Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R-5-(4-Carbamoyl-5-aminoimidazol-1-yl-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR, a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained

  8. A biomimetic physiological model for human adipose tissue by adipocytes and endothelial cell cocultures with spatially controlled distribution.

    Science.gov (United States)

    Yao, Rui; Du, Yanan; Zhang, Renji; Lin, Feng; Luan, Jie

    2013-08-01

    An in vitro model that recapitulates the characteristics of native human adipose tissue would largely benefit pathology studies and therapy development. In this paper, we fabricated a physiological model composed of both human adipocytes and endothelial cells with spatially controlled distribution that biomimics the structure and composition of human adipose tissue. Detailed studies into the cell-cell interactions between the adipocytes and endothelial cells revealed a mutual-enhanced effect which resembles the in vivo routine. Furthermore, comparisons between planar coculture and model coculture demonstrated improved adipocyte function as well as endothelial cell proliferation under the same conditions. This research provided a reliable model for human adipose tissue development studies and potential obesity-related therapy development.

  9. Induction of adipocyte-like phenotype in human mesenchymal stem cells by hypoxia

    DEFF Research Database (Denmark)

    Fink, Trine; Abildtrup, Lisbeth Ann; Fogd, Kirsten;

    2004-01-01

    inclusions was observed. The effect of hypoxia on the expression of adipocyte-specific genes was determined by real-time reverse transcription polymerase chain reaction. Interestingly, neither of the two central regulators of adipogenesis--the transcription factors peroxisome proliferator-activated receptor......Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most...

  10. Endogenous oils derived from human adipocytes are potent adjuvants that promote IL-1α-dependent inflammation.

    Science.gov (United States)

    Tynan, Graham A; Hearnden, Claire H; Oleszycka, Ewa; Lyons, Claire L; Coutts, Graham; O'Connell, Jean; Corrigan, Michelle A; Lynch, Lydia; Campbell, Matthew; Callanan, John J; Mok, Kenneth H; Geoghegan, Justin; O'Farrelly, Cliona; Allan, Stuart M; Roche, Helen M; O'Shea, Donal B; Lavelle, Ed C

    2014-06-01

    Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological "danger signal." Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund's incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α-dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α-dependent proinflammatory responses in vivo.

  11. L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein β Signaling Pathway in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiang-Zhu Xie

    2016-01-01

    Conclusions: OxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

  12. The perivascular phenotype and behaviors of dedifferentiated cells derived from human mature adipocytes.

    Science.gov (United States)

    Song, Ning; Kou, Liang; Lu, Xiao-Wen; Sugawara, Atsunori; Shimizu, Yutaka; Wu, Min-Ke; Du, Li; Wang, Hang; Sato, Soh; Shen, Jie-Fei

    2015-02-13

    Derived from mature adipocytes, dedifferentiated fat (DFAT) cells represent a special group of multipotent cells. However, their phenotype and cellular nature remain unclear. Our study found that human DFAT cells adopted perivascular characteristics and behaviors. Flow cytometry and immunofluorescent staining revealed that human DFAT cells positively expressed markers highly related to perivascular cell lineages, such as CD140b, NG2 and desmin, but were negative for common endothelial markers, including CD31, CD34, and CD309. Furthermore, DFAT cells displayed vascular network formation ability in Matrigel, and they noticeably promoted and stabilized the vessel structures formed by human umbilical vascular endothelial cells (HUVECs) in vitro. These results provide novel evidence on the pericyte nature of human DFAT cells, further supporting the recent model for the perivascular origin of adult stem cells, in which tissue-specific progenitor cells in mesenchymal tissues associate with blood vessels, exhibiting perivascular characteristics and functions.

  13. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ninomiya, Yuichi [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Sugahara-Yamashita, Yzumi; Nakachi, Yutaka; Tokuzawa, Yoshimi; Okazaki, Yasushi [Division of Functional Genomics and Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241 (Japan); Nishiyama, Masahiko, E-mail: yamacho@saitama-med.ac.jp [Translational Research Center, Saitama International Medical, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan)

    2010-04-02

    Human mesenchymal stem cells (hMSCs) derived from bone marrow are multipotent stem cells that can regenerate mesenchymal tissues such as adipose, bone or muscle. It is thought that hMSCs can be utilized as a cell resource for tissue engineering and as human models to study cell differentiation mechanisms, such as adipogenesis, osteoblastogenesis and so on. Since it takes 2-3 weeks for hMSCs to differentiate into adipocytes using conventional culture methods, the development of methods to induce faster differentiation into adipocytes is required. In this study we optimized the culture conditions for adipocyte induction to achieve a shorter cultivation time for the induction of adipocyte differentiation in bone marrow-derived hMSCs. Briefly, we used a cocktail of dexamethasone, insulin, methylisobutylxanthine (DIM) plus a peroxisome proliferator-activated receptor {gamma} agonist, rosiglitazone (DIMRo) as a new adipogenic differentiation medium. We successfully shortened the period of cultivation to 7-8 days from 2-3 weeks. We also found that rosiglitazone alone was unable to induce adipocyte differentiation from hMSCs in vitro. However, rosiglitazone appears to enhance hMSC adipogenesis in the presence of other hormones and/or compounds, such as DIM. Furthermore, the inhibitory activity of TGF-{beta}1 on adipogenesis could be investigated using DIMRo-treated hMSCs. We conclude that our rapid new culture method is very useful in measuring the effect of molecules that affect adipogenesis in hMSCs.

  14. MicroRNA profiling links miR-378 to enhanced adipocyte lipolysis in human cancer cachexia.

    Science.gov (United States)

    Kulyté, Agné; Lorente-Cebrián, Silvia; Gao, Hui; Mejhert, Niklas; Agustsson, Thorhallur; Arner, Peter; Rydén, Mikael; Dahlman, Ingrid

    2014-02-01

    Cancer cachexia is associated with pronounced adipose tissue loss due to, at least in part, increased fat cell lipolysis. MicroRNAs (miRNAs) have recently been implicated in controlling several aspects of adipocyte function. To gain insight into the possible impact of miRNAs on adipose lipolysis in cancer cachexia, global miRNA expression was explored in abdominal subcutaneous adipose tissue from gastrointestinal cancer patients with (n = 10) or without (n = 11) cachexia. Effects of miRNA overexpression or inhibition on lipolysis were determined in human in vitro differentiated adipocytes. Out of 116 miRNAs present in adipose tissue, five displayed distinct cachexia-associated expression according to both microarray and RT-qPCR. Four (miR-483-5p/-23a/-744/-99b) were downregulated, whereas one (miR-378) was significantly upregulated in cachexia. Adipose expression of miR-378 associated strongly and positively with catecholamine-stimulated lipolysis in adipocytes. This correlation is most probably causal because overexpression of miR-378 in human adipocytes increased catecholamine-stimulated lipolysis. In addition, inhibition of miR-378 expression attenuated stimulated lipolysis and reduced the expression of LIPE, PLIN1, and PNPLA2, a set of genes encoding key lipolytic regulators. Taken together, increased miR-378 expression could play an etiological role in cancer cachexia-associated adipose tissue loss via effects on adipocyte lipolysis.

  15. The Action of D-Dopachrome Tautomerase as an Adipokine in Adipocyte Lipid Metabolism

    OpenAIRE

    2012-01-01

    Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-rela...

  16. Comparison of Markers and Functional Attributes of Human Adipose-Derived Stem Cells and Dedifferentiated Adipocyte Cells from Subcutaneous Fat of an Obese Diabetic Donor.

    Science.gov (United States)

    Watson, James E; Patel, Niketa A; Carter, Gay; Moor, Andrea; Patel, Rekha; Ghansah, Tomar; Mathur, Abhishek; Murr, Michel M; Bickford, Paula; Gould, Lisa J; Cooper, Denise R

    2014-03-01

    Objective: Adipose tissue is a robust source of adipose-derived stem cells (ADSCs) that may be able to provide secreted factors that promote the ability of wounded tissue to heal. However, adipocytes also have the potential to dedifferentiate in culture to cells with stem cell-like properties that may improve their behavior and functionality for certain applications. Approach: ADSCs are adult mesenchymal stem cells that are cultured from the stromal vascular fraction of adipose tissue. However, adipocytes are capable of dedifferentiating into cells with stem cell properties. In this case study, we compare ADSC and dedifferentiated fat (DFAT) cells from the same patient and fat depot for mesenchymal cell markers, embryonic stem cell markers, ability to differentiate to adipocytes and osteoblasts, senescence and telomerase levels, and ability of conditioned media (CM) to stimulate migration of human dermal fibroblasts (HDFs). Innovation and Conclusions: ADSCs and DFAT cells displayed identical levels of CD90, CD44, CD105, and were CD34- and CD45-negative. They also expressed similar levels of Oct4, BMI1, KLF4, and SALL4. DFAT cells, however, showed higher efficiency in adipogenic and osteogenic capacity. Telomerase levels of DFAT cells were double those of ADSCs, and senescence declined in DFAT cells. CM from both cell types altered the migration of fibroblasts. Despite reports of ADSCs from a number of human depots, there have been no comparisons of the ability of dedifferentiated DFAT cells from the same donor and depot to differentiate or modulate migration of HDFs. Since ADSCs were from an obese diabetic donor, reprogramming of DFAT cells may help improve a patient's cells for regenerative medicine applications.

  17. Melanocortin agonists stimulate lipolysis in human adipose tissue explants but not in adipocytes.

    Science.gov (United States)

    Møller, Cathrine Laustrup; Pedersen, Steen B; Richelsen, Bjørn; Conde-Frieboes, Kilian W; Raun, Kirsten; Grove, Kevin L; Wulff, Birgitte Schjellerup

    2015-10-12

    The central melanocortin system is broadly involved in the regulation of mammalian nutrient utilization. However, the function of melanocortin receptors (MCRs) expressed directly in peripheral metabolic tissues is still unclear. The objective of this study was to investigate the lipolytic capacity of MC1-5R in differentiated adipocytes versus intact white adipose tissue. Non-selective MCR agonist α-MSH, MC5R-selective agonist PG-901 and MC4R-selective agonist LY2112688 significantly stimulated lipolysis in intact white adipose tissue, whereas stimulation of MCRs in differentiated adipocytes failed to do so. The lipolytic response of MC5R was decreased in intact human white adipose tissue when co-treating with β-adrenergic antagonist propranolol, suggesting that the effect may be dependent on neuronal innervation via noradrenalin release. When developing an anti-obesity therapeutic drug with selective MC4R/MC5R properties, effects on lipolysis in white adipose tissue may be physiologically relevant.

  18. Long-acting insulin analog detemir displays reduced effects on adipocyte differentiation of human subcutaneous and visceral adipose stem cells.

    Science.gov (United States)

    Cignarelli, A; Perrini, S; Nigro, P; Ficarella, R; Barbaro, M; Peschechera, A; Porro, S; Natalicchio, A; Laviola, L; Puglisi, F; Giorgino, F

    2016-04-01

    Since treatment with insulin detemir results in a lower weight gain compared to human insulin, we investigated whether detemir is associated with lower ability to promote adipogenesis and/or lipogenesis in human adipose stem cells (ASC). Human ASC isolated from both the subcutaneous and visceral adipose tissues were differentiated for 30 days in the presence of human insulin or insulin detemir. Nile Red and Oil-Red-O staining were used to quantify the rate of ASC conversion to adipocytes and lipid accumulation, respectively. mRNA expression levels of early genes, including Fos and Cebpb, as well as of lipogenic and adipogenic genes, were measured at various phases of differentiation by qRT-PCR. Activation of insulin signaling was assessed by immunoblotting. ASC isolated from subcutaneous and visceral adipose tissue were less differentiated when exposed to insulin detemir compared to human insulin, showing lower rates of adipocyte conversion, reduced triglyceride accumulation, and impaired expression of late-phase adipocyte marker genes, such as Pparg2, Slc2a4, Adipoq, and Cidec. However, no differences in activation of insulin receptor, Akt and Erk and induction of the early genes Fos and Cebpb were observed between insulin detemir and human insulin. Insulin detemir displays reduced induction of the Pparg2 adipocyte master gene and diminished effects on adipocyte differentiation and lipogenesis in human subcutaneous and visceral ASC, in spite of normal activation of proximal insulin signaling reactions. These characteristics of insulin detemir may be of potential relevance to its weight-sparing effects observed in the clinical setting. Copyright © 2015 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.

  19. Reappraisal of bicarbonate secretion by the human oesophagus.

    OpenAIRE

    Mertz-Nielsen, A; Hillingsø, J; Bukhave, K; Rask-Madsen, J

    1997-01-01

    BACKGROUND AND AIMS: Administration of omeprazole to healthy volunteers was recently reported to increase proximal duodenal mucosal bicarbonate secretion. As human oesophagus also secretes bicarbonate, the hypothesis was tested that omeprazole may stimulate oesophageal bicarbonate secretion and thus contribute to the therapeutic efficacy of the drug in gastro-oesophageal reflux disease. SUBJECTS AND METHODS: In nine healthy volunteers, oesophageal "steady state" perfusion of a 10 cm open segm...

  20. Acetylcholine regulates ghrelin secretion in humans

    NARCIS (Netherlands)

    F. Broglio (Fabio); E. Ghigo (Ezio); C. Gottero; F. Prodam (Flavia); S. Destefanis; A. Benso; C. Gauna (Carlotta); L.J. Hofland (Leo); E. Arvat; A-J. van der Lely (Aart-Jan); P.M. van Koetsveld (Peter)

    2004-01-01

    textabstractGhrelin secretion has been reportedly increased by fasting and energy restriction but decreased by food intake, glucose, insulin, and somatostatin. However, its regulation is still far from clarified. The cholinergic system mediates some ghrelin actions, e.g.

  1. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    DEFF Research Database (Denmark)

    Ali; Hamam; Alfayez;

    2016-01-01

    The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including...... abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell...... or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating...

  2. G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase.

    Science.gov (United States)

    Schweiger, Martina; Paar, Margret; Eder, Christina; Brandis, Janina; Moser, Elena; Gorkiewicz, Gregor; Grond, Susanne; Radner, Franz P W; Cerk, Ines; Cornaciu, Irina; Oberer, Monika; Kersten, Sander; Zechner, Rudolf; Zimmermann, Robert; Lass, Achim

    2012-11-01

    The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL), which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 switch gene-2 (G0S2). CGI-58 activates and G0S2 inhibits ATGL activity. In contrast to mice, the functional role of G0S2 in human adipocyte lipolysis is poorly characterized. Here we show that overexpression or silencing of G0S2 in human SGBS adipocytes decreases and increases lipolysis, respectively. Human G0S2 is upregulated during adipocyte differentiation and inhibits ATGL activity in a dose-dependent manner. Interestingly, C-terminally truncated ATGL mutants, which fail to localize to lipid droplets, translocate to the lipid droplet upon coexpression with G0S2, suggesting that G0S2 anchors ATGL to lipid droplets independent of ATGL's C-terminal lipid binding domain. Taken together, our results indicate that G0S2 also regulates human lipolysis by affecting enzyme activity and intracellular localization of ATGL. Increased lipolysis is known to contribute to the pathogenesis of insulin resistance, and G0S2 expression has been shown to be reduced in poorly controlled type 2 diabetic patients. Our data indicate that downregulation of G0S2 in adipose tissue could represent one of the underlying causes leading to increased lipolysis in the insulin-resistant state.

  3. White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways

    Directory of Open Access Journals (Sweden)

    V. Barquissau

    2016-05-01

    Conclusions: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

  4. Effects of Rosmarinus officinalis extract on human primary omental preadipocytes and adipocytes.

    Science.gov (United States)

    Stefanon, Bruno; Pomari, Elena; Colitti, Monica

    2015-07-01

    The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this in vitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase 4, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, wingless-type MMTV integration site family, member 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-143. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement.

  5. Insulin/glucose induces natriuretic peptide clearance receptor in human adipocytes: a metabolic link with the cardiac natriuretic pathway.

    Science.gov (United States)

    Bordicchia, M; Ceresiani, M; Pavani, M; Minardi, D; Polito, M; Wabitsch, M; Cannone, V; Burnett, J C; Dessì-Fulgheri, P; Sarzani, R

    2016-07-01

    Cardiac natriuretic peptides (NP) are involved in cardiorenal regulation and in lipolysis. The NP activity is largely dependent on the ratio between the signaling receptor NPRA and the clearance receptor NPRC. Lipolysis increases when NPRC is reduced by starving or very-low-calorie diet. On the contrary, insulin is an antilipolytic hormone that increases sodium retention, suggesting a possible functional link with NP. We examined the insulin-mediated regulation of NP receptors in differentiated human adipocytes and tested the association of NP receptor expression in visceral adipose tissue (VAT) with metabolic profiles of patients undergoing renal surgery. Differentiated human adipocytes from VAT and Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte cell line were treated with insulin in the presence of high-glucose or low-glucose media to study NP receptors and insulin/glucose-regulated pathways. Fasting blood samples and VAT samples were taken from patients on the day of renal surgery. We observed a potent insulin-mediated and glucose-dependent upregulation of NPRC, through the phosphatidylinositol 3-kinase pathway, associated with lower lipolysis in differentiated adipocytes. No effect was observed on NPRA. Low-glucose medium, used to simulate in vivo starving conditions, hampered the insulin effect on NPRC through modulation of insulin/glucose-regulated pathways, allowing atrial natriuretic peptide to induce lipolysis and thermogenic genes. An expression ratio in favor of NPRC in adipose tissue was associated with higher fasting insulinemia, HOMA-IR, and atherogenic lipid levels. Insulin/glucose-dependent NPRC induction in adipocytes might be a key factor linking hyperinsulinemia, metabolic syndrome, and higher blood pressure by reducing NP effects on adipocytes. Copyright © 2016 the American Physiological Society.

  6. Female adipocyte androgen synthesis and the effects of insulin

    Directory of Open Access Journals (Sweden)

    David Cadagan

    2014-01-01

    Full Text Available The metabolic syndrome is a cluster of metabolic disorders characterized by insulin resistance and hyperinsulinaemia, and its presence can increase the risk of cardiovascular disease significantly. The metabolic syndrome is associated with increased circulating androgen levels in women, which may originate from the ovaries and adrenal glands. Adipocytes are also able to synthesise steroid hormones, and this output has been hypothesised to increase with elevated insulin plasma concentrations. However, the contribution of the adipocytes to the circulating androgen levels in women with metabolic syndrome is limited and the effects of insulin are not fully understood. The aim of this study was to investigate the presence of steroid precursors and synthetic enzymes in human adipocyte biopsies as markers of possible adipocyte androgen synthesis. We examined pre and mature adipocytes taken from tissue biopsies of abdominal subcutaneous adipose tissue of participating women from the Department of Obstetrics and Gynaecology, of the Royal Derby Hospital. The results showed the potential for localised adipocyte androgen synthesis through the presence of the androgen precursor progesterone, as well as the steroid-converting enzyme 17α-hydroxylase. Furthermore, we found the controlled secretion of androstenedione in vitro and that insulin treatment caused levels to increase. Continued examination of a localised source of androgen production is therefore of clinical relevance due to its influence on adipocyte metabolism, its negative impact on female steroidogenic homeostasis, and the possible aggravation this may have when associated to obesity and obesity related metabolic abnormalities such as hyperinsulinaemia.

  7. A catalogue of human secreted proteins and its implications

    Directory of Open Access Journals (Sweden)

    Shivakumar Keerthikumar

    2016-11-01

    Full Text Available Under both normal and pathological conditions, cells secrete variety of proteins through classical and non-classical secretory pathways into the extracellular space. Majority of these proteins represent pathophysiology of the cell from which it is secreted. Recently, though more than 92% of the protein coding genes has been mapped by human proteome map project, but number of those proteins that constitutes secretome of the cell still remains elusive. Secreted proteins or the secretome can be accessible in bodily fluids and hence are considered as potential biomarkers to discriminate between healthy and diseased individuals. In order to facilitate the biomarker discovery and to further aid clinicians and scientists working in these arenas, we have compiled and catalogued secreted proteins from the human proteome using integrated bioinformatics approach. In this study, nearly 14% of the human proteome is likely to be secreted through classical and non-classical secretory pathways. Out of which, ~38% of these secreted proteins were found in extracellular vesicles including exosomes and shedding microvesicles. Among these secreted proteins, 94% were detected in human bodily fluids including blood, plasma, serum, saliva, semen, tear and urine. We anticipate that this high confidence list of secreted proteins could serve as a compendium of candidate biomarkers. In addition, the catalogue may provide functional insights in understanding the molecular mechanisms involved in various physiological and pathophysiological conditions of the cell.

  8. bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

    OpenAIRE

    Xinghui Song; Yanwei Li; Xiao Chen; Guoli Yin; Qiong Huang; Yingying Chen; Guowei Xu; Linlin Wang

    2014-01-01

    In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and exp...

  9. Taurine chloramine modulates the expression of adipokines through inhibition of the STAT-3 signaling pathway in differentiated human adipocytes.

    Science.gov (United States)

    Kim, Kyoung Soo; Ji, Hye-In; Chung, Hyunju; Kim, Chakyeun; Lee, Sang Hoon; Lee, Yeon-Ah; Yang, Hyung-In; Yoo, Myung Chul; Hong, Seung Jae

    2013-12-01

    To examine the possible role of taurine chloramine (TauCl) in modulating the expression of adipokines in adipose tissue associated with obesity, we evaluated the effect of TauCl in human differentiated adipocytes in response to IL-1β. To study the physiological effects of TauCl on adipokine expression, differentiated adipocytes were treated with IL-1β in the presence or absence of TauCl at concentrations ranging from 200 to 600 μM for 7 days. Cell culture supernatants and total RNA were analyzed by ELISA and real-time PCR, respectively, to determine protein and mRNA levels of adipokines, including adiponectin, leptin, IL-6, and IL-8. Levels of proteins involved in relevant signaling pathways were investigated by western blotting. Stimulation with IL-1β significantly decreased levels of adiponectin and leptin in adipocytes, but increased levels of IL-6 and IL-8 in a dose-dependent manner. Treatment with TauCl significantly reversed the modulation of adipokine expression by inhibiting STAT-3 signaling in IL-1β-stimulated adipocytes, independent of MAPK signaling. TauCl treatment more significantly modulated the expression of adipokines in adipocytes stimulated with IL-1β than that of non-stimulated adipocytes, suggesting that TauCl plays a significant role in modulating the expression of adipokines under inflammatory conditions. In conclusion, TauCl and other taurine derivatives that inhibit the STAT-3 signaling pathway can modulate expression of adipokines and thus may be useful as therapeutic agents for obesity-related diseases.

  10. Both inflammatory and classical lipolytic pathways are involved in lipopolysaccharide-induced lipolysis in human adipocytes.

    Science.gov (United States)

    Grisouard, Jean; Bouillet, Elisa; Timper, Katharina; Radimerski, Tanja; Dembinski, Kaethi; Frey, Daniel M; Peterli, Ralph; Zulewski, Henryk; Keller, Ulrich; Müller, Beat; Christ-Crain, Mirjam

    2012-02-01

    High fat diet-induced endotoxaemia triggers low-grade inflammation and lipid release from adipose tissue. This study aims to unravel the cellular mechanisms leading to the lipopolysaccharide (LPS) effects in human adipocytes. Subcutaneous pre-adipocytes surgically isolated from patients were differentiated into mature adipocytes in vitro. Lipolysis was assessed by measurement of glycerol release and mRNA expression of pro-inflammatory cytokines were evaluated by real-time PCR. Treatment with LPS for 24 h induced a dose-dependent increase in interleukin (IL)-6 and IL-8 mRNA expression. At 1 µg/ml LPS, IL-6 and IL-8 were induced to 19.5 ± 1.8-fold and 662.7 ± 91.5-fold (P < 0.01 vs basal), respectively. From 100 ng/ml to 1 µg/ml, LPS-induced lipolysis increased to a plateau of 3.1-fold above basal level (P < 0.001 vs basal). Co-treatment with inhibitors of inhibitory kappa B kinase kinase beta (IKKβ) or NF-κB inhibited LPS-induced glycerol release. Co-treatment with the protein kinase A (PKA) inhibitor H-89, the lipase inhibitor orlistat or the hormone-sensitive lipase (HSL) inhibitor CAY10499 abolished the lipolytic effects of LPS. Co-treatment with the MAPK inhibitor, U0126 also reduced LPS-induced glycerol release. Inhibition of lipolysis by orlistat or CAY10499 reduced LPS-induced IL-6 and IL-8 mRNA expression. Induction of lipolysis by the synthetic catecholamine isoproterenol or the phosphodiesterase type III inhibitor milrinone did not alter basal IL-6 and IL-8 mRNA expression after 24 treatments whereas these compounds enhanced LPS-induced IL-6 and IL-8 mRNA expression. Both the inflammatory IKKβ/NF-κB pathway and the lipolytic PKA/HSL pathways mediate LPS-induced lipolysis. In turn, LPS-induced lipolysis reinforces the expression of pro-inflammatory cytokines and, thereby, triggers its own lipolytic activity.

  11. Metformin induces glucose uptake in human preadipocyte-derived adipocytes from various fat depots.

    Science.gov (United States)

    Fischer, M; Timper, K; Radimerski, T; Dembinski, K; Frey, D M; Zulewski, H; Keller, U; Müller, B; Christ-Crain, M; Grisouard, J

    2010-04-01

    To evaluate the effect of metformin on basal and insulin-induced glucose uptake in subcutaneous and visceral preadipocyte-derived adipocytes from obese and non-obese patients, preadipocytes were obtained from subcutaneous and visceral fat depots during abdominal surgery. Differentiation efficiency was evaluated by measurement of intracellular triglyceride accumulation. Preadipocyte-derived adipocytes were treated with metformin (1 mM) for 24 h with or without the addition of insulin (100 nM) for 20 min and glucose uptake was measured. In cells from each donor, intracellular triglyceride accumulation was more abundant in subcutaneous preadipocyte-derived adipocytes than in visceral preadipocyte-derived adipocytes (p < 0.001). Insulin stimulated glucose uptake in subcutaneous preadipocyte-derived adipocytes from both non-obese and obese patients (p < 0.001 vs. basal). In visceral preadipocyte-derived adipocytes, insulin did not increase basal glucose uptake. In subcutaneous preadipocyte-derived adipocytes from non-obese and obese patients, metformin alone increased glucose uptake to 2.7 +/- 0.2 (p < 0.001) and 2.1 +/- 0.1 fold (p < 0.001) respectively. Metformin increased glucose uptake in visceral preadipocyte-derived adipocytes from non-obese (1.7 +/- 0.1 fold vs. basal, p < 0.001) and obese (2.0 +/- 0.2 fold vs. basal, p < 0.001) patients. Combined treatment with metformin and insulin increased glucose uptake in subcutaneous preadipocyte-derived adipocytes from both non-obese and obese patients (p < 0.001 vs. insulin alone). In preadipocyte-derived adipocytes glucose uptake is induced by metformin independent of the fat depot origin of the preadipocytes (subcutaneous or visceral) and the obesity state of the patients (non-obese or obese). In adipocytes, metformin seems to induce glucose uptake independent of insulin suggesting an alternative mechanism of action of this drug.

  12. Original Research: Hydroxytyrosol, an ingredient of olive oil, reduces triglyceride accumulation and promotes lipolysis in human primary visceral adipocytes during differentiation.

    Science.gov (United States)

    Stefanon, Bruno; Colitti, Monica

    2016-10-01

    Hydroxytyrosol has various pharmacological properties, including anti-oxidative stress and anti-inflammatory activities, preventing hyperglycemia, insulin resistance, and the metabolic syndrome. The present study is focused on the anti-adipogenic and lipolytic activity of hydroxytyrosol on primary human visceral adipocytes. Pre-adipocytes were analyzed after 10 (P10) and 20 (P20) days of treatment during differentiation and after 7 (A7) days of treatment when they reached mature shape. The treatment with hydroxytyrosol extract significantly (P lipolysis in P20 cells in comparison to control cells and A7 mature adipocytes. Hydroxytyrosol-treated P20 cells significantly (P lipolysis and apoptotic activity in primary human visceral pre-adipocytes during differentiation and does not affect already mature adipocytes. © 2016 by the Society for Experimental Biology and Medicine.

  13. Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.

    Directory of Open Access Journals (Sweden)

    Julie Lessard

    Full Text Available Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1 were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity.

  14. Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.

    Science.gov (United States)

    Lessard, Julie; Pelletier, Mélissa; Biertho, Laurent; Biron, Simon; Marceau, Simon; Hould, Frédéric-Simon; Lebel, Stéfane; Moustarah, Fady; Lescelleur, Odette; Marceau, Picard; Tchernof, André

    2015-01-01

    Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin) was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1) were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity.

  15. Acetylcholine regulates ghrelin secretion in humans

    NARCIS (Netherlands)

    F. Broglio (Fabio); E. Ghigo (Ezio); C. Gottero; F. Prodam (Flavia); S. Destefanis; A. Benso; C. Gauna (Carlotta); L.J. Hofland (Leo); E. Arvat; A-J. van der Lely (Aart-Jan); P.M. van Koetsveld (Peter)

    2004-01-01

    textabstractGhrelin secretion has been reportedly increased by fasting and energy restriction but decreased by food intake, glucose, insulin, and somatostatin. However, its regulation is still far from clarified. The cholinergic system mediates some ghrelin actions, e.g. stimulatio

  16. Genome-wide profiling of H3K56 acetylation and transcription factor binding sites in human adipocytes.

    Directory of Open Access Journals (Sweden)

    Kinyui Alice Lo

    Full Text Available The growing epidemic of obesity and metabolic diseases calls for a better understanding of adipocyte biology. The regulation of transcription in adipocytes is particularly important, as it is a target for several therapeutic approaches. Transcriptional outcomes are influenced by both histone modifications and transcription factor binding. Although the epigenetic states and binding sites of several important transcription factors have been profiled in the mouse 3T3-L1 cell line, such data are lacking in human adipocytes. In this study, we identified H3K56 acetylation sites in human adipocytes derived from mesenchymal stem cells. H3K56 is acetylated by CBP and p300, and deacetylated by SIRT1, all are proteins with important roles in diabetes and insulin signaling. We found that while almost half of the genome shows signs of H3K56 acetylation, the highest level of H3K56 acetylation is associated with transcription factors and proteins in the adipokine signaling and Type II Diabetes pathways. In order to discover the transcription factors that recruit acetyltransferases and deacetylases to sites of H3K56 acetylation, we analyzed DNA sequences near H3K56 acetylated regions and found that the E2F recognition sequence was enriched. Using chromatin immunoprecipitation followed by high-throughput sequencing, we confirmed that genes bound by E2F4, as well as those by HSF-1 and C/EBPα, have higher than expected levels of H3K56 acetylation, and that the transcription factor binding sites and acetylation sites are often adjacent but rarely overlap. We also discovered a significant difference between bound targets of C/EBPα in 3T3-L1 and human adipocytes, highlighting the need to construct species-specific epigenetic and transcription factor binding site maps. This is the first genome-wide profile of H3K56 acetylation, E2F4, C/EBPα and HSF-1 binding in human adipocytes, and will serve as an important resource for better understanding adipocyte

  17. Glucocorticoids antagonize tumor necrosis factor-α-stimulated lipolysis and resistance to the antilipolytic effect of insulin in human adipocytes.

    Science.gov (United States)

    Lee, Mi-Jeong; Fried, Susan K

    2012-11-01

    High concentrations of TNF within obese adipose tissue increase basal lipolysis and antagonize insulin signaling. Adipocytes of the obese are also exposed to elevated levels of glucocorticoids (GCs), which antagonize TNF actions in many cell types. We tested the hypothesis that TNF decreases sensitivity to the antilipolytic effect of insulin and that GCs antagonize this effect in differentiated human adipocytes. Lipolysis and expression levels of lipolytic proteins were measured after treating adipocytes with TNF, dexamethasone (DEX), or DEX + TNF for up to 48 h. TNF not only increased basal lipolysis, it caused resistance to the antilipolytic effects of insulin in human adipocytes. DEX alone did not significantly affect lipolysis. Cotreatment with DEX blocked TNF induction of basal lipolysis and insulin resistance by antagonizing TNF stimulation of PKA-mediated phosphorylation of hormone-sensitive lipase (HSL) at Ser⁵⁶³ and Ser⁶⁶⁰ and perilipin. TNF did not affect perilipin, HSL, or phosphodiesterase-3B mass but paradoxically suppressed adipose tissue triglyceride lipase expression, and this effect was blocked by DEX. The extent to which GCs can restrain the lipolytic actions of TNF may both diminish the potentially deleterious effects of excess lipolysis and contribute to fat accumulation in obesity.

  18. Superparamagnetic iron oxide nanoparticles alter expression of obesity and T2D-associated risk genes in human adipocytes

    Science.gov (United States)

    Sharifi, S.; Daghighi, S.; Motazacker, M. M.; Badlou, B.; Sanjabi, B.; Akbarkhanzadeh, A.; Rowshani, A. T.; Laurent, S.; Peppelenbosch, M. P.; Rezaee, F.

    2013-07-01

    Adipocytes hypertrophy is the main cause of obesity and its affliction such as type 2 diabetes (T2D). Since superparamagnetic iron oxide nanoparticles (SPIONs) are used for a wide range of biomedical/medical applications, we aimed to study the effect of SPIONs on 22 and 29 risk genes (Based on gene wide association studies) for obesity and T2D in human adipocytes. The mRNA expression of lipid and glucose metabolism genes was changed upon the treatment of human primary adipocytes with SPIONs. mRNA of GULP1, SLC30A8, NEGR1, SEC16B, MTCH2, MAF, MC4R, and TMEM195 were severely induced, whereas INSIG2, NAMPT, MTMR9, PFKP, KCTD15, LPL and GNPDA2 were down-regulated upon SPIONs stimulation. Since SEC16B gene assist the phagocytosis of apoptotic cells and this gene were highly expressed upon SPIONs treatment in adipocytes, it is logic to assume that SPIONs may play a crucial role in this direction, which requires more consideration in the future.

  19. mTOR inhibition with rapamycin causes impaired insulin signalling and glucose uptake in human subcutaneous and omental adipocytes.

    Science.gov (United States)

    Pereira, Maria J; Palming, Jenny; Rizell, Magnus; Aureliano, Manuel; Carvalho, Eugénia; Svensson, Maria K; Eriksson, Jan W

    2012-05-15

    Rapamycin is an immunosuppressive agent used after organ transplantation, but its molecular effects on glucose metabolism needs further evaluation. We explored rapamycin effects on glucose uptake and insulin signalling proteins in adipocytes obtained via subcutaneous (n=62) and omental (n=10) fat biopsies in human donors. At therapeutic concentration (0.01 μM) rapamycin reduced basal and insulin-stimulated glucose uptake by 20-30%, after short-term (15 min) or long-term (20 h) culture of subcutaneous (n=23 and n=10) and omental adipocytes (n=6 and n=7). Rapamycin reduced PKB Ser473 and AS160 Thr642 phosphorylation, and IRS2 protein levels in subcutaneous adipocytes. Additionally, it reduced mTOR-raptor, mTOR-rictor and mTOR-Sin1 interactions, suggesting decreased mTORC1 and mTORC2 formation. Rapamycin also reduced IR Tyr1146 and IRS1 Ser307/Ser616/Ser636 phosphorylation, whereas no effects were observed on the insulin stimulated IRS1-Tyr and TSC2 Thr1462 phosphorylation. This is the first study to show that rapamycin reduces glucose uptake in human adipocytes through impaired insulin signalling and this may contribute to the development of insulin resistance associated with rapamycin therapy.

  20. The fat controller: adipocyte development.

    Directory of Open Access Journals (Sweden)

    Jacqueline M Stephens

    Full Text Available Obesity is a condition characterized by excess adipose tissue that results from positive energy balance and is the most common metabolic disorder in the industrialized world. The obesity epidemic shows no sign of slowing, and it is increasingly a global problem. Serious clinical problems associated with obesity include an increased risk for type 2 diabetes, atherosclerosis, and cancer. Hence, understanding the origin and development of adipocytes and adipose tissue will be critical to the analysis and treatment of metabolic diseases. Historically, albeit incorrectly, adipocytes were thought to be inert cells whose singular function was lipid storage. It is now known that adipocytes have other critical functions; the most important include sensitivity to insulin and the ability to produce and secrete adipocyte-specific endocrine hormones that regulate energy homeostasis in other tissues. Today, adipocytes are recognized as critical regulators of whole-body metabolism and known to be involved in the pathogenesis of a variety of metabolic diseases. All cells come from other cells and many cells arise from precursor cells. Adipocytes are not created from other adipocytes, but they arise from precursor cells. In the last two decades, scientists have discovered the function of many proteins that influence the ability of precursor cells to become adipocytes. If the expansion of the adipose tissue is the problem, it seems logical that adipocyte development inhibitors could be a viable anti-obesity therapeutic. However, factors that block adipocyte development and limit adipocyte expansion also impair metabolic health. This notion may be counterintuitive, but several lines of evidence support the idea that blocking adipocyte development is unhealthy. For this reason it is clear that we need a better understanding of adipocyte development.

  1. Somatomammotrophic cells in GH-secreting and PRL-secreting human pituitary adenomas.

    Science.gov (United States)

    Bassetti, M; Brina, M; Spada, A; Giannattasio, G

    1989-11-01

    A morphological study has been carried out on 20 GH-secreting adenomas removed from acromegalic normoprolactinemic patients, on 29 PRL-secreting adenomas removed from hyperprolactinemic patients without signs of acromegaly and on one normal human anterior pituitary gland collected at autopsy. The protein A-gold immunoelectron microscopic technique has been utilized in order to verify the presence of mixed cells producing both GH and PRL (somatomammotrophs) in these pituitary tissues. In the normal pituitary a considerable number of somatomammotrophs (15-20%) was found, thus supporting the idea that these cells are normal components of the human anterior pituitary gland. In 10 GH-secreting adenomas and in 10 PRL-secreting adenomas somatomammotrophs were present in a variable number (from 4 to 20% of the whole cell population in GH adenomas and from 1 to 47% in PRL tumors). It can be concluded therefore that these cells, largely present in all GH/PRL-secreting adenomas, can also be found in GH-secreting and PRL-secreting tumors without clinical evidence of a mixed secretion. Adenomatous somatomammotrophs displayed ultrastructural features of adenomatous somatotrophs and mammotrophs (prominent Golgi complexes, abundant rough endoplasmic reticulum, irregular nuclei). The size and the number of granules were variable. In some cells GH and PRL were stored in distinct secretory granules, in others in mixed granules or both in mixed and distinct granules, thus suggesting that in adenomatous somatomammotrophs the efficiency of the mechanisms of sorting of the two hormones varies from one cell to another.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Human placenta secretes apolipoprotein B-100-containing lipoproteins

    DEFF Research Database (Denmark)

    Munk-Madsen, Eva; Lindegaard, Marie Louise Skakkebæk; Andersen, Claus B;

    2004-01-01

    early during pregnancy in the placenta. To examine whether the human placenta produces lipoproteins, we examined apoB and microsomal triglyceride transfer protein (MTP) mRNA expression in placental biopsies. ApoB and MTP are mandatory for assembly and secretion of apoB-containing lipoproteins. Both...... genes were expressed in placenta and microsomal extracts from human placenta contained triglyceride transfer activity, indicating expression of bioactive MTP. To detect lipoprotein secretion, biopsies from term placentas were placed in medium with [(35)S]methionine and [(35)S]cysteine for 3-24 h. Upon...... lipoproteins secreted from placental tissue showed spherical particles with a diameter of 47 +/- 10 nm. These results demonstrate that human placenta expresses both apoB and MTP and consequently synthesize and secrete apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel pathway...

  3. Inhibitory effect of calcitonin on pure human pancreatic secretion.

    Directory of Open Access Journals (Sweden)

    Tanaka,Juntaro

    1989-06-01

    Full Text Available The inhibitory effect of calcitonin on human pancreatic secretion was evaluated to examine whether the different results reported earlier between humans, cats and dogs can be ascribed to the different sensitivity of these species to calcitonin, as suggested by some investigators. Pancreatic juice was obtained by endoscopic cannulation of the pancreatic duct from 11 patients with relapsing pancreatitis during intravenous infusion of secretin (1 U/kg/h plus caerulein (0.04 microgram/kg/h. After steady secretion was attained 20 min after the beginning of collection, five 2-min fractions were obtained before, and ten 2-min fractions were obtained after intravenous infusion of calcitonin (1 IU/kg/h. The pre- and post-calcitonin fractions from each patient were compared by Student's t-test. Calcitonin inhibited the secretory volume (26.8 to 65.6% and bicarbonate secretion (21.4 to 62.0% in 8 patients, and amylase (48.4 to 89.5% and lipase secretion (47.4 to 90.5% in all patients. The present studies reconfirmed that prominent inhibition of enzyme secretion occurs in humans. A new finding was that significant inhibition of the secretory volume and bicarbonate secretion occurs in humans. The inhibitory effects of calcitonin in humans did not appear to differ from those in cats and dogs, when evaluated similarly with the use of pure pancreatic juice.

  4. Fat-specific Protein 27 (FSP27) Interacts with Adipose Triglyceride Lipase (ATGL) to Regulate Lipolysis and Insulin Sensitivity in Human Adipocytes*

    Science.gov (United States)

    Grahn, Tan Hooi Min; Kaur, Rajween; Yin, Jun; Schweiger, Martina; Sharma, Vishva Mitra; Lee, Mi-Jeong; Ido, Yasuo; Smas, Cynthia M.; Zechner, Rudolf; Lass, Achim; Puri, Vishwajeet

    2014-01-01

    In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120–220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120–220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes. PMID:24627478

  5. Fat-specific protein 27 (FSP27) interacts with adipose triglyceride lipase (ATGL) to regulate lipolysis and insulin sensitivity in human adipocytes.

    Science.gov (United States)

    Grahn, Tan Hooi Min; Kaur, Rajween; Yin, Jun; Schweiger, Martina; Sharma, Vishva Mitra; Lee, Mi-Jeong; Ido, Yasuo; Smas, Cynthia M; Zechner, Rudolf; Lass, Achim; Puri, Vishwajeet

    2014-04-25

    In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120-220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120-220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.

  6. Activation of IRF1 in Human Adipocytes Leads to Phenotypes Associated with Metabolic Disease.

    Science.gov (United States)

    Friesen, Max; Camahort, Raymond; Lee, Youn-Kyoung; Xia, Fang; Gerszten, Robert E; Rhee, Eugene P; Deo, Rahul C; Cowan, Chad A

    2017-05-09

    The striking rise of obesity-related metabolic disorders has focused attention on adipocytes as critical mediators of disease phenotypes. To better understand the role played by excess adipose in metabolic dysfunction it is crucial to decipher the transcriptional underpinnings of the low-grade adipose inflammation characteristic of diseases such as type 2 diabetes. Through employing a comparative transcriptomics approach, we identified IRF1 as differentially regulated between primary and in vitro-derived genetically matched adipocytes. This suggests a role as a mediator of adipocyte inflammatory phenotypes, similar to its function in other tissues. Utilizing adipose-derived mesenchymal progenitors we subsequently demonstrated that expression of IRF1 in adipocytes indeed contributes to upregulation of inflammatory processes, both in vitro and in vivo. This highlights IRF1's relevance to obesity-related inflammation and the resultant metabolic dysregulation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Stress of endoplasmic reticulum modulates differentiation and lipogenesis of human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Koc, Michal; Mayerová, Veronika; Kračmerová, Jana [Franco-Czech Laboratory for Clinical Research on Obesity, Third Faculty of Medicine, Prague (Czech Republic); Department of Sport Medicine, Third Faculty of Medicine, Charles University in Prague, CZ-100 00 (Czech Republic); Mairal, Aline [Franco-Czech Laboratory for Clinical Research on Obesity, Third Faculty of Medicine, Prague (Czech Republic); Inserm, UMR1048, Obesity Research Laboratory, Institute of Metabolic and Cardiovascular Diseases, 31432 Toulouse, Cedex 4 (France); Mališová, Lucia; Štich, Vladimír [Franco-Czech Laboratory for Clinical Research on Obesity, Third Faculty of Medicine, Prague (Czech Republic); Department of Sport Medicine, Third Faculty of Medicine, Charles University in Prague, CZ-100 00 (Czech Republic); Langin, Dominique [Franco-Czech Laboratory for Clinical Research on Obesity, Third Faculty of Medicine, Prague (Czech Republic); Inserm, UMR1048, Obesity Research Laboratory, Institute of Metabolic and Cardiovascular Diseases, 31432 Toulouse, Cedex 4 (France); University of Toulouse, UMR1048, Paul Sabatier University, 31432 Toulouse, Cedex 4 (France); Toulouse University Hospitals, Department of Clinical Biochemistry, 31059 Toulouse, Cedex 9 (France); Rossmeislová, Lenka, E-mail: Lenka.Rossmeislova@lf3.cuni.cz [Franco-Czech Laboratory for Clinical Research on Obesity, Third Faculty of Medicine, Prague (Czech Republic); Department of Sport Medicine, Third Faculty of Medicine, Charles University in Prague, CZ-100 00 (Czech Republic)

    2015-05-08

    Background: Adipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. Methods: Preadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. Results: Exposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1–24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. Conclusions: Acute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes. - Highlights: • High intensity ERS inhibits lipogenic capacity of adipocytes. • ERS impairs adipogenesis when present in early stages of adipogenesis. • Lipogenesis in mature adipocytes is not

  8. Trans-10, cis-12 conjugated linoleic acid decreases de novo lipid synthesis in human adipocytes

    DEFF Research Database (Denmark)

    Obsen, Thomas; Faergeman, Nils J; Chung, Soonkyu;

    2012-01-01

    Conjugated linoleic acid (CLA) reduces adiposity in vivo. However, mechanisms mediating these changes are unclear. Therefore, we treated cultures of human adipocytes with trans-10, cis-12 (10,12) CLA, cis-9, trans-11 (9,11) CLA or other trans fatty acids (FA), and measured indices of lipid......]-oleic or [(14)C]-linoleic acids. When using [(14)C]-acetic acid and [(14)C]-pyruvic acid as substrates, 30 μM 10,12 CLA, but not 9,11 CLA, decreased de novo synthesis of triglyceride, free FA, diacylglycerol, cholesterol esters, cardiolipin, phospholipids and ceramides within 3-24 h. Treatment with 30 μM 10......,12 CLA, but not 9,11 CLA, decreased total cellular lipids within 3 days and the ratio of monounsaturated FA (MUFA) to saturated FA, and increased C18:0 acyl-CoA levels within 24 h. Consistent with these data, stearoyl-CoA desaturase (SCD)-1 mRNA and protein levels were down-regulated by 10,12 CLA within...

  9. Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

    Directory of Open Access Journals (Sweden)

    Peraldi Pascal

    2008-02-01

    Full Text Available Abstract Background Multipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue. Results Our results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis. Conclusion These results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.

  10. Reappraisal of bicarbonate secretion by the human oesophagus

    DEFF Research Database (Denmark)

    Mertz-Nielsen, A; Hillingsø, J; Bukhave, Klaus

    1997-01-01

    BACKGROUND AND AIMS: Administration of omeprazole to healthy volunteers was recently reported to increase proximal duodenal mucosalbicarbonate secretion. As human oesophagus also secretes bicarbonate, the hypothesis was tested that omeprazole may stimulate oesophagealbicarbonate secretion and thus....../day omeprazole for three days and 80 mg intravenous omeprazole before perfusionor 600 mg/day ranitidine for three days and 50 mg/h intravenously during the perfusion. Saliva and samples of aspirate from the perfusedoesophagus and stomach were collected and bicarbonate concentrations were measured. RESULTS......: The median rates (95% confidence intervals)of intrinsic oesophageal bicarbonate secretion, corrected for contaminating salivary and gastric bicarbonate, were 89 (33-150) and 121 (63-203)mumol/h/10 cm (p > 0.5) in omeprazole and ranitidine treated subjects respectively. Salivary and gastric bicarbonate...

  11. Adipocyte differentiation and leptin expression

    DEFF Research Database (Denmark)

    Hwang, C S; Loftus, T M; Mandrup, S

    1997-01-01

    , most notably those of the C/EBP and PPAR families, which combine to regulate each other and to control the expression of adipocyte-specific genes. One such gene, i.e. the obese gene, was recently identified and found to encode a hormone, referred to as leptin, that plays a major role in the regulation...... of energy intake and expenditure. The hormonal and transcriptional control of adipocyte differentiation is discussed, as is the role of leptin and other factors secreted by the adipocyte that participate in the regulation of adipose homeostasis....

  12. The secret of neuroscience boom: Are there secret human experiments in Latin América?

    Directory of Open Access Journals (Sweden)

    David Salinas Flores

    2016-01-01

    Full Text Available About 6 years ago there sparked a phenomenon in science called the neuroscientific boom. Neurologists underpin this phenomenon to cost reduction techniques such as electroencephalograms and to improved noninvasive technology such as functional MRI. But the human brain, the most complex organ in the universe, has not yet been fully investigated with the existing noninvasive technologies. Thus, there is a suspicion that the real reason for this boom is a secret, forced, and illicit human experimentation in Latin America. Physicians should investigate, be alert, and report these potential unethical human experiments to prevent any further damage to the public health of the citizens of Latin societies.

  13. Second messenger signaling mechanisms of the brown adipocyte thermogenic program: an integrative perspective.

    Science.gov (United States)

    Shi, Fubiao; Collins, Sheila

    2017-09-26

    β-adrenergic receptors (βARs) are well established for conveying the signal from catecholamines to adipocytes. Acting through the second messenger cyclic adenosine monophosphate (cAMP) they stimulate lipolysis and also increase the activity of brown adipocytes and the 'browning' of adipocytes within white fat depots (so-called 'brite' or 'beige' adipocytes). Brown adipose tissue mitochondria are enriched with uncoupling protein 1 (UCP1), which is a regulated proton channel that allows the dissipation of chemical energy in the form of heat. The discovery of functional brown adipocytes in humans and inducible brown-like ('beige' or 'brite') adipocytes in rodents have suggested that recruitment and activation of these thermogenic adipocytes could be a promising strategy to increase energy expenditure for obesity therapy. More recently, the cardiac natriuretic peptides and their second messenger cyclic guanosine monophosphate (cGMP) have gained attention as a parallel signaling pathway in adipocytes, with some unique features. In this review, we begin with some important historical work that touches upon the regulation of brown adipocyte development and physiology. We then provide a synopsis of some recent advances in the signaling cascades from β-adrenergic agonists and natriuretic peptides to drive thermogenic gene expression in the adipocytes and how these two pathways converge at a number of unexpected points. Finally, moving from the physiologic hormonal signaling, we discuss yet another level of control downstream of these signals: the growing appreciation of the emerging roles of non-coding RNAs as important regulators of brown adipocyte formation and function. In this review, we discuss new developments in our understanding of the signaling mechanisms and factors including new secreted proteins and novel non-coding RNAs that control the function as well as the plasticity of the brown/beige adipose tissue as it responds to the energy needs and environmental

  14. Human placenta secretes apolipoprotein B-100-containing lipoproteins

    DEFF Research Database (Denmark)

    Munk-Madsen, Eva; Lindegaard, Marie Louise Skakkebæk; Andersen, Claus B

    2004-01-01

    Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very...... of lipid transfer from the mother to the developing fetus....

  15. Gene-chip studies of adipogenesis-regulated microRNAs in mouse primary adipocytes and human obesity

    Directory of Open Access Journals (Sweden)

    Gallagher Iain J

    2011-03-01

    Full Text Available Abstract Background Adipose tissue abundance relies partly on the factors that regulate adipogenesis, i.e. proliferation and differentiation of adipocytes. While components of the transcriptional program that initiates adipogenesis is well-known, the importance of microRNAs in adipogenesis is less well studied. We thus set out to investigate whether miRNAs would be actively modulated during adipogenesis and obesity. Methods Several models exist to study adipogenesis in vitro, of which the cell line 3T3-L1 is the most well known, albeit not the most physiologically appropriate. Thus, as an alternative, we produced EXIQON microarray of brown and white primary murine adipocytes (prior to and following differentiation to yield global profiles of miRNAs. Results We found 65 miRNAs regulated during in vitro adipogenesis in primary adipocytes. We evaluated the similarity of our responses to those found in non-primary cell models, through literature data-mining. When comparing primary adipocyte profiles, with those of cell lines reported in the literature, we found a high degree of difference in 'adipogenesis' regulated miRNAs suggesting that the model systems may not be accurately representing adipogenesis. The expression of 10 adipogenesis-regulated miRNAs were studied using real-time qPCR and then we selected 5 miRNAs, that showed robust expression, were profiled in subcutaneous adipose tissue obtained from 20 humans with a range of body mass indices (BMI, range = 21-48, and all samples have U133+2 Affymetrix profiles provided. Of the miRNAs tested, mir-21 was robustly expressed in human adipose tissue and positively correlated with BMI (R2 = 0.49, p Conclusion In conclusion, we provide a preliminary analysis of miRNAs associated with primary cell in vitro adipogenesis and demonstrate that the inflammation-associated miRNA, mir-21 is up-regulated in subcutaneous adipose tissue in human obesity. Further, we provide a novel transcriptomics database of

  16. Metabolic signatures of cultured human adipocytes from metabolically healthy versus unhealthy obese individuals.

    Directory of Open Access Journals (Sweden)

    Anja Böhm

    Full Text Available Among obese subjects, metabolically healthy and unhealthy obesity (MHO/MUHO can be differentiated: the latter is characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Aim of this study was, to identify adipocyte-specific metabolic signatures and functional biomarkers for MHO versus MUHO.10 insulin-resistant (IR vs. 10 insulin-sensitive (IS non-diabetic morbidly obese (BMI >40 kg/m2 Caucasians were matched for gender, age, BMI, and percentage of body fat. From subcutaneous fat biopsies, primary preadipocytes were isolated and differentiated to adipocytes in vitro. About 280 metabolites were investigated by a targeted metabolomic approach intracellularly, extracellularly, and in plasma.Among others, aspartate was reduced intracellularly to one third (p = 0.0039 in IR adipocytes, pointing to a relative depletion of citric acid cycle metabolites or reduced aspartate uptake in MUHO. Other amino acids, already known to correlate with diabetes and/or obesity, were identified to differ between MUHO's and MHO's adipocytes, namely glutamine, histidine, and spermidine. Most species of phosphatidylcholines (PCs were lower in MUHO's extracellular milieu, though simultaneously elevated intracellularly, e.g., PC aa C32∶3, pointing to increased PC synthesis and/or reduced PC release. Furthermore, altered arachidonic acid (AA metabolism was found: 15(S-HETE (15-hydroxy-eicosatetraenoic acid; 0 vs. 120pM; p = 0.0014, AA (1.5-fold; p = 0.0055 and docosahexaenoic acid (DHA, C22∶6; 2-fold; p = 0.0033 were higher in MUHO. This emphasizes a direct contribution of adipocytes to local adipose tissue inflammation. Elevated DHA, as an inhibitor of prostaglandin synthesis, might be a hint for counter-regulatory mechanisms in MUHO.We identified adipocyte-inherent metabolic alterations discriminating between MHO and MUHO.

  17. Biophysical assessment of human aquaporin-7 as a water and glycerol channel in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Ana Madeira

    Full Text Available The plasma membrane aquaporin-7 (AQP7 has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.

  18. Secretion of human interferon alpha 2b by Streptomyces lividans.

    Science.gov (United States)

    Pimienta, E; Fando, R; Sánchez, J C; Vallin, C

    2002-02-01

    Biologically active human interferon alpha 2b (HuIFNalpha-2b) was secreted into the culture medium by Streptomyces lividans transformed with recombinant plasmids coding for HuIFNalpha-2b fused to the Streptomyces exfoliatus M11 lipase A signal sequence. Levels were low, 15 or 100 ng/ml, depending on the plasmid used. Neither processed nor unprocessed HuIFNalpha-2b was detected in cell lysates of the transformants secreting the recombinant product. However, the secreted recombinant product was found to partially degrade when cultures reached the stationary phase by the action of an, as yet, unidentified mycelium-associated factor. Experimental evidence suggests that the degrading factor is related to mycelium-associated proteolytic activity.

  19. White-to-brown metabolic conversion of human adipocytes by JAK inhibition

    NARCIS (Netherlands)

    Moisan, A.; Lee, Y. K.; Zhang, J.D.; Hudak, C.S.; Meyer, C.A.; Prummer, M.; Zoffmann, S.; Truong, H.H.; Ebeling, M.; Kiialainen, A.; Gerard, R.; Xia, F.; Schinzel, R.T.; Amrein, K.E.; Cowan, C.A.

    2015-01-01

    The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been

  20. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

    Science.gov (United States)

    Viguerie-Bascands, N; Saulnier-Blache, J S; Dandine, M; Dauzats, M; Daviaud, D; Langin, D

    1999-03-05

    Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans. Copyright 1999 Academic Press.

  1. The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism.

    Directory of Open Access Journals (Sweden)

    Takeo Iwata

    Full Text Available Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL and acetyl-CoA carboxylase (ACC, in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT, suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA, which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through

  2. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1979-01-01

    Efforts were directed towards maintenance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro. The production of human growth hormone (hGH) by this means would be of benefit for the treatment of certain human hypopituitary diseases such as dwarfism. One of the primary approaches was the testing of agents which may logically be expected to increase hGH release. The progress towards this goal is summarized. Results from preliminary experiments dealing with electrophoresis of pituitary cell for the purpose of somatotroph separation are described.

  3. Purification and cultivation of human pituitary growth hormone secreting cells

    Science.gov (United States)

    Hymer, W. C.

    1978-01-01

    The maintainance of actively secreting human pituitary growth hormone cells (somatotrophs) in vitro was studied. The primary approach was the testing of agents which may be expected to increase the release of the human growth hormone (hGH). A procedure for tissue procurement is described along with the methodologies used to dissociate human pituitary tissue (obtained either at autopsy or surgery) into single cell suspensions. The validity of the Biogel cell column perfusion system for studying the dynamics of GH release was developed and documented using a rat pituitary cell system.

  4. Selective novel inverse agonists for human GPR43 augment GLP-1 secretion.

    Science.gov (United States)

    Park, Bi-Oh; Kim, Seong Heon; Kong, Gye Yeong; Kim, Da Hui; Kwon, Mi So; Lee, Su Ui; Kim, Mun-Ock; Cho, Sungchan; Lee, Sangku; Lee, Hyun-Jun; Han, Sang-Bae; Kwak, Young Shin; Lee, Sung Bae; Kim, Sunhong

    2016-01-15

    GPR43/Free Fatty Acid Receptor 2 (FFAR2) is known to be activated by short-chain fatty acids and be coupled to Gi and Gq family of heterotrimeric G proteins. GPR43 is mainly expressed in neutrophils, adipocytes and enteroendocrine cells, implicated to be involved in inflammation, obesity and type 2 diabetes. However, several groups have reported the contradictory data about the physiological functions of GPR43, so that its roles in vivo remain unclear. Here, we demonstrate that a novel compound of pyrimidinecarboxamide class named as BTI-A-404 is a selective and potent competitive inverse agonist of human GPR43, but not the murine ortholog. Through structure-activity relationship (SAR), we also found active compound named as BTI-A-292. These regulators increased the cyclic AMP level and reduced acetate-induced cytoplasmic Ca(2+) level. Furthermore, we show that they modulated the downstream signaling pathways of GPR43, such as ERK, p38 MAPK, and NF-κB. It was surprising that two compounds augmented the secretion of glucagon-like peptide 1 (GLP-1) in NCI-H716 cell line. Collectively, these novel and specific competitive inhibitors regulate all aspects of GPR43 signaling and the results underscore the therapeutic potential of them.

  5. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Bingbing Jia

    Full Text Available Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA and exchange protein directly activated by cAMP (Epac in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS. We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2 (PGI(2 may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX. Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

  6. microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

    DEFF Research Database (Denmark)

    Hamam, D; Ali, D; Vishnubalaji, R

    2014-01-01

    MSC, and utilized bioinformatics as well as functional and biochemical assays, and identified several novel miRNAs differentially expressed during adipogenesis. Among these, miR-320 family (miR-320a, 320b, 320c, 320d and 320e) were ~2.2-3.0-fold upregulated. Overexpression of miR-320c in hMSC enhanced adipocytic......, MIB1 (mindbomb E3 ubiquitin protein ligase 1), PAX6 (paired box 6), YWHAH and ZWILCH. siRNA-mediated silencing of those genes enhanced adipocytic differentiation of hMSC, thus corroborating an important role for those genes in miR-320c-mediated adipogenesis. Concordant with that, lentiviral......-mediated stable expression of miR-320c at physiological levels (~1.5-fold) promoted adipocytic and suppressed osteogenic differentiation of hMSC. Luciferase assay validated RUNX2 (Runt-related transcription factor 2) as a bona fide target for miR-320 family. Therefore, our data suggest miR-320 family as possible...

  7. Evaluation of the synuclein-γ (SNCG gene as a PPARγ target in murine adipocytes, dorsal root ganglia somatosensory neurons, and human adipose tissue.

    Directory of Open Access Journals (Sweden)

    Tamara N Dunn

    Full Text Available Recent evidence in adipocytes points to a role for synuclein-γ in metabolism and lipid droplet dynamics, but interestingly this factor is also robustly expressed in peripheral neurons. Specific regulation of the synuclein-γ gene (Sncg by PPARγ requires further evaluation, especially in peripheral neurons, prompting us to test if Sncg is a bona fide PPARγ target in murine adipocytes and peripheral somatosensory neurons derived from the dorsal root ganglia (DRG. Sncg mRNA was decreased in 3T3-L1 adipocytes (~68% by rosiglitazone, and this effect was diminished by the PPARγ antagonist T0070907. Chromatin immunoprecipitation experiments confirmed PPARγ protein binding at two promoter sequences of Sncg during 3T3-L1 adipogenesis. Rosiglitazone did not affect Sncg mRNA expression in murine cultured DRG neurons. In subcutaneous human WAT samples from two cohorts treated with pioglitazone (>11 wks, SNCG mRNA expression was reduced, albeit highly variable and most evident in type 2 diabetes. Leptin (Lep expression, thought to be coordinately-regulated with Sncg based on correlations in human adipose tissue, was also reduced in 3T3-L1 adipocytes by rosiglitazone. However, Lep was unaffected by PPARγ antagonist, and the LXR agonist T0901317 significantly reduced Lep expression (~64% while not impacting Sncg. The results support the concept that synuclein-γ shares some, but not all, gene regulators with leptin and is a PPARγ target in adipocytes but not DRG neurons. Regulation of synuclein-γ by cues such as PPARγ agonism in adipocytes is logical based on recent evidence for an important role for synuclein-γ in the maintenance and dynamics of adipocyte lipid droplets.

  8. Differential genes in adipocytes induced from polycystic and non-polycystic ovary syndrome-derived human embryonic stem cells.

    Science.gov (United States)

    Wang, Fang; Liu, Wei-Wei; Chen, Xue-Mei; Kong, Hui-Juan; Li, Jing; Sun, Ying-Pu

    2014-06-01

    We explored the molecular mechanisms of obesity and insulin resistance in patients with polycystic ovary syndrome (PCOS) using a human embryonic stem cell model (hESCs). Three PCOS-derived and one non-PCOS-derived hESC lines were induced into adipocytes, and then total RNA was extracted. The differentially expressed PCOS-derived and non-PCOS-derived adipocytes genes were identified using the Boao Biological human V 2.0 whole genome oligonucleotide microarray. Signals of interest were then validated by real-time PCR. A total of 153 differential genes were expressed of which 91 genes were up-regulated and 62 down-regulated. Nuclear receptor subfamily 0, group B, member 2 (NR0B2) was an up-regulated gene, and the GeneChip CapitalBio® Molecule Annotation System V4.0 indicated that it was associated with obesity and diabetes (Ratio ≥ 2.0X). Multiple genes are involved in PCOS. Nuclear receptor subfamily 0, group B, member 2 may play a role in obesity and insulin resistance in patients with PCOS.

  9. Periprostatic adipocytes act as a driving force for prostate cancer progression in obesity.

    Science.gov (United States)

    Laurent, Victor; Guérard, Adrien; Mazerolles, Catherine; Le Gonidec, Sophie; Toulet, Aurélie; Nieto, Laurence; Zaidi, Falek; Majed, Bilal; Garandeau, David; Socrier, Youri; Golzio, Muriel; Cadoudal, Thomas; Chaoui, Karima; Dray, Cedric; Monsarrat, Bernard; Schiltz, Odile; Wang, Yuan Yuan; Couderc, Bettina; Valet, Philippe; Malavaud, Bernard; Muller, Catherine

    2016-01-12

    Obesity favours the occurrence of locally disseminated prostate cancer in the periprostatic adipose tissue (PPAT) surrounding the prostate gland. Here we show that adipocytes from PPAT support the directed migration of prostate cancer cells and that this event is strongly promoted by obesity. This process is dependent on the secretion of the chemokine CCL7 by adipocytes, which diffuses from PPAT to the peripheral zone of the prostate, stimulating the migration of CCR3 expressing tumour cells. In obesity, higher secretion of CCL7 by adipocytes facilitates extraprostatic extension. The observed increase in migration associated with obesity is totally abrogated when the CCR3/CCL7 axis is inhibited. In human prostate cancer tumours, expression of the CCR3 receptor is associated with the occurrence of aggressive disease with extended local dissemination and a higher risk of biochemical recurrence, highlighting the potential benefit of CCR3 antagonists in the treatment of prostate cancer.

  10. Aldose reductases influence prostaglandin F2α levels and adipocyte differentiation in male mouse and human species.

    Science.gov (United States)

    Pastel, Emilie; Pointud, Jean-Christophe; Loubeau, Gaëlle; Dani, Christian; Slim, Karem; Martin, Gwenaëlle; Volat, Fanny; Sahut-Barnola, Isabelle; Val, Pierre; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2015-05-01

    Aldose reductases (AKR1B) are widely expressed oxidoreductases whose physiological function remains elusive. Some isoforms are genuine prostaglandin F2α (PGF2α) synthases, suggesting they might influence adipose homeostasis because PGF2α inhibits adipogenesis. This was shown by Akr1b7 gene ablation in the mouse, which resulted in increased adiposity related to a lower PGF2α content in fat. Yet humans have no ortholog gene for Akr1b7, so the role of aldose reductases in human adipose homeostasis remains to be explored. We analyzed expression of genes encoding human and mouse aldose reductase isoforms in adipose tissues and differentiating adipocytes to assess conserved mechanisms regulating PGF2α synthesis and adipogenesis. The Akr1b3 gene encoded the most abundant isoform in mouse adipose tissue, whereas Akr1b7 encoded the only isoform enriched in the stromal vascular fraction. Most mouse aldose reductase gene expression peaked in early adipogenesis of 3T3-L1 cells and diminished with differentiation. In contrast with its mouse ortholog Akr1b3, AKR1B1 expression increased throughout differentiation of human multipotent adipose-derived stem cells, paralleling PGF2α release, whereas PGF2α receptor (FP) levels collapsed in early differentiation. Pharmacological inhibition of aldose reductase using Statil altered PGF2α production and enhanced human multipotent adipose-derived stem adipocyte differentiation. As expected, the adipogenic effects of Statil were counteracted by an FP agonist (cloprostenol). Thus, in both species aldose reductase-dependent PGF2α production could be important in early differentiation to restrict adipogenesis. PGF2α antiadipogenic signaling could then be toned down through the FP receptor or aldose reductases down-regulation in human and mouse cells, respectively. Our data suggest that aldose reductase inhibitors could have obesogenic potential.

  11. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed;

    2012-01-01

    Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA......) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence......(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long...

  12. Reduced phosphorylation of AS160 contributes to glucocorticoid-mediated inhibition of glucose uptake in human and murine adipocytes.

    Science.gov (United States)

    Ngo, Sherry; Barry, Janelle B; Nisbet, Janelle C; Prins, Johannes B; Whitehead, Jonathan P

    2009-04-10

    Excess glucocorticoids induce insulin resistance and reduce glucose uptake although the underlying mechanisms are unclear. Here we demonstrate that Dex (1 microM for 24h) inhibits basal and insulin (1 nM) stimulated glucose uptake in human and murine adipocytes by 50% with a concomitant reduction in the levels of GLUT1/4 at the plasma membrane but no change in total GLUT1/4 levels. Expression and phosphorylation of proximal insulin signalling molecules (IRS1, PI3K, AKT) was unaffected by Dex as was phosphorylation of mTOR and FOXO1. In contrast, phosphorylation of AKT substrate 160kDa (AS160) at T642, which is essential for 14-3-3 recruitment and GLUT4 translocation, was reduced by 50% in basal and insulin-stimulated cells and this was mirrored by decreased 14-3-3 association. Co-treatment with the glucocorticoid receptor antagonist RU486 (10 microM) abrogated the Dex effect on AS160-T642 phosphorylation and restored glucose uptake by 80%. These data suggest Dex inhibits glucose uptake in adipocytes, at least in part, by reducing AS160 phosphorylation and interaction with 14-3-3.

  13. Reactive oxygen species facilitate translocation of hormone sensitive lipase to the lipid droplet during lipolysis in human differentiated adipocytes.

    Directory of Open Access Journals (Sweden)

    Sarah A Krawczyk

    Full Text Available In obesity, there is an increase in reactive oxygen species (ROS within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies.

  14. Reactive oxygen species facilitate translocation of hormone sensitive lipase to the lipid droplet during lipolysis in human differentiated adipocytes.

    Science.gov (United States)

    Krawczyk, Sarah A; Haller, Jorge F; Ferrante, Tom; Zoeller, Raphael A; Corkey, Barbara E

    2012-01-01

    In obesity, there is an increase in reactive oxygen species (ROS) within adipose tissue caused by increases in inflammation and overnutrition. Hormone sensitive lipase (HSL) is part of the canonical lipolytic pathway and critical for complete lipolysis. This study hypothesizes that ROS is a signal that integrates regulation of lipolysis by targeting HSL. Experiments were performed with human differentiated adipocytes from the subcutaneous depot. Antioxidants were employed as a tool to decrease ROS, and it was found that scavenging ROS with diphenyliodonium, N-acetyl cysteine, or resveratrol decreased lipolysis in adipocytes. HSL phosphorylation of a key serine residue, Ser552, as well as translocation of this enzyme from the cytosol to the lipid droplet upon lipolytic stimulation were both abrogated by scavenging ROS. The phosphorylation status of other serine residues on HSL were not affected. These findings are significant because they document that ROS contributes to the physiological regulation of lipolysis via an effect on translocation. Such regulation could be useful in developing new obesity therapies.

  15. TLR-3 is present in human adipocytes, but its signalling is not required for obesity-induced inflammation in adipose tissue in vivo.

    Directory of Open Access Journals (Sweden)

    Dov B Ballak

    Full Text Available Innate immunity plays a pivotal role in obesity-induced low-grade inflammation originating from adipose tissue. Key receptors of the innate immune system including Toll-like receptors-2 and -4 (TLRs are triggered by nutrient excess to promote inflammation. The role of other TLRs in this process is largely unknown. In addition to double-stranded viral mRNA, TLR-3 can also recognize mRNA from dying endogenous cells, a process that is frequently observed within obese adipose tissue. Here, we identified profound expression of TLR-3 in adipocytes and investigated its role during diet-induced obesity. Human adipose tissue biopsies (n=80 and an adipocyte cell-line were used to study TLR-3 expression and function. TLR-3-/- and WT animals were exposed to a high-fat diet (HFD for 16 weeks to induce obesity. Expression of TLR-3 was significantly higher in human adipocytes compared to the non-adipocyte cells part of the adipose tissue. In vitro, TLR-3 expression was induced during differentiation of adipocytes and stimulation of the receptor led to elevated expression of pro-inflammatory cytokines. In vivo, TLR-3 deficiency did not significantly influence HFD-induced obesity, insulin sensitivity or inflammation. In humans, TLR-3 expression in adipose tissue did not correlate with BMI or insulin sensitivity (HOMA-IR. Together, our results demonstrate that TLR-3 is highly expressed in adipocytes and functionally active. However, TLR-3 appears to play a redundant role in obesity-induced inflammation and insulin resistance.

  16. Adipocyte induced arterial calcification is prevented with sodium thiosulfate

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Neal X., E-mail: xuechen@iupui.edu [Divison of Nephrology, Indiana University School of Medicine, Indianapolis, IN (United States); O’Neill, Kalisha; Akl, Nader Kassis [Divison of Nephrology, Indiana University School of Medicine, Indianapolis, IN (United States); Moe, Sharon M. [Divison of Nephrology, Indiana University School of Medicine, Indianapolis, IN (United States); Roudebush VA Medical Center, Indianapolis, IN (United States)

    2014-06-20

    Highlights: • High phosphorus can induce calcification of adipocytes, even when fully differentiated. • Adipocytes can induce vascular calcification in an autocrine manner. • Sodium thiosulfate inhibits adipocyte calcification. - Abstract: Background: Calcification can occur in fat in multiple clinical conditions including in the dermis, breasts and in the abdomen in calciphylaxis. All of these are more common in patients with advanced kidney disease. Clinically, hyperphosphatemia and obesity are risk factors. Thus we tested the hypothesis that adipocytes can calcify in the presence of elevated phosphorus and/or that adipocytes exposed to phosphorus can induce vascular smooth muscle cell (VSMC) calcification. Methods: 3T3-L1 preadipocytes were induced into mature adipocytes and then treated with media containing high phosphorus. Calcification was assessed biochemically and PCR performed to determine the expression of genes for osteoblast and adipocyte differentiation. Adipocytes were also co-cultured with bovine VSMC to determine paracrine effects, and the efficacy of sodium thiosulfate was determined. Results: The results demonstrated that high phosphorus induced the calcification of differentiated adipocytes with increased expression of osteopontin, the osteoblast transcription factor Runx2 and decreased expression of adipocyte transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (CEBPα), indicating that high phosphorus led to a phenotypic switch of adipocytes to an osteoblast like phenotype. Sodium thiosulfate, dose dependently decreased adipocyte calcification and inhibited adipocyte induced increase of VSMC calcification. Co-culture studies demonstrated that adipocytes facilitated VSMC calcification partially mediated by changes of secretion of leptin and vascular endothelial growth factor (VEGF) from adipocytes. Conclusion: High phosphorus induced calcification of mature adipocytes, and

  17. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    Science.gov (United States)

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  18. Reappraisal of bicarbonate secretion by the human oesophagus

    DEFF Research Database (Denmark)

    Mertz-Nielsen, A; Hillingsø, Jens; Bukhave, K

    1997-01-01

    measured. RESULTS: The median rates (95% confidence intervals) of intrinsic oesophageal bicarbonate secretion, corrected for contaminating salivary and gastric bicarbonate, were 89 (33-150) and 121 (63-203) mumol/h/10 cm (p > 0.5) in omeprazole and ranitidine treated subjects respectively. Salivary...... and gastric bicarbonate contaminating the oesophagus accounted for 14% and 3%, respectively, of total oesophageal bicarbonate output. CONCLUSIONS: Bicarbonate secretory capacity of the human oesophagus is less than previously assumed, and the clinical relevance of intrinsic oesophageal bicarbonate for mucosal...

  19. Involvement of a cGMP-dependent pathway in the natriuretic peptide-mediated hormone-sensitive lipase phosphorylation in human adipocytes.

    Science.gov (United States)

    Sengenes, Coralie; Bouloumie, Anne; Hauner, Hans; Berlan, Michel; Busse, Rudi; Lafontan, Max; Galitzky, Jean

    2003-12-05

    Our previous studies have demonstrated that natriuretic peptides (NPs), peptide hormones with natriuretic, diuretic, and vasodilating properties, exert a potent control on the lipolysis in human adipocytes via the activation of the type A guanylyl cyclase receptor (1, 2). In the current study we investigated the intracellular mechanisms involved in the NP-stimulated lipolytic effect in human preadipocytes and adipocytes. We demonstrate that the atrial NP (ANP)-induced lipolysis in human adipocytes was associated with an enhanced serine phosphorylation of the hormone-sensitive lipase (HSL). Both ANP-mediated lipolysis and HSL phosphorylation were inhibited in the presence of increasing concentrations of the guanylyl cyclase inhibitor LY-83583. ANP did not modulate the activity of the cAMP-dependent protein kinase (PKA). Moreover, H-89, a PKA inhibitor, did not affect the ANP-induced lipolysis. On primary cultures of human preadipocytes, the ANP-mediated lipolytic effect was dependent on the differentiation process. On differentiated human preadipocytes, ANP-mediated lipolysis, associated with an increased phosphorylation of HSL and of perilipin A, was strongly decreased by treatment with the inhibitor of the cGMP-dependent protein kinase I (cGKI), Rp-8-pCPT-cGMPS. Thus, ANP-induced lipolysis in human adipocytes is a cGMP-dependent pathway that induces the phosphorylation of HSL and perilipin A via the activation of cGKI. The present study shows that lipolysis in human adipocytes can be controlled by an independent cGKI-mediated signaling as well as by the classical cAMP/PKA pathway.

  20. Evaluation of the synuclein-y (SNCG) gene as a PPARy target in murine adipocytes, dorsal root ganglia somatosensory neurons, and human adipose tissue

    Science.gov (United States)

    Synuclein-gamma is highly expressed in both adipocytes and peripheral nervous system (PNS) somatosensory neurons. Its mRNA is induced during adipogenesis, increased in obese human white adipose tissue (WAT), may be coordinately regulated with leptin, and is decreased following treatment of murine 3T...

  1. Effect of silent adipocyte fatty acid-binding protein gene on secretion of adipocyte-synthesized triglyceride and adiponectin%沉默脂肪细胞型脂肪酸结合蛋白基因对脂肪细胞合成甘油三酯及脂联素分泌的影响

    Institute of Scientific and Technical Information of China (English)

    吴洁; 钟敏; 邹瑾

    2012-01-01

    Objective To study the effect of 3T3-L1 adipocyte fatty acid-binding protein(A-FABP) gene on secretion of adipocyte-synthesized triglyceride and adiponectin by constructing its micro RNA expression vector using RNAi technology. Methods A microRMA expression vector for A-FABP gene was constructed and transfected into 3T3-L1 adipocytes. Expression of A-FABP mRNA and protein was detected by RT-PCR and Western blot, respectively. A model of silent A-FABP gene was established. Free fatty acid(FFA) cells(0. 5 mmol/L) and mature adpocytes were incubated for 24 h and divided into control group,FFA group,RNAi group,and RNAi + FFA group. Triglyceride and adiponectin levels were measured by ELISA. Expression of A-FABP and adiponectin mRNA was detected by RT-PCR. Results The microRNA expression vector after transfected into 3T3-L1 adipocytes could significantly inhibit the expression of A-FABP mRNA and protein. The synthesis of triglyceride increased significantly while the secretion of adiponectin decreased significantly with the increasing fatty acid concentration in adipocytes(P<0. 05). The triglyceride level was significantly lower while the adiponectin secretion level and the adiponectin mRNA expression level were significantly higher in RNAi and RNAi+ groups than in control and fatty acid groups(P<0. 05). Conclusion Silent A-FABP gene may become a new target for preventing and treating atherosclerosis and diabetes mellitus.%目的 应用RNA干扰(RNAi)技术,构建针对3T3-L1脂肪细胞型脂肪酸结合蛋白(A-FABP)的microRNA表达载体,研究沉默A-FABP基因的表达对脂肪细胞合成甘油三酯及脂联素分泌的影响.方法 构建靶向A-FABP基因的microRNA表达载体转染3T3-L1脂肪细胞后,RT PCR及Western blot法检测A-FABP mRNA 及蛋白表达.建立A-FABP基因沉默细胞模型,0.5 mmol/L游离脂肪酸和成熟脂肪细胞共孵育24 h,分为对照组、脂肪酸组、RNAi组、RNAi+脂肪酸组.ELISA法分别检测甘油三酯及脂联

  2. Reduced expression of collagen VI alpha 3 (COL6A3) confers resistance to inflammation-induced MCP1 expression in adipocytes.

    Science.gov (United States)

    Gesta, Stephane; Guntur, Kalyani; Majumdar, Ishita Deb; Akella, Syamala; Vishnudas, Vivek K; Sarangarajan, Rangaprasad; Narain, Niven R

    2016-08-01

    Collagen VI alpha 3 (COL6A3) is associated with insulin resistance and adipose tissue inflammation. In this study, the role of COL6A3 in human adipocyte function was characterized. Immortalized human preadipocyte cell lines stably expressing control or COL6A3 shRNA were used to study adipocyte function and inflammation. COL6A3 knockdown increased triglyceride content, lipolysis, insulin-induced Akt phosphorylation, and mRNA expression of key adipogenic genes (peroxisome proliferator-activated receptor-γ, glucose transporter, adiponectin, and fatty acid binding protein), indicating increased adipocyte function and insulin sensitivity. However, COL6A3 knockdown decreased basal adipocyte chemokine (C-C motif) ligand 2 [CCL2, monocyte chemoattractant protein (MCP1)] mRNA expression, reduced secreted protein levels, and abrogated tumor necrosis factor-α- and lipopolysaccharide-induced MCP1 mRNA expression. In addition, while control adipocytes co-cultured with THP1 macrophages showed a threefold increase in adipocyte MCP1 mRNA expression, in COL6A3 knockdown adipocytes MCP1 mRNA expression was unaltered by co-culturing. Lastly, in normal differentiated adipocytes, matrix metalloproteinase-11 treatment reduced expression of COL6A3 protein, MCP1 mRNA, MCP1 secretion, and abrogated tumor necrosis factor-α- and lipopolysaccharide-induced MCP1 mRNA expression and protein secretion. COL6A3 knockdown in adipocytes leads to the development of a unique state of inflammatory resistance via suppression of MCP1 induction. © 2016 The Obesity Society.

  3. Hesperetin inhibit adipocyte differentiation and enhance Bax- and p21-mediated adipolysis in human mesenchymal stem cell adipogenesis.

    Science.gov (United States)

    Subash-Babu, Pandurangan; Alshatwi, Ali A

    2015-03-01

    We aimed to explore the antiadipogenic and adipolysis effect of hesperetin in human mesenchymal stem cells (hMSCs)-induced adipogenesis. IC50 value of hesperetin was higher for hMSCs such as 149.2 ± 13.2 μmol for 24 h and 89.4 ± 11.4 μmol in 48 h, whereas in preadipocytes was 87.6 ± 9.5 μmol and 72.4 ± 5.6 μmol in 24 h and 48 h, respectively. Hesperetin treatment (5, 10, and 20 μmol) to adipogenesis-induced hMSCs (Group 1) and preadipocytes (Group 2) resulted in a significantly (p p21 expression in Group 2 compared to untreated preadipocytes. hMSCs cultured in adipogenic medium along with hesperetin significantly inhibited adipocyte differentiation and increased the proapoptotic gene expression levels in preadipocyte. Our result indicates the antiadipogenic and adipolysis effects of hesperetin.

  4. Basal Secretion of Lysozyme from Human Airways in Vitro

    Directory of Open Access Journals (Sweden)

    Patricia Roger

    1999-01-01

    Full Text Available The aim of this study was to examine the basal release of lysozyme from isolated human lung tissues. Measurements of lysozyme in the fluids derived from lung preparations were performed using a rate-of-lysis assay subsequent to acidification of the biological samples. Lysozyme released from bronchial preparations into fluids was greater than that observed for parenchymal tissues. The lysozyme quantities detected in bronchial fluids were not modified by removal of the surface epithelium. Furthermore, the quantities of lysozyme in bronchial fluids was correlated with the size of the bronchial preparations. These results suggest that the lysozyme was principally secreted by the human bronchi (submucosal layer rather than by parenchyma tissues and that a greater release was observed in the proximal airways.

  5. Human pituitary and placental hormones control human insulin-like growth factor II secretion in human granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramasharma, K.; Li, C.H.

    1987-05-01

    Human granulosa cells cultured with calf serum actively proliferated for 18-20 generations and secreted progesterone into the medium; progesterone levels appeared to decline with increase in generation number. Cells cultured under serum-free conditions secreted significant amounts of progesterone and insulin-like growth factor II (IGF-II). The progesterone secretion was enhanced by the addition of human follitropin, lutropin, and chorionic gonadotropin but not by growth hormone. These cells, when challenged to varying concentrations of human growth hormone, human chorionic somatomammotropin, human prolactin, chorionic gonadotropin, follitropin, and lutropin, secreted IGF-II into the medium as measured by specific IGF-II RIA. Among these human hormones, chorionic gonadotropin, follitropin, and lutropin were most effective in inducing IGF-II secretion from these cells. When synthetic lutropin-releasing hormone and ..cap alpha..-inhibin-92 were tested, only lutropin-releasing hormone was effective in releasing IGF-II. The results described suggest that cultured human granulosa cells can proliferate and actively secrete progesterone and IGF-II into the medium. IGF-II production in human granulosa cells was influenced by a multi-hormonal complex including human growth hormone, human chorionic somatomammotropin, and prolactin.

  6. Polyphenol-rich grape powder extract (GPE) attenuates inflammation in human macrophages and in human adipocytes exposed to macrophage-conditioned media.

    Science.gov (United States)

    Overman, A; Bumrungpert, A; Kennedy, A; Martinez, K; Chuang, C-C; West, T; Dawson, B; Jia, W; McIntosh, M

    2010-05-01

    Obesity-associated inflammation is characterized by an increased abundance of macrophages (MPhis) in white adipose tissue (WAT), leading to the production of inflammatory cytokines, chemokines and prostaglandins (PGs) that can cause insulin resistance. Grape powder extract (GPE) is rich in phenolic phytochemicals that possess anti-oxidant and anti-inflammatory properties. We examined the ability of GPE to prevent lipopolysaccharide (LPS)-mediated inflammation in human MPhis and silence the cross-talk between human MPhis and adipocytes. We investigated the effect of GPE pretreatment on LPS-mediated activation of mitogen activated protein kinases (MAPKs), nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1), and induction of inflammatory genes in human MPhis (that is, differentiated U937 cells). In addition, we determined the effect of GPE pretreatment of MPhis on inflammation and insulin resistance in primary human adipocytes incubated with LPS-challenged MPhi-conditioned medium (MPhi-CM). Pretreatment of MPhis with GPE attenuated LPS-induction of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-1beta; chemokines, such as IL-8 and interferon-gamma inducible protein-10 (IP-10); and a marker of PG production, cyclooxygenase-2 (COX-2). Grape powder extract also attenuated LPS activation of MAPKs, NF-kappaB and AP-1 (c-Jun), as evidenced by decreased (1) phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38; (2) degradation of IkappaBalpha and activation of an NF-kappaB reporter construct; and (3) phosphorylation of c-Jun and Elk-1. Using LPS-challenged MPhi-CM, GPE pretreatment attenuated MPhi-mediated inflammatory gene expression, activation of an NF-kappaB reporter and suppression of insulin-stimulated glucose uptake in human adipocytes. Collectively, these data demonstrate that GPE attenuates LPS-mediated inflammation in MPhis, possibly by decreasing the activation of MAPKs, NF-kappaB and AP-1

  7. Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites

    Directory of Open Access Journals (Sweden)

    Sandelin Albin

    2011-03-01

    Full Text Available Abstract Background The transcription factors peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer binding protein α (C/EBPα are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes. Results Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors. Conclusions This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.

  8. Obestatin regulates adipocyte function and protects against diet-induced insulin resistance and inflammation.

    Science.gov (United States)

    Granata, Riccarda; Gallo, Davide; Luque, Raul M; Baragli, Alessandra; Scarlatti, Francesca; Grande, Cristina; Gesmundo, Iacopo; Córdoba-Chacón, Jose; Bergandi, Loredana; Settanni, Fabio; Togliatto, Gabriele; Volante, Marco; Garetto, Stefano; Annunziata, Marta; Chanclón, Belén; Gargantini, Eleonora; Rocchietto, Stefano; Matera, Lina; Datta, Giacomo; Morino, Mario; Brizzi, Maria Felice; Ong, Huy; Camussi, Giovanni; Castaño, Justo P; Papotti, Mauro; Ghigo, Ezio

    2012-08-01

    The metabolic actions of the ghrelin gene-derived peptide obestatin are still unclear. We investigated obestatin effects in vitro, on adipocyte function, and in vivo, on insulin resistance and inflammation in mice fed a high-fat diet (HFD). Obestatin effects on apoptosis, differentiation, lipolysis, and glucose uptake were determined in vitro in mouse 3T3-L1 and in human subcutaneous (hSC) and omental (hOM) adipocytes. In vivo, the influence of obestatin on glucose metabolism was assessed in mice fed an HFD for 8 wk. 3T3-L1, hSC, and hOM preadipocytes and adipocytes secreted obestatin and showed specific binding for the hormone. Obestatin prevented apoptosis in 3T3-L1 preadipocytes by increasing phosphoinositide 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK)1/2 signaling. In both mice and human adipocytes, obestatin inhibited isoproterenol-induced lipolysis, promoted AMP-activated protein kinase phosphorylation, induced adiponectin, and reduced leptin secretion. Obestatin also enhanced glucose uptake in either the absence or presence of insulin, promoted GLUT4 translocation, and increased Akt phosphorylation and sirtuin 1 (SIRT1) protein expression. Inhibition of SIRT1 by small interfering RNA reduced obestatin-induced glucose uptake. In HFD-fed mice, obestatin reduced insulin resistance, increased insulin secretion from pancreatic islets, and reduced adipocyte apoptosis and inflammation in metabolic tissues. These results provide evidence of a novel role for obestatin in adipocyte function and glucose metabolism and suggest potential therapeutic perspectives in insulin resistance and metabolic dysfunctions.

  9. Intravenous Glucose Acutely Stimulates Intestinal Lipoprotein Secretion in Healthy Humans.

    Science.gov (United States)

    Xiao, Changting; Dash, Satya; Morgantini, Cecilia; Lewis, Gary F

    2016-07-01

    Increased production of intestinal triglyceride-rich lipoproteins (TRLs) contributes to dyslipidemia and increased risk of atherosclerotic cardiovascular disease in insulin resistance and type 2 diabetes. We have previously demonstrated that enteral glucose enhances lipid-stimulated intestinal lipoprotein particle secretion. Here, we assessed whether glucose delivered systemically by intravenous infusion also enhances intestinal lipoprotein particle secretion in humans. On 2 occasions, 4 to 6 weeks apart and in random order, 10 healthy men received a constant 15-hour intravenous infusion of either 20% glucose to induce hyperglycemia or normal saline as control. Production of TRL-apolipoprotein B48 (apoB48, primary outcomes) and apoB100 (secondary outcomes) was assessed during hourly liquid-mixed macronutrient formula ingestion with stable isotope enrichment and multicompartmental modeling, under pancreatic clamp conditions to limit perturbations in pancreatic hormones (insulin and glucagon) and growth hormone. Compared with saline infusion, glucose infusion induced both hyperglycemia and hyperinsulinemia, increased plasma triglyceride levels, and increased TRL-apoB48 concentration and production rate (Plipoprotein production. Hyperglycemia may contribute to intestinal lipoprotein overproduction in type 2 diabetes. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02607839. © 2016 American Heart Association, Inc.

  10. Acetylcholine regulates pancreastatin secretion from the human pancreastatin-producing cell line (QGP-1N).

    Science.gov (United States)

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Kono, A

    1991-07-01

    Studies were made of pancreastatin (PST) secretion from a human PST-producing cell line (QGP-1N) in response to various secretagogues. Cells with immunoreactivity for PST were observed in monolayer cultures of QGP-1N cells. Carbachol stimulated PST secretion and the intracellular Ca2+ mobilization concentration dependently in the range of 10(-6)-10(-4) M. The PST secretion and Ca2+ mobilization induced by carbachol were inhibited by atropine. The calcium ionophore (A23187) stimulated PST secretion. However, cholecystokinin and gastrin-releasing peptide did not stimulate either PST secretion or Ca2+ mobilization. Secretin also did not stimulate PST secretion. The glucose concentration in the culture medium had no effect on PST secretion. These results suggest that PST secretion is mainly regulated by acetylcholine through a muscarinic receptor, and that an increase in intracellular Ca2+ plays an important role in stimulus-secretion coupling in QGP-1N cells.

  11. Secreted Isoform of Human Lynx1 (SLURP-2)

    DEFF Research Database (Denmark)

    Lyukmanova, E N; Shulepko, M A; Shenkarev, Z O

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we......-evoked currents at concentrations via...... interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the 'classical' orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs....

  12. Analysis of in vitro secretion profiles from adipose-derived cell populations

    Directory of Open Access Journals (Sweden)

    Blaber Sinead P

    2012-08-01

    Full Text Available Abstract Background Adipose tissue is an attractive source of cells for therapeutic purposes because of the ease of harvest and the high frequency of mesenchymal stem cells (MSCs. Whilst it is clear that MSCs have significant therapeutic potential via their ability to secrete immuno-modulatory and trophic cytokines, the therapeutic use of mixed cell populations from the adipose stromal vascular fraction (SVF is becoming increasingly common. Methods In this study we have measured a panel of 27 cytokines and growth factors secreted by various combinations of human adipose-derived cell populations. These were 1. co-culture of freshly isolated SVF with adipocytes, 2. freshly isolated SVF cultured alone, 3. freshly isolated adipocytes alone and 4. adherent adipose-derived mesenchymal stem cells (ADSCs at passage 2. In addition, we produced an ‘in silico’ dataset by combining the individual secretion profiles obtained from culturing the SVF with that of the adipocytes. This was compared to the secretion profile of co-cultured SVF and adipocytes. Two-tailed t-tests were performed on the secretion profiles obtained from the SVF, adipocytes, ADSCs and the ‘in silico’ dataset and compared to the secretion profiles obtained from the co-culture of the SVF with adipocytes. A p-value of  Results A co-culture of SVF and adipocytes results in a distinct secretion profile when compared to all other adipose-derived cell populations studied. This illustrates that cellular crosstalk during co-culture of the SVF with adipocytes modulates the production of cytokines by one or more cell types. No biologically relevant differences were detected in the proteomes of SVF cultured alone or co-cultured with adipocytes. Conclusions The use of mixed adipose cell populations does not appear to induce cellular stress and results in enhanced secretion profiles. Given the importance of secreted cytokines in cell therapy, the use of a mixed cell population such as the

  13. Insulin elevates leptin secretion and mRNA levels via cyclic AMP in 3T3-L1 adipocytes deprived of glucose

    Directory of Open Access Journals (Sweden)

    Tomomi Tsubai

    2016-11-01

    Conclusion: Insulin alone stimulates leptin secretion and elevates leptin mRNA levels via cAMP under the lack of glucose metabolism, while glucose is a significant and ambivalent effector on the insulin effects of leptin.

  14. Differentiating of banked human umbilical cord blood-derived mesenchymal stem cells into insulin-secreting cells.

    Science.gov (United States)

    Phuc, Pham Van; Nhung, Truong Hai; Loan, Dang Thi Tung; Chung, Doan Chinh; Ngoc, Phan Kim

    2011-01-01

    Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are multipotent cells. They are able to differentiate into functional cells from not only mesoderm but also endoderm. Many researches showed that cells derived from fresh human UCB could transdifferentiate into insulin-secreting cells. In this study, transdifferentiating potential of cryopreserved human UCB-derived MSCs into insulin-secreting cell was investigated. Fresh human UCB was enriched the mononuclear cells by Ficoll-Paque centrifugation. The mononuclear cell population was cryopreserved in cryo-medium containing Iscove's modified Dulbecco's media (IMDM) with 10% DMSO at -196°C for 1 yr. After thawing, mononuclear cells were cultured to isolate MSCs in medium IMDM with 20% FBS supplemented with growth factors. At the fifth passages, MSCs were confirmed by flow cytometry about expression of CD13, CD14, CD34, CD45, CD166, and HLA-DR markers; after that, they were induced to differentiate into adipocytes and osteoblasts. After inducing with specific medium for islet differentiation, there were many clusters of cell like islet at day 14-28. Using real-time reverse transcription polymerase chain reaction (RT-PCR) to analyze the expression of functional genes, the result showed that Nestin, Pdx-1, Ngn3, Ils-1, Pax6, Pax4, Nkx2.2, Nkx6.1, Glut-2, Insulin genes expressed. The results showed that MSCs derived from banked cord blood can differentiate into functional pancreatic islet-like cells in vitro. If human MSCs, especially MSCs from banked cord blood of diabetes patients themselves can be isolated, proliferated, differentiated into functional pancreatic islet-like cells, and transplanted back into them (autologous transplantation), their high-proliferation potency and rejection avoidance will provide one promising therapy for diabetes.

  15. Association of lipidome remodeling in the adipocyte membrane with acquired obesity in humans.

    Directory of Open Access Journals (Sweden)

    Kirsi H Pietiläinen

    2011-06-01

    Full Text Available Identification of early mechanisms that may lead from obesity towards complications such as metabolic syndrome is of great interest. Here we performed lipidomic analyses of adipose tissue in twin pairs discordant for obesity but still metabolically compensated. In parallel we studied more evolved states of obesity by investigating a separated set of individuals considered to be morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the obese twin individuals had increased proportions of palmitoleic and arachidonic acids in their adipose tissue, including increased levels of ethanolamine plasmalogens containing arachidonic acid. Information gathered from these experimental groups was used for molecular dynamics simulations of lipid bilayers combined with dependency network analysis of combined clinical, lipidomics, and gene expression data. The simulations suggested that the observed lipid remodeling maintains the biophysical properties of lipid membranes, at the price, however, of increasing their vulnerability to inflammation. Conversely, in morbidly obese subjects, the proportion of plasmalogens containing arachidonic acid in the adipose tissue was markedly decreased. We also show by in vitro Elovl6 knockdown that the lipid network regulating the observed remodeling may be amenable to genetic modulation. Together, our novel approach suggests a physiological mechanism by which adaptation of adipocyte membranes to adipose tissue expansion associates with positive energy balance, potentially leading to higher vulnerability to inflammation in acquired obesity. Further studies will be needed to determine the cause of this effect.

  16. The activity of the endocannabinoid metabolising enzyme fatty acid amide hydrolase in subcutaneous adipocytes correlates with BMI in metabolically healthy humans

    Directory of Open Access Journals (Sweden)

    Alexander Stephen PH

    2011-08-01

    Full Text Available Abstract Background The endocannabinoid system (ECS is a ubiquitously expressed signalling system, with involvement in lipid metabolism and obesity. There are reported changes in obesity of blood concentrations of the endocannabinoids anandamide (AEA and 2-arachidonoylglcyerol (2-AG, and of adipose tissue expression levels of the two key catabolic enzymes of the ECS, fatty acid amide hydrolase (FAAH and monoacylglycerol lipase (MGL. Surprisingly, however, the activities of these enzymes have not been assayed in conditions of increasing adiposity. The aim of the current study was to investigate whether FAAH and MGL activities in human subcutaneous adipocytes are affected by body mass index (BMI, or other markers of adiposity and metabolism. Methods Subcutaneous abdominal mature adipocytes, fasting blood samples and anthropometric measurements were obtained from 28 metabolically healthy subjects representing a range of BMIs. FAAH and MGL activities were assayed in mature adipocytes using radiolabelled substrates. Serum glucose, insulin and adipokines were determined using ELISAs. Results MGL activity showed no relationship with BMI or other adiposity indices, metabolic markers (fasting serum insulin or glucose or serum adipokine levels (adiponectin, leptin or resistin. In contrast, FAAH activity in subcutaneous adipocytes correlated positively with BMI and waist circumference, but not with skinfold thickness, metabolic markers or serum adipokine levels. Conclusions In this study, novel evidence is provided that FAAH activity in subcutaneous mature adipocytes increases with BMI, whereas MGL activity does not. These findings support the hypothesis that some components of the ECS are upregulated with increasing adiposity in humans, and that AEA and 2-AG may be regulated differently.

  17. Human Parotid Gland Alpha-Amylase Secretion as a Function of Chronic Hyperbaric Exposure

    Science.gov (United States)

    1979-01-01

    parotid ...Pullman, WA 99163 Gilman, S. C, G. J. Fischer, R. J. Biersner, R. D. Thornton, and D. A. Miller. 1979. Human parotid gland alpha-amylase secretion...as a function of chronic hyperbaric exposure. Undersea Biomed. Res. 6(3):303-307.—Secretion of a-amylase by the human parotid gland increased

  18. Adipocyte differentiation of human bone marrow-derived stromal cells is modulated by microRNA-155, microRNA-221, and microRNA-222.

    Science.gov (United States)

    Skårn, Magne; Namløs, Heidi M; Noordhuis, Paul; Wang, Meng-Yu; Meza-Zepeda, Leonardo A; Myklebost, Ola

    2012-04-10

    Human mesenchymal stromal cells (hMSCs) are capable of limited self-renewal and multilineage differentiation in vitro. Several studies have demonstrated that microRNAs (miRNAs, miRs), post-transcriptional modifiers of mRNA stability and protein translation, play crucial roles in the regulation of these complex processes. To gain knowledge regarding the role of miRNAs in human adipocyte differentiation, we examined the miRNA expression profile of the immortalized human bone marrow-derived stromal cell line hMSC-Tert20. Such a model system has the advantage of a reproducible and consistent phenotype while maintaining important properties of the primary donor cells, including the potential to differentiate to adipocytes, osteoblasts, and chondrocytes. We identified 12 miRNAs that were differentially expressed during adipogenesis, of which several have been previously shown to play important roles in adipocyte biology. Among these, the expression of miRNA-155, miRNA-221, and miRNA-222 decreased during the adipogenic program of both immortalized and primary hMSCs, suggesting that they act as negative regulators of differentiation. Interestingly, ectopic expression of the miRNAs significantly inhibited adipogenesis and repressed induction of the master regulators PPARγ and CCAAT/enhancer-binding protein alpha. Our study provides the first experimental evidence that miRNA-155, miRNA-221, and miRNA-222 have an important function in human adipocyte differentiation, and that their downregulation is necessary to relieve the repression of genes crucial for this process.

  19. Mammary adipocytes stimulate breast cancer invasion through metabolic remodeling of tumor cells

    Science.gov (United States)

    Wang, Yuan Yuan; Attané, Camille; Milhas, Delphine; Dirat, Béatrice; Dauvillier, Stéphanie; Guerard, Adrien; Gilhodes, Julia; Lazar, Ikrame; Alet, Nathalie; Laurent, Victor; Le Gonidec, Sophie; Hervé, Caroline; Bost, Frédéric; Ren, Guo Sheng; Bono, Françoise; Escourrou, Ghislaine; Prentki, Marc; Nieto, Laurence; Valet, Philippe

    2017-01-01

    In breast cancer, a key feature of peritumoral adipocytes is their loss of lipid content observed both in vitro and in human tumors. The free fatty acids (FFAs), released by adipocytes after lipolysis induced by tumor secretions, are transferred and stored in tumor cells as triglycerides in lipid droplets. In tumor cell lines, we demonstrate that FFAs can be released over time from lipid droplets through an adipose triglyceride lipase–dependent (ATGL-dependent) lipolytic pathway. In vivo, ATGL is expressed in human tumors where its expression correlates with tumor aggressiveness and is upregulated by contact with adipocytes. The released FFAs are then used for fatty acid β-oxidation (FAO), an active process in cancer but not normal breast epithelial cells, and regulated by coculture with adipocytes. However, in cocultivated cells, FAO is uncoupled from ATP production, leading to AMPK/acetyl-CoA carboxylase activation, a circle that maintains this state of metabolic remodeling. The increased invasive capacities of tumor cells induced by coculture are completely abrogated by inhibition of the coupled ATGL-dependent lipolysis/FAO pathways. These results show a complex metabolic symbiosis between tumor-surrounding adipocytes and cancer cells that stimulate their invasiveness, highlighting ATGL as a potential therapeutic target to impede breast cancer progression. PMID:28239646

  20. Omeprazole promotes proximal duodenal mucosal bicarbonate secretion in humans.

    Science.gov (United States)

    Mertz-Nielsen, A; Hillingsø, J; Bukhave, K; Rask-Madsen, J

    1996-01-01

    The proton pump inhibitor, omeprazole, surprisingly resulted in higher rates of proximal duodenal mucosal bicarbonate secretion than previously reported using an H2 receptor antagonist for gastric acid inhibition. Gastroduodenal perfusions were performed in healthy volunteers to evaluate whether this incidental finding is explained by more potent gastric acid inhibition by omeprazole or might be caused by the different mode of drug action. Basal and stimulated gastric and duodenal bicarbonate secretion rates were measured in the same subjects in control experiments (n = 17) and after pretreatment with high dose omeprazole (n = 17) and ranitidine (n = 9), respectively, by use of a technique permitting simultaneous measurements. Concentrations of bicarbonate were measured in the respective effluents by the method of back titration. Both omeprazole and ranitidine completely inhibited gastric acid secretion (pH 6.9 v 6.8; p > 0.05). Omeprazole caused higher rates of basal (mean (SEM)) (597 (48) v 351 (39) mumol/h; p 0.05) duodenal bicarbonate secretion compared with control experiments. Also the combination of omeprazole and ranitidine increased (p = 0.05) duodenal bicarbonate secretion, while ranitidine alone caused no change in either basal or stimulated secretion. In the stomach basal as well as vagally stimulated bicarbonate secretion was independent of the means of acid inhibition. These results show that the proton pump inhibitor, omeprazole, promotes proximal duodenal mucosal bicarbonate secretion apparently independent of its gastric acid inhibitory effect. The mechanism of action remains speculative.

  1. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

    Science.gov (United States)

    McDonough, Patrick M; Ingermanson, Randall S; Loy, Patricia A; Koon, Erick D; Whittaker, Ross; Laris, Casey A; Hilton, Jeffrey M; Nicoll, James B; Buehrer, Benjamin M; Price, Jeffrey H

    2011-06-01

    Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.

  2. [The adipocyte in the history of slimming agents].

    Science.gov (United States)

    Franchi, J; Pellicier, F; André, P; Schnebert, S

    2003-07-01

    Nowadays, in industrialised societies, it is fashionable for women to be slim. However, throughout history, this has not always been the case, especially as "cellulite" (cellulitis) was full of typically feminine symbols. The ideal feminine silhouette has changed with the rhythm of cultures. Cellulitis is an inappropriate term used by women to describe curves which they judge to be too plump and not very aesthetic, mostly around the thighs and hips. This lipodystrophy of the adipose tissue represents approximately 25% of a woman's body weight. It is clinically characterised by an "orange peel" skin surface, which is a result of the excessive development of the volume of the adipocytes organised in lobules within the walls of the unstretchable conjunctive tissue. This phenomenon is associated with an insufficiency of the venous tonus and an increase in the capillary permeability, which both contribute to an increase in the infiltration of water in the tissue. In reality, the understanding of cellulite has truly progressed with research based on adipocyte functions. An adipocyte is a metabolically active cell which plays a central role in the control of the energetic balance of the organism. In order to assume this role, it possesses all the enzymatic equipment necessary for synthesis (lipogenesis) and for triglyceride storage, mobilisation and liberation as free fatty acids (lipolysis). During these last few years, as well as this role as an energetic reserve which manages lipogenesis/lipolysis balance, the adipocyte has acquired the status of an endocrine and paracrine cell through the identification of numerous secreted factors. When we look back at the history of slimming products launched on the market since the 1980's, we can notice the role of the adipocyte tool and understand its functions in the choice of active ingredients, the development of complementary actions, the importance of the texture, the evolution of methods used to evaluate the efficacy on

  3. On the formation of lipid droplets in human adipocytes: the organization of the perilipin-vimentin cortex.

    Directory of Open Access Journals (Sweden)

    Hans Heid

    Full Text Available We report on the heterogeneity and diversity of lipid droplets (LDs in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN family, we could distinguish between endogenously derived LDs (endogenous LDs positive for perilipin from exogenously induced LDs (exogenous LDs positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

  4. Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase.

    Directory of Open Access Journals (Sweden)

    Victòria Ceperuelo-Mallafré

    Full Text Available Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.

  5. Congenital and environmental factors associated with adipocyte dysregulation as defects of insulin resistance.

    Science.gov (United States)

    Wang, Chao-Ping; Chung, Fu-Mei; Shin, Shyi-Jang; Lee, Yau-Jiunn

    2007-01-01

    The metabolic syndrome refers to insulin resistance and its associated cluster of related cardiovascular metabolic risk factors including type 2 diabetes, hypertension, dyslipidemia and central obesity. Although many hypotheses and facts have been proposed to explain the interaction between genetic and environmental causes of this syndrome, the primary etiology of the metabolic syndrome is adipose tissue dysregulation. Firstly, the thrifty genotype and phenotype hypothesis may explain the endemic increase in type 2 diabetes and cardiovascular disease in developing countries and elucidates the congenital susceptibility and environmental triggering of the metabolic syndrome. Secondly, over-nutrition leads to fatty acid (FA) accumulation in adipocytes and to an overflow to ectopic fat storage organs. This causes functional changes in adipocytes shifting the intra-cellular metabolic pathway toward insulin resistance. Thirdly, obese subjects exhibit increased fat cell size and over-secretion of biologic adipocytokines. Fourthly, failure to adequately develop adipose tissue mass, as seen in lipodystrophy cases, causes severe insulin resistance and diabetes. Lastly, similar to human type 2 diabetes, Psammonys obesus, a desert rat which feeds mainly on low-calorie vegetation, develops the metabolic syndrome when given a diet of calorie rich food. The above evidence indicates adipocyte dysregulation and secretion of FA as well as certain molecules from overloaded adipocytes/adipokines contribute to the pathogenesis of impaired insulin secretion and insulin resistance, endothelial dysfunction, a pro-inflammatory state and promote progression of atherosclerosis. The metabolic syndrome is a modern disease resulting adipocyte dysmetabolism resulting from the paradox of the slow human evolution combined with rapid environmental changes.

  6. Anti-diabetic effects of Caulerpa lentillifera: stimulation of insulin secretion in pancreatic β-cells and enhancement of glucose uptake in adipocytes

    OpenAIRE

    Sharma, Bhesh Raj; Rhyu, Dong Young

    2014-01-01

    Objective: To evaluate anti-diabetic effect of Caulerpa lentillifera (C. lentillifera). Methods: The inhibitory effect of C. lentillifera extract on dipeptidyl peptidase-IV and α-glucosidase enzyme was measured in a cell free system. Then, interleukin-1β and interferon-γ induced cell death and insulin secretion were measured in rat insulinoma (RIN) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and ELISA kit, respectively. Glucose uptake and glucose transporter...

  7. Salt-inducible kinase 2 and -3 are downregulated in adipose tissue from obese or insulin-resistant individuals: implications for insulin signalling and glucose uptake in human adipocytes.

    Science.gov (United States)

    Säll, Johanna; Pettersson, Annie M L; Björk, Christel; Henriksson, Emma; Wasserstrom, Sebastian; Linder, Wilhelm; Zhou, Yuedan; Hansson, Ola; Andersson, Daniel P; Ekelund, Mikael; Degerman, Eva; Stenkula, Karin G; Laurencikiene, Jurga; Göransson, Olga

    2017-02-01

    Salt-inducible kinases (SIKs) are related to the metabolic regulator AMP-activated protein kinase (AMPK). SIK2 is abundant in adipose tissue. The aims of this study were to investigate the expression of SIKs in relation to human obesity and insulin resistance, and to evaluate whether changes in the expression of SIKs might play a causal role in the development of disturbed glucose uptake in human adipocytes. SIK mRNA and protein was determined in human adipose tissue or adipocytes, and correlated to clinical variables. SIK2 and SIK3 expression and phosphorylation were analysed in adipocytes treated with TNF-α. Glucose uptake, GLUT protein levels and localisation, phosphorylation of protein kinase B (PKB/Akt) and the SIK substrate histone deacetylase 4 (HDAC4) were analysed after the SIKs had been silenced using small interfering RNA (siRNA) or inhibited using a pan-SIK-inhibitor (HG-9-91-01). We demonstrate that SIK2 and SIK3 mRNA are downregulated in adipose tissue from obese individuals and that the expression is regulated by weight change. SIK2 is also negatively associated with in vivo insulin resistance (HOMA-IR), independently of BMI and age. Moreover, SIK2 protein levels and specific kinase activity display a negative correlation to BMI in human adipocytes. Furthermore, SIK2 and SIK3 are downregulated by TNF-α in adipocytes. Silencing or inhibiting SIK1-3 in adipocytes results in reduced phosphorylation of HDAC4 and PKB/Akt, less GLUT4 at the plasma membrane, and lower basal and insulin-stimulated glucose uptake in adipocytes. This is the first study to describe the expression and function of SIKs in human adipocytes. Our data suggest that SIKs might be protective in the development of obesity-induced insulin resistance, with implications for future treatment strategies.

  8. Human Beta Cells Produce and Release Serotonin to Inhibit Glucagon Secretion from Alpha Cells

    OpenAIRE

    Joana Almaça; Judith Molina; Danusa Menegaz; Pronin, Alexey N.; Alejandro Tamayo; Vladlen Slepak; Per-Olof Berggren; Alejandro Caicedo

    2016-01-01

    In the pancreatic islet, serotonin is an autocrine signal increasing beta cell mass during metabolic challenges such as those associated with pregnancy or high-fat diet. It is still unclear whether serotonin is relevant for regular islet physiology and hormone secretion. Here, we show that human beta cells produce and secrete serotonin when stimulated with increases in glucose concentration. Serotonin secretion from beta cells decreases cyclic AMP (cAMP) levels in neighboring alpha cells via ...

  9. ROCKing cytokine secretion balance in human T cells.

    Science.gov (United States)

    Zanin-Zhorov, Alexandra; Waksal, Samuel D

    2015-04-01

    Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.

  10. Indomethacin decreases gastroduodenal mucosal bicarbonate secretion in humans

    DEFF Research Database (Denmark)

    Mertz-Nielsen, A; Hillingsø, Jens; Bukhave, K

    1995-01-01

    healthy volunteers. Bicarbonate and PGE2 were measured in the gastroduodenal effluents by back-titration and radioimmunoassay, respectively. RESULTS: Vagal stimulation and duodenal luminal acidification (0.1 M HCl; 20 ml; 5 min) increased gastroduodenal bicarbonate secretion (p

  11. In vitro study of the effect of diesterified alkoxyglycerols with conjugated linoleic acid on adipocyte inflammatory mediators

    Directory of Open Access Journals (Sweden)

    Reglero Guillermo

    2010-04-01

    Full Text Available Abstract Background Adipocytes contribute to inflammation and the innate immune response through expression of inflammatory mediators. High levels of these mediators have been related to chronic inflammation state and insulin resistance, cardiovascular diseases and diabetes type 2, among other disorders. 3-octadecylglycerol (batyl alcohol has been described as an inflammatory agent, whereas Conjugated Linoleic Acid (CLA is considered effective against obesity. In this study we examined the anti-inflammatory activity and mechanisms of modified alkoxyglycerols. Tumor necrosis factor (TNF-α activated mature adipocytes were used as cellular model of inflammation. Secreted levels and gene expressions of some inflammatory mediators, such as the adipokines, interleukin (IL-1β, IL-6 and IL-10; and the levels of leptin and adiponectin hormones were quantified in presence and absence of alkoxyglycerols and when human adipocyte cells were or not activated by TNF-α. The aim of this study is to describe the effects of nonesterified alkoxyglycerols, CLA and diesterified alkoxyglycerols with CLA (DEA-CLA and check if they present beneficial properties using an in vitro model of some chronic diseases related to the inflammatory process, such as obesity, using human mature adipocytes activated with TNF-α. Results Our data suggest that DEA-CLA, product of the esterification between the CLA and batyl alcohol, present beneficial effects on adipocytes close to observed and described for CLA (i.e. decrease of IL-1β and no adverse effects as observed for batyl alcohol (i.e. decrease of IL-10. In addition, DEA-CLA presented similar activity to CLA showing a trend to increase the secreted levels of adiponectin and decreasing the secreted levels of leptin. Conclusions CLA and DEA-CLA modify adipocyte inflammatory mediators and also could play a role on energy homeostasis through depletion of leptin levels.

  12. Relative shrinkage of adipocytes by paraffin in proportion to plastic embedding in human adipose tissue before and after weight loss.

    Science.gov (United States)

    Verhoef, Sanne P M; van Dijk, Paul; Westerterp, Klaas R

    2013-01-01

    Adipocyte size is a major modulator of endocrine functioning of adipose tissue and methods allowing accurate determination of adipocyte size are important to study energy metabolism. The aim of this study was to assess the relative shrinkage of adipocytes before and after weight loss by comparing adipose tissue from the same subjects embedded in paraffin and plastic. 18 healthy subjects (5 males and 13 females) aged 20-50 y with a BMI of 28-38 kg/m² followed a very low energy diet for 8 weeks. Adipose tissue biopsies were taken prior to and after weight loss and were processed for paraffin and plastic sections. Parameters of adipocyte size were determined with computer image analysis. Mean adipocyte size was smaller in paraffin compared to plastic embedded tissue both before (66 ± 4 vs. 103 ± 5 μm, P paraffin embedded tissue in proportion to plastic embedded tissue was not significantly different before and after weight loss (73 and 69%, respectively). Shrinkage due to the type of embedding of the adipose tissue can be ignored when comparing before and after weight loss. Plastic embedding of adipose tissue provides more accurate and sensitive results. © 2013 Asian Oceanian Association for the Study of Obesity . Published by Elsevier Ltd. All rights reserved.

  13. Biliary Secretion of Quasi-Enveloped Human Hepatitis A Virus

    Directory of Open Access Journals (Sweden)

    Asuka Hirai-Yuki

    2016-12-01

    Full Text Available Hepatitis A virus (HAV is an unusual picornavirus that is released from cells cloaked in host-derived membranes. These quasi-enveloped virions (eHAV are the only particle type circulating in blood during infection, whereas only nonenveloped virions are shed in feces. The reason for this is uncertain. Hepatocytes, the only cell type known to support HAV replication in vivo, are highly polarized epithelial cells with basolateral membranes facing onto hepatic (blood sinusoids and apical membranes abutting biliary canaliculi from which bile is secreted to the gut. To assess whether eHAV and nonenveloped virus egress from cells via vectorially distinct pathways, we studied infected polarized cultures of Caco-2 and HepG2-N6 cells. Most (>99% progeny virions were released apically from Caco-2 cells, whereas basolateral (64% versus apical (36% release was more balanced with HepG2-N6 cells. Both apically and basolaterally released virions were predominantly enveloped, with no suggestion of differential vectorial release of eHAV versus naked virions. Basolateral to apical transcytosis of either particle type was minimal (<0.02%/h in HepG2-N6 cells, arguing against this as a mechanism for differences in membrane envelopment of serum versus fecal virus. High concentrations of human bile acids converted eHAV to nonenveloped virions, whereas virus present in bile from HAV-infected Ifnar1−/−Ifngr1−/− and Mavs−/− mice banded over a range of densities extending from that of eHAV to that of nonenveloped virions. We conclude that nonenveloped virions shed in feces are derived from eHAV released across the canalicular membrane and stripped of membranes by the detergent action of bile acids within the proximal biliary canaliculus.

  14. Adipocytes promote prostate cancer stem cell self-renewal through amplification of the cholecystokinin autocrine loop.

    Science.gov (United States)

    Tang, Kai-Dun; Liu, Ji; Jovanovic, Lidija; An, Jiyuan; Hill, Michelle M; Vela, Ian; Lee, Terence Kin-Wah; Ma, Stephanie; Nelson, Colleen; Russell, Pamela J; Clements, Judith A; Ling, Ming-Tat

    2016-01-26

    Obesity has long been linked with prostate cancer progression, although the underlying mechanism is still largely unknown. Here, we report that adipocytes promote the enrichment of prostate cancer stem cells (CSCs) through a vicious cycle of autocrine amplification. In the presence of adipocytes, prostate cancer cells actively secrete the peptide hormone cholecystokinin (CCK), which not only stimulates prostate CSC self-renewal, but also induces cathepsin B (CTSB) production of the adipocytes. In return, CTSB facilitates further CCK secretion by the cancer cells. More importantly, inactivation of CCK receptor not only suppresses CTSB secretion by the adipocytes, but also synergizes the inhibitory effect of CTSB inhibitor on adipocyte-promoted prostate CSC self-renewal. In summary, we have uncovered a novel mechanism underlying the mutual interplay between adipocytes and prostate CSCs, which may help explaining the role of adipocytes in prostate cancer progression and provide opportunities for effective intervention.

  15. Genome-wide analysis of gene expression during adipogenesis in human adipose-derived stromal cells reveals novel patterns of gene expression during adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Melvin Anyasi Ambele

    2016-05-01

    Full Text Available We have undertaken an in-depth transcriptome analysis of adipogenesis in human adipose-derived stromal cells (ASCs induced to differentiate into adipocytes in vitro. Gene expression was assessed on days 1, 7, 14 and 21 post-induction and genes differentially expressed numbered 128, 218, 253 and 240 respectively. Up-regulated genes were associated with blood vessel development, leukocyte migration, as well as tumor growth, invasion and metastasis. They also shared common pathways with certain obesity-related pathophysiological conditions. Down-regulated genes were enriched for immune response processes. KLF15, LMO3, FOXO1 and ZBTB16 transcription factors were up-regulated throughout the differentiation process. CEBPA, PPARG, ZNF117, MLXIPL, MMP3 and RORB were up-regulated only on days 14 and 21, which coincide with the maturation of adipocytes and could possibly serve as candidates for controlling fat accumulation and the size of mature adipocytes. In summary, we have identified genes that were up-regulated only on days 1 and 7 or days 14 and 21 that could serve as potential early and late-stage differentiation markers.

  16. Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes

    Science.gov (United States)

    Sanjabi, Bahram; Dashty, Monireh; Özcan, Behiye; Akbarkhanzadeh, Vishtaseb; Rahimi, Mehran; Vinciguerra, Manlio; van Rooij, Felix; Al-Lahham, Saad; Sheedfar, Fareeba; van Kooten, Theo G.; Spek, C. Arnold; Rowshani, Ajda T.; van der Want, Johannes; Klaassen, Rene; Sijbrands, Eric; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2015-03-01

    Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

  17. Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes

    Science.gov (United States)

    Sanjabi, Bahram; Dashty, Monireh; Özcan, Behiye; Akbarkhanzadeh, Vishtaseb; Rahimi, Mehran; Vinciguerra, Manlio; van Rooij, Felix; Al-Lahham, Saad; Sheedfar, Fareeba; van Kooten, Theo G.; Spek, C. Arnold; Rowshani, Ajda T.; van der Want, Johannes; Klaassen, Rene; Sijbrands, Eric; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2015-01-01

    Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health. PMID:25743104

  18. Maintenance of osteoblastic and adipocytic differentiation potential with age and osteoporosis in human marrow stromal cell cultures

    DEFF Research Database (Denmark)

    Justesen, J; Dokkedahl, Karin Stenderup; Eriksen, E F

    2002-01-01

    Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP) is the ......Osteoblasts and adipocytes share a common precursor cell in the bone marrow stroma, termed marrow stromal cell (MSC). As the volume of bone adipose tissue increases in vivo with age, we hypothesized that decreased bone formation observed during aging and in patients with osteoporosis (OP...

  19. Differentially expressed protein markers in human submandibular and sublingual secretions.

    Science.gov (United States)

    Hu, Shen; Denny, Patricia; Denny, Paul; Xie, Yongming; Loo, Joseph A; Wolinsky, Lawrence E; Li, Yang; McBride, Jim; Ogorzalek Loo, Rachel R; Navazesh, Mavash; Wong, David T

    2004-11-01

    Proteome analysis of secretions from individual salivary glands is important for understanding the health of the oral cavity and pathogenesis of certain diseases. However, cross-contamination of submandibular (SM) and sublingual (SL) glandular secretions can occur. The close anatomic relationship of the SM and SL ductal orifices can lead to such contamination. Additionally, these glands may share common ducts. To insure the purity of SM/SL secretions for proteomic analysis, it is important to develop unique biomarkers which could be used to verify the integrity of the individual glandular saliva. In this study, a proteomics approach based on mass spectrometry and gel electrophoresis techniques was utilized to identify and verify a set of proteins (cystatin C, calgranulin B and MUC5B mucin), which are differentially expressed in SM/SL secretions. SM/SL fluids were obtained from nine healthy subjects. Cystatin C was found to be an SM-selective protein as it was found in all SM fluids but not detected in two SL fluids. MUC5B mucin and calgranulin B, on the other hand, were found to be SL-selective proteins. All SL samples contained MUC5B mucin, whereas MUC5B mucin was not detected in four SM samples. Eight of the SL samples contained calgranulin B; however, calgranulin B was absent in eight SM samples. This set of protein markers, especially calgranulin B, can be used to determine the purity of SM/SL samples, and therefore identify potential individuals who do not exhibit cross-contaminated SM/SL secretions, an important requirement for subsequent proteome analysis of pure SM and SL secretions.

  20. CFTR is involved in the regulation of glucagon secretion in human and rodent alpha cells.

    Science.gov (United States)

    Edlund, Anna; Pedersen, Morten Gram; Lindqvist, Andreas; Wierup, Nils; Flodström-Tullberg, Malin; Eliasson, Lena

    2017-12-01

    Glucagon is the main counterregulatory hormone in the body. Still, the mechanism involved in the regulation of glucagon secretion from pancreatic alpha cells remains elusive. Dysregulated glucagon secretion is common in patients with Cystic Fibrosis (CF) that develop CF related diabetes (CFRD). CF is caused by a mutation in the Cl(-) channel Cystic fibrosis transmembrane conductance regulator (CFTR), but whether CFTR is present in human alpha cells and regulate glucagon secretion has not been investigated in detail. Here, both human and mouse alpha cells showed CFTR protein expression, whereas CFTR was absent in somatostatin secreting delta cells. CFTR-current activity induced by cAMP was measured in single alpha cells. Glucagon secretion at different glucose levels and in the presence of forskolin was increased by CFTR-inhibition in human islets, whereas depolarization-induced glucagon secretion was unaffected. CFTR is suggested to mainly regulate the membrane potential through an intrinsic alpha cell effect, as supported by a mathematical model of alpha cell electrophysiology. In conclusion, CFTR channels are present in alpha cells and act as important negative regulators of cAMP-enhanced glucagon secretion through effects on alpha cell membrane potential. Our data support that loss-of-function mutations in CFTR contributes to dysregulated glucagon secretion in CFRD.

  1. Omeprazole promotes proximal duodenal mucosal bicarbonate secretion in humans

    DEFF Research Database (Denmark)

    Mertz-Nielsen, Anette; Hillingsø, J; Bukhave, Klaus

    1996-01-01

    dose omeprazole (n=17) and ranitidine (n=9), respectively, by use of a technique permitting simultaneous measurements. Concentrations of bicarbonate were measured in the respective effluents by the method of back titration. Both omeprazole and ranitidine completely inhibited gastric acid secretion (p...

  2. G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

    NARCIS (Netherlands)

    Schweiger, M.; Paar, M.; Eder, C.; Brandis, J.; Moser, E.; Gorkiewisz, G.; Grond, S.; Radner, F.P.W.; Cerk, I.; Cornaciu, I.; Oberer, M.; Kersten, A.H.; Zechner, R.; Zimmermann, M.B.; Lass, A.

    2012-01-01

    The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL)5, which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 sw

  3. The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells.

    Science.gov (United States)

    Zych, J; Stimamiglio, M A; Senegaglia, A C; Brofman, P R S; Dallagiovanna, B; Goldenberg, S; Correa, A

    2013-05-01

    Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

  4. Superparamagnetic iron oxide nanoparticles alter expression of obesity and T2D-associated risk genes in human adipocytes

    NARCIS (Netherlands)

    Sharifi, S.; Daghighi, S.; Motazacker, M. M.; Badlou, B.; Sanjabi, B.; Akbarkhanzadeh, A.; Rowshani, A. T.; Laurent, S.; Peppelenbosch, M. P.; Rezaee, F.

    2013-01-01

    Adipocytes hypertrophy is the main cause of obesity and its affliction such as type 2 diabetes (T2D). Since superparamagnetic iron oxide nanoparticles (SPIONs) are used for a wide range of biomedical/medical applications, we aimed to study the effect of SPIONs on 22 and 29 risk genes (Based on gene

  5. G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

    NARCIS (Netherlands)

    Schweiger, M.; Paar, M.; Eder, C.; Brandis, J.; Moser, E.; Gorkiewisz, G.; Grond, S.; Radner, F.P.W.; Cerk, I.; Cornaciu, I.; Oberer, M.; Kersten, A.H.; Zechner, R.; Zimmermann, M.B.; Lass, A.

    2012-01-01

    The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL)5, which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1 sw

  6. Superparamagnetic iron oxide nanoparticles alter expression of obesity and T2D-associated risk genes in human adipocytes

    NARCIS (Netherlands)

    Sharifi, S.; Daghighi, S.; Motazacker, M. M.; Badlou, B.; Sanjabi, B.; Akbarkhanzadeh, A.; Rowshani, A. T.; Laurent, S.; Peppelenbosch, M. P.; Rezaee, F.

    2013-01-01

    Adipocytes hypertrophy is the main cause of obesity and its affliction such as type 2 diabetes (T2D). Since superparamagnetic iron oxide nanoparticles (SPIONs) are used for a wide range of biomedical/medical applications, we aimed to study the effect of SPIONs on 22 and 29 risk genes (Based on gene

  7. G0/G1 switch gene-2 regulates human adipocyte lipolysis by affecting activity and localization of adipose triglyceride lipase

    NARCIS (Netherlands)

    Schweiger, M.; Paar, M.; Eder, C.; Brandis, J.; Moser, E.; Gorkiewisz, G.; Grond, S.; Radner, F.P.W.; Cerk, I.; Cornaciu, I.; Oberer, M.; Kersten, A.H.; Zechner, R.; Zimmermann, M.B.; Lass, A.

    2012-01-01

    The hydrolysis of triglycerides in adipocytes, termed lipolysis, provides free fatty acids as energy fuel. Murine lipolysis largely depends on the activity of adipose triglyceride lipase (ATGL)5, which is regulated by two proteins annotated as comparative gene identification-58 (CGI-58) and G0/G1

  8. Parallel secretion of pancreastatin and somatostatin from human pancreastatin producing cell line (QGP-1N).

    Science.gov (United States)

    Funakoshi, A; Tateishi, K; Kitayama, N; Jimi, A; Matsuoka, Y; Kono, A

    1993-05-01

    In this investigation we studied pancreastatin (PST) secretion from a human PST producing cell line (QGP-1N) in response to various secretagogues. Immunocytochemical study revealed the immunoreactivity of PST and somatostatin (SMT) in the same cells of a monolayer culture. Ki-ras DNA point mutation on codon 12 was found. Carbachol stimulated secretion of PST and SMT and intracellular Ca2+ mobilization in the range of 10(-6)-10(-4) M. The secretion and Ca2+ mobilization were inhibited by atropine, a muscarinic receptor antagonist. Phorbol ester and calcium ionophore (A23187) stimulated secretion of PST and SMT. The removal of extracellular calcium suppressed both secretions throughout stimulation with 10(-5) M carbachol. Fluoride, a well-known activator of guanine nucleotide binding (G) protein, stimulated intracellular Ca2+ mobilization and secretion of PST and SMT in a dose-dependent manner in the range of 5-40 mM. Also, 10(-5) M carbachol and 20 mM fluoride stimulated inositol 1,4,5-triphosphate production. However, cholecystokinin and gastrin-releasing peptide did not stimulate Ca2+ mobilization or secretion of the two peptides. These results suggest that secretion of PST and SMT from QGP-1N cells is regulated mainly by acetylcholine in a parallel fashion through muscarinic receptors coupled to the activation of polyphosphoinositide breakdown by a G-protein and that increases in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  9. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin.

    Science.gov (United States)

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2015-05-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure.

  10. Fibroblast growth factor 21 improves insulin sensitivity and synergizes with insulin in human adipose stem cell-derived (hASC adipocytes.

    Directory of Open Access Journals (Sweden)

    Darwin V Lee

    Full Text Available Fibroblast growth factor 21 (FGF21 has evolved as a major metabolic regulator, the pharmacological administration of which causes weight loss, insulin sensitivity and glucose control in rodents and humans. To understand the molecular mechanisms by which FGF21 exerts its metabolic effects, we developed a human in vitro model of adipocytes to examine crosstalk between FGF21 and insulin signaling. Human adipose stem cell-derived (hASC adipocytes were acutely treated with FGF21 alone, insulin alone, or in combination. Insulin signaling under these conditions was assessed by measuring tyrosine phosphorylation of insulin receptor (InsR, insulin receptor substrate-1 (IRS-1, and serine 473 phosphorylation of Akt, followed by a functional assay using 14C-2-deoxyglucose [14C]-2DG to measure glucose uptake in these cells. FGF21 alone caused a modest increase of glucose uptake, but treatment with FGF21 in combination with insulin had a synergistic effect on glucose uptake in these cells. The presence of FGF21 also effectively lowered the insulin concentration required to achieve the same level of glucose uptake compared to the absence of FGF21 by 10-fold. This acute effect of FGF21 on insulin signaling was not due to IR, IGF-1R, or IRS-1 activation. Moreover, we observed a substantial increase in basal S473-Akt phosphorylation by FGF21 alone, in contrast to the minimal shift in basal glucose uptake. Taken together, our data demonstrate that acute co-treatment of hASC-adipocytes with FGF21 and insulin can result in a synergistic improvement in glucose uptake. These effects were shown to occur at or downstream of Akt, or separate from the canonical insulin signaling pathway.

  11. Action of N-acylated ambroxol derivatives on secretion of chloride ions in human airway epithelia.

    Science.gov (United States)

    Yamada, Takahiro; Takemura, Yoshizumi; Niisato, Naomi; Mitsuyama, Etsuko; Iwasaki, Yoshinobu; Marunaka, Yoshinori

    2009-03-13

    We report the effects of new N-acylated ambroxol derivatives (TEI-588a, TEI-588b, TEI-589a, TEI-589b, TEI-602a and TEI-602b: a, aromatic amine-acylated derivative; b, aliphatic amine-acylated derivative) induced from ambroxol (a mucolytic agent to treat human lung diseases) on Cl(-) secretion in human submucosal serous Calu-3 cells under a Na(+)/K(+)/2Cl(-) cotransporter-1 (NKCC1)-mediated hyper-secreting condition. TEI-589a, TEI-589b and TEI-602a diminished hyper-secretion of Cl(-) by diminishing the activity of NKCC1 without blockade of apical Cl(-) channel (TEI-589a>TEI-602a>TEI-589b), while any other tested compounds including ambroxol had no effects on Cl(-) secretion. These indicate that the inhibitory action of an aromatic amine-acylated derivative on Cl(-) secretion is stronger that that of an aliphatic amine-acylated derivative, and that 3-(2,5-dimethyl)furoyl group has a strong action in inhibition of Cl(-) secretion than cyclopropanoyl group. We here indicate that TEI-589a, TEI-589b and TEI-602a reduce hyper-secretion to an appropriate level in the airway, providing a possibility that the compound can be an effective drug in airway obstructive diseases including COPD by reducing the airway resistance under a hyper-secreting condition.

  12. Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

    Directory of Open Access Journals (Sweden)

    Maaike Kockx

    Full Text Available Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs or directly target the GTPase domain (Dyngo or Dynole series, dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  13. Fructose Alters Intermediary Metabolism of Glucose in Human Adipocytes and Diverts Glucose to Serine Oxidation in the One–Carbon Cycle Energy Producing Pathway

    OpenAIRE

    Vijayalakshmi Varma; Boros, László G; Nolen, Greg T.; Ching-Wei Chang; Martin Wabitsch; Beger, Richard D.; Jim Kaput

    2015-01-01

    Increased consumption of sugar and fructose as sweeteners has resulted in the utilization of fructose as an alternative metabolic fuel that may compete with glucose and alter its metabolism. To explore this, human Simpson-Golabi-Behmel Syndrome (SGBS) preadipocytes were differentiated to adipocytes in the presence of 0, 1, 2.5, 5 or 10 mM of fructose added to a medium containing 5 mM of glucose representing the normal blood glucose concentration. Targeted tracer [1,2-13C2]-d-glucose fate asso...

  14. Omeprazole promotes proximal duodenal mucosal bicarbonate secretion in humans.

    OpenAIRE

    Mertz-Nielsen, A; Hillingsø, J; Bukhave, K; Rask-Madsen, J

    1996-01-01

    The proton pump inhibitor, omeprazole, surprisingly resulted in higher rates of proximal duodenal mucosal bicarbonate secretion than previously reported using an H2 receptor antagonist for gastric acid inhibition. Gastroduodenal perfusions were performed in healthy volunteers to evaluate whether this incidental finding is explained by more potent gastric acid inhibition by omeprazole or might be caused by the different mode of drug action. Basal and stimulated gastric and duodenal bicarbonate...

  15. Dibutyltin-induced alterations of interleukin 1beta secretion from human immune cells.

    Science.gov (United States)

    Brown, Shyretha; Tehrani, Shahin; Whalen, Margaret M

    2017-02-01

    Dibutyltin (DBT) is used to stabilize polyvinyl chloride plastics (including pipes that distribute drinking water) and as a de-worming agent in poultry. DBT is found in human blood, and DBT exposures alter the secretion of tumor necrosis factor alpha and interferon gamma from lymphocytes. Interleukin (IL)-1β is a proinflammatory cytokine that regulates cellular growth, tissue restoration and immune response regulation. IL-1β plays a role in increasing invasiveness of certain tumors. This study reveals that exposures to DBT (24 h, 48 h and 6 days) modify the secretion of IL-1β from increasingly reconstituted preparations of human immune cells (highly enriched human natural killer cells, monocyte-depleted [MD] peripheral blood mononuclear cells [PBMCs], PBMCs, granulocytes and a preparation combining both PBMCs and granulocytes). DBT altered IL-1β secretion from all cell preparations. Higher concentrations of DBT (5 and 2.5 μm) decreased the secretion of IL-1β, while lower concentrations of DBT (0.1 and 0.05 μm) increased the secretion of IL-1β. Selected signaling pathways were examined in MD-PBMCs to determine if they play a role in DBT-induced elevations of IL-1β secretion. Pathways examined were IL-1β converting enzyme (caspase 1), mitogen-activated protein kinases and nuclear factor kappa B. Caspase 1 and mitogen-activated protein kinase pathways appear to be utilized by DBT in increasing IL-1β secretion. These results indicate that DBT alters IL-1β secretion from human immune cells in an ex. vivo system utilizing several IL-1β regulating signaling pathways. Thus, DBT may have the potential to alter IL-1β secretion in an in vivo system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Impact of 3-Amino-1,2,4-Triazole (3-AT)-Derived Increase in Hydrogen Peroxide Levels on Inflammation and Metabolism in Human Differentiated Adipocytes

    Science.gov (United States)

    Ruiz-Ojeda, Francisco Javier; Gomez-Llorente, Carolina; Aguilera, Concepción María; Gil, Angel; Rupérez, Azahara Iris

    2016-01-01

    Obesity is characterized by an excessive accumulation of fat in adipose tissue, which is associated with oxidative stress and chronic inflammation. Excessive H2O2 levels are degraded by catalase (CAT), the activity of which is decreased in obesity. We investigated the effects of inhibition of catalase activity on metabolism and inflammation by incubating human differentiated adipocytes with 10 mM 3-amino-1,2,4-triazole (3-AT) for 24 h. As expected, the treatment decreased CAT activity and increased intracellular H2O2 levels significantly. Glutathione peroxidase (GPX) activity was also reduced, and the gene expression levels of the antioxidant enzymes GPX4 and peroxiredoxins (1, 3 and 5) were inhibited. Interestingly, this occurred along with lower mRNA levels of the transcription factors nuclear factor (erythroid 2-like 2) and forkhead box O, which are involved in redox homeostasis. However, superoxide dismutase activity and expression were increased. Moreover, 3-AT led to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and increased tumor necrosis alpha and interleukin 6 protein and gene expression levels, while lowering peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein levels. These alterations were accompanied by an altered glucose and lipid metabolism. Indeed, adipocytes treated with 3-AT showed reduced basal glucose uptake, reduced glucose transporter type 4 gene and protein expression, reduced lipolysis, reduced AMP-activated protein kinase activation and reduced gene expression of lipases. Our results indicate that increased H2O2 levels caused by 3-AT treatment impair the antioxidant defense system, lower PPARγ expression and initiate inflammation, thus affecting glucose and lipid metabolism in human differentiated adipocytes. PMID:27023799

  17. Impact of 3-Amino-1,2,4-Triazole (3-AT-Derived Increase in Hydrogen Peroxide Levels on Inflammation and Metabolism in Human Differentiated Adipocytes.

    Directory of Open Access Journals (Sweden)

    Francisco Javier Ruiz-Ojeda

    Full Text Available Obesity is characterized by an excessive accumulation of fat in adipose tissue, which is associated with oxidative stress and chronic inflammation. Excessive H2O2 levels are degraded by catalase (CAT, the activity of which is decreased in obesity. We investigated the effects of inhibition of catalase activity on metabolism and inflammation by incubating human differentiated adipocytes with 10 mM 3-amino-1,2,4-triazole (3-AT for 24 h. As expected, the treatment decreased CAT activity and increased intracellular H2O2 levels significantly. Glutathione peroxidase (GPX activity was also reduced, and the gene expression levels of the antioxidant enzymes GPX4 and peroxiredoxins (1, 3 and 5 were inhibited. Interestingly, this occurred along with lower mRNA levels of the transcription factors nuclear factor (erythroid 2-like 2 and forkhead box O, which are involved in redox homeostasis. However, superoxide dismutase activity and expression were increased. Moreover, 3-AT led to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation and increased tumor necrosis alpha and interleukin 6 protein and gene expression levels, while lowering peroxisome proliferator-activated receptor gamma (PPARγ mRNA and protein levels. These alterations were accompanied by an altered glucose and lipid metabolism. Indeed, adipocytes treated with 3-AT showed reduced basal glucose uptake, reduced glucose transporter type 4 gene and protein expression, reduced lipolysis, reduced AMP-activated protein kinase activation and reduced gene expression of lipases. Our results indicate that increased H2O2 levels caused by 3-AT treatment impair the antioxidant defense system, lower PPARγ expression and initiate inflammation, thus affecting glucose and lipid metabolism in human differentiated adipocytes.

  18. Adipose tissue and adipocyte dysregulation.

    Science.gov (United States)

    Lafontan, M

    2014-02-01

    Obesity-associated insulin resistance is a complex disorder involving a number of candidate molecules, pathways and transduction systems possessing potential causal actions. Inflammation in adipose tissue (AT) is one mechanism proposed to explain the development of insulin resistance, while identification of factors that lead to or cause AT dysfunction when it reaches its limit of expansion represents an important challenge. Pathological expansion of AT is characterized by changes in its blood flow, and the presence of enlarged and dysfunctional adipocytes that begin an inflammatory campaign of altered adipokine and cytokine secretions. Adipocyte senescence, necrosis and death are associated with increased immune cell and macrophage infiltration of AT in obesity. This can boost inflammation and reinforce fat cell dysfunction and death. In addition, pathological fat mass expansion is also related to limited recruitment of fat cell progenitors able to proliferate and differentiate into healthy small fat cells to compensate for cell death and preserve adipocyte numbers. Limiting vascular development and enhancing fibrotic processes worsen inflammation towards chronic irreversibility. The AT expandability hypothesis states that failure of AT expansion is one of the key factors linking positive energy balance and cardiometabolic risks, not obesity per se. Besides the usual treatment of obesity based on behavioral approaches (specific dietary/nutritional approaches together with increased physical activity), a number of questions remain concerning the possible recovery of metabolic health after inflammation-preventing interventions. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  19. Pharmacologic concentrations of linezolid modify oxidative phosphorylation function and adipocyte secretome

    Directory of Open Access Journals (Sweden)

    Laura Llobet

    2017-10-01

    Full Text Available The oxidative phosphorylation system is important for adipocyte differentiation. Therefore, xenobiotics inhibitors of the oxidative phosphorylation system could affect adipocyte differentiation and adipokine secretion. As adipokines impact the overall health status, these xenobiotics may have wide effects on human health. Some of these xenobiotics are widely used therapeutic drugs, such as ribosomal antibiotics. Because of its similarity to the bacterial one, mitochondrial translation system is an off-target for these compounds. To study the influence of the ribosomal antibiotic linezolid on adipokine production, we analyzed its effects on adipocyte secretome. Linezolid, at therapeutic concentrations, modifies the levels of apolipoprotein E and several adipokines and proteins related with the extracellular matrix. This antibiotic also alters the global methylation status of human adipose tissue-derived stem cells and, therefore, its effects are not limited to the exposure period. Besides their consequences on other tissues, xenobiotics acting on the adipocyte oxidative phosphorylation system alter apolipoprotein E and adipokine production, secondarily contributing to their systemic effects.

  20. Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

    Science.gov (United States)

    Lechner, Stefan; Mitterberger, Maria C; Mattesich, Monika; Zwerschke, Werner

    2013-01-01

    We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of

  1. The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    J. Zych

    2013-05-01

    Full Text Available Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs derived from bone marrow (BM-MSCs and adipose tissue (ADSCs using the chromatin-modifying agents trichostatin A (TSA, a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC, a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

  2. TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells.

    Science.gov (United States)

    Mitrovic, Sandra; Nogueira, Cristina; Cantero-Recasens, Gerard; Kiefer, Kerstin; Fernández-Fernández, José M; Popoff, Jean-François; Casano, Laetitia; Bard, Frederic A; Gomez, Raul; Valverde, Miguel A; Malhotra, Vivek

    2013-05-28

    Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion. DOI:http://dx.doi.org/10.7554/eLife.00658.001.

  3. The Rab11 Effector Protein FIP1 Regulates Adiponectin Trafficking and Secretion

    Science.gov (United States)

    Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

    2013-01-01

    Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321

  4. The rab11 effector protein FIP1 regulates adiponectin trafficking and secretion.

    Science.gov (United States)

    Carson, Brian P; Del Bas, Josep Maria; Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia

    2013-01-01

    Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.

  5. The rab11 effector protein FIP1 regulates adiponectin trafficking and secretion.

    Directory of Open Access Journals (Sweden)

    Brian P Carson

    Full Text Available Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.

  6. Lubiprostone stimulates secretion from tracheal submucosal glands of sheep, pigs, and humans

    OpenAIRE

    Joo, N. S.; Wine, J. J.; Cuthbert, A. W.

    2009-01-01

    Lubiprostone, a putative ClC-2 chloride channel opener, has been investigated for its effects on airway epithelia (tracheas). Lubiprostone is shown to increase submucosal gland secretion in pigs, sheep, and humans and to increase short-circuit current (SCC) in the surface epithelium of pigs and sheep. Use of appropriate blocking agents and ion-substitution experiments shows anion secretion is the driving force for fluid formation in both glands and surface epithelium. From SCC concentration-r...

  7. Hsp60 is actively secreted by human tumor cells.

    Directory of Open Access Journals (Sweden)

    Anna M Merendino

    Full Text Available BACKGROUND: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. METHODOLOGY/PRINCIPAL FINDINGS: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.

  8. In vitro evaluation of different methods of handling human liposuction aspirate and their effect on adipocytes and adipose derived stem cells.

    Science.gov (United States)

    Palumbo, Paola; Miconi, Gianfranca; Cinque, Benedetta; La Torre, Cristina; Lombardi, Francesca; Zoccali, Giovanni; Orsini, Gino; Leocata, Pietro; Giuliani, Maurizio; Cifone, Maria Grazia

    2015-08-01

    Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue-derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10-20-30 min) with the centrifugation technique at different speeds (90-400-1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, "middle layer", and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45-) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available.

  9. Role of ENPP1 on adipocyte maturation.

    Directory of Open Access Journals (Sweden)

    Jian Liang

    Full Text Available BACKGROUND: It is recognized that the ability of adipose tissue to expand in response to energy excess, i.e. adipocyte maturation, is important in determining systemic abnormalities in glucose and lipid metabolism. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1, also known as PC-1 has been recently reported to be involved in the pathogenesis of insulin resistance and related diseases. However, its role on adipose tissue physiology as a mechanism of systemic insulin resistance is not understood. This study was performed to evaluate whether ENPP1 is regulated during adipogenesis and whether over-expression in adipocytes can affect adipocyte maturation, a potential novel mechanism of ENPP1-related insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: ENPP1 expression was found down-regulated during 3T3-L1 maturation, and over-expression of human ENPP1 in 3T3-L1 (pQCXIP-ENPP1 vector resulted in adipocyte insulin resistance and in defective adipocyte maturation. Adipocyte maturation was more efficient in mesenchymal embryonal cells from ENPP1 knockout mice than from wild-type. CONCLUSIONS: We identify ENPP1 as a novel mechanism of defective adipocyte maturation. This mechanism could contribute to the pathogenesis of insulin resistance in absence of obesity.

  10. Cadmium modulates adipocyte functions in metallothionein-null mice

    Energy Technology Data Exchange (ETDEWEB)

    Kawakami, Takashige; Nishiyama, Kaori; Kadota, Yoshito; Sato, Masao; Inoue, Masahisa; Suzuki, Shinya, E-mail: suzukis@ph.bunri-u.ac.jp

    2013-11-01

    Our previous study has demonstrated that exposure to cadmium (Cd), a toxic heavy metal, causes a reduction of adipocyte size and the modulation of adipokine expression. To further investigate the significance of the Cd action, we studied the effect of Cd on the white adipose tissue (WAT) of metallothionein null (MT{sup −/−}) mice, which cannot form atoxic Cd–MT complexes and are used for evaluating Cd as free ions, and wild type (MT{sup +/+}) mice. Cd administration more significantly reduced the adipocyte size of MT{sup −/−} mice than that of MT{sup +/+} mice. Cd exposure also induced macrophage recruitment to WAT with an increase in the expression level of Ccl2 (MCP-1) in the MT{sup −/−} mice. The in vitro exposure of Cd to adipocytes induce triglyceride release into culture medium, decrease in the expression levels of genes involved in fatty acid synthesis and lipid hydrolysis at 24 h, and at 48 h increase in phosphorylation of the lipid-droplet-associated protein perilipin, which facilitates the degradation of stored lipids in adipocytes. Therefore, the reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. Taken together, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and may accelerate the risk of developing insulin resistance and type 2 diabetes. - Highlights: • Cd causes a marked reduction in adipocyte size in MT-null mice. • Cd enhances macrophage migration into adipose tissue and disrupt adipokine secretion. • MT gene alleviates Cd-induced adipocyte dysfunctions. • Cd enhances the degradation of stored lipids in adipocytes, mediated by perilipin. • Cd induces unusually small adipocytes and the abnormal expression of adipokines.

  11. Glucagon-like peptide 2 inhibits ghrelin secretion in humans

    DEFF Research Database (Denmark)

    Banasch, Matthias; Bulut, Kerem; Hagemann, Dirk;

    2006-01-01

    INTRODUCTION: The growth hormone secretagogue receptor ligand ghrelin is known to play a pivotal role in the central nervous control of energy homeostasis. Circulating ghrelin levels are high under fasting conditions and decline after meal ingestion, but the mechanisms underlying the postprandial...... drop in ghrelin levels are poorly understood. In the present study we addressed, whether (1) exogenous GLP-2 administration decreases ghrelin levels and (2) what other endogenous factors are related to ghrelin secretion under fasting conditions. PATIENTS AND METHODS: Fifteen healthy male volunteers...... were studied with the intravenous infusion of GLP-2 (2 pmol l(-1) min(-1)) or placebo over 120 min in the fasting state. Plasma concentrations of glucose, insulin, C-peptide, glucagon, intact GLP-2 and ghrelin were determined. RESULTS: During the infusion of GLP-2, plasma concentrations of intact GLP-2...

  12. 硫化氢对脂肪肝脂细胞脂联素分泌的调节%Regulatory effect of hydrogen sulfide on adiponectin secretion in fatty liver adipocytes

    Institute of Scientific and Technical Information of China (English)

    王新国; 丁虹; 黄利华; 黄谦

    2013-01-01

    Objective To observe whether hydrogen sulfide modulates adipocytes to secrete adiponectin.Methods Thirty-two male SD rats were randomly divided into normal diet group (n =8),high-fat diet group (n =8),high-fat + cysteine group (n =8),and high-fat + propargylglycine (cystathionine-γ-lyase inhibitor) group (n =8).The fatty liver animal model was established using the high fat diet; meanwhile,L-cysteine was applied to the animals to produce endogenous hydrogen sulfide and propargylglycine to inhibit the production of endogenous hydrogen sulfide.Fatty degeneration of the hver of animals and plasma within the adiponectin concentration was observed after 8 weeks.The omental adipose tissues were excluded and digested using collagenase Ⅰ to isolate fat cells.Sodium hydrosulfide and propargylglycine was applied to stimulate the cells in vitro for 7 days before the detection of the cell supernatant adiponectin content.Results Liver steatosis was established after highfat diet for eight weeks.Steatosis was significantly extenuated after the application of L-cysteine and aggravated by propargylglycine.Plasma hydrogen sulfide and adiponectin levels in fat diet group animals were (21.13 ± 7.06) mmol/L and (3.16 ± 1.15) mg/L respectively,which significantly decreased when compared with the normal diet group with the levels of hydrogen sulfide (29.13 ± 13.06) mmol/L and adiponectin (8.98 ± 2.84) mg/L (P=0.0229,P=0.0062).Hydrogen sulfide [(35.47 ±9.04) mmol/L] and adiponectin [(6.54 ± 1.38) mg/L] levels in the high fat + cysteine group significantly increased (P =0.0032,P =0.0131).However,hydrogen sulfide [(16.65 ±8.79) mmol/L] and adiponectin [(2.50±0.91) mg/L] in high fat + propargylglycine group were significantly decreased (P =0.0191,P =0.0021).Hydrogen sulfide was not significantly differem in the supematam of adipocyte isolated from normal diet group [(26.77 ± 12.65) mmol/L] and from high-fat diet group [(28.76 ±9.09) mmol/L] (P =0.0927),but that in the sodium

  13. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  14. Dehydration-induced vasopressin secretion in humans: involvement of the histaminergic system.

    Science.gov (United States)

    Kjaer, A; Knigge, U; Jørgensen, H; Warberg, J

    2000-12-01

    In rats, the hypothalamic neurotransmitter histamine participates in regulation of vasopressin secretion and seems to be of physiological importance, because blockade of the histaminergic system reduces dehydration-induced vasopressin secretion. We investigated whether histamine is also involved in regulation of vasopressin secretion during dehydration in humans. We found that 40 h of dehydration gradually increased plasma osmolality by 10 mosmol/kg and induced a fourfold increase in vasopressin levels. Pretreatment with the H(2)-receptor antagonists cimetidine or ranitidine significantly reduced the dehydration-induced increase in vasopressin levels approximately 40% after 34 and 37 h of dehydration, whereas this was not the case with the H(1)-receptor antagonist mepyramine. Dehydration reduced aldosterone secretion by approximately 50%. This effect of dehydration was reduced by both H(1)- and H(2)-receptor blockade after 16 and/or 34 h of dehydration. We conclude that vasopressin secretion in response to dehydration in humans is under the regulatory influence of histamine and that the effect seems to be mediated via H(2)-receptors. In addition, the regulation of aldosterone secretion during dehydration also seems to involve the histaminergic system via H(1) and H(2) receptors.

  15. Lubiprostone stimulates secretion from tracheal submucosal glands of sheep, pigs, and humans.

    Science.gov (United States)

    Joo, N S; Wine, J J; Cuthbert, A W

    2009-05-01

    Lubiprostone, a putative ClC-2 chloride channel opener, has been investigated for its effects on airway epithelia (tracheas). Lubiprostone is shown to increase submucosal gland secretion in pigs, sheep, and humans and to increase short-circuit current (SCC) in the surface epithelium of pigs and sheep. Use of appropriate blocking agents and ion-substitution experiments shows anion secretion is the driving force for fluid formation in both glands and surface epithelium. From SCC concentration-response relations, it is shown that for apical lubiprostone K(d) = 10.5 nM with a Hill slope of 1.08, suggesting a single type of binding site and, from the speed of the response, close to the apical surface, confirmed the rapid blockade by Cd ions. Responses to lubiprostone were reversible and repeatable, responses being significantly larger with ventral compared with dorsal epithelium. Submucosal gland secretion rates following basolateral lubiprostone were, respectively, 0.2, 0.5, and 0.8 nl gl(-1) min(-1) in humans, sheep, and pigs. These rates dwarf any contribution surface secretion adds to the accumulation of surface liquid under the influence of lubiprostone. Lubiprostone stimulated gland secretion in two out of four human cystic fibrosis (CF) tissues and in two of three disease controls, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis (COPD/IPF), but in neither type of tissue was the increase significant. Lubiprostone was able to increase gland secretion rates in normal human tissue in the continuing presence of a high forskolin concentration. Lubiprostone had no spasmogenic activity on trachealis muscle, making it a potential agent for increasing airway secretion that may have therapeutic utility.

  16. ChIP-seq profiling of the active chromatin marker H3K4me3 and PPARγ, CEBPα and LXR target genes in human SGBS adipocytes

    Science.gov (United States)

    Galhardo, Mafalda; Sinkkonen, Lasse; Berninger, Philipp; Lin, Jake; Sauter, Thomas; Heinäniemi, Merja

    2014-01-01

    Transcription factors (TFs) represent key factors to establish a cellular phenotype. It is known that several TFs could play a role in disease, yet less is known so far how their targets overlap. We focused here on identifying the most highly induced TFs and their putative targets during human adipogenesis. Applying chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) in the human SGBS pre-adipocyte cell line, we identified genes with binding sites in their vicinity for the three TFs studied, PPARγ, CEBPα and LXR. Here we describe the experimental design and quality controls in detail for the deep sequencing data and related results published by Galhardo et al. in Nucleic Acids Research 2014 [1] associated with the data uploaded to NCBI Gene Expression Omnibus (GSE41578). PMID:26484099

  17. ChIP-seq profiling of the active chromatin marker H3K4me3 and PPARγ, CEBPα and LXR target genes in human SGBS adipocytes

    Directory of Open Access Journals (Sweden)

    Mafalda Galhardo

    2014-12-01

    Full Text Available Transcription factors (TFs represent key factors to establish a cellular phenotype. It is known that several TFs could play a role in disease, yet less is known so far how their targets overlap. We focused here on identifying the most highly induced TFs and their putative targets during human adipogenesis. Applying chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq in the human SGBS pre-adipocyte cell line, we identified genes with binding sites in their vicinity for the three TFs studied, PPARγ, CEBPα and LXR. Here we describe the experimental design and quality controls in detail for the deep sequencing data and related results published by Galhardo et al. in Nucleic Acids Research 2014 [1] associated with the data uploaded to NCBI Gene Expression Omnibus (GSE41578.

  18. Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

    DEFF Research Database (Denmark)

    Ali, D.; Hamam, R.; Alfayez, M.;

    2016-01-01

    abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell...... proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition...

  19. Farnesol Has an Anti-obesity Effect in High-Fat Diet-Induced Obese Mice and Induces the Development of Beige Adipocytes in Human Adipose Tissue Derived-Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Hye-Lin Kim

    2017-09-01

    Full Text Available Brown adipocytes dissipate energy as heat and hence have an important therapeutic capacity for obesity. Development of brown-like adipocytes (also called beige is also another attractive target for obesity treatment. Here, we investigated the effect of farnesol, an isoprenoid, on adipogenesis in adipocytes and on the browning of white adipose tissue (WAT as well as on the weight gain of high-fat diet (HFD-induced obese mice. Farnesol inhibited adipogenesis and the related key regulators including peroxisome proliferator-activated receptor γ (PPARγ and CCAAT/enhancer binding protein α through the up-regulation of AMP-activated protein kinase in 3T3-L1 murine adipocytes and human adipose tissue-derived mesenchymal stem cells (hAMSCs. Farnesol markedly increased the expression of uncoupling protein 1 and PPARγ coactivator 1 α in differentiated hAMSCs. In addition, farnesol limited the weight gain in HFD obese mice and induced the development of beige adipocytes in both inguinal and epididymal WAT. These results suggest that farnesol could be a potential therapeutic agent for obesity treatment.

  20. Cocoa procyanidins and human cytokine transcription and secretion.

    Science.gov (United States)

    Mao, T; Van De Water, J; Keen, C L; Schmitz, H H; Gershwin, M E

    2000-08-01

    We examined whether cocoa, in its isolated procyanidin fractions (monomer through decamer), would modulate cytokine production at the levels of transcription and protein secretion in both resting and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). In resting cells, interleukin (IL)-1beta and IL-4 gene expression from cocoa-treated cells varied markedly among the subjects tested. However, at the protein level, the larger fractions (pentamer through decamer) stimulated a dramatic increase in IL-1beta concentration (up to ninefold) with increasing degree of polymerization. Similarly, these larger fractions augmented IL-4 concentration by as much as 2 pg/ml, whereas the control displayed levels nearly undetectable. In the presence of PHA, gene expression also seemed to be most affected by the larger procyanidin fractions. The pentameric through decameric fractions increased IL-1 beta expression by 7-19% compared with PHA control, whereas the hexameric through decameric fractions significantly inhibited PHA-induced IL-4 transcription in the range of 71-86%. This observation at the transcription level for IL-1 beta was reflected at the protein level in PHA-stimulated PBMC. Significant reductions in mitogen-induced IL-4 production were also seen at the protein level with the hexamer, heptamer and octamer. Individual oligomeric cocoa fractions were unstimulatory for IL-2 in resting PBMC. However, when induced with PHA, the pentamer, hexamer and heptamer fractions caused a 61-73% inhibition in IL-2 gene expression. This study offers additional data for the consideration of the health benefits of dietary polyphenols from a wide variety of foods, including those benefits associated specifically with cocoa and chocolate consumption.

  1. Lipid droplets hypertrophy: A crucial determining factor in insulin regulation by adipocytes

    NARCIS (Netherlands)

    B. Sanjabi (B.); M. Dashty (Monireh); B. Özcan (Behiye); V. Akbarkhanzadeh (Vishtaseb); M. Rahimi (Mehran); M. Vinciguerra (Manlio); F. Van Rooij (Felix); S.H. Al-Lahhman (Sa'ad); F. Sheedfar (Fareeba); T.G. Van Kooten (Theo G.); C.A. Spek (Arnold); A.T. Rowshani (Ajda); J.J.L. van der Want (Johannes); R. Klaassen (Rene); E.J.G. Sijbrands (Eric); M.P. Peppelenbosch (Maikel); F. Rezaee (Farhad)

    2015-01-01

    textabstractLipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitut

  2. Dextran sodium sulfate enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe.

    Science.gov (United States)

    Mukaiyama, Hiroyuki; Giga-Hama, Yuko; Tohda, Hideki; Takegawa, Kaoru

    2009-11-01

    The effect of medium supplementation on heterologous production of human serum transferrin (hTF) in the fission yeast Schizosaccharomyces pombe has been investigated. The productivity of recombinant hTF was low in wild-type S. pombe cells. To overcome this impediment, culture media supplements were screened for their ability to improve secretion of hTF. Casamino acids (CAA), which have been reported to increase heterologous protein productivity in Pichia pastoris, improved the secretion hTF by more than fourfold. An anion surfactant deoxycholate or polyethylene glycol also improved the secretion hTF. Interestingly, dextran sodium sulfate (DSS), a poly-anion surfactant, was found to enhance production of secreted hTF better than any other supplement tested. Addition of DSS in the presence of 2% CAA exhibited a synergistic effect on increasing hTF secretion, resulting in an increase of about sevenfold relative to conventional conditions. Cell growth was not found to be affected by the addition of DSS or CAA. DSS may act as a surfactant and may also facilitate the anchoring of liposomes, and these properties may contribute to efficient secretion or exocytosis through the plasma membrane.

  3. Gram-Negative Bacterial Lipopolysaccharide Stimulates Activin A Secretion from Human Amniotic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yumiko Abe

    2013-01-01

    Full Text Available Activin A is involved in inflammation. The present study was performed to clarify if lipopolysaccharide, a component of Gram-negative bacteria, stimulates activin A secretion from human amniotic epithelial cells and to determine if activin A plays a role in amnionitis. Fetal membranes were obtained during elective cesarean sections performed in full-term pregnancies of patients without systemic disease, signs of premature delivery, or fetal complications. Amniotic epithelial cells were isolated by trypsinization. The activin A concentrations in the culture media were measured by enzyme-linked immunosorbent assay, and cell proliferation was assessed by 5-bromo-2′-deoxyuridine incorporation. Amniotic epithelial cells secreted activin A in a cell density-dependent manner, and lipopolysaccharide (10 μg/mL enhanced the secretion at each cell density. Lipopolysaccharide (10–50 μg/mL also stimulated activin A secretion in a dose-dependent manner. Contrary to the effect of activin A secretion, lipopolysaccharide inhibited cell proliferation in amniotic epithelial cells. The present study suggests that lipopolysaccharide stimulation of activin A secretion may be a mechanism in the pathogenesis of amnionitis.

  4. Inhibition of mitochondrial gene transcription suppresses neurotensin secretion in the human carcinoid cell line BON.

    Science.gov (United States)

    Li, Nan; Wang, Qingding; Li, Jing; Wang, Xiaofu; Hellmich, Mark R; Rajaraman, Srinivasan; Greeley, George H; Townsend, Courtney M; Evers, B Mark

    2005-02-01

    Mitochondria, organelles essential for ATP production, play a central role in a number of cellular functions, including the regulation of insulin secretion. Neurotensin (NT), an important regulatory intestinal hormone, has been implicated in fatty acid translocation, gut motility and secretion, and intestinal cell growth; however, mechanisms regulating NT secretion have not been entirely defined. The purpose of this study was to determine the effect of inhibition of mitochondrial gene transcription on NT secretion. BON cells, a novel human carcinoid cell line that produces and secretes NT peptide and expresses the gene encoding NT (designated NT/N), were treated with ethidium bromide (EB; 0.05, 0.1, and 0.4 microg/ml), an inhibitor of DNA and RNA synthesis, or vehicle over a time course (1-4 days). Cells were then stimulated with either ACh (100 microM) or phorbol 12 myristate,13-acetate (PMA, 10 nM) for 30 min. Media and cells were extracted, and NT peptide measured by RIA. Treatment with EB had no effect on BON cell viability or cell cycle distribution over the 4-day course. In contrast, EB treatment produced a dose-dependent reduction of mitochondrial gene expression; however, NT/N gene expression was not altered. Mitochondrial inhibition by EB treatment suppressed NT secretion induced by ACh and PMA, both in a dose-dependent manner. EB-mediated inhibition of NT secretion and mitochondrial gene expression was reversed with removal of EB. Our results demonstrate that inhibition of mitochondrial gene transcription suppresses both ACh- and PMA-stimulated NT release. These findings are the first to demonstrate that mitochondrial function is important for agonist-mediated NT secretion.

  5. THE ROLE OF CALCIUM ION IN THE PATHOGENESIS OF HUMAN PITUITARY GH-SECRETING ADENOMAS

    Institute of Scientific and Technical Information of China (English)

    邓洁英; 史轶蘩; 尹娟娟

    1996-01-01

    To study the role of Ca2+ in the pathogenesis of pituitary growth hormone secreting adenornas,the function of Ca2+ in 23 cases of human pituitary GH-secreting adenoma was investigated in monolayer cell culture.It was found that Ca2- channel blockers nicardipin and nifedipin inhibited hasal and growth hormone releasing hormone (GRH)-stimulated GH secretion in 87.5% and 100.0% of the GH adenomas.respectively,demonstrating that in most human pituitary GH agonist octreotide regulated the processes of GH secretion via Ca2+ had defects in different steps including receptor.postreceptor Ca2+ channel and Ca2+-GH secreting coupling in 6(66.6%)and 5(55.5%) cases of 9 GH adenomas respectively.Among them,the defects in GRH receptor and SRIF regulated Ca2+ channel are the main causes of the dysfunction of GH adenomas.These defects may be related to GH hypersecretion in GH adenomas.Our data provides advance evidences for intrinsic defects of GH adenomas.

  6. Non-immunoglobulin fraction of human milk protects rabbits against enterotoxin-induced intestinal fluid secretion.

    OpenAIRE

    Otnaess, A B; Svennerholm, A M

    1982-01-01

    Human milk was fractionated by ammonium sulphate precipitation and column chromatography. A milk fraction depleted of secretory immunoglobulin A and with an apparent molecular weight of greater than 400,000 inhibited fluid secretion induced by cholera toxin and Escherichia coli heat-labile toxin in rabbit ileal loops.

  7. Human pituitary tissue secretes a potent growth factor for chondrocyte proliferation.

    Science.gov (United States)

    Kasper, S; Friesen, H G

    1986-01-01

    We report the secretion from human pituitary tumor fragments in organ culture of a potent mitogen for chondrocyte proliferation. Primary human pituitary cell and organ cultures were established from pituitary fragments obtained from patients with acromegaly, prolactinomas, and nonfunctional adenomas. The conditioned culture medium contained a mitogenic factor(s) that stimulated rabbit fetal chondrocyte proliferation, causing up to an 8-fold increase in cell number when added to Ham's F-10 medium in the presence of 10% fetal bovine serum. Blood leaking into the surgical field after the adenomectomy is known to contain very high concentrations of pituitary hormones. Serum samples, obtained from this venous "ooze" collected at the site of pituitary surgery, also were found to contain chondrocyte growth-promoting activity. Some venous serum samples stimulated chondrocyte proliferation in a dose-dependent manner down to a 1:10 dilution of 1 microliter serum, indicating that the material being secreted was very potent indeed. Gel filtration on Sephadex G-100 and analytical gel isoelectric focusing of culture media or serum samples from the pituitary fossa demonstrated that the growth factor secreted from the pituitary tumor fragments as well as from the venous serum is similar, if not identical, to chondrocyte growth factor (mol wt, 43,000; pI 7.6-7.9) purified from human pituitaries collected at autopsy. These results suggest that the chondrocyte growth-promoting factor(s) may not only be secreted by pituitary tumor fragments but by normal human pituitary tissue as well.

  8. Stimulation of mucin secretion from human bronchial epithelial cells by mast cell chymase

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Jian ZHENG

    2004-01-01

    AIM: To investigate the effect ofchymase on the mucin secretion from human bronchial epithelial cells. METHODS:Primarily-cultured human bronchial epithelial (PCHBE) cells and normal human bronchial epithelial (NHBE) cells were cultured with chymase or other stimulus in a mixture of bronchial epithelial growth medium (BEGM) and Dulbecco's modified Eagle's medium (DMEM), and the quantities of stimulatory mucin release were recorded.MUC5AC mucin was measured with an ELISA and dolichos biflorus agglutinin (DBA) mucin was determined with an enzyme linked DBA assay. RESULTS: A dose-dependent secretion of DBA mucin from PCHBE cells was observed with chymase with a maximum secretion of 98 % above baseline being achieved following 3 h incubation.The action of chymase started from 1 h, peaked at 3 h and dramatically decreased at 20 h following incubation.Chymase was able to also stimulate approximately 38 % increase in MUC5AC mucin release from PCHBE cells, and about 121% increase in DBA mucin release from NHBE cells. A chymase inhibitor soybean trypsin inhibitor (SBTI)was able to inhibit up to 85 % chymase induced mucin release, indicating that the enzymatic activity was essential for the actions of chymase on bronchial epithelial cells. CONCLUSION: Chymase is a potent stimulus of mucin secretion from human bronchial epithelial cells. It can contribute to mucus hypersecretion process in the patients with chronic obstructive pulmonary disease or asthma.

  9. Cephalic phase secretion of insulin and other enteropancreatic hormones in humans

    DEFF Research Database (Denmark)

    Veedfald, Simon; Plamboeck, Astrid; Deacon, Carolyn F

    2016-01-01

    Enteropancreatic hormone secretion is thought to include a cephalic phase, but the evidence in humans is ambiguous. We studied vagally induced gut hormone responses with and without muscarinic blockade in 10 glucose-clamped healthy men (age: 24.5 ± 0.6 yr, means ± SE; body mass index: 24.0 ± 0.5 ...

  10. Secretion and apparent activation of human hepatic lipase requires proper oligosaccharide processing in the endoplasmic reticulum

    NARCIS (Netherlands)

    A.J.M. Verhoeven (Adrie); B.P. Neve (Bernadette); H. Jansen (Hans)

    1999-01-01

    textabstractHuman hepatic lipase (HL) is a glycoprotein with four N-linked oligosaccharide side chains. The importance of glycosylation for the secretion of catalytically active HL was studied in HepG2 cells by using inhibitors of intracellular trafficking, N-glycosylat

  11. Chronic TNFalpha and cAMP pre-treatment of human adipocytes alter HSL, ATGL and perilipin to regulate basal and stimulated lipolysis.

    Science.gov (United States)

    Bézaire, Véronic; Mairal, Aline; Anesia, Rodica; Lefort, Corinne; Langin, Dominique

    2009-09-17

    We examined the effects of chronic TNFalpha and dibutyryl-cAMP (Db-cAMP) pre-treatment on the lipolytic machinery of human hMADS adipocytes. TNFalpha decreased adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein content and triglycerides (TG)-hydrolase activity but increased basal lipolysis due to a marked reduction in perilipin (PLIN) protein content. Conversely, Db-cAMP increased ATGL and HSL protein content but prevented PLIN phosphorylation, the net result being accentuated basal lipolysis. In forskolin-stimulated conditions, TNFalpha and Db-cAMP pre-treatment decreased stimulated TG-hydrolase activity and impaired PLIN phosphorylation. Together, this resulted in a severely attenuated response to forskolin-stimulated lipolysis.

  12. Inflammation and insulin resistance induced by trans-10, cis-12 conjugated linoleic acid depend on intracellular calcium levels in primary cultures of human adipocytes

    DEFF Research Database (Denmark)

    Kennedy, Arion; Martinez, Kristina; Chung, Soonkyu

    2010-01-01

    10,12 CLA-mediated production of reactive oxygen species (ROS), activation of ERK1/2 and cJun-NH2-terminal kinase (JNK), and induction of inflammatory genes. 10,12 CLA-mediated binding of NFkappaB to the promoters of interleukin (IL)-8 and cyclooxygenase (COX)-2 and induction of calcium......We previously demonstrated that trans-10, cis-12 (10,12) conjugated linoleic acid (CLA) induced inflammation and insulin resistance in primary human adipocytes by activating nuclear factor kappaB (NFkappaB) and extracellular signal-related kinase (ERK) signaling. In this study, we demonstrated...... that the initial increase in intracellular calcium ([Ca2+]i) mediated by 10,12 CLA was attenuated by TMB-8, an inhibitor of calcium release from the endoplasmic reticulum (ER), by BAPTA, an intracellular calcium chelator, and by D609, a phospholipase C (PLC) inhibitor. Moreover, BAPTA, TMB-8, and D609 attenuated...

  13. Reduced UCP-1 content in in vitro differentiated beige/brite adipocytes derived from preadipocytes of human subcutaneous white adipose tissues in obesity.

    Directory of Open Access Journals (Sweden)

    Andrew L Carey

    Full Text Available INTRODUCTION: Brown adipose tissue (BAT is a potential therapeutic target to reverse obesity. The purpose of this study was to determine whether primary precursor cells isolated from human adult subcutaneous white adipose tissue (WAT can be induced to differentiate in-vitro into adipocytes that express key markers of brown or beige adipose, and whether the expression level of such markers differs between lean and obese young adult males. METHODS: Adipogenic precursor cells were isolated from lean and obese individuals from subcutaneous abdominal WAT biopsies. Cells were grown to confluence, differentiated for 2.5 weeks then harvested for measurement of gene expression and UCP1 protein. RESULTS: There was no difference between groups with respect to differentiation into adipocytes, as indicated by oil red-O staining, rates of lipolysis, and expression of adipogenic genes (FABP4, PPARG. WAT genes (HOXC9, RB1 were expressed equally in the two groups. Post differentiation, the beige adipose specific genes CITED1 and CD137 were significantly increased in both groups, but classic BAT markers ZIC1 and LHX8 decreased significantly. Cell lines from both groups also equally increased post-differentiation expression of the thermogenic-responsive gene PPARGC1A (PGC-1α. UCP1 gene expression was undetectable prior to differentiation, however after differentiation both gene expression and protein content were increased in both groups and were significantly greater in cultures from lean compared with obese individuals (p<0.05. CONCLUSION: Human subcutaneous WAT cells can be induced to attain BAT characteristics, but this capacity is reduced in WAT cells from obese individuals.

  14. [Therapeutic potential of human mesenchymal stromal cells secreted components: a problem with standartization].

    Science.gov (United States)

    Sagaradze, G D; Grigorieva, O A; Efimenko, A Yu; Chaplenko, A A; Suslina, S N; Sysoeva, V Yu; Kalinina, N I; Akopyan, Zh A; Tkachuk, V A

    2015-01-01

    Regenerative medicine approaches, such as replacement of damaged tissue by ex vivo manufactured constructions or stimulation of endogenous reparative and regenerative processes to treat different diseases, are actively developing. One of the major tools for regenerative medicine are stem and progenitor cells, including multipotent mesenchymal stem/stromal cells (MSC). Because the paracrine action of bioactive factors secreted by MSC is considered as a main mechanism underlying MSC regenerative effects, application of MSC extracellular secreted products could be a promising approach to stimulate tissue regeneration; it also has some advantages compared to the injection of the cells themselves. However, because of the complexity of composition and multiplicity of mechanisms of action distinguished the medicinal products based on bioactive factors secreted by human MSC from the most of pharmaceuticals, it is important to develop the approaches to their standardization and quality control. In the current study, based on the literature data and guidelines as well as on our own experimental results, we provided rationalization for nomenclature and methods of quality control for the complex of extracellular products secreted by human adipose-derived MSC on key indicators, such as "Identification", "Specific activity" and "Biological safety". Developed approaches were tested on the samples of conditioned media contained products secreted by MSC isolated from subcutaneous adipose tissue of 30 donors. This strategy for the standardization of innovative medicinal products and biomaterials based on the bioactive extracellular factors secreted by human MSC could be applicable for a wide range of bioactive complex products, produced using the different types of stem and progenitor cells.

  15. Intermittent treatment with parathyroid hormone (PTH) as well as a non-peptide small molecule agonist of the PTH1 receptor inhibits adipocyte differentiation in human bone marrow stromal cells.

    Science.gov (United States)

    Rickard, David J; Wang, Fei-Lan; Rodriguez-Rojas, Ana-Maria; Wu, Zining; Trice, Wen J; Hoffman, Sandra J; Votta, Bartholomew; Stroup, George B; Kumar, Sanjay; Nuttall, Mark E

    2006-12-01

    Whereas continuous PTH infusion increases bone resorption and bone loss, intermittent PTH treatment stimulates bone formation, in part, via reactivation of quiescent bone surfaces and reducing osteoblast apoptosis. We investigated the possibility that intermittent and continuous PTH treatment also differentially regulates osteogenic and adipocytic lineage commitment of bone marrow stromal progenitor/mesenchymal stem cells (MSC). The MSC were cultured under mildly adipogenic conditions in medium supplemented with dexamethasone, insulin, isobutyl-methylxanthine and troglitazone (DIIT), and treated with 50 nM human PTH(1-34) for either 1 h/day or continuously (PTH replenished every 48 h). After 6 days, cells treated with PTH for 1 h/day retained their normal fibroblastic appearance whereas those treated continuously adopted a polygonal, irregular morphology. After 12-18 days numerous lipid vacuole and oil red O-positive adipocytes had developed in cultures treated with DIIT alone, or with DIIT and continuous PTH. In contrast, adipocyte number was reduced and alkaline phosphatase staining increased in the cultures treated with DIIT and 1 h/day PTH, indicating suppression of adipogenesis and possible promotion of early osteoblastic differentiation. Furthermore, intermittent but not continuous PTH treatment suppressed markers of differentiated adipocytes such as mRNA expression of lipoprotein lipase and PPARgamma as well as glycerol 3-phosphate dehydrogenase activity. All of these effects of intermittent PTH were also produced by a 1 h/day treatment with AH3960 (30 microM), a small molecule, non-peptide agonist of the PTH1 receptor. AH3960, like PTH, activates both the cAMP and calcium signaling pathways. Treatment with the adenylyl cyclase activator forskolin for 1 h/day, mimicked the anti-adipogenic effect of intermittent PTH, whereas pretreatment with the protein kinase-A inhibitor H89 prior to intermittent PTH resulted in almost complete conversion to adipocytes. In

  16. Cytokine secretion profiles of human keratinocytes during Trichophyton tonsurans and Arthroderma benhamiae infections.

    Science.gov (United States)

    Shiraki, Yumi; Ishibashi, Yoshio; Hiruma, Masataro; Nishikawa, Akemi; Ikeda, Shigaku

    2006-09-01

    Dermatophytes cause intractable superficial infections in humans. Arthroderma benhamiae, a zoophilic dermatophyte, triggers severe inflammatory responses in humans, while Trichophyton tonsurans, an anthropophilic dermatophyte, triggers minimal ones. Cytokines and other factors derived from keratinocytes play important roles in inflammatory and immune responses in the skin. The authors performed an in vitro investigation to determine the human keratinocyte cytokine profiles during dermatophyte infection. The human keratinocyte cell line PHK16-0b was infected with A. benhamiae or T. tonsurans for 24 h, and the cytokines secreted were analysed using a human cytokine antibody array. Marked differences were observed in the cytokine profiles of the cells infected with the two dermatophytes. A. benhamiae infection resulted in the secretion of a broad spectrum of cytokines, including proinflammatory cytokines, chemokines, and immunomodulatory cytokines. In contrast, T. tonsurans-infected keratinocytes secreted only limited cytokines, including eotaxin-2, interleukin (IL)-8 and IL-16. cDNA microarray analysis confirmed that A. benhamiae infection upregulated genes encoding IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-13, IL-15, IL-16, IL-17 and interferon (IFN)-gamma, while T. tonsurans infection upregulated only a few genes, such as those encoding IL-1beta and IL-16. RT-PCR demonstrated that infection by both dermatophytes enhanced IL-8 mRNA expression in keratinocytes. These results suggest that A. benhamiae-induced secretion of several cytokines from keratinocytes may be involved in a severe inflammatory response, and that the limited cytokine secretion from keratinocytes in response to T. tonsurans infection may result in a minimal inflammatory response in the skin. These cytokine profiles may aid in proving the clinical features of dermatophytosis.

  17. Precocious puberty due to human chorionic gonadotropin secreting germinoma

    Directory of Open Access Journals (Sweden)

    Daiane J Nascimento

    2012-01-01

    Full Text Available This study aims to report a rare case of precocious puberty (PP due to a human chorionic gonadotropin (hCG-producing germinoma located in the suprasellar region. A 10-year-old male patient presented with sexual precocity, headache, drowsiness, loss of appetite, and papilledema. Significant acceleration of bone age in relation to chronological age, high serum total testosterone levels, and hypopituitarism (unresponsiveness to stimulation test were observed. Magnetic resonance imaging (MRI of the brain showed a large suprasellar tumor and triventricular dilatation. High hCG levels were found in both blood and cerebrospinal fluid. Hormone replacement therapy and transcranial surgery associated with radiotherapy were performed, with complete regression of sexual characteristics and normal laboratory tests post-operatively. Clinical and laboratory findings, in addition to MRI scans, led to the diagnosis of an hCG-producing tumor and PP, which represents a rare report in the literature.

  18. Functional characterization of serotonin receptor subtypes in human duodenal secretion

    DEFF Research Database (Denmark)

    Engelmann, Bodil Elisabeth; Bindslev, Niels; Poulsen, Steen Seier;

    2006-01-01

    of dyspeptic patients with or without Helicobacter pylori infection, and to determine the 5-HT receptor subtypes functionally involved. Biopsies from the second part of duodenum were obtained from 43 dyspeptic patients during routine endoscopy. Biopsies were mounted in modified Ussing chambers with air suction......: ketanserin, ondansetron, or SB-204070 (1-butyl-4 piperidinmethyl-8-amino-7-chloro-2,3-dihydro-1,4-benzodioxin-5-carboxylate HCl). Histological examination was performed on duodenal biopsies. Helicobacter urease testing and histological examination determined Helicobacter pylori infection. 5-HT induced a dose......-dependent and bumetanide-sensitive short-circuit current, which was independent of the presence of Helicobacter pylori infection. All the three 5-HT receptor antagonists failed to significantly effect basal and 5-HT-induced short-circuit current. Our results indicate that in human duodenum 1) 5-HT is a potent stimulator...

  19. A role for SPARC in the moderation of human insulin secretion.

    Directory of Open Access Journals (Sweden)

    Lorna W Harries

    Full Text Available AIMS/HYPOTHESIS: We have previously shown the implication of the multifunctional protein SPARC (Secreted protein acidic and rich in cysteine/osteonectin in insulin resistance but potential effects on beta-cell function have not been assessed. We therefore aimed to characterise the effect of SPARC on beta-cell function and features of diabetes. METHODS: We measured SPARC expression by qRT-PCR in human primary pancreatic islets, adipose tissue, liver and muscle. We then examined the relation of SPARC with glucose stimulated insulin secretion (GSIS in primary human islets and the effect of SPARC overexpression on GSIS in beta cell lines. RESULTS: SPARC was expressed at measurable levels in human islets, adipose tissue, liver and skeletal muscle, and demonstrated reduced expression in primary islets from subjects with diabetes compared with controls (p< = 0.05. SPARC levels were positively correlated with GSIS in islets from control donors (p< = 0.01. Overexpression of SPARC in cultured beta-cells resulted in a 2.4-fold increase in insulin secretion in high glucose conditions (p< = 0.01. CONCLUSIONS: Our data suggest that levels of SPARC are reduced in islets from donors with diabetes and that it has a role in insulin secretion, an effect which appears independent of SPARC's modulation of obesity-induced insulin resistance in adipose tissue.

  20. Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells

    Science.gov (United States)

    Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.

    1985-01-01

    The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.

  1. Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

    Directory of Open Access Journals (Sweden)

    Xuefei Song

    2015-01-01

    Full Text Available Purpose. The purpose of this study was to determine the impact of cross-linking (CXL on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham’s F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2 during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA. Results. Following CXL, cell viability and proliferation decreased (P0.06. Five hours after CXL, FGFb secretion increased significantly (P=0.037; however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P>0.12. Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours, normalizing after 24 hours.

  2. Stimulation tests of human growth hormone secretion by insulin, lysine vasopressin, pyrogen and glucagon

    Directory of Open Access Journals (Sweden)

    Ogawa,Norio

    1974-06-01

    Full Text Available Firstly, comparisons have been made of the secretion of human growth hormone (HGH that was induced by insulin, lysine vasopressin and pyrogen injections in order to study whether these substances can be utilized as a rapid test of HGH secretion. In insulin test, a fall of the fasting blood glucose level by 28.6% or more seemed to be sufficient to provoke adequate HGH elevation, and 9.4 ng/ml or higher HGH increment was recognized as being normal, because lysine vasopressin and pyrogen produce varying degrees of side-effects and are less specific and unpredictable in the release of HGH. Secondly, the pharmacologic effects and mechanism of action of exogenous glucagon upon the HGH secretion were studied. In normal subjects after one mg sc glucagon, there was a mean peak blood glucose level of 142. 4±3.l mg/lOO ml at 30 min, HGH levels reached a mean peak level of 22. 6±4. 8 ng/ml at 150 min, and no false negative response was noted. In patients with hypopituitarism, there was no positive response in plasma HGH levels after the sc glucagon. The present study revealed that the rise and subsequent fall of blood glucose are not the sole mechanism responsible for the effct of glucagon on HGH secretion, and that the HGH secretion in response to the sc glucagon was not triggered by cathecholamine via the stimulation of the adrenal medulla.

  3. Secretion of Human Protein C in Mouse Milk

    Directory of Open Access Journals (Sweden)

    Chae-Won Park

    2015-03-01

    Full Text Available To determine the production of recombinant human protein C (rec-hPC in milk, we created two homozygous mice lines for the goat β-casein/hPC transgene. Females and males of both lines (#10 and #11 displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands.

  4. Fructose Alters Intermediary Metabolism of Glucose in Human Adipocytes and Diverts Glucose to Serine Oxidation in the One-Carbon Cycle Energy Producing Pathway.

    Science.gov (United States)

    Varma, Vijayalakshmi; Boros, László G; Nolen, Greg T; Chang, Ching-Wei; Wabitsch, Martin; Beger, Richard D; Kaput, Jim

    2015-06-16

    Increased consumption of sugar and fructose as sweeteners has resulted in the utilization of fructose as an alternative metabolic fuel that may compete with glucose and alter its metabolism. To explore this, human Simpson-Golabi-Behmel Syndrome (SGBS) preadipocytes were differentiated to adipocytes in the presence of 0, 1, 2.5, 5 or 10 mM of fructose added to a medium containing 5 mM of glucose representing the normal blood glucose concentration. Targeted tracer [1,2-13C2]-d-glucose fate association approach was employed to examine the influence of fructose on the intermediary metabolism of glucose. Increasing concentrations of fructose robustly increased the oxidation of [1,2-13C2]-d-glucose to 13CO2 (p < 0.000001). However, glucose-derived 13CO2 negatively correlated with 13C labeled glutamate, 13C palmitate, and M+1 labeled lactate. These are strong markers of limited tricarboxylic acid (TCA) cycle, fatty acid synthesis, pentose cycle fluxes, substrate turnover and NAD+/NADP+ or ATP production from glucose via complete oxidation, indicating diminished mitochondrial energy metabolism. Contrarily, a positive correlation was observed between glucose-derived 13CO2 formed and 13C oleate and doses of fructose which indicate the elongation and desaturation of palmitate to oleate for storage. Collectively, these results suggest that fructose preferentially drives glucose through serine oxidation glycine cleavage (SOGC pathway) one-carbon cycle for NAD+/NADP+ production that is utilized in fructose-induced lipogenesis and storage in adipocytes.

  5. Fructose Alters Intermediary Metabolism of Glucose in Human Adipocytes and Diverts Glucose to Serine Oxidation in the One–Carbon Cycle Energy Producing Pathway

    Directory of Open Access Journals (Sweden)

    Vijayalakshmi Varma

    2015-06-01

    Full Text Available Increased consumption of sugar and fructose as sweeteners has resulted in the utilization of fructose as an alternative metabolic fuel that may compete with glucose and alter its metabolism. To explore this, human Simpson-Golabi-Behmel Syndrome (SGBS preadipocytes were differentiated to adipocytes in the presence of 0, 1, 2.5, 5 or 10 mM of fructose added to a medium containing 5 mM of glucose representing the normal blood glucose concentration. Targeted tracer [1,2-13C2]-d-glucose fate association approach was employed to examine the influence of fructose on the intermediary metabolism of glucose. Increasing concentrations of fructose robustly increased the oxidation of [1,2-13C2]-d-glucose to 13CO2 (p < 0.000001. However, glucose-derived 13CO2 negatively correlated with 13C labeled glutamate, 13C palmitate, and M+1 labeled lactate. These are strong markers of limited tricarboxylic acid (TCA cycle, fatty acid synthesis, pentose cycle fluxes, substrate turnover and NAD+/NADP+ or ATP production from glucose via complete oxidation, indicating diminished mitochondrial energy metabolism. Contrarily, a positive correlation was observed between glucose-derived 13CO2 formed and 13C oleate and doses of fructose which indicate the elongation and desaturation of palmitate to oleate for storage. Collectively, these results suggest that fructose preferentially drives glucose through serine oxidation glycine cleavage (SOGC pathway one-carbon cycle for NAD+/NADP+ production that is utilized in fructose-induced lipogenesis and storage in adipocytes.

  6. Control of Adipocyte Differentiation in Different Fat Depots; Implications for Pathophysiology or Therapy

    Directory of Open Access Journals (Sweden)

    Xiuquan eMa

    2015-01-01

    Full Text Available Adipocyte differentiation and its impact on restriction or expansion of particular adipose tissue depots has physiological and pathophysiological significance in view of the different functions of these depots. Brown or beige fat [BAT] expansion can enhance thermogenesis, lipid oxidation, insulin sensitivity and glucose tolerance; conversely expanded visceral fat [VAT] is associated with insulin resistance, low grade inflammation, dyslipidaemia and cardiometabolic risk. The largest depot, subcutaneous white fat [WAT], has important beneficial characteristics including storage of lipid out of harms way and secretion of adipokines, especially leptin and adiponectin, with positive metabolic effects including lipid oxidation, energy utilisation, enhanced insulin action and an anti-inflammatory role. The absence of these functions in lipodystrophies leads to major metabolic disturbances. An ability to expand WAT adipocyte differentiation would seem an important defence mechanism against the detrimental effects of energy excess and limit harmful accumulation of lipid in ectopic sites, such as liver and muscle.Adipocyte differentiation involves a transcriptional cascade with PPARg being most important in WAT but less so in VAT, with increased angiogenesis also critical. The transcription factor, Islet1, is fairly specific to VAT and in vitro inhibits adipocyte differentiation. The physiological importance of Islet1 requires further study. Basic control of differentiation is similar in BAT but important differences include the effect of PGC-1a on mitochondrial biosynthesis and upregulation of UCP1; also PRDM16 plays a pivotal role in expression of the BAT phenotype.Modulation of the capacity or function of these different adipose tissue depots, by altering adipocyte differentiation or other means, holds promise for interventions that can be helpful in human disease, particularly cardiometabolic disorders associated with the world wide explosion of

  7. Impaired response of mature adipocytes of diabetic mice to hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Seok Jong, E-mail: seok-hong@northwestern.edu; Jin, Da P.; Buck, Donald W.; Galiano, Robert D.; Mustoe, Thomas A., E-mail: tmustoe@nmh.org

    2011-10-01

    Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.

  8. Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

    OpenAIRE

    Bozec, Aline; Hannemann, Nicole

    2016-01-01

    Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our pr...

  9. Effect of Cytokines Secreted by Human Adipose Stromal Cells on Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    LI Bingong; ZENG Qiutang; WANG Hongxiang; MAO Xiaobo

    2006-01-01

    To isolate and culture adipose stromal cells (ASCs), and study the effect of cytokines secreted by ASCs on endothelial cells, human adipose tissue was digested with collagenase type Ⅰ solution and ASCs were derived by culture. The cells surface phenotype was examined by flow cytometry. ELISA was used to detect the secretion of VEGF, HGF, SDF-1 α and RT-PCR was employed to detect the expression of their mRNA. Then the ASC medium was utilized to culture human umbilical vein endothelial cells ECV304. Cells were counted by hemacytometer to determine the proliferation and Annexin V/PI was employed for the examination of the apoptosis rate of ECV304. ASCs were derived by culture and expressed CD34, CD105 while they did not express CD31 or CD45. ASCs secreted cytokines such as VEGF, HGF and SDF-1 α so the ASC medium could stimulate proliferation and counteract apoptosis of endothelial cells (P<0.05). Bcl-2 mRNA was also found to be up-regulated in the endothelial cells. It is concluded that ASCs can secrete cytokines and has significant effect on the proliferation of endothelial cells and apoptosis.

  10. Characterization of age-related gene expression profiling in bone marrow and epididymal adipocytes

    Directory of Open Access Journals (Sweden)

    Ueno Masami

    2011-05-01

    Full Text Available Abstract Background While an increase in bone marrow adiposity is associated with age-related bone disease, the function of bone marrow adipocytes has not been studied. The aim of this study was to characterize and compare the age-related gene expression profiles in bone marrow adipocytes and epididymal adipocytes. Results A total of 3918 (13.7% genes were differentially expressed in bone marrow adipocytes compared to epididymal adipocytes. Bone marrow adipocytes revealed a distinct gene profile with low expression of adipocyte-specific genes peroxisome proliferator-activated receptor gamma (PPARγ, fatty acid binding protein 4 (FABP4, perilipin (Plin1, adipsin (CFD and high expression of genes associated with early adipocyte differentiation (CCAAT/enhancer binding protein beta (C/EBPβ, regulator of G-protein signaling 2 (RGS2. In addition, a number of genes including secreted frizzled related protein 4 (SFRP4, tumor necrosis factor α (TNFα, transforming growth factor beta 1(TGFβ1, G-protein coupled receptor 109A (GPR109A and interleukin 6 (IL-6, that could affect adipose-derived signaling to bone are markedly increased in bone marrow adipocytes. Age had a substantial effect on genes associated with mitochondria function and inflammation in bone marrow adipocytes. Twenty seven genes were significantly changed with age in both adipocyte depots. Among these genes, IL6 and GPR109A were significantly reduced with age in both adipocyte depots. Conclusions Overall, gene profiling reveals a unique phenotype for primary bone marrow adipocytes characterized by low adipose-specific gene expression and high expression of inflammatory response genes. Bone marrow and epididymal adipocytes share a common pathway in response to aging in mice, but age has a greater impact on global gene expression in epididymal than in bone marrow adipocytes. Genes that are differentially expressed at greater levels in the bone marrow are highly regulated with age.

  11. Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

    Science.gov (United States)

    Wawrzyniak, Ivan; Texier, Catherine; Poirier, Philippe; Viscogliosi, Eric; Tan, Kevin S W; Delbac, Frédéric; El Alaoui, Hicham

    2012-09-01

    Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

  12. RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains

    DEFF Research Database (Denmark)

    Haas, Claus; Hanson, E; Anjos, M J;

    2014-01-01

    The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3...... housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating...... laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were...

  13. The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

    Directory of Open Access Journals (Sweden)

    Niv Bachnoff

    2011-01-01

    Full Text Available A PKA consensus phosphorylation site S1928 at the α11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α11.2 or α11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s at the C-tail of α11.2, the pore forming subunit of CaV1.2.

  14. Secretion of protein and epidermal growth factor (EGF) by transplanted human pancreas.

    Science.gov (United States)

    Konturek, J W; Buesing, M; Hopt, U T; Stachura, J; Becker, H D; Konturek, S J

    1992-08-01

    Epidermal growth factor (EGF) has been localized in human salivary and Brunner's glands and found to stimulate the proliferation of gastrointestinal and pancreatic tissues in animals, but little is known about EGF in human pancreas. This study was designed to determine the distribution and release of EGF in the pancreas and to assess the secretion of EGF and protein by the transplanted human pancreas. The peroxidase antiperoxidase (PAP) immunocytochemical method with anti-hEGF showed that EGF was restricted mainly to the excretory cells lining pancreatic ducts. The EGF immunoreactivity in the pancreatic tissue averaged about 15 +/- 0.5 micrograms/g of tissue wt. The concentration and output of EGF in the pancreatic juice were, respectively, about 3.4 +/- 0.7 ng/mL and 68 + 12 ng/h in basal secretion collected from the whole pancreatic transplant. A significant increase in EGF release from this transplant started about 2 h after its reperfusion and was accompanied by a parallel increase in protein output. Injection of iv secretion (1 U/kg) resulted in a transient rise in EGF output, probably as a result of washout by increased vol flow, whereas HCCK (1 U/kg) caused more prolonged release of EGF accompanied by a marked stimulation of protein secretion. Ingestion of a mixed meal caused an immediate and sustained increment in EGF output, and protein output showed a more protracted increase, reaching its peak in the second postprandial hour. Fractionation of an extract of pancreatic juice on G-5O Sephadex superfine column revealed that EGF immunoreactivity emerged as a major peak in the same position as authentic human EGF (hEGF).(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line.

    Science.gov (United States)

    Traber, M G; Kayden, H J; Rindler, M J

    1987-11-01

    Lipoprotein secretion by Caco-2 cells, a human intestinal cell line, was studied in cells grown on inserts containing a Millipore filter (0.45 micron), separating secretory products from the apical and basolateral membranes into separate chambers. Under these conditions, as observed by electron microscopy, the cells formed a monolayer of columnar epithelial cells with microvilli on the apical surface and tight junctions between cells. The electrical resistances of the cell monolayers were 250-500 ohms/cm2. Both 14C-labeled lipids and 35S-labeled proteins were used to assess lipoprotein secretion. After a 24-hr incubation with [14C]oleic acid, 60-80% of the secreted triglyceride (TG) was in the basolateral chamber; 40% of the TG was present in the d less than 1.006 g/ml (chylomicron + VLDL) fraction and 50% in the 1.006 less than d less than 1.063 g/ml (LDL) fraction. After a 4-hr incubation with [35S]methionine, apolipoproteins were found to be major secretory products with 75-100% secreted to the basolateral chamber. Apolipoproteins B-100, B-48, E, A-I, A-IV, and C-III were identified by immunoprecipitation. The d less than 1.006 g/ml fraction was found to contain all of the major apolipoproteins, while the LDL fraction contained primarily apoB-100 and apoE; the HDL (1.063 less than d less than 1.21 g/ml) fraction principally contained apoA-I and apoA-IV. Mn-heparin precipitated all of the [35S]methionine-labeled apoB-100 and B-48 and a majority of the other apolipoproteins, and 80% of the [14C]oleic acid-labeled triglyceride, but only 15% of the phospholipid, demonstrating that Caco-2 cells secrete triglyceride-rich lipoproteins containing apoB. Secretion of lipoproteins was dependent on the lipid content of the medium; prior incubation with lipoprotein-depleted serum specifically reduced the secretion of lipoproteins, while addition of both LDL and oleic acid to the medium maintained the level of apoB-100, B-48, and A-IV secretion to that observed in the control

  16. Human rhinovirus 16 causes Golgi apparatus fragmentation without blocking protein secretion.

    Science.gov (United States)

    Mousnier, Aurelie; Swieboda, Dawid; Pinto, Anaïs; Guedán, Anabel; Rogers, Andrew V; Walton, Ross; Johnston, Sebastian L; Solari, Roberto

    2014-10-01

    The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. Importance: The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be

  17. Habituation of adult Magellanic penguins to human visitation as expressed through behavior and corticosterone secretion.

    Science.gov (United States)

    Walker, Brian G; Boersma, P Dee; Wingfield, John C

    2006-02-01

    Ecotourism is increasing worldwide; hence, it is important to know how wildlife are affected behaviorally and physiologically by human visitation. We studied the effects of human visitation on the Magellanic Penguins (Spheniscus magellanicus) at Punta Tombo, Argentina, by monitoring changes in defensive head turns and plasma corticosterone (a hormone secreted in response to stress) for penguins with and without a history of tourist visitation. Habituation to human visitation was rapid. In penguins with no previous exposure to tourists, the number of defensive head turns and level of plasma corticosterone decreased significantly within 5 days of one 15-minute visit/day. Penguins living in tourist-visited and undisturbed areas secreted more corticosterone when captured and restrained than penguins visited by a person. Penguins in tourist areas, however did not show as strong a corticosterone response to capture and restraint as did penguins in areas without tourists. This difference was due to a decreased capability of the adrenocortical tissue to secrete corticosterone in tourist-visited birds. Although our data show no direct negative effects of tourism on Magellanic Penguins at Punta Tombo, consequences of a modification of physiological capabilities (e.g., adrenocortical function) may not become apparent until much later in life. The physiological differences between tourist-visited and undisturbed groups of Magellanic Penguins emphasize the importance of monitoring the effects of anthropogenic disturbances on wildlife at multiple levels.

  18. Adipose tissue and adipocytes support tumorigenesis and metastasis.

    Science.gov (United States)

    Nieman, Kristin M; Romero, Iris L; Van Houten, Bennett; Lengyel, Ernst

    2013-10-01

    Adipose tissue influences tumor development in two major ways. First, obese individuals have a higher risk of developing certain cancers (endometrial, esophageal, and renal cell cancer). However, the risk of developing other cancers (melanoma, rectal, and ovarian) is not altered by body mass. In obesity, hypertrophied adipose tissue depots are characterized by a state of low grade inflammation. In this activated state, adipocytes and inflammatory cells secrete adipokines and cytokines which are known to promote tumor development. In addition, the adipocyte mediated conversion of androgens to estrogen specifically contributes to the development of endometrial cancer, which shows the greatest relative risk (6.3-fold) increase between lean and obese individuals. Second, many tumor types (gastric, breast, colon, renal, and ovarian) grow in the anatomical vicinity of adipose tissue. During their interaction with cancer cells, adipocytes dedifferentiate into pre-adipocytes or are reprogrammed into cancer-associated adipocytes (CAA). CAA secrete adipokines which stimulate the adhesion, migration, and invasion of tumor cells. Cancer cells and CAA also engage in a dynamic exchange of metabolites. Specifically, CAA release fatty acids through lipolysis which are then transferred to cancer cells and used for energy production through β-oxidation. The abundant availability of lipids from adipocytes in the tumor microenvironment, supports tumor progression and uncontrolled growth. Given that adipocytes are a major source of adipokines and energy for the cancer cell, understanding the mechanisms of metabolic symbiosis between cancer cells and adipocytes, should reveal new therapeutic possibilities. This article is part of a Special Issue entitled Lipid Metabolism in Cancer. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. The effect of lesions of the sympathoadrenal system on training induced adaptations in adipocytes and pancreatic islets in rats

    DEFF Research Database (Denmark)

    Stallknecht, B; Roesdahl, M; Vinten, J

    1996-01-01

    in sham adrenodemedullated rats (P swim trained for 10 weeks or remained sedentary. Insulin stimulated 3-O-[14C]methylglucose transport......Physical training increases insulin stimulated glucose uptake in adipocytes and decreases insulin secretion from pancreatic islets. The mechanism behind these adaptations is not known. Because in acute exercise adrenergic activity influences both adipocytes and pancreatic islets, the sympathetic...... was measured in adipocytes from epididymal fat pads, and insulin secretion and glucose metabolism were measured in glucose stimulated pancreatic islets. Training increased insulin stimulated glucose transport in adipocytes (P

  20. Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion

    Science.gov (United States)

    Cataldo, Luis R.; Gutierrez, Juan; Santos, José L.; Casas, Mariana; Contreras-Ferrat, Ariel E.; Moro, Cedric; Bouzakri, Karim

    2017-01-01

    Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle) glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines). We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS). In conditioned media from human myotubes incubated with/without insulin (100 nmol/L) for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p < 0.05). Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets. PMID:28286777

  1. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells.

    Science.gov (United States)

    Vogt, Rhonda R; Unda, Richard; Yeh, Lee-Chuan C; Vidro, Eileen K; Lee, John C; Tsin, Andrew T

    2006-08-01

    Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

  2. Secretion of respiratory syncytial virus inhibitors and antibody in human milk throughout lactation.

    Science.gov (United States)

    Toms, G L; Gardner, P S; Pullan, C R; Scott, M; Taylor, C

    1980-01-01

    Neutralising inhibitors to respiratory syncytial (RS) virus have been demonstrated in the whey of most samples of human milk tested. Although high titres were secreted in colostra of some mothers (1/10-1/2,560; median 1/40) inhibitor levels in milk collected after the first week of lactation were uniformly low (median 1/10). High neutralising titres correlated with high colostral levels of specific antiviral IgA but, unlike neutralising activity, IgA antiviral antibody persisted in the milk of only four of 18 mothers. Similarly, antiviral IgG and IgM antibodies were not generally detected after the first post-partum week. Differences in antibody secretion among mothers did not correlate with differences in total protein or total immunoglobulin secretion, and appeared to reflect maternal immune status. In one mother a marked rise in specific antiviral IgA and IgG secretions during the second and third months of lactation suggested a response to virus infection. The relevance of maternal immunity and colostral and milk antiviral antibody to protection of breast-fed babies from RS-virus bronchiolitis is discussed.

  3. Effect of Human Myotubes-Derived Media on Glucose-Stimulated Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Maria L. Mizgier

    2017-01-01

    Full Text Available Fasting to postprandial transition requires a tight adjustment of insulin secretion to its demand, so tissue (e.g., skeletal muscle glucose supply is assured while hypo-/hyperglycemia are prevented. High muscle glucose disposal after meals is pivotal for adapting to increased glycemia and might drive insulin secretion through muscle-released factors (e.g., myokines. We hypothesized that insulin influences myokine secretion and then increases glucose-stimulated insulin secretion (GSIS. In conditioned media from human myotubes incubated with/without insulin (100 nmol/L for 24 h, myokines were qualitatively and quantitatively characterized using an antibody-based array and ELISA-based technology, respectively. C57BL6/J mice islets and Wistar rat beta cells were incubated for 24 h with control and conditioned media from noninsulin- and insulin-treated myotubes prior to GSIS determination. Conditioned media from insulin-treated versus nontreated myotubes had higher RANTES but lower IL6, IL8, and MCP1 concentration. Qualitative analyses revealed that conditioned media from noninsulin- and insulin-treated myotubes expressed 32 and 23 out of 80 myokines, respectively. Islets incubated with conditioned media from noninsulin-treated myotubes had higher GSIS versus control islets (p<0.05. Meanwhile, conditioned media from insulin-treated myotubes did not influence GSIS. In beta cells, GSIS was similar across conditions. In conclusion, factors being present in noninsulin-stimulated muscle cell-derived media appear to influence GSIS in mice islets.

  4. Ultrastructural Evidence of Exosome Secretion by Progenitor Cells in Adult Mouse Myocardium and Adult Human Cardiospheres

    Directory of Open Access Journals (Sweden)

    Lucio Barile

    2012-01-01

    Full Text Available The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34+ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an “off-the-shelf” product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  5. Autotaxin Is Regulated by Glucose and Insulin in Adipocytes.

    Science.gov (United States)

    D'Souza, Kenneth; Kane, Daniel A; Touaibia, Mohamed; Kershaw, Erin E; Pulinilkunnil, Thomas; Kienesberger, Petra C

    2017-04-01

    Autotaxin (ATX) is an adipokine that generates the bioactive lipid, lysophosphatidic acid. Despite recent studies implicating adipose-derived ATX in metabolic disorders including obesity and insulin resistance, the nutritional and hormonal regulation of ATX in adipocytes remains unclear. The current study examined the regulation of ATX in adipocytes by glucose and insulin and the role of ATX in adipocyte metabolism. Induction of insulin resistance in adipocytes with high glucose and insulin concentrations increased ATX secretion, whereas coincubation with the insulin sensitizer, rosiglitazone, prevented this response. Moreover, glucose independently increased ATX messenger RNA (mRNA), protein, and activity in a time- and concentration-dependent manner. Glucose also acutely upregulated secreted ATX activity in subcutaneous adipose tissue explants. Insulin elicited a biphasic response. Acute insulin stimulation increased ATX activity in a PI3Kinase-dependent and mTORC1-independent manner, whereas chronic insulin stimulation decreased ATX mRNA, protein, and activity. To examine the metabolic role of ATX in 3T3-L1 adipocytes, we incubated cells with the ATX inhibitor, PF-8380, for 24 hours. Whereas ATX inhibition increased the expression of peroxisome proliferator-activated receptor-γ and its downstream targets, insulin signaling and mitochondrial respiration were unaffected. However, ATX inhibition enhanced mitochondrial H2O2 production. Taken together, this study suggests that ATX secretion from adipocytes is differentially regulated by glucose and insulin. This study also suggests that inhibition of autocrine/paracrine ATX-lysophosphatidic acid signaling does not influence insulin signaling or mitochondrial respiration, but increases reactive oxygen species production in adipocytes. Copyright © 2017 Endocrine Society.

  6. B Lymphocyte Stimulator (BLyS) Is Expressed in Human Adipocytes In Vivo and Is Related to Obesity but Not to Insulin Resistance

    OpenAIRE

    Nike Müller; Schulte, Dominik M.; Susann Hillebrand; Kathrin Türk; Jochen Hampe; Clemens Schafmayer; Mario Brosch; Witigo von Schönfels; Markus Ahrens; Rainald Zeuner; Schröder, Johann O.; Matthias Blüher; Christian Gutschow; Sandra Freitag-Wolf; Marta Stelmach-Mardas

    2014-01-01

    Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found ...

  7. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  8. Practice and application of the construction of the human's secret science museum in medical colleges

    Institute of Scientific and Technical Information of China (English)

    Mu Zhaoxin; Sui Yuelin; Lu Lanhong

    2015-01-01

    Human anatomy showroom is an important place for students to contact the theory with prac-tice, at the same time is also a window to exchange with other schools. How to make the high quality anatomy teaching resources to service the public? How to provide a place for medical practitioners' to continue learning? It is a subject that many anatomical workers have been studied all the way. This study is based on the human anatomy laboratory, and developing from basic construction, space layout to the design of Showcase and exhibition booth, sample production, writing materials and other aspects. Every link has been carefully argument and arranged. It puts science, knowledge, interest and artistry together. So we can build up the human's secret science museum to allow people to cognize human`s inner construction and open to primary and middle school students and the public. It has finally obtained good social and economic benefits.

  9. Fat intake leads to differential response of rat adipocytes to glucose, insulin and ascorbic acid.

    Science.gov (United States)

    Garcia-Diaz, Diego F; Campion, Javier; Arellano, Arianna V; Milagro, Fermin I; Moreno-Aliaga, Maria J; Martinez, J Alfredo

    2012-04-01

    Antioxidant-based treatments have emerged as novel and interesting approaches to counteract fat accumulation in obesity and associated metabolic disturbances. Adipocytes from rats that were fed on chow or high-fat diet (HFD) for 50 d were isolated (primary adipocytes) and incubated (72 h) on low (LG; 5.6 mmol/L) or high (HG; 25 mmol/L) glucose levels, in the presence or absence of 1.6 nmol/L insulin and 200 μmol/L vitamin C (VC). Adipocytes from HFD-fed animals presented lower insulin-induced glucose uptake, lower lactate and glycerol release, and lower insulin-induced secretion of some adipokines as compared with controls. HG treatment restored the blunted response to insulin regarding apelin secretion in adipocytes from HFD-fed rats. VC treatment inhibited the levels of nearly all variables, irrespective of the adipocytes' dietary origin. The HG treatment reduced adipocyte viability, and VC protected from this toxic effect, although more drastically in control adipocytes. Summing up, in vivo chow or HFD intake determines a differential response to insulin and glucose treatments that appears to be dependent on the insulin-resistance status of the adipocytes, while VC modifies some responses from adipocytes independently of the previous dietary intake of the animals.

  10. Proteomic analysis of the secretions of Pseudallescheria boydii, a human fungal pathogen with unknown genome.

    Science.gov (United States)

    da Silva, Bianca Alcântara; Sodré, Cátia Lacerda; Souza-Gonçalves, Ana Luiza; Aor, Ana Carolina; Kneipp, Lucimar Ferreira; Fonseca, Beatriz Bastos; Rozental, Sonia; Romanos, Maria Teresa Villela; Sola-Penna, Mauro; Perales, Jonas; Kalume, Dário Eluan; dos Santos, André Luis Souza

    2012-01-01

    Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P

  11. Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming human beta cell function

    Science.gov (United States)

    Rodriguez-Diaz, Rayner; Dando, Robin; Jacques-Silva, M. Caroline; Fachado, Alberto; Molina, Judith; Abdulreda, Midhat; Ricordi, Camillo; Roper, Stephen D.; Berggren, Per-Olof; Caicedo, Alejandro

    2011-01-01

    Acetylcholine is a neurotransmitter that plays a major role in the function of the insulin secreting pancreatic beta cell1,2. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species1,3,4, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of the human islet provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. We anticipate these results to revise models about neural input and cholinergic signaling in the endocrine pancreas. Cholinergic signaling within the islet represents a potential therapeutic target in diabetes5, highlighting the relevance of this advance to future drug development. PMID:21685896

  12. The production and secretion of complement component C1q by human mast cells.

    Science.gov (United States)

    van Schaarenburg, Rosanne A; Suurmond, Jolien; Habets, Kim L L; Brouwer, Mieke C; Wouters, Diana; Kurreeman, Fina A S; Huizinga, Tom W J; Toes, René E M; Trouw, Leendert A

    2016-10-01

    C1q is the initiation molecule of the classical pathway of the complement system and is produced by macrophages and immature dendritic cells. As mast cells share the same myeloid progenitor cells, we have studied whether also mast cells can produce and secrete C1q. Mast cells were generated in vitro from CD34+ progenitor cells from buffy coats or cord blood. Fully differentiated mast cells were shown by both RNA sequencing and qPCR to express C1QA, C1QB and C1QC. C1q produced by mast cells has a similar molecular make-up as serum C1q. Reconstituting C1q depleted serum with mast cell supernatant in haemolytic assays, indicated that C1q secreted by mast cells is functionally active. The level of C1q in supernatants produced under basal conditions was considerably enhanced upon stimulation with LPS, dexamethasone in combination with IFN- γ or via FcεRI triggering. Mast cells in human tissues stained positive for C1q in both healthy and in inflamed tissue. Moreover, mast cells in healthy and diseased skin appear to be the predominant C1q positive cells. Together, our data reveal that mast cells are able to produce and secrete functional active C1q and indicate mast cells as a local source of C1q in human tissue. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. The Putative Role of Human Peritoneal Adipocytes in the Fight against Bacteria: Synthesis of the Antimicrobial Active Peptide DEFA1-3

    OpenAIRE

    2010-01-01

    Background: Spontaneous peritonitis is a rather rare event, even in peritoneal dialysis (PD). As defensins are natural antimicrobial peptides, we hypothesized that adipocytes as the major constituents of the omentum could play an important role in protecting against infection by generating defensin (DEFA1-3). Methods: We isolated adipocytes from the omentum majus and conducted qualitative and quantitative RT-PCR and immunohistochemical analyses. Results: For the first time described, we were ...

  14. Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis.

    Science.gov (United States)

    Bozec, Aline; Hannemann, Nicole

    2016-06-03

    Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1α protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

  15. Validation of methods for measurement of insulin secretion in humans in vivo

    DEFF Research Database (Denmark)

    Kjems, L L; Christiansen, E; Vølund, A;

    2000-01-01

    of these mathematical techniques for quantification of insulin secretion have been tested in dogs, but not in humans. In the present studies, we examined the validity of both methods to recover the known infusion rates of insulin and C-peptide mimicking ISR during an oral glucose tolerance test. ISR from both......To detect and understand the changes in beta-cell function in the pathogenesis of type 2 diabetes, an accurate and precise estimation of prehepatic insulin secretion rate (ISR) is essential. There are two common methods to assess ISR, the deconvolution method (by Eaton and Polonsky......)-considered the "gold standard"-and the combined model (by Vølund et al.). The deconvolution method is a 2-day method, which generally requires separate assessment of C-peptide kinetics, whereas the combined model is a single-day method that uses insulin and C-peptide data from a single test of interest. The validity...

  16. Validation of methods for measurement of insulin secretion in humans in vivo

    DEFF Research Database (Denmark)

    Kjems, L L; Christiansen, E; Vølund, A;

    2000-01-01

    To detect and understand the changes in beta-cell function in the pathogenesis of type 2 diabetes, an accurate and precise estimation of prehepatic insulin secretion rate (ISR) is essential. There are two common methods to assess ISR, the deconvolution method (by Eaton and Polonsky...... of these mathematical techniques for quantification of insulin secretion have been tested in dogs, but not in humans. In the present studies, we examined the validity of both methods to recover the known infusion rates of insulin and C-peptide mimicking ISR during an oral glucose tolerance test. ISR from both...... the combined model and the deconvolution method were accurate, i.e., recovery of true ISR was not significantly different from 100%. Furthermore, both maximal and total ISRs from the combined model were strongly correlated to those obtained by the deconvolution method (r = 0.89 and r = 0.82, respectively...

  17. Cytokine secretion and NK cell activity in human ADAM17 deficiency.

    Science.gov (United States)

    Tsukerman, Pinchas; Eisenstein, Eli M; Chavkin, Maor; Schmiedel, Dominik; Wong, Eitan; Werner, Marion; Yaacov, Barak; Averbuch, Diana; Molho-Pessach, Vered; Stepensky, Polina; Kaynan, Noa; Bar-On, Yotam; Seidel, Einat; Yamin, Rachel; Sagi, Irit; Elpeleg, Orly; Mandelboim, Ofer

    2015-12-29

    Genetic deficiencies provide insights into gene function in humans. Here we describe a patient with a very rare genetic deficiency of ADAM17. We show that the patient's PBMCs had impaired cytokine secretion in response to LPS stimulation, correlating with the clinical picture of severe bacteremia from which the patient suffered. ADAM17 was shown to cleave CD16, a major NK killer receptor. Functional analysis of patient's NK cells demonstrated that his NK cells express normal levels of activating receptors and maintain high surface levels of CD16 following mAb stimulation. Activation of individual NK cell receptors showed that the patient's NK cells are more potent when activated directly by CD16, albeit no difference was observed in Antibody Depedent Cytotoxicity (ADCC) assays. Our data suggest that ADAM17 inhibitors currently considered for clinical use to boost CD16 activity should be cautiously applied, as they might have severe side effects resulting from impaired cytokine secretion.

  18. Effect of phorbol and glucose on insulin secretion from the human fetal pancreas.

    Science.gov (United States)

    Tuch, B E; Williams, P F; Handelsman, D; Dunlop, M; Grigoriou, S; Turtle, J R

    1987-04-01

    It has been reported previously that 12-0-tetradecanoylphorbol-13-acetate is capable of stimulating the release of insulin from adult and neonatal pancreatic tissue. The data from this study show that this agent at a concentration of 1.3 uM, in the presence of 2.8 mM glucose, was unable to cause significant secretion of insulin from cultured human fetal pancreatic explants. By contrast 20 mM glucose was able to cause a small but significant immediate increase in secretion of insulin, but was unable to maintain this response beyond ten minutes. When the two agents were combined, a synergistic effect was seen throughout the entire 50 minute period of stimulation. The reason for this synergism is unclear since, whilst both secretagogues were able to cause a rise in the levels of diacylglycerol, together no extra effect was observed.

  19. Quercetin Impacts Expression of Metabolism- and Obesity-Associated Genes in SGBS Adipocytes.

    Science.gov (United States)

    Leiherer, Andreas; Stoemmer, Kathrin; Muendlein, Axel; Saely, Christoph H; Kinz, Elena; Brandtner, Eva M; Fraunberger, Peter; Drexel, Heinz

    2016-05-12

    Obesity is characterized by the rapid expansion of visceral adipose tissue, resulting in a hypoxic environment in adipose tissue which leads to a profound change of gene expression in adipocytes. As a consequence, there is a dysregulation of metabolism and adipokine secretion in adipose tissue leading to the development of systemic inflammation and finally resulting in the onset of metabolic diseases. The flavonoid quercetin as well as other secondary plant metabolites also referred to as phytochemicals have anti-oxidant, anti-inflammatory, and anti-diabetic effects known to be protective in view of obesity-related-diseases. Nevertheless, its underlying molecular mechanism is still obscure and thus the focus of this study was to explore the influence of quercetin on human SGBS (Simpson Golabi Behmel Syndrome) adipocytes' gene expression. We revealed for the first time that quercetin significantly changed expression of adipokine (Angptl4, adipsin, irisin and PAI-1) and glycolysis-involved (ENO2, PFKP and PFKFB4) genes, and that this effect not only antagonized but in part even overcompensated the effect mediated by hypoxia in adipocytes. Thus, these results are explained by the recently proposed hypothesis that the protective effect of quercetin is not solely due to its free radical-scavenging activity but also to a direct effect on mitochondrial processes, and they demonstrate that quercetin might have the potential to counteract the development of obesity-associated complications.

  20. Effects of Methylmercury exposure in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Theresa Vertigan

    2017-02-01

    Full Text Available Mercury-containing compounds are environmental pollutants that have become increasingly consequential in the Arctic regions of North America due to processes of climate change increasing their release and availability at northern latitudes. Currently, the form of mercury known to be most detrimental to human health is methylmercury, CH3Hg+, which is found in the environment and accumulates in the tissues of piscivores, including those consumed by Alaska Natives through subsistence gathering. Much is known about the neurotoxicity of methylmercury after exposure to high concentrations, but little is known about toxicity to other tissues and cell types, particularly for long-term exposure and the lower concentrations that would occur through fish consumption. Effects of methylmercury exposure on 3T3-L1 adipocytes in culture were assessed using assays for cytotoxicity and an ELISA assay for vascular endothelial growth factor (VEGF, a signaling molecule shown to be important for maintaining metabolic status in adipose tissue. Results showed that exposure to methylmercury leads to significant toxicity in adipocytes at exposures of 100 ng/mL during later stages of differentiation, but lower methylmercury concentrations produced little to no toxicity. Results also showed that VEGF secretion is elevated in adipocytes exposed to methylmercury after the process of differentiating into mature, fat-storing cells. These results provide a basis for further exploration into metabolic consequences of methylmercury exposure on specific cell types and cell models.

  1. DNA microarray analysis of genes differentially expressed in adipocyte differentiation

    Indian Academy of Sciences (India)

    Chunyan Yin; Yanfeng Xiao; Wei Zhang; Erdi Xu; Weihua Liu; Xiaoqing Yi; Ming Chang

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a ≥ 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RT-PCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  2. DNA microarray analysis of genes differentially expressed in adipocyte differentiation.

    Science.gov (United States)

    Yin, Chunyan; Xiao, Yanfeng; Zhang, Wei; Xu, Erdi; Liu, Weihua; Yi, Xiaoqing; Chang, Ming

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a greater than or equal to 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RTPCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR?2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  3. Development of Gonadotropin-Releasing Hormone-Secreting Neurons from Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Carina Lund

    2016-08-01

    Full Text Available Gonadotropin-releasing hormone (GnRH neurons regulate human puberty and reproduction. Modeling their development and function in vitro would be of interest for both basic research and clinical translation. Here, we report a three-step protocol to differentiate human pluripotent stem cells (hPSCs into GnRH-secreting neurons. Firstly, hPSCs were differentiated to FOXG1, EMX2, and PAX6 expressing anterior neural progenitor cells (NPCs by dual SMAD inhibition. Secondly, NPCs were treated for 10 days with FGF8, which is a key ligand implicated in GnRH neuron ontogeny, and finally, the cells were matured with Notch inhibitor to bipolar TUJ1-positive neurons that robustly expressed GNRH1 and secreted GnRH decapeptide into the culture medium. The protocol was reproducible both in human embryonic stem cells and induced pluripotent stem cells, and thus provides a translational tool for investigating the mechanisms of human puberty and its disorders.

  4. DPP4 gene variation affects GLP-1 secretion, insulin secretion, and glucose tolerance in humans with high body adiposity

    DEFF Research Database (Denmark)

    Böhm, Anja; Wagner, Robert; Machicao, Fausto

    2017-01-01

    OBJECTIVE: Dipeptidyl-peptidase 4 (DPP-4) cleaves and inactivates the insulinotropic hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide, collectively termed incretins. DPP-4 inhibitors entered clinical practice as approved therapeutics for type-2 diabetes in 2006. However......-BMI interaction effects were detected (p = 0.8). After stratification for body fat content, the SNP negatively affected glucose-stimulated GLP-1 levels (p = 0.0229), insulin secretion (p = 0.0061), and glucose tolerance (p = 0.0208) in subjects with high body fat content only. CONCLUSIONS: A common variant, i......, inter-individual variance in the responsiveness to DPP-4 inhibitors was reported. Thus, we asked whether genetic variation in the DPP4 gene affects incretin levels, insulin secretion, and glucose tolerance in participants of the TÜbingen Family study for type-2 diabetes (TÜF). RESEARCH DESIGN...

  5. Ethanol extract of lotus (Nelumbo nucifera) root exhibits an anti-adipogenic effect in human pre-adipocytes and anti-obesity and anti-oxidant effects in rats fed a high-fat diet.

    Science.gov (United States)

    You, Jeong Soon; Lee, Yun Ju; Kim, Kyoung Soo; Kim, Sung Hoon; Chang, Kyung Ja

    2014-03-01

    Lotus (Nelumbo Nucifera) root, a well-known medicinal plant in Asia, is reported to have various therapeutic benefits, including anti-diabetes, anti-hypertension, and anti-hyperlipidaemia. We hypothesized that the ethanol extract of lotus root (ELR) would exhibit an anti-adipogenic effect in human pre-adipocytes as well as anti-obesity and anti-oxidant effects in rats fed a high-fat diet. Treatment with ELR in human pre-adipocytes resulted in inhibition of lipid accumulation and attenuated expression of adipogenic transcription factors such as peroxisome proliferator-activated receptor gamma and adipocyte marker genes, such as glucose transporter 4 and leptin. Administration of ELR resulted in a significant decrease in relative weights of adipose tissues in rats fed a high-fat diet. Consumption of a high-fat diet resulted in an increase in serum total cholesterol (TC) and triglyceride (TG) levels; however, administration of ELR resulted in a decrease in the levels of TC and TG. Administration of ELR resulted in a decrease in the level of serum leptin and insulin. Administration of ELR in rats fed a high-fat diet resulted in a decrease in hepatic thiobarbituric acid reactive substance content, elevated by a high-fat diet and an increase in superoxide dismutase activity and hepatic glutathione content. These results suggest that lotus root exerts anti-oxidant and anti-obesity effects and could be used as a functional and nutraceutical ingredient in combatting obesity-related diseases.

  6. Adipocyte activation of cancer stem cell signaling in breast cancer

    Institute of Scientific and Technical Information of China (English)

    Benjamin; Wolfson; Gabriel; Eades; Qun; Zhou

    2015-01-01

    Signaling within the tumor microenvironment has a critical role in cancer initiation and progression. Adipocytes, one of the major components of the breast microenvironment,have been shown to provide pro-tumorigenic signals that promote cancer cell proliferation and invasiveness in vitro and tumorigenicity in vivo. Adipocyte secreted factors such as leptin and interleukin-6(IL-6) have a paracrine effect on breast cancer cells. In adipocyte-adjacent breast cancer cells, the leptin and IL-6 signaling pathways activate janus kinase 2/signal transducer and activatorof transcription 5, promoting the epithelial-mesenchymal transition, and upregulating stemness regulators such as Notch, Wnt and the Sex determining region Y-box 2/octamer binding transcription factor 4/Nanog signaling axis. In this review we will summarize the major signaling pathways that regulate cancer stem cells in breast cancer and describe the effects that adipocyte secreted IL-6 and leptin have on breast cancer stem cell signaling. Finally we will introduce a new potential treatment paradigm of inhibiting the adipocyte-breast cancer cell signaling via targeting the IL-6 or leptin pathways.

  7. Nonlinear dynamics in pulsatile secretion of parathyroid hormone in normal human subjects

    Science.gov (United States)

    Prank, Klaus; Harms, Heio; Brabant, Georg; Hesch, Rolf-Dieter; Dämmig, Matthias; Mitschke, Fedor

    1995-03-01

    In many biological systems, information is transferred by hormonal ligands, and it is assumed that these hormonal signals encode developmental and regulatory programs in mammalian organisms. In contrast to the dogma of endocrine homeostasis, it could be shown that the biological information in hormonal networks is not only present as a constant hormone concentration in the circulation pool. Recently, it has become apparent that hormone pulses contribute to this hormonal pool, which modulates the responsiveness of receptors within the cell membrane by regulation of the receptor synthesis, movement within the membrane layer, coupling to signal transduction proteins and internalization. Phase space analysis of dynamic parathyroid hormone (PTH) secretion allowed the definition of a (in comparison to normal subjects) relatively quiet ``low dynamic'' secretory pattern in osteoporosis, and a ``high dynamic'' state in hyperparathyroidism. We now investigate whether this pulsatile secretion of PTH in healthy men exhibits characteristics of nonlinear determinism. Our findings suggest that this is conceivable, although on the basis of presently available data and techniques, no proof can be established. Nevertheless, pulsatile secretion of PTH might be a first example of nonlinear deterministic dynamics in an apparently irregular hormonal rhythm in human physiology.

  8. Effects of aging on the human ovary: the secretion of immunoreactive alpha-inhibin and progesterone.

    Science.gov (United States)

    Pellicer, A; Marí, M; de los Santos, M J; Simón, C; Remohí, J; Tarín, J J

    1994-04-01

    To investigate the changes induced by age in the function and secretory pattern of the human ovary. Immunoreactive alpha-inhibin, E2, and P secretion in vivo and in vitro have been compared in two different populations. Prospective study. Women undergoing IVF-ET were divided into two groups according to age: group 1 (32.0 +/- 0.7 years; mean +/- SEM) and group 2 (40.3 +/- 0.3 years). In vitro fertilization program at the Instituto Valenciano de Infertilidad. A total of 33 infertile women with regular menses, undergoing IVF-ET. Follicle aspiration performed by transvaginal ultrasound. Four follicles per patient were aspirated in individual plastic tubes. Granulosa-luteal cells isolated with Percoll columns and cultured in vitro up to 4 days in the presence of hCG. In vitro fertilization parameters, serum levels of E2, immunoreactive alpha-inhibin, and P, as well as the secretion of immunoreactive alpha-inhibin and P by the cultured granulosa-luteal cells. Serum immunoreactive alpha-inhibin levels the day of ovum pick-up were significantly lower in group 2 compared with group 1. Incubation of cells for 96 hours showed a significantly higher ability to accumulate immunoreactive alpha-inhibin in group 1 than 2. Human chorionic gonadotropin stimulated immunoreactive alpha-inhibin production after 96 hours. Cells from younger women displayed a significantly higher ability to secrete P than cells from older women. Human chorionic gonadotropin was able to significantly stimulate P production in group 1. These results confirm previous observations showing a reduced production of immunoreactive alpha-inhibin and steroids of ovaries from older women and suggest that a reduced cellular function, rather than a decrease in the follicular population, is the main mechanism by which these changes are produced.

  9. Induction of mucin secretion from human bronchial tissue and epithelial cells by rhinovirus and lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Shao-heng HE; Jian ZHENG; Ming-ke DUAN

    2004-01-01

    AIM: To examine the effects of rhinovirus and lipopolysaccharide (LPS) on mucin secretion from bronchial tissue and epithelial cells in vitro. METHODS: Human small bronchial tissue fragments (HSBTF) and human bronchial epithelial cells (HBEC) were cultured with rhinovirus 16 and LPS, respectively and culture supernatants were collected for mucin measurement. To determine mucin levels in the culture supernatants, a MUC5AC enzyme linked immunosorbent assay and an enzyme linked lectin assay procedure with dolichos bifiorus agglutinin (DBA)were developed, and mucin release was expressed as percentage increased (or decreased) secretion over baseline level. RESULTS: A concentration-dependent release of DBA mucin and MUC5AC mucin were observed when HSBTF were infected with various concentrations of rhinovirus 16 at 37 ℃. The maximum-induced DBA mucin and MUC5AC mucin release were approximately 258 % and 83 % over baseline. The response of HSBTF to rhinovirus was completely abolished by metabolic inhibitors. Rhinovirus was also able to induce a concentrationdependent release of DBA mucin and MUC5AC mucin from primarily cultured HBEC. LPS 100 mg/L was able to provoke up to approximately 19 % and 54 % increase in DBA and MUC5AC mucin release over baseline, respectively from HSBTF, and 3.1% and 57 % increase from HBEC at 20 h. Soybean trypsin inhibitor (SBTI) 30 mg/L was able to inhibit LPS-induced mucin release from HSBTF and HBEC. CONCLUSION: Rhinovirus is able to induce mucin secretion from human bronchial tissue and bronchial epithelial cells in vitro. LPS can induce MUC5AC mucin release from HSBTF and HBEC.

  10. Regulation of MMP-3 expression and secretion by the chemokine eotaxin-1 in human chondrocytes

    Directory of Open Access Journals (Sweden)

    Chao Pin-Zhir

    2011-11-01

    Full Text Available Abstract Background Osteoarthritis (OA is characterized by the degradation of articular cartilage, marked by the breakdown of matrix proteins. Studies demonstrated the involvement of chemokines in this process, and some may potentially serve as diagnostic markers and therapeutic targets; however, the underlying signal transductions are not well understood. Methods We investigated the effects of the CC chemokine eotaxin-1 (CCL11 on the matrix metalloproteinase (MMP expression and secretion in the human chondrocyte cell line SW1353 and primary chondrocytes. Results Eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Inhibitors of extracellular signal-regulated kinase (ERK and p38 kinase were able to repress eotaxin-1-induced MMP-3 expression. On the contrary, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPs, a competitive cAMP antagonist for cAMP receptors, and H-89, a protein kinase A (PKA inhibitor, markedly enhanced eotaxin-1-induced MMP-3 expression. These results suggest that MMP-3 expression is specifically mediated by the G protein-coupled eotaxin-1 receptor activities. Interestingly, little amount of MMP-3 protein was detected in the cell lysates of eotaxin-1-treated SW1353 cells, and most of MMP-3 protein was in the culture media. Furthermore we found that the eotaxin-1-dependent MMP-3 protein secretion was regulated by phospholipase C (PLC-protein kinase C (PKC cascade and c-Jun N-terminal kinase (JNK/mitogen-activated protein (MAP kinase pathways. These data indicate a specific regulation of MMP-3 secretion also by eotaxin-1 receptor activities. Conclusions Eotaxin-1 not only induces MMP-3 gene expression but also promotes MMP-3 protein secretion through G protein-coupled eotaxin-1 receptor activities. Chemokines, such as eotaxin-1, could be a potential candidate in the diagnosis and treatment of arthritis.

  11. A novel Gymnema sylvestre extract stimulates insulin secretion from human islets in vivo and in vitro.

    Science.gov (United States)

    Al-Romaiyan, A; Liu, B; Asare-Anane, H; Maity, C R; Chatterjee, S K; Koley, N; Biswas, T; Chatterji, A K; Huang, G-C; Amiel, S A; Persaud, S J; Jones, P M

    2010-09-01

    Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

  12. Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from human lung mast cells.

    Science.gov (United States)

    Leung, K B; Flint, K C; Brostoff, J; Hudspith, B N; Johnson, N M; Lau, H Y; Liu, W L; Pearce, F L

    1988-01-01

    Sodium cromoglycate and nedocromil sodium produced a dose dependent inhibition of histamine secretion from human pulmonary mast cells obtained by bronchoalveolar lavage and by enzymatic dissociation of lung parenchyma. Both compounds were significantly more active against the lavage cells than against the dispersed lung cells, and nedocromil sodium was an order of magnitude more effective than sodium cromoglycate against both cell types. Tachyphylaxis was observed with the parenchymal cells but not with the lavage cells. Nedocromil sodium and sodium cromoglycate also inhibited histamine release from the lavage cells of patients with sarcoidosis and extrinsic asthma. PMID:2462755

  13. Effects of parabens on adipocyte differentiation.

    Science.gov (United States)

    Hu, Pan; Chen, Xin; Whitener, Rick J; Boder, Eric T; Jones, Jeremy O; Porollo, Aleksey; Chen, Jiangang; Zhao, Ling

    2013-01-01

    Parabens are a group of alkyl esters of p-hydroxybenzoic acid that include methylparaben, ethylparaben, propylparaben, butylparaben, and benzylparaben. Paraben esters and their salts are widely used as preservatives in cosmetics, toiletries, food, and pharmaceuticals. Humans are exposed to parabens through the use of such products from dermal contact, ingestion, and inhalation. However, research on the effects of parabens on health is limited, and the effects of parabens on adipogenesis have not been systematically studied. Here, we report that (1) parabens promote adipogenesis (or adipocyte differentiation) in murine 3T3-L1 cells, as revealed by adipocyte morphology, lipid accumulation, and mRNA expression of adipocyte-specific markers; (2) the adipogenic potency of parabens is increased with increasing length of the linear alkyl chain in the following potency ranking order: methyl- parabens, and the structurally related benzoic acid (without the OH group) are inactive in promoting 3T3-L1 adipocyte differentiation; (3) parabens activate glucocorticoid receptor and/or peroxisome proliferator-activated receptor γ in 3T3-L1 preadipocytes; however, no direct binding to, or modulation of, the ligand binding domain of the glucocorticoid receptor by parabens was detected by glucocorticoid receptor competitor assays; and lastly, (4) parabens, butyl- and benzylparaben in particular, also promote adipose conversion of human adipose-derived multipotent stromal cells. Our results suggest that parabens may contribute to obesity epidemic, and the role of parabens in adipogenesis in vivo needs to be examined further.

  14. 10e12z CLA alters adipocyte differentiation and adipocyte cytokine expression and induces macrophage proliferation.

    Science.gov (United States)

    Belda, Benjamin J; Thompson, Jerry T; Eser, Pinar O; Vanden Heuvel, John P

    2012-05-01

    The trans-10, cis-12 (10e12z) conjugated linoleic acid (CLA) isomer of CLA is responsible for loss of lipid storage or adipose tissue in vitro or in vivo. This isomer also induces inflammatory signaling in both mouse and human adipocytes in vitro. However, when these events occur and whether they are significant enough to affect other cell types are unclear. In these experiments, the 3T3-L1 cell line has been used to examine the interaction between inflammatory signaling and decreased differentiation or lipid storage induced by 10e12z CLA. In assays measuring both lipid accumulation and gene expression, differentiating 3T3-L1 cells exhibit concurrent induction of inflammatory signaling, as measured by cyclooxygenase-2 expression, and a decrease in adipocyte marker gene expression. Furthermore, in fully differentiated adipocytes, as identified in microarray assays and confirmed with real-time polymerase chain reaction, 10e12z CLA also significantly affected expression of both matrix metalloprotein-3 (MMP-3), collagen VI α 3 ColVI alpha 3 (VIα3) and the cytokine epiregulin, demonstrating that the effects of 10e12z broadly impact adipocyte function. In agreement with other experimental systems, 10e12z CLA inhibited RAW 264.7 cell proliferation; however, in response to adipocyte-conditioned media, 10e12z-CLA-treated adipocytes induced proliferation of this cell line, suggesting that the effect of 10e12z CLA is context dependent. These results are largely consistent with the known activation of the inflammatory mediator nuclear factor-κB in adipocytes in vitro and in vivo by 10e12z CLA treatment and demonstrate that adipose is an important target tissue of this isomer that impacts other cell types.

  15. TNF-α alters the inflammatory secretion profile of human first trimester placenta.

    Science.gov (United States)

    Siwetz, Monika; Blaschitz, Astrid; El-Heliebi, Amin; Hiden, Ursula; Desoye, Gernot; Huppertz, Berthold; Gauster, Martin

    2016-04-01

    Implantation and subsequent placental development depend on a well-orchestrated interaction between fetal and maternal tissues, involving a fine balanced synergistic cross-talk of inflammatory and immune-modulating factors. Tumor necrosis factor (TNF)-α has been increasingly recognized as pivotal factor for successful pregnancy, although high maternal TNF-α levels are associated with a number of adverse pregnancy conditions including gestational hypertension and gestational diabetes mellitus. This study describes effects of exogenously applied TNF-α, mimicking increased maternal TNF-α levels, on the secretion profile of inflammation associated factors in human first trimester villous placenta. Conditioned culture media from first trimester villous placental explants were analyzed by inflammation antibody arrays and ELISA after 48 h culture in the presence or absence of TNF-α. Inflammation antibody arrays identified interleukin (IL)-6, IL-8, chemokine (C-C motif) ligand 2 (CCL2), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) as the most abundantly secreted inflammation-associated factors under basal culture conditions. In the presence of TNF-α, secretion of GM-CSF, CCL5, and IL-10 increased, whereas IL-4 and macrophage CSF levels decreased compared with controls. ELISA analysis verified antibody arrays by showing significantly increased synthesis and release of GM-CSF and CCL5 by placental explants in response to TNF-α. Immunohistochemistry localized GM-CSF in the villous trophoblast compartment, whereas CCL5 was detected in maternal platelets adhering to perivillous fibrin deposits on the villous surface. mRNA-based in situ padlock probe approach localized GM-CSF and CCL5 transcripts in the villous trophoblast layer and the villous stroma. Results from this study suggest that the inflammatory secretion profile of human first trimester placenta shifts towards increased levels of GM-CSF, CCL5, and IL10 in response to elevated maternal

  16. Reevaluation of Fatty Acid Receptor 1 as a Drug Target for the Stimulation of Insulin Secretion in Humans

    Science.gov (United States)

    Wagner, Robert; Kaiser, Gabriele; Gerst, Felicia; Christiansen, Elisabeth; Due-Hansen, Maria E.; Grundmann, Manuel; Machicao, Fausto; Peter, Andreas; Kostenis, Evi; Ulven, Trond; Fritsche, Andreas; Häring, Hans-Ulrich; Ullrich, Susanne

    2013-01-01

    The role of free fatty acid receptor 1 (FFAR1/GPR40) in glucose homeostasis is still incompletely understood. Small receptor agonists stimulating insulin secretion are undergoing investigation for the treatment of type 2 diabetes. Surprisingly, genome-wide association studies did not discover diabetes risk variants in FFAR1. We reevaluated the role of FFAR1 in insulin secretion using a specific agonist, FFAR1-knockout mice and human islets. Nondiabetic individuals were metabolically phenotyped and genotyped. In vitro experiments indicated that palmitate and a specific FFAR1 agonist, TUG-469, stimulate glucose-induced insulin secretion through FFAR1. The proapoptotic effect of chronic exposure of β-cells to palmitate was independent of FFAR1. TUG-469 was protective, whereas inhibition of FFAR1 promoted apoptosis. In accordance with the proapoptotic effect of palmitate, in vivo cross-sectional observations demonstrated a negative association between fasting free fatty acids (NEFAs) and insulin secretion. Because NEFAs stimulate secretion through FFAR1, we examined the interaction of genetic variation in FFAR1 with NEFA and insulin secretion. The inverse association of NEFA and secretion was modulated by rs1573611 and became steeper for carriers of the minor allele. In conclusion, FFAR1 agonists support β-cell function, but variation in FFAR1 influences NEFA effects on insulin secretion and therefore could affect therapeutic efficacy of FFAR1 agonists. PMID:23378609

  17. Leukotriene B4 potentiates CpG signaling for enhanced cytokine secretion by human leukocytes.

    Science.gov (United States)

    Gaudreault, Eric; Gosselin, Jean

    2009-08-15

    TLRs are known to be important in innate host defense against a variety of microbial infections. In particular, TLR9 has been associated with immune defense against different foreign organisms by recognition of unmethylated DNA sequences. In this report, we provide evidence that leukotriene B(4) (LTB(4)) has the capacity to modulate TLR9 expression on human neutrophils. The effect of LTB(4) was found to be specific, because related leukotrienes such as LTC(4) and LTD(4) or neutrophil agonists IL-8 and C5a failed to modulate TLR9 expression in neutrophils. Using fluorochrome-tagged CpG DNA, we observed that LTB(4) treatment also increased TLR9 ligand binding in neutrophils. Moreover, LTB(4) stimulation potentiates CpG-mediated signaling via an endosome-independent mechanism in human neutrophils, leading to enhanced secretion of proinflammatory cytokines. The increase in cytokine secretion by LTB(4) following CpG stimulation of neutrophils was associated with the activation of TGF-beta-activated kinase (TAK-1) as well as p38 and c-Jun (JNK) kinases. In contrast, in PBMC LTB(4) leads to an increase in cytokine secretion following CpG stimulation but via a MyD88- and endosome-dependent mechanism. As observed in neutrophils, PBMC stimulation with LTB(4) in the presence of CpG also results in enhanced TAK-1, p38, and JNK phosphorylation/activation. These data provide new evidence underlying the immunomodulatory properties of LTB(4) leading to antimicrobial defense.

  18. [Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion].

    Science.gov (United States)

    Zhu, Fu-xiang; Yang, Shu-de; Liu, Ze-long; Miao, Jing; Qu, Hui-ge; Chi, Xiao-yan

    2010-10-01

    This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

  19. Cultured human astrocytes secrete large cholesteryl ester- andtriglyceride-rich lipoproteins along with endothelial lipase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lin; Liu, Yanzhu; Forte, Trudy M.; Chisholm, Jeffrey W.; Parks, John S.; Shachter, Neil S.

    2003-12-01

    We cultured normal human astrocytes and characterized their secreted lipoproteins. Human astrocytes secreted lipoproteins in the size range of plasma VLDL (Peak 1), LDL (Peak 2), HDL (Peak 3) and a smaller peak (Peak 4), as determined by gel filtration chromatography, nondenaturing gradient gel electrophoresis and transmission electron microscopy. Cholesterol enrichment of astrocytes led to a particular increase in Peak 1. Almost all Peak 2, 3 and 4 cholesterol and most Peak 1 cholesterol was esterified (unlike mouse astrocyte lipoproteins, which exhibited similar peaks but where cholesterol was predominantly non-esterified). Triglycerides were present at about 2/3 the level of cholesterol. LCAT was detected along with two of its activators, apolipoprotein (apo) A-IV and apoC-I. ApoA-I and apoA-II mRNA and protein were absent. ApoJ was present equally in all peaks but apoE was present predominantly in peaks 3 and 4. ApoB was not detected. The electron microscopic appearance of Peak 1 lipoproteins suggested partial lipolysis leading to the detection of a heparin-releasable triglyceride lipase consistent with endothelial lipase. The increased neuronal delivery of lipids from large lipoprotein particles, for which apoE4 has greater affinity than does apoE3, may be a mechanism whereby the apoE {var_epsilon}4 allele contributes to neurodegenerative risk.

  20. Secreted human adipose leptin decreases mitochondrial respiration in HCT116 colon cancer cells.

    Science.gov (United States)

    Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

    2013-01-01

    Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, pobese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues.

  1. Secreted human adipose leptin decreases mitochondrial respiration in HCT116 colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Einav Yehuda-Shnaidman

    Full Text Available Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration and extracellular acidification rate (ECAR, mostly lactate production via glycolysis were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese subjects decreased basal (40%, p<0.05 and maximal (50%, p<0.05 OCR and gene expression of mitochondrial proteins and Bax without affecting cell viability or expression of glycolytic enzymes. Similar changes could be recapitulated by incubating cells with leptin, whereas, leptin-receptor specific antagonist inhibited the reduced OCR induced by conditioned media from obese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues.

  2. Effect of Rat Schwann Cell Secretion on Proliferation and Differentiation of Human Neural Stem Cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and b-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were b-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.

  3. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle.

    Science.gov (United States)

    Hoier, Birgitte; Prats, Clara; Qvortrup, Klaus; Pilegaard, Henriette; Bangsbo, Jens; Hellsten, Ylva

    2013-09-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations of teased muscle fibers by transmission electron microscopy (TEM) and confocal microscopy (CM). Muscle interstitial fluid was sampled from microdialysis probes placed in the thigh muscle. TEM and CM analysis revealed two primary sites of localization of VEGF: in vesicles located in the subsarcolemmal regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P<0.05) after exercise. The observation of more VEGF vesicles close to sarcolemma after exercise, combined with a 5-fold increase (P<0.05) in VEGF in the interstitial fluid, suggest that VEGF-containing vesicles redistribute to sarcolemma and that VEGF is secreted to the extracellular fluid. This study provides the first evidence in humans for a mechanism by which skeletal muscle fibers can control capillary growth by releasing VEGF from intracellular vesicles during contraction.

  4. Human umbilical cord-derived mesenchymal stem cells can secrete insulin in vitro and in vivo.

    Science.gov (United States)

    Boroujeni, Zahra Niki; Aleyasin, Ahmad

    2014-01-01

    Diabetes mellitus is characterized by autoimmune destruction of pancreatic beta cells, leading to decreased insulin production. Differentiation of mesenchymal stem cells (MSCs) into insulin-producing cells offers novel ways of diabetes treatment. MSCs can be isolated from the human umbilical cord tissue and differentiate into insulin-secreting cells. Human umbilical cord-derived stem cells (hUDSCs) were obtained after birth, selected by plastic adhesion, and characterized by flow cytometric analysis. hUDSCs were transduced with nonintegrated lentivirus harboring PDX1 (nonintegrated LV-PDX1) and was cultured in differentiation medium in 21 days. Pancreatic duodenum homeobox protein-1 (PDX1) is a transcription factor in pancreatic development. Significant expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2), and somatostatin were detected by quantitative RT-PCR (P insulin proteins were shown by immunocytochemistry analysis. Insulin secretion of hUDSCs(PDX1+) in the high-glucose medium was 1.8 μU/mL. They were used for treatment of diabetic rats and could decrease the blood glucose level from 400 mg/dL to a normal level in 4 days. In conclusion, our results demonstrated that hUDSCs are able to differentiate into insulin-producing cells by transduction with nonintegrated LV-PDX1. These hUDSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

  5. The spectrophotometric sulfo-phospho-vanillin assessment of total lipids in human meibomian gland secretions.

    Science.gov (United States)

    McMahon, Anne; Lu, Hua; Butovich, Igor A

    2013-05-01

    Human meibomian gland secretions (meibum) are the major lipid component of the human preocular tear film. The predominant lipid classes found in meibum include waxes (WE), cholesteryl esters (CE), and varying amounts of cholesterol (Chl). The classical sulfo-phospho-vanillin assay (SPVA), adapted for a microplate reader, was used to quantitate lipids in meibum. To account for varying reactivities of different lipids in SPVA, a model meibomian lipid mixture (MMx) that approximated the WE/CE/Chl composition of meibum was developed and used to quantitate meibomian lipids. The overall SPV responses of MMx and meibum were found to be close, with similar intermediate and final reaction products for both. Saturated WE that had not been expected to be reactive were found to be SPV-positive. A reaction mechanism for these compounds in SPVA which involves the formation of alkenyl ethers is proposed and discussed. Tested proteins were non-reactive in SPVA. Thus, by comparing the results of gravimetric analyses of meibum samples with the results of a properly calibrated SPVA, it was estimated that the SPV-reactive lipid content of dry meibum in tested samples was about 78 % (w/w). The SPV method can also be adopted for analyzing other types of complex lipids secretions, such as sebum, as well as whole lipid extracts from other lipid-enriched organs and tissues, if proper standards are chosen.

  6. Human iPSC-Derived Immature Astroglia Promote Oligodendrogenesis by Increasing TIMP-1 Secretion

    Directory of Open Access Journals (Sweden)

    Peng Jiang

    2016-05-01

    Full Text Available Astrocytes, once considered passive support cells, are increasingly appreciated as dynamic regulators of neuronal development and function, in part via secreted factors. The extent to which they similarly regulate oligodendrocytes or proliferation and differentiation of oligodendrocyte progenitor cells (OPCs is less understood. Here, we generated astrocytes from human pluripotent stem cells (hiPSC-Astros and demonstrated that immature astrocytes, as opposed to mature ones, promote oligodendrogenesis in vitro. In the PVL mouse model of neonatal hypoxic/ischemic encephalopathy, associated with cerebral palsy in humans, transplanted immature hiPSC-Astros promoted myelinogenesis and behavioral outcome. We further identified TIMP-1 as a selectively upregulated component secreted from immature hiPSC-Astros. Accordingly, in the rat PVL model, intranasal administration of conditioned medium from immature hiPSC-Astros promoted oligodendrocyte maturation in a TIMP-1-dependent manner. Our findings suggest stage-specific developmental interactions between astroglia and oligodendroglia and have important therapeutic implications for promoting myelinogenesis.

  7. Activation of AMPK inhibits cholera toxin stimulated chloride secretion in human and murine intestine.

    Directory of Open Access Journals (Sweden)

    Ailín C Rogers

    Full Text Available Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR, is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK, can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK. In order to substantiate our findings on the whole tissue level, short-circuit current (SCC was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.

  8. Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

    Science.gov (United States)

    Dirix, Violette; Mielcarek, Nathalie; Debrie, Anne-Sophie; Willery, Eve; Alonso, Sylvie; Versheure, Virginie; Mascart, Françoise; Locht, Camille

    2014-07-01

    In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

  9. Induction of Chemokine Secretion and Monocyte Migration by Human Choroidal Melanocytes in Response to Proinflammatory Cytokines

    DEFF Research Database (Denmark)

    Jehs, Tina; Faber, Carsten; Udsen, Maja S.

    2016-01-01

    Purpose: To determine to which extent inflammatory cytokines affect chemokine secretion by primary human choroidal melanocytes (HCMs), their capacity to attract monocytes, and whether HCMs are able to influence the proliferation of activated T cells. Methods: Primary cultures of HCMs were...... established from eyes of 13 donors. Human choroidal melanocytes were stimulated with IFN-γ and TNF-α or with supernatant from activated T cells (T-cell–conditioned media [TCM]). Gene expression analysis was performed by using microarrays. Protein levels were quantified with ELISA or cytometric bead array....... Supernatants of HCMs were assessed for the capability to attract monocytes in a transwell plate. Proliferation of activated T cells was assessed in a direct coculture with HCMs by a [3H]-thymidine incorporation assay. Results: Stimulation of HCMs with TCM or IFN-γ and TNF-α resulted in increased expression...

  10. Identification and quantification of serum proteins secreted into the normal human jejunum

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Hegnhøj, J H

    1990-01-01

    The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates....... Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions....... The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins...

  11. Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

    Science.gov (United States)

    Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

    2013-01-01

    Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, prespiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

  12. Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells

    OpenAIRE

    Ramaesh, T; Ramaesh, K; Riley, S C; West, J.D.; Dhillon, B

    2012-01-01

    Matrix metalloproteinase-9 (MMP-9) secreted by corneal epithelial cells has a role in the remodelling of extracellular matrix and migration of epithelial cells. Elevated levels of MMP-9 activity in the ocular surface may be involved in the pathogenesis of corneal diseases. N-acetylcysteine (NAC) has been used to treat corneal diseases, including recurrent epithelial erosions. In this study, its effects on the MMP-9 secretion and human corneal epithelial (HCE) cell migration were evaluated in ...

  13. Featured Article: Dexamethasone and rosiglitazone are sufficient and necessary for producing functional adipocytes from mesenchymal stem cells

    Science.gov (United States)

    Ezquer, Fernando; Espinosa, Maximiliano; Arango-Rodriguez, Martha; Puebla, Carlos; Sobrevia, Luis; Conget, Paulette

    2015-01-01

    The final product of adipogenesis is a functional adipocyte. This mature cell acquires the necessary machinery for lipid metabolism, loses its proliferation potential, increases its insulin sensitivity, and secretes adipokines. Multipotent mesechymal stromal cells have been recognized as a source of adipocytes both in vivo and in vitro. The in vitro adipogenic differentiation of human MSC (hMSC) has been induced up to now by using a complex stimulus which includes dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin (a classical cocktail) and evaluated according to morphological changes. The present work was aimed at demonstrating that the simultaneous activation of dexamethasone’s canonical signaling pathways, through the glucocorticoid receptor and CCAAT-enhancer-binding proteins (C/EBPs) and rosiglitazone through peroxisome proliferator-activated receptor gamma (PPAR-gamma) is sufficient yet necessary for inducing hMSC adipogenic differentiation. It was also ascertained that hMSC exposed just to dexamethasone and rosiglitazone (D&R) differentiated into cells which accumulated neutral lipid droplets, expressed C/EBP-alpha, PPAR-gamma, aP2, lipoprotein lipase, acyl-CoA synthetase, phosphoenolpyruvate carboxykinase, adiponectin, and leptin genes but did not proliferate. Glucose uptake was dose dependent on insulin stimulus and high levels of adipokines were secreted (i.e. displaying not only the morphology but also expressing mature adipocytes’ specific genes and functional characteristics). This work has demonstrated that (i) the activating C/EBPs and PPAR-gamma signaling pathways were sufficient to induce adipogenic differentiation from hMSC, (ii) D&R producing functional adipocytes from hMSC, (iii) D&R induce adipogenic differentiation from mammalian MSC (including those which are refractory to classical adipogenic differentiation stimuli). D&R would thus seem to be a useful tool for MSC characterization, studying adipogenesis pathways and

  14. S-CMC-Lys-dependent stimulation of electrogenic glutathione secretion by human respiratory epithelium.

    Science.gov (United States)

    Guizzardi, F; Rodighiero, S; Binelli, A; Saino, S; Bononi, E; Dossena, S; Garavaglia, M L; Bazzini, C; Bottà, G; Conese, M; Daffonchio, L; Novellini, R; Paulmichl, M; Meyer, G

    2006-01-01

    Glutathione (GSH) is one of the most important defense mechanisms against oxidative stress in the respiratory epithelial lining fluid. Considering that GSH secretion in respiratory cells has been postulated to be at least partially electrogenic, and that the mucoregulator S-carbocysteine lysine salt monohydrate (S-CMC-Lys) can cause an activation of epithelial Cl(-) conductance, the purpose of this study was to verify whether S-CMC-Lys is able to stimulate GSH secretion. Experiments have been performed by patch-clamp technique, by high-performance liquid chromatography (HPLC) assay, and by Western blot analysis on cultured lines of human respiratory cells (WI-26VA4 and CFT1-C2). In whole-cell configuration, after cell exposure to 100 microM S-CMC-Lys, a current due to an outward GSH flux was observed, which was inhibitable by 5-nitro-2-(3-phenylpropylamino)-benzoate and glibenclamide. This current was not observed in CFT1-C2 cells, where a functional cystic fibrosis transmembrane conductance regulator (CFTR) is lacking. Inside-out patch-clamp experiments (GSH on the cytoplasm side, Cl(-) on the extracellular side) showed the activity of a channel, which was able to conduct current in both directions: the single channel conductance was 2-4 pS, and the open probability (P(o)) was low and voltage-independent. After preincubation with 100 microM S-CMC-Lys, there was an increase in P(o), in the number of active channels present in each patch, and in the relative permeability to GSH vs Cl(-). Outwardly directed efflux of GSH could also be increased by protein kinase A, adenosine 5'-triphosphate, and cyclic adenosine monophosphate (cAMP) added to the cytoplasmic side (whole-cell configuration). The increased secretion of GSH observed in the presence of S-CMC-Lys or 8-bromoadenosine-3',5'-cyclic monophosphate was also confirmed by HPLC assay of GSH on a confluent monolayer of respiratory cells. Western blot analysis confirmed the presence of CFTR in WI-26VA4 cells. This

  15. Adipocyte Derived Paracrine Mediators of Mammary Ductal Morphogenesis Controlled by Retinoic Acid Receptors

    Science.gov (United States)

    Marzan, Christine V.; Kupumbati, Tara S.; Bertran, Silvina P.; Samuels, TraceyAnn; Leibovitch, Boris; Lopez, Rafael Mira y; Ossowski, Liliana; Farias, Eduardo F.

    2010-01-01

    We generated a transgenic (Tg)-mouse model expressing a dominant negative-(DN)-RARα, (RARαG303E) under adipocytes-specific promoter to explore the paracrine role of adipocyte retinoic acid receptors (RARs) in mammary morphogenesis. Transgenic adipocytes had reduced level of RARα, β and γ, which coincided with a severely underdeveloped pubertal and mature ductal tree with profoundly decreased epithelial cell proliferation. Transplantation experiments of mammary epithelium and of whole mammary glands implicated a fat-pad dependent paracrine mechanism in the stunted phenotype of the epithelial-ductal tree. Co-cultures of primary adipocytes, or in vitro differentiated adipocyte cell line, with mammary epithelium showed that when activated, adipocyte RARs contribute to generation of secreted proliferative and pro-migratory factors. Gene expression microarrays revealed a large number of genes regulated by adipocyte-RARs. Among them, pleiotrophin (PTN) was identified as the paracrine effectors of epithelial cell migration. Its expression was found to be strongly inhibited by DN-RARα, an inhibition relieved by pharmacological doses of all-trans retinoic acid (atRA) in culture and in vivo. Moreover, adipocyte-PTHR, another atRA responsive gene, was found to be an up-stream regulator of PTN. Overall, these results support the existence of a novel paracrine loop controlled by adipocyte-RAR that regulates the mammary ductal tree morphogenesis. PMID:20974122

  16. White, brown and pink adipocytes: the extraordinary plasticity of the adipose organ.

    Science.gov (United States)

    Giordano, Antonio; Smorlesi, Arianna; Frontini, Andrea; Barbatelli, Giorgio; Cinti, Saverio

    2014-05-01

    In mammals, adipocytes are lipid-laden cells making up the parenchyma of the multi-depot adipose organ. White adipocytes store lipids for release as free fatty acids during fasting periods; brown adipocytes burn glucose and lipids to maintain thermal homeostasis. A third type of adipocyte, the pink adipocyte, has recently been characterised in mouse subcutaneous fat depots during pregnancy and lactation. Pink adipocytes are mammary gland alveolar epithelial cells whose role is to produce and secrete milk. Emerging evidence suggests that they derive from the transdifferentiation of subcutaneous white adipocytes. The functional response of the adipose organ to a range of metabolic and environmental challenges highlights its extraordinary plasticity. Cold exposure induces an increase in the 'brown' component of the organ to meet the increased thermal demand; in states of positive energy balance, the 'white' component expands to store excess nutrients; finally, the 'pink' component develops in subcutaneous depots during pregnancy to ensure litter feeding. At the cell level, plasticity is provided not only by stem cell proliferation and differentiation but also, distinctively, by direct transdifferentiation of fully differentiated adipocytes by the stimuli that induce genetic expression reprogramming and through it a change in phenotype and, consequently function. A greater understanding of adipocyte transdifferentiation mechanisms would have the potential to shed light on their biology as well as inspire novel therapeutic strategies against metabolic syndrome (browning) and breast cancer (pinking).

  17. The human fetus preferentially secretes corticosterone, rather than cortisol, in response to intra-partum stressors.

    Directory of Open Access Journals (Sweden)

    Katherine E Wynne-Edwards

    Full Text Available CONTEXT: Fetal stress is relevant to newborn outcomes. Corticosterone is rarely quantified in human clinical endocrinology and is found at much lower concentrations than cortisol. However, fetal corticosterone is a candidate hormone as a fetal stress signal. OBJECTIVE: Test the hypothesis that preferential fetal corticosterone synthesis occurs in response to fetal intra-partum stress. DESIGN: Cross-sectional comparison of paired serum corticosteroid concentrations in umbilical artery and vein from 300 women providing consent at admission to a General Hospital Labor and Delivery unit. Pre-term and multiple births were excluded, leaving 265 healthy deliveries. MAIN OUTCOME MEASURES: Corticosterone and cortisol concentrations determined by LC-MS/MS for umbilical cord venous (V and arterial (A samples and used to calculate fetal synthesis (A-V and proportional fetal synthesis ([A-V]/V. Chart-derived criteria stratified samples by type of delivery, maternal regional analgesia, augmentation of contractions, and clinical rationale for emergent Caesarian delivery. RESULTS: Cortisol concentrations were higher than corticosterone concentrations; however, the fetus preferentially secretes corticosterone (148% vs 49% proportional increase for cortisol and differentially secretes corticosterone as fetal stress increases. Fetal corticosterone synthesis is elevated after passage through the birth canal relative to Caesarian deliveries. For vaginal deliveries, augmentation of contractions does not affect corticosteroid concentrations whereas maternal regional analgesia decreases venous (maternal concentrations and increases fetal synthesis. Fetal corticosterone synthesis is also elevated after C-section indicated by cephalopelvic disproportion after labor, whereas cortisol is not. CONCLUSIONS: The full-term fetus preferentially secretes corticosterone in response to fetal stress during delivery. Fetal corticosterone could serve as a biomarker of fetal stress.

  18. The human fetus preferentially secretes corticosterone, rather than cortisol, in response to intra-partum stressors.

    Science.gov (United States)

    Wynne-Edwards, Katherine E; Edwards, Heather E; Hancock, Trina M

    2013-01-01

    Fetal stress is relevant to newborn outcomes. Corticosterone is rarely quantified in human clinical endocrinology and is found at much lower concentrations than cortisol. However, fetal corticosterone is a candidate hormone as a fetal stress signal. Test the hypothesis that preferential fetal corticosterone synthesis occurs in response to fetal intra-partum stress. Cross-sectional comparison of paired serum corticosteroid concentrations in umbilical artery and vein from 300 women providing consent at admission to a General Hospital Labor and Delivery unit. Pre-term and multiple births were excluded, leaving 265 healthy deliveries. Corticosterone and cortisol concentrations determined by LC-MS/MS for umbilical cord venous (V) and arterial (A) samples and used to calculate fetal synthesis (A-V) and proportional fetal synthesis ([A-V]/V). Chart-derived criteria stratified samples by type of delivery, maternal regional analgesia, augmentation of contractions, and clinical rationale for emergent Caesarian delivery. Cortisol concentrations were higher than corticosterone concentrations; however, the fetus preferentially secretes corticosterone (148% vs 49% proportional increase for cortisol) and differentially secretes corticosterone as fetal stress increases. Fetal corticosterone synthesis is elevated after passage through the birth canal relative to Caesarian deliveries. For vaginal deliveries, augmentation of contractions does not affect corticosteroid concentrations whereas maternal regional analgesia decreases venous (maternal) concentrations and increases fetal synthesis. Fetal corticosterone synthesis is also elevated after C-section indicated by cephalopelvic disproportion after labor, whereas cortisol is not. The full-term fetus preferentially secretes corticosterone in response to fetal stress during delivery. Fetal corticosterone could serve as a biomarker of fetal stress.

  19. Adipocyte-derived monocyte chemotactic protein-1 (MCP-1) promotes prostate cancer progression through the induction of MMP-2 activity.

    Science.gov (United States)

    Ito, Yusuke; Ishiguro, Hitoshi; Kobayashi, Naohito; Hasumi, Hisashi; Watanabe, Masatoshi; Yao, Masahiro; Uemura, Hiroji

    2015-07-01

    Obesity is known to be associated with prostate cancer development and progression, but the detailed mechanism is not clear. Monocyte chemotactic protein-1 (MCP-1) is secreted from cancer cells, stromal cells, and adipocytes, and it is involved in prostate cancer progression. Here we investigated the biological role of MCP-1 secreted from adipocytes for prostate cancer cells. Human pre-adipocytes (HPAds) were cultured and differentiated to mature adipocytes. Conditioned medium (CM) from HPAd cells was obtained using phenol red-free RPMI1640 medium. We performed a cytokine membrane array analysis to detect cytokines in the CM. To characterize the physiological function of MCP-1 in the CM, we performed an MTT-assay, a wound-healing and invasion assay with anti-MCP-1 antibody using three prostate cancer cell lines: DU145, LNCaP, and PC-3. Matrix metalloproteinase (MMP)-2 and MMP-9 activities were evaluated by gelatin zymography. A qPCR and Western blotting were used to examine the mRNA and protein expression levels of MMP-2. The cytokine membrane array of the CM showed a strong signal of MCP-1compared to the control medium, and we thus focused our attention on MCP-1 in the CM. The CM up-regulated the cancer cell proliferation, and the neutralization by anti-MCP-1 antibody inhibited the proliferative effect of the prostate cancer cell lines. The CM greatly increased the invasive activity in the prostate cancer cell lines, and anti-MCP-1 antibody decreased the invasiveness. Gelatin zymography revealed that the CM markedly enhanced the enzymatic activity of MMP-2, and anti-MCP-1 antibody down-regulated its effect. MMP-2 mRNA expression was undetected and the MMP-2 protein level was unchanged between the control medium and CM in DU145 cells. MCP-1 from adipocytes enhances the growth and invasion activity of prostate cancer cells. The inhibition of MCP-1 derived from adipocytes might be an effective treatment for prostate cancer. © 2015 Wiley Periodicals, Inc.

  20. Extra Fructose in the Growth Medium Fuels Lipogenesis of Adipocytes

    Directory of Open Access Journals (Sweden)

    Armin Robubi

    2014-01-01

    Full Text Available Fructose in excessive amounts exerts negative effects on insulin sensitivity, blood pressure, and liver metabolism. These adverse outcomes were attributed to its disturbances of key metabolic pathways in the liver. Recently, possible consequences of high fructose levels directly on adipocytes in vivo have been considered. We have cultured adipocytes in growth media containing 1 g/L fructose additionally to glucose and monitored the cells fate. Cells developed lipid vesicles much earlier with fructose and showed altered kinetics of the expression of mRNAs involved in lipogenesis and hexose uptake. Adiponectin secretion, too, peaked earlier in fructose containing media than in media with glucose only. From these data it can be speculated that similar effects of fructose containing diets happen in vivo also. Apart from toxic action on liver cells, adipocytes might be stimulated to take up extra fructose and generate new lipid vesicles, further dysregulating energy homeostasis.

  1. DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens

    Directory of Open Access Journals (Sweden)

    Dolores L. Guzmán-Herrador

    2017-08-01

    Full Text Available We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

  2. Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis

    DEFF Research Database (Denmark)

    Lasica, Anna M; Goulas, Theodoros; Mizgalska, Danuta;

    2016-01-01

    Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T...

  3. Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

    Science.gov (United States)

    Besse, A; Trimoreau, F; Faucher, J L; Praloran, V; Denizot, Y

    1999-07-08

    Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

  4. Resistin induces lipolysis and suppresses adiponectin secretion in cultured human visceral adipose tissue.

    Science.gov (United States)

    Chen, Neng; Zhou, Lingmei; Zhang, Zixiang; Xu, Jiaying; Wan, Zhongxiao; Qin, Liqiang

    2014-11-01

    Resistin is an adipokine secreted from adipose tissue, which is likely involved in the development of obesity and insulin resistance via its interaction with other organs, as well as affecting adipose tissue function. The impact of resistin treatment on lipolysis and adiponectin secretion in human visceral adipose tissue is currently unknown. Mesenteric adipose tissue samples were obtained from 14 male subjects [age 54±6 yr, body mass index (BMI) 23.59±0.44 kg/m(2)] undergoing abdominal surgeries. Adipose tissues were cultured and treated with resistin (100 ng/mL, 24h) in the absence or presence of different signaling inhibitors: H89 (1 μM), PD98059 (25 μM) and SB201290 (20 μM) for glycerol and non-esterified fatty acid (NEFA) measurement. Adiponectin level from media at 24 h was also measured via ELISA. Adipose tissue minces after resistin incubation (100 ng/mL, 24 h) were also collected for further Western blotting analysis. Resistin resulted in significant induction of glycerol (3.62±0.57 vs. 5.30±1.11 mmol/L/g tissue, ptissue, ptissue, ptissues via its effect on adipose tissue function.

  5. Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

    Directory of Open Access Journals (Sweden)

    Jessica Wahlgren

    Full Text Available It has previously been shown that nano-meter sized vesicles (30-100 nm, exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3⁺ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3⁺ T cells have on resting CD3⁺ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3⁺ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3⁺ T cells together with IL-2 were able to generate proliferation in autologous resting CD3⁺ T cells. The CD3⁺ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8⁺ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3⁺ T cells communicate with resting autologous T cells via exosomes.

  6. The secretion of high molecular weight cathepsin B from cultured human liver cancers.

    Directory of Open Access Journals (Sweden)

    Ohsawa,Toshiya

    1989-02-01

    Full Text Available The biochemical characteristics of cathepsin B secreted from cultured human liver cancer cells were examined. The enzyme activity of culture medium against a synthetic substrate, N-carbobenzoxy-L-arginyl-L-arginine-4-methyl-coumaryl-7-amide, was dependent on the addition of cysteine, and the optimal pH was found to be 6.0. No activity was observed when the enzyme source was fresh medium not used for culture. These results suggest that the enzyme released from liver cancer cells is the thiol-protease cathepsin B. The molecular weight of the enzyme with 90% of the total activity was 40,000. Two cathepsin B molecules were found in liver tissue from patients with hepatocellular carcinoma (HCC; one was equivalent in size to the secreted enzyme, and a smaller one was the same as normal liver cathepsin B (27,000, which was also obtained from HCC-bearing cirrhotic liver. These results demonstrate that two molecules of cathepsin B are synthesized in liver cancer, and that the larger one is released into the surrounding tissue.

  7. Quantitative analysis of secretome from adipocytes regulated by insulin

    Institute of Scientific and Technical Information of China (English)

    Hu Zhou; Yuanyuan Xiao; Rongxia Li; Shangyu Hong; Sujun Li; Lianshui Wang; Rong Zeng; Kan Liao

    2009-01-01

    Adipocyte is not only a central player involved in storage and release of energy, but also in regulation of energy metabolism in other organs via secretion of pep-tides and proteins. During the pathogenesis of insulin resistance and type 2 diabetes, adipocytes are subjected to the increased levels of insulin, which may have a major impact on the secretion of adipokines. We have undertaken cleavable isotope-coded affinity tag (clCAT) and label-free quantitation approaches to identify and quantify secretory factors that are differen-tially secreted by 3T3-LI adipocytes with or without insulin treatment. Combination of clCAT and label-free results, there are 317 proteins predicted or annotated as secretory proteins. Among these secretory proteins, 179 proteins and 53 proteins were significantly up-regulated and down-regulated, respectively. A total of 77 reported adipokines were quantified in our study, such as adiponectin, cathepsin D, cystatin C, resistin, and transferrin. Western blot analysis of these adipo-kines confirmed the quantitative results from mass spectrometry, and revealed individualized secreting pat-terns of these proteins by increasing insulin dose. In addition, 240 proteins were newly identified and quanti-fied as secreted proteins from 3T3-L1 adipocytes in our study, most of which were up-regulated upon insulin treatment. Further comprehensive bioinformatics analysis revealed that the secretory proteins in extra-cellular matrix-receptor interaction pathway and glycan structure degradation pathway were significantly up-regulated by insulin stimulation.

  8. Vitriols do guarantee an efficacious reduction of the human sweat when secreted from eccrine glands

    Directory of Open Access Journals (Sweden)

    Lorenzo Martini

    2016-01-01

    Full Text Available Introduction: Eccrine glands in human body are designed to secrete a salty solution containing variable percentages of urea and/or lactic acid and/or lactates. Aim: We want to demonstrate that it is possible to determine the preponderance of the former or of the latter in order to achieve the astringency of the same eccrine glands using vitriols, that is white or blue vitriol (zinc sulphate or copper sulphate. Results and Conclusion: The results are really thrilling, and it is suggestive to notice that gymnastical exercises and climate temperatures may or not influence the production of urea and/or lactic acid, nevertheless vitriols are exceptional to achieve the desired expectations, as far as astringency and anti-perspiration is concerned.

  9. Engineering human cells for in vivo secretion of antibody and non-antibody therapeutic proteins.

    Science.gov (United States)

    Sánchez-Martín, David; Sanz, Laura; Álvarez-Vallina, Luis

    2011-12-01

    Purified proteins such as antibodies are widely used as therapeutic agents in clinical medicine. However, clinical-grade proteins for therapeutic use require sophisticated technologies and are extremely expensive to produce. In vivo secretion of therapeutic proteins by genetically engineered human cells may advantageously replace injection of highly purified proteins. The use of gene transfer methods circumvents problems related to large-scale production and purification and offers additional benefits by achieving sustained concentrations of therapeutic protein with a syngenic glycosylation pattern that make the protein potentially less immunogenic. The feasibility of the in vivo production of therapeutic proteins by diverse cells/tissues has now been demonstrated using different techniques, such as ex vivo genetically modified cells and in vivo gene transfer mediated by viral vectors.

  10. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus

    2013-01-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...... of teased muscle fibers by transmission electron microscopy (TEM) and confocal microscopy (CM). Muscle interstitial fluid was sampled from microdialysis probes placed in the thigh muscle. TEM and CM analysis revealed two primary sites of localization of VEGF: in vesicles located in the subsarcolemmal...... regions and between the contractile elements within the muscle fibers; and in pericytes situated on the skeletal muscle capillaries. Quantitation of the subsarcolemmal density of VEGF vesicles, calculated on top of myonuclei, in the muscle fibers revealed a ∼50% increase (P...

  11. Pleiotropic effects of cancer cells' secreted factors on human stromal (mesenchymal) stem cells

    DEFF Research Database (Denmark)

    Al-toub, Mashael; Almusa, Abdulaziz; Almajed, Mohammed

    2013-01-01

    INTRODUCTION: Studying cancer tumors' microenvironment may reveal a novel role in driving cancer progression and metastasis. The biological interaction between stromal (mesenchymal) stem cells (MSCs) and cancer cells remains incompletely understood. Herein, we investigated the effects of tumor...... cells' secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. METHODS: Morphological changes were assessed using fluorescence microscopy....... Changes in gene expression were assessed using Agilent microarray and qRT-PCR. GeneSpring 12.1 and DAVID tools were used for bioinformatic and signaling pathway analyses. Cell migration was assessed using a transwell migration system. SB-431542, PF-573228 and PD98059 were used to inhibit transforming...

  12. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Christina Kopp

    2014-11-01

    Full Text Available The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001 and the mRNA abundances of GPR109A (p ≤ 0.05 and chemerin (p ≤ 0.01. Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001. The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

  13. α-Naphthoflavone Increases Lipid Accumulation in Mature Adipocytes and Enhances Adipocyte-Stimulated Endothelial Tube Formation

    Directory of Open Access Journals (Sweden)

    Mei-Lin Wang

    2015-04-01

    Full Text Available The aryl hydrocarbon receptor (AhR is a ligand-activated factor that regulates biological effects associated with obesity. The AhR agonists, such as environmental contaminants 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD and β-naphthoflavone (BNF, inhibit preadipocyte differentiation and interfere with the functions of adipose tissue, whereas the antagonist may have opposite or protective effects in obesity. This study investigated the effects of α-naphthoflavone (α-NF, an AhR antagonist, on adipogenesis- and angiogenesis-associated factors in mature adipocytes and on cross-talk of mature adipocytes with endothelial cells (ECs. Besides, the roles of the AhR on lipid accumulation and on secretion of vascular endothelial growth factor were also determined by introducing siRNA of AhR. Differentiated 3T3-L1 cells were treated with α-naphthoflavone (α-NF (1–5 μM for 16 h. Lipid accumulation and the expressions of AhR-associated factors in the cells were determined. The interaction between adipocytes and ECs was investigated by cultivating ECs with conditioned medium (CM from α-NF-treated mature adipocytes, followed by the determination of endothelial tube formation. The results showed that α-NF significantly increased triglyceride (TG accumulation in mature adipocytes, which was associated with increased expression of hormone-sensitive lipase (HSL, estrogen receptor (ER, as well as decreased expression of AhR, AhR nuclear translocator (ARNT, cytochrome P4501B1 (CYP1B1, and nuclear factor erythroid-2-related factor (NRF-2 proteins. In addition, CM stimulated formation of tube-like structures in ECs, and α-NF further enhanced such stimulation in association with modulated the secretions of various angiogenic mediators by mature adipocytes. Similarly, increased TG accumulation and vascular endothelial growth factor (VEGF secretion were observed in AhR-knockout cells. In conclusion, α-NF increased TG accumulation in mature adipocytes and

  14. Role of adipose secreted factors and kisspeptin in the metabolic control of gonadotropin secretion and puberty

    Science.gov (United States)

    Factors secreted by adipose tissue continue to be discovered. Evidence indicates a strong link between neural influences and adipocyte expression and secretion of a wide array of cytokines, neurotrophic factors, growth factors, binding proteins, and neuropeptides. These “adipokines” are linked to im...

  15. IL-3 and IL-33 induces IL-9 secretion from human basophils

    DEFF Research Database (Denmark)

    Poulsen, Britta C.; Jensen, Bettina Margrethe; Blom, Lars

    Background IL-9 has been suggested to play an important role in chronic inflammation due to its effect on remodeling and cell recruitment. Since basophils can be found in inflamed tissue, we wanted to test if human basophils are capable of secreting IL-9 under the influence of growth or tissue...... factors like IL-3 or IL-33, respectively. Methods Basophils, purified from buffy coat blood by negative selection (purity > 95%), were cultured for 24 hours in RPMI culture medium (1x106 cells/ml) supplemented with 5% human serum, and IL-3 (10 ng/ml) and/or IL-33 (50 ng/ml). The cells were then analysed...... by FACS for ST2 expression where basophils were gated as FceRIa+CD3-CD14-CD19-CD56-. The supernatant was tested for IL-9 using a multiplex magnetic bead assay. Results Human basophils have recently been described to express the IL-33 receptor ST2. In agreement with previous findings, our results showed...

  16. RFX6 Regulates Insulin Secretion by Modulating Ca2+ Homeostasis in Human β Cells

    Directory of Open Access Journals (Sweden)

    Vikash Chandra

    2014-12-01

    Full Text Available Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca2+-channel genes resulting in the reduction in L-type Ca2+-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca2+-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.

  17. E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte

    Directory of Open Access Journals (Sweden)

    Christine M. Kusminski

    2015-10-01

    Conclusion: We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

  18. Role of adipocyte-derived lipoprotein lipase in adipocyte hypertrophy

    Directory of Open Access Journals (Sweden)

    Orlando Robert A

    2007-10-01

    Full Text Available Abstract Background A major portion of available fatty acids for adipocyte uptake is derived from lipoprotein lipase (LPL-mediated hydrolysis of circulating lipoprotein particles. In vivo studies aimed at identifying the precise role of adipocyte-derived LPL in fat storage function of adipose tissue have been unable to provide conclusive evidence due to compensatory mechanisms that activate endogenous fatty acid synthesis. To address this gap in knowledge, we have measured the effect of reducing adipocyte LPL expression on intracellular lipid accumulation using a well-established cultured model of adipocyte differentiation. Methods siRNA specific for mouse LPL was transfected into 3T3-L1 adipocytes. Expression of LPL was measured by quantitative real-time PCR and cell surface-associated LPL enzymatic activity was measured by colorimetric detection following substrate (p-nitrophenyl butyrate hydrolysis. Apolipoprotein CII and CIII expression ratios were also measured by qRT-PCR. Intracellular lipid accumulation was quantified by Nile Red staining. Results During differentiation of 3T3-L1 pre-adipocytes, LPL mRNA expression increases 6-fold resulting in a 2-fold increase in cell surface-associated LPL enzymatic activity. Parallel to this increase in LPL expression, we found that intracellular lipids increased ~10-fold demonstrating a direct correlation between adipocyte-derived LPL expression and lipid storage. We next reduced LPL expression in adipocytes using siRNA transfections to directly quantify the contributions of adipocyte-derived LPL to lipid storage, This treatment reduced LPL mRNA expression and cell surface-associated LPL enzymatic activity to ~50% of non-treated controls while intracellular lipid levels were reduced by 80%. Exogenous addition of purified LPL (to restore extracellular lipolytic activity or palmitate (as a source of free fatty acids to siRNA-treated cells restored intracellular lipid levels to those measured for non

  19. Characterization of stimulus-secretion coupling in the human pancreatic EndoC-βH1 beta cell line.

    Directory of Open Access Journals (Sweden)

    Lotta E Andersson

    Full Text Available Studies on beta cell metabolism are often conducted in rodent beta cell lines due to the lack of stable human beta cell lines. Recently, a human cell line, EndoC-βH1, was generated. Here we investigate stimulus-secretion coupling in this cell line, and compare it with that in the rat beta cell line, INS-1 832/13, and human islets.Cells were exposed to glucose and pyruvate. Insulin secretion and content (radioimmunoassay, gene expression (Gene Chip array, metabolite levels (GC/MS, respiration (Seahorse XF24 Extracellular Flux Analyzer, glucose utilization (radiometric, lactate release (enzymatic colorimetric, ATP levels (enzymatic bioluminescence and plasma membrane potential and cytoplasmic Ca2+ responses (microfluorometry were measured. Metabolite levels, respiration and insulin secretion were examined in human islets.Glucose increased insulin release, glucose utilization, raised ATP production and respiratory rates in both lines, and pyruvate increased insulin secretion and respiration. EndoC-βH1 cells exhibited higher insulin secretion, while plasma membrane depolarization was attenuated, and neither glucose nor pyruvate induced oscillations in intracellular calcium concentration or plasma membrane potential. Metabolite profiling revealed that glycolytic and TCA-cycle intermediate levels increased in response to glucose in both cell lines, but responses were weaker in EndoC-βH1 cells, similar to those observed in human islets. Respiration in EndoC-βH1 cells was more similar to that in human islets than in INS-1 832/13 cells.Functions associated with early stimulus-secretion coupling, with the exception of plasma membrane potential and Ca2+ oscillations, were similar in the two cell lines; insulin secretion, respiration and metabolite responses were similar in EndoC-βH1 cells and human islets. While both cell lines are suitable in vitro models, with the caveat of replicating key findings in isolated islets, EndoC-βH1 cells have the

  20. Yeast modulation of human dendritic cell cytokine secretion: an in vitro study.

    Science.gov (United States)

    Smith, Ida M; Christensen, Jeffrey E; Arneborg, Nils; Jespersen, Lene

    2014-01-01

    Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current

  1. Yeast modulation of human dendritic cell cytokine secretion: an in vitro study.

    Directory of Open Access Journals (Sweden)

    Ida M Smith

    Full Text Available Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications

  2. Early B-cell Factor 1 Regulates Adipocyte Morphology and Lipolysis in White Adipose Tissue

    OpenAIRE

    Gao, Hui; Mejhert, Niklas; Fretz, Jackie A.; Arner, Erik; Lorente-Cebrián, Silvia; Ehrlund, Anna; Dahlman-Wright, Karin; Gong, Xiaowei; Strömblad, Staffan; Douagi, Iyadh; Laurencikiene, Jurga; Dahlman, Ingrid; Daub, Carsten O.; Rydén, Mikael; Mark C Horowitz

    2014-01-01

    White adipose tissue (WAT) morphology characterized by hypertrophy (i.e. fewer but larger adipocytes) associates with increased adipose inflammation, lipolysis, insulin resistance and risk of diabetes. However, the causal relationships and the mechanisms controlling WAT morphology are unclear. Herein, we identified EBF1 as an adipocyte-expressed transcription factor with decreased expression/activity in WAT hypertrophy. In human adipocytes, the regulatory targets of EBF1 were enriched for gen...

  3. Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Pawin Pongkorpsakol

    2017-06-01

    Full Text Available Intestinal Cl− secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl− secretion in human intestinal epithelial (T84 cells. FFA inhibited cAMP-dependent Cl− secretion in T84 cell monolayers with IC50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl− channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl− secretion with IC50 of ∼10 μM. FFA inhibited activities of Ca2+-activated Cl− channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+–K+–Cl− cotransporters and Na+–K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl− secretion by suppressing activities of both apical Cl− channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.

  4. Angiopoietin-like 4 (Angptl4) protein is a physiological mediator of intracellular lipolysis in murine adipocytes.

    Science.gov (United States)

    Gray, Nora E; Lam, Lily N; Yang, Karen; Zhou, Anna Y; Koliwad, Suneil; Wang, Jen-Chywan

    2012-03-09

    Intracellular triacylglycerol (TG) hydrolysis and fatty acid release by the white adipose tissue (WAT) during a fast is stimulated by counter-regulatory factors acting in concert, although how adipocytes integrate these lipolytic inputs is unknown. We tested the role of angiopoietin-like 4 (Angptl4), a secreted protein induced by fasting or glucocorticoid treatment, in modulating intracellular adipocyte lipolysis. Glucocorticoid receptor blockade prevented fasting-induced tissue Angptl4 expression and WAT TG hydrolysis in mice, and TG hydrolysis induced by fasts of 6 or 24 h was greatly reduced in mice lacking Angptl4 (Angptl4(-/-)). Glucocorticoid treatment mimicked the lipolytic effects of fasting, although with slower kinetics, and this too required Angptl4. Thus, fasting-induced WAT TG hydrolysis requires glucocorticoid action and Angptl4. Both fasting and glucocorticoid treatment also increased WAT cAMP levels and downstream phosphorylation of lipolytic enzymes. Angptl4 deficiency markedly reduced these effects, suggesting that Angptl4 may stimulate lipolysis by modulating cAMP-dependent signaling. In support of this, cAMP levels and TG hydrolysis were reduced in primary Angptl4(-/-) murine adipocytes treated with catecholamines, which stimulate cAMP-dependent signaling to promote lipolysis, and was restored by treatment with purified human ANGPTL4. Remarkably, human ANGPTL4 treatment alone increased cAMP levels and induced lipolysis in these cells. Pharmacologic agents revealed that Angptl4 modulation of cAMP-dependent signaling occurs upstream of adenylate cyclase and downstream of receptor activation. We show that Angptl4 is a glucocorticoid-responsive mediator of fasting-induced intracellular lipolysis and stimulates cAMP signaling in adipocytes. Such a role is relevant to diseases of aberrant lipolysis, such as insulin resistance.

  5. Lithocholic acid attenuates cAMP-dependent Cl- secretion in human colonic epithelial T84 cells.

    Science.gov (United States)

    Ao, Mei; Domingue, Jada C; Khan, Nabihah; Javed, Fatima; Osmani, Kashif; Sarathy, Jayashree; Rao, Mrinalini C

    2016-06-01

    Bile acids (BAs) play a complex role in colonic fluid secretion. We showed that dihydroxy BAs, but not the monohydroxy BA lithocholic acid (LCA), stimulate Cl(-) secretion in human colonic T84 cells (Ao M, Sarathy J, Domingue J, Alrefai WA, Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013). In this study, we explored the effect of LCA on the action of other secretagogues in T84 cells. While LCA (50 μM, 15 min) drastically (>90%) inhibited FSK-stimulated short-circuit current (Isc), it did not alter carbachol-stimulated Isc LCA did not alter basal Isc, transepithelial resistance, cell viability, or cytotoxicity. LCA's inhibitory effect was dose dependent, acted faster from the apical membrane, rapid, and not immediately reversible. LCA also prevented the Isc stimulated by the cAMP-dependent secretagogues 8-bromo-cAMP, lubiprostone, or chenodeoxycholic acid (CDCA). The LCA inhibitory effect was BA specific, since CDCA, cholic acid, or taurodeoxycholic acid did not alter FSK or carbachol action. While LCA alone had no effect on intracellular cAMP concentration ([cAMP]i), it decreased FSK-stimulated [cAMP]i by 90%. Although LCA caused a small increase in intracellular Ca(2+) concentration ([Ca(2+)]i), chelation by BAPTA-AM did not reverse LCA's effect on Isc LCA action does not appear to involve known BA receptors, farnesoid X receptor, vitamin D receptor, muscarinic acetylcholine receptor M3, or bile acid-specific transmembrane G protein-coupled receptor 5. LCA significantly increased ERK1/2 phosphorylation, which was completely abolished by the MEK inhibitor PD-98059. Surprisingly PD-98059 did not reverse LCA's effect on Isc Finally, although LCA had no effect on basal Isc, nystatin permeabilization studies showed that LCA both stimulates an apical cystic fibrosis transmembrane conductance regulator Cl(-) current and inhibits a basolateral K(+) current. In summary, 50 μM LCA greatly inhibits cAMP-stimulated Cl(-) secretion, making low doses of LCA of

  6. Adipocytes activate mitochondrial fatty acid oxidation and autophagy to promote tumor growth in colon cancer

    Science.gov (United States)

    Wen, Yang-An; Xing, Xiaopeng; Harris, Jennifer W; Zaytseva, Yekaterina Y; Mitov, Mihail I; Napier, Dana L; Weiss, Heidi L; Mark Evers, B; Gao, Tianyan

    2017-01-01

    Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells. PMID:28151470

  7. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    Science.gov (United States)

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

  8. Intracellular Ca2+ signals in human-derived pancreatic somatostatin-secreting cells (QGP-1N).

    Science.gov (United States)

    Squires, P E; Amiranoff, B; Dunne, M J

    1994-10-01

    Single-cell microfluorimetry techniques have been used to examine the effects of acetylcholine (0.1-100 microM) on the intracellular free calcium ion concentration ([Ca2+]i) in a human-derived pancreatic somatostatin-secreting cell line, QGP-1N. When applied to the bath solution, acetylcholine was found to evoke a marked and rapid increase in [Ca2+]i at all concentrations tested. These responses were either sustained, or associated with the generation of complex patterns of [Ca2+]i transients. Overall, the pattern of response was concentration related. In general, 0.1-10 microM acetylcholine initiated a series of repetitive oscillations in cytoplasmic Ca2+, whilst at higher concentrations the responses consisted of a rapid rise in [Ca2+]i followed by a smaller more sustained increase. Without external Ca2+, 100 microM acetylcholine caused only a transient rise in [Ca2+]i, whereas lower concentrations of the agonist were able to initiate, but not maintain, [Ca2+]i oscillations. Acetylcholine-evoked Ca2+ signals were abolished by atropine (1-10 microM), verapamil (100 microM) and caffeine (20 mM). Nifedipine failed to have any significant effect upon agonist-evoked increases in [Ca2+]i, whilst 50 mM KCl, used to depolarise the cell membrane, only elicited a transient increase in [Ca2+]i. Ryanodine (50-500 nM) and caffeine (1-20 mM) did not increase basal Ca2+ levels, but the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)-hydroquinone (TBQ) and thapsigargin both elevated [Ca2+]i levels. These data demonstrate for the first time cytosolic Ca2+ signals in single isolated somatostatin-secreting cells of the pancreas. We have demonstrated that acetylcholine will evoke both Ca2+ influx and Ca2+ mobilisation, and we have partially addressed the subcellular mechanism responsible for these events.

  9. Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria.

    LENUS (Irish Health Repository)

    Sibartie, Shomik

    2009-01-01

    BACKGROUND: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays. RESULTS: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion. CONCLUSION: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

  10. Adipose tissue and adipocytes supports tumorigenesis and metastasis#

    OpenAIRE

    Nieman, Kristin M; Romero, Iris L.; Van Houten, Bennett; Lengyel, Ernst

    2013-01-01

    Adipose tissue influences tumor development in two major ways. First, obese individuals have a higher risk of developing certain cancers (endometrial, esophageal, and renal cell cancer). However, the risk of developing other cancers (melanoma, rectal, and ovarian) is not altered by body mass. In obesity, hypertrophied adipose tissue depots are characterized by a state of low grade inflammation. In this activated state, adipocytes and inflammatory cells secrete adipokines and cytokines which a...

  11. Lipolytic effect of a polyphenolic citrus dry extract of red orange, grapefruit, orange (SINETROL) in human body fat adipocytes. Mechanism of action by inhibition of cAMP-phosphodiesterase (PDE).

    Science.gov (United States)

    Dallas, Constantin; Gerbi, Alain; Tenca, Guillaume; Juchaux, Franck; Bernard, François-Xavier

    2008-10-01

    The present study investigated the lipolytic (break of fat stored) effect of a citrus-based polyphenolic dietary supplement (SINETROL) at human adipocytes (ex vivo), body fat (clinical) and biochemical levels (inhibition of phosphodiesterase). Free fatty acids (FFA) release was used as indicator of human adipocyte lipolysis and SINETROL activity has been compared with known lipolytic products (isoproterenol, theopylline and caffeine). SINETROL stimulated significantly the lipolytic activity in a range of 6 fold greater than the control. Moreover, SINETROL has 2.1 greater activity than guarana 12% caffeine while its content in caffeine is 3 times lower. Clinically, two groups of 10 volunteers with BMI relevant of overweight were compared during 4 and 12 weeks with 1.4 g/day SINETROL and placebo supplementation. In the SINETROL Group the body fat (%) decreased with a significant difference of 5.53% and 15.6% after 4 and 12 weeks, respectively, while the body weight (kg) decreased with a significant difference of 2.2 and 5.2 kg after 4 and 12 weeks, respectively. These observed effects are linked to SINETROL polyphenolic composition and its resulting synergistic activity. SINETROL is a potent inhibitor of cAMP-phosphodiesterase (PDE) (97%) compared to other purified compounds (cyanidin-3 glycoside, narangin, caffeine). These results suggest that SINETROL has a strong lipolytic effect mediated by cAMP-PDE inhibition. SINETROL may serve to prevent obesity by decreasing BMI.

  12. Secreted proteoglycans directly mediate human embryonic stem cell-basic fibroblast growth factor 2 interactions critical for proliferation.

    Science.gov (United States)

    Levenstein, Mark E; Berggren, W Travis; Lee, Ji Eun; Conard, Kevin R; Llanas, Rachel A; Wagner, Ryan J; Smith, Lloyd M; Thomson, James A

    2008-12-01

    Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface, and their removal from CM impairs proliferation. Finally, we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography, immunoblotting, and mass spectrometry-based proteomic analysis, we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture.

  13. Fructose stimulates GLP-1 but not GIP secretion in mice, rats, and humans

    DEFF Research Database (Denmark)

    Kuhre, Rune Ehrenreich; Gribble, Fiona M; Hartmann, Bolette

    2014-01-01

    glucose potently stimulated GIP release, fructose was without effect. Similar patterns were found in the mouse and rat, with both fructose and glucose stimulating GLP-1 secretion, whereas only glucose caused GIP secretion. In GLUTag cells, a murine cell line used as model for L cells, fructose...... was metabolized and stimulated GLP-1 secretion dose-dependently (EC50 = 0.155 mM) by ATP-sensitive potassium channel closure and cell depolarization. Because fructose elicits GLP-1 secretion without simultaneous release of glucagonotropic GIP, the pathways underlying fructose-stimulated GLP-1 release might...... be useful targets for type 2 diabetes mellitus and obesity drug development....

  14. Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.

    OpenAIRE

    Hellerman, J G; Cone, R C; Potts, J. T.; Rich, A; Mulligan, R C; Kronenberg, H M

    1984-01-01

    In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recomb...

  15. Human amniotic membrane-derived stromal cells (hAMSC) interact depending on breast cancer cell type through secreted molecules.

    Science.gov (United States)

    Kim, Sun-Hee; Bang, So Hee; Kang, So Yeong; Park, Ki Dae; Eom, Jun Ho; Oh, Il Ung; Yoo, Si Hyung; Kim, Chan-Wha; Baek, Sun Young

    2015-02-01

    Human amniotic membrane-derived stromal cells (hAMSC) are candidates for cell-based therapies. We examined the characteristics of hAMSC including the interaction between hAMSC and breast cancer cells, MCF-7, and MDA-MB-231. Human amniotic membrane-derived stromal cells showed typical MSC properties, including fibroblast-like morphology, surface antigen expression, and mesodermal differentiation. To investigate cell-cell interaction via secreted molecules, we cultured breast cancer cells in hAMSC-conditioned medium (hAMSC-CM) and analyzed their proliferation, migration, and secretome profiles. MCF-7 and MDA-MB-231 cells exposed to hAMSC-CM showed increased proliferation and migration. However, in hAMSC-CM, MCF-7 cells proliferated significantly faster than MDA-MB-231 cells. When cultured in hAMSC-CM, MCF-7 cells migrated faster than MDA-MB-231 cells. Two cell types showed different profiles of secreted factors. MCF-7 cells expressed much amounts of IL-8, GRO, and MCP-1 in hAMSC-CM. Human amniotic membrane-derived stromal cells interact with breast cancer cells through secreted molecules. Factors secreted by hAMSCs promote the proliferation and migration of MCF-7 breast cancer cells. For much safe cell-based therapies using hAMSC, it is necessary to study carefully about interaction between hAMSC and cancer cells.

  16. An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency

    Directory of Open Access Journals (Sweden)

    Hempel Franziska

    2012-09-01

    Full Text Available Abstract Background Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins. Results In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient. Conclusions Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins.

  17. Brown and brite adipocytes: Same function, but different origin and response.

    Science.gov (United States)

    Chu, Dinh-Toi; Gawronska-Kozak, Barbara

    2017-07-01

    Inducing brown adipocytes in white adipose tissues is a promising target to combat obesity and its related disorders in human beings. This goal has been especially encouraged by new important discoveries of human brown adipose tissues. The accumulating evidence confirms the presence of active brown adipocytes, not only in newborns, but also in adult humans. In rodents, there are two populations of the Ucp1-expressing adipocytes with well characterized-thermogenic functions, classical interscapular brown adipocytes and brite/beige adipocytes (brown adipocytes that are induced in white adipose tissues). Importantly, the anatomical localization, gene expression profiling and functional characterization of Ucp1-expressing fat cells indicates brite and brown adipocytes coexist in human beings. Therefore, the research directions of brown and brite adipogenesis provide lead to potential new therapies to fight obesity and its related metabolic diseases in human being. The objectives of this review are (1) to discuss the fate of primary adipocytes based on tissue origins, and (2) to discuss mechanisms of brown and brite adipogenesis which could lead to their different responses to browning reagents. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  18. Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum.

    Science.gov (United States)

    Nevalainen, Timo J; Eerola, Leena I; Rintala, Esa; Laine, V Jukka O; Lambeau, Gérard; Gelb, Michael H

    2005-04-15

    Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0

  19. Regulation of vascular tone by adipocytes

    Directory of Open Access Journals (Sweden)

    Van de Voorde Johan

    2011-03-01

    Full Text Available Abstract Recent studies have shown that adipose tissue is an active endocrine and paracrine organ secreting several mediators called adipokines. Adipokines include hormones, inflammatory cytokines and other proteins. In obesity, adipose tissue becomes dysfunctional, resulting in an overproduction of proinflammatory adipokines and a lower production of anti-inflammatory adipokines. The pathological accumulation of dysfunctional adipose tissue that characterizes obesity is a major risk factor for many other diseases, including type 2 diabetes, cardiovascular disease and hypertension. Multiple physiological roles have been assigned to adipokines, including the regulation of vascular tone. For example, the unidentified adipocyte-derived relaxing factor (ADRF released from adipose tissue has been shown to relax arteries. Besides ADRF, other adipokines such as adiponectin, omentin and visfatin are vasorelaxants. On the other hand, angiotensin II and resistin are vasoconstrictors released by adipocytes. Reactive oxygen species, leptin, tumour necrosis factor α, interleukin-6 and apelin share both vasorelaxing and constricting properties. Dysregulated synthesis of the vasoactive and proinflammatory adipokines may underlie the compromised vascular reactivity in obesity and obesity-related disorders.

  20. Secretion of neurotensin from a human pancreatic islet cell carcinoma cell line (QGP-1N).

    Science.gov (United States)

    Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

    1993-12-10

    Effects of various secretagogues on secretion of neurotensin from a pancreatic islet cell carcinoma cell line (QGP-1N) were examined. Carbachol stimulated secretion of neurotensin concentration-dependently in the range of 10(-6) - 10(-4) M. The neurotensin secretion stimulated with 10(-5) M carbachol was completely inhibited by atropine at 10(-5) M. Phorbol ester and calcium ionophore (A23187) stimulated secretion of neurotensin. The removal of extracellular Ca2+ suppressed the secretion through the stimulation with 10(-5) M carbachol. Fluoride, an activator of guanine nucleotide-binding (G) protein, stimulated secretion of neurotensin. Neurotensin released into culture medium through stimulation with carbachol coeluted with neurotensin 1-13 on a gel-chromatography. Our results suggest that secretion of neurotensin from QGP-1N cells is mainly regulated by acetylcholine through muscarinic receptors coupled to G protein and that an increase in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  1. Role of incretin hormones in the regulation of insulin secretion in diabetic and nondiabetic humans

    DEFF Research Database (Denmark)

    Holst, Jens Juul; Gromada, Jesper

    2004-01-01

    of GIP is near normal, whereas the secretion of GLP-1 is decreased. On the other hand, the insulintropic effect of GLP-1 is preserved, whereas the effect of GIP is greatly reduced, mainly because of a complete loss of the normal GIP-induced potentiation of second-phase insulin secretion. These two...

  2. CDK4 is an essential insulin effector in adipocytes

    Science.gov (United States)

    Lagarrigue, Sylviane; Lopez-Mejia, Isabel C.; Denechaud, Pierre-Damien; Escoté, Xavier; Castillo-Armengol, Judit; Jimenez, Veronica; Chavey, Carine; Giralt, Albert; Lai, Qiuwen; Zhang, Lianjun; Martinez-Carreres, Laia; Delacuisine, Brigitte; Annicotte, Jean-Sébastien; Blanchet, Emilie; Huré, Sébastien; Abella, Anna; Tinahones, Francisco J.; Vendrell, Joan; Dubus, Pierre; Bosch, Fatima; Kahn, C. Ronald; Fajas, Lluis

    2015-01-01

    Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4R24C). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT. PMID:26657864

  3. CDK4 is an essential insulin effector in adipocytes.

    Science.gov (United States)

    Lagarrigue, Sylviane; Lopez-Mejia, Isabel C; Denechaud, Pierre-Damien; Escoté, Xavier; Castillo-Armengol, Judit; Jimenez, Veronica; Chavey, Carine; Giralt, Albert; Lai, Qiuwen; Zhang, Lianjun; Martinez-Carreres, Laia; Delacuisine, Brigitte; Annicotte, Jean-Sébastien; Blanchet, Emilie; Huré, Sébastien; Abella, Anna; Tinahones, Francisco J; Vendrell, Joan; Dubus, Pierre; Bosch, Fatima; Kahn, C Ronald; Fajas, Lluis

    2016-01-01

    Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4(R24C)). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT.

  4. Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Jiong Cai; Fang Li; Shizhen Wang

    2008-01-01

    BACKGROUND: Studies have shown that monoclonal or polyclonal antibody injections ofamyloid β peptide arc effective in removing amyloid β peptide overload in the brain.OBJECTIVE: Based on successful screening of a human single-chain fragment variable antibody specific to amyloid β peptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide.DESIGN, TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006.MATERIALS: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library.METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a Teasy plasmid for pT-seFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA β was cut by EcoRl and Notl endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvA β expression vector, which was confirmed by gene sequencing. Linearized pPICgK-scFvA β was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5 % methanol to express human single-chain fragment variable antibody specific to amyloid β peptide.MAIN OUTCOME MEASURES: Protein electrophoresis was used to identify PCR products, gene sequencing was uscd to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris.RESULTS: Gene sequencing confirmed pPICgK-scFvA β orientation. Rccomhinants were obtained by lineadzed pPIC9K-scFvA β transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size

  5. A somatostatin-secreting cell line established from a human pancreatic islet cell carcinoma (somatostatinoma): release experiment and immunohistochemical study.

    Science.gov (United States)

    Iguchi, H; Hayashi, I; Kono, A

    1990-06-15

    Production and secretion of somatostatin (SRIF) were studied using a carcinoembryonic antigen (CEA)-producing cell line (QGP-1) established from a human pancreatic islet cell carcinoma. High concentrations of SRIF (274 +/- 51 ng/mg of protein, mean +/- SD, n = 5) and CEA (3083 +/- 347 ng/mg of protein, mean +/- SD, n = 5) were present in QGP-1 cells, and the basal secretion rates of SRIF and CEA by the cells (n = 5) were 46.4 +/- 4.8 and 1690 +/- 78 pg/10(5) cells/h, respectively. Immunohistochemical studies revealed the presence of SRIF in xenografts of QGP-1 cells and colocalization of SRIF and CEA. Secretion of SRIF by QGP-1 cells was stimulated in the presence of high K+ (50 mmol) and theophylline (10 mmol), but arginine (10 mmol) and glucose (300 mg/dl) had no effect on the SRIF secretion. The QGP-1 cell line may be useful for studying the regulation mechanism of SRIF secretion.

  6. Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF.

    Science.gov (United States)

    Weiser, W Y; Remold, H G; David, J R

    1985-01-01

    Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

  7. Alendronate inhibits cell invasion and MMP-2 secretion in human chondrosarcoma cell line

    Institute of Scientific and Technical Information of China (English)

    Te-jen LAI; Yi-chin FONG; Sheng-feng HSU; Temao LI; Horng-chaung HSU; Jaung-geng LIN; Chin-jung HSU; Ming-chih CHOU; Meng-chih LEE; Shun-fa YANG

    2007-01-01

    Aim: Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of the present study was to investigate the effect of alendronate, a bisphosphonate, on the invasion and migration of human chondrosarcoma cells (JJ012). Methods: JJ012 cells were treated with alendronate of various concentrations up to 100 μmol/L for a speci-fied period, and then gelatin zymography and matrigel invasion assay was per-formed to study the effects of alendronate on matrix metalloproteinase (MMP)-2 activity and the invasion ability of JJ012 cells, respectively. Results: Our data showed that alendronate exerted a dose- and time-dependent inhibitory effect on the invasion and migration of JJ012 cells. Furthermore, gelatin zymography and RT-PCR showed that alendronate treatment decreased the activity and mRNA levels of MMP-2 in a concentration-dependent manner. Conclusion: Our find-ings suggest that alendronate may reduce MMP-2 secretion at the transcriptional and translational levels, and inhibit the invasion of chondrosarcoma cell. Therefore,alendronate may be a potential candidate for the systemic therapy of chondro-sarcomas, as well as other malignant diseases.

  8. Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Yélian Marc Bossou

    2017-06-01

    Full Text Available Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1 and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity.

  9. Mechanism and Function of Type IV Secretion During Infection of the Human Host.

    Science.gov (United States)

    Gonzalez-Rivera, Christian; Bhatty, Minny; Christie, Peter J

    2016-06-01

    Bacterial pathogens employ type IV secretion systems (T4SSs) for various purposes to aid in survival and proliferation in eukaryotic hosts. One large T4SS subfamily, the conjugation systems, confers a selective advantage to the invading pathogen in clinical settings through dissemination of antibiotic resistance genes and virulence traits. Besides their intrinsic importance as principle contributors to the emergence of multiply drug-resistant "superbugs," detailed studies of these highly tractable systems have generated important new insights into the mode of action and architectures of paradigmatic T4SSs as a foundation for future efforts aimed at suppressing T4SS machine function. Over the past decade, extensive work on the second large T4SS subfamily, the effector translocators, has identified a myriad of mechanisms employed by pathogens to subvert, subdue, or bypass cellular processes and signaling pathways of the host cell. An overarching theme in the evolution of many effectors is that of molecular mimicry. These effectors carry domains similar to those of eukaryotic proteins and exert their effects through stealthy interdigitation of cellular pathways, often with the outcome not of inducing irreversible cell damage but rather of reversibly modulating cellular functions. This article summarizes the major developments for the actively studied pathogens with an emphasis on the structural and functional diversity of the T4SSs and the emerging common themes surrounding effector function in the human host.

  10. Galectin-9 enhances cytokine secretion, but suppresses survival and degranulation, in human mast cell line.

    Directory of Open Access Journals (Sweden)

    Reiji Kojima

    Full Text Available Galectin-9 (Gal-9, a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.

  11. Maintenance of human pluripotent stem cells using 4SP-hFGF2-secreting STO cells.

    Science.gov (United States)

    Lee, Won-Young; Kim, Jumi; Gil, Chang-Hyun; Lee, Jae-Ho; Song, Hyuk; Kim, Jae-Hwan; Chung, Hyung-Min

    2011-11-01

    Human embryonic stem cells (hESCs) are typically cultured on fibroblast feeder cells or in fibroblast conditioned medium supplemented with fibroblast growth factor 2 (FGF2, also known as bFGF). FGF signaling appears to be important for hESC self-renewal and is required to enable the culture of hESCs in an undifferentiated state. In this study, we generated a transgenic fibroblast feeder line stably expressing a secretable FGF4 signal peptide tagged hFGF2 (4SP-hFGF2). The expression of this transgene functionally replaced the requirement for exogenous FGF2 when using these cells as feeders for the maintenance of hESCs. Under these conditions, hESCs maintained the typical marker of pluripotency assessed after long term culture, while still retaining the capacity for differentiation to all three germ layers. This transgene could be applied to mass produce 4SP-hFGF2 protein, serving to be an economical and effective strategy for culturing pluripotent stem cells as feeder cells. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions.

    Science.gov (United States)

    Denny, Paul; Hagen, Fred K; Hardt, Markus; Liao, Lujian; Yan, Weihong; Arellanno, Martha; Bassilian, Sara; Bedi, Gurrinder S; Boontheung, Pinmannee; Cociorva, Daniel; Delahunty, Claire M; Denny, Trish; Dunsmore, Jason; Faull, Kym F; Gilligan, Joyce; Gonzalez-Begne, Mireya; Halgand, Frédéric; Hall, Steven C; Han, Xuemei; Henson, Bradley; Hewel, Johannes; Hu, Shen; Jeffrey, Sherry; Jiang, Jiang; Loo, Joseph A; Ogorzalek Loo, Rachel R; Malamud, Daniel; Melvin, James E; Miroshnychenko, Olga; Navazesh, Mahvash; Niles, Richard; Park, Sung Kyu; Prakobphol, Akraporn; Ramachandran, Prasanna; Richert, Megan; Robinson, Sarah; Sondej, Melissa; Souda, Puneet; Sullivan, Mark A; Takashima, Jona; Than, Shawn; Wang, Jianghua; Whitelegge, Julian P; Witkowska, H Ewa; Wolinsky, Lawrence; Xie, Yongming; Xu, Tao; Yu, Weixia; Ytterberg, Jimmy; Wong, David T; Yates, John R; Fisher, Susan J

    2008-05-01

    Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

  13. Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

    Institute of Scientific and Technical Information of China (English)

    TANG Zehai; LIU Xinyue; ZOU Ping

    2007-01-01

    This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor (VEGF) and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concen- trations (12.5-200 μmol/L) for different time lengths (12-48 h). The proliferation of the U937 leu- kemic cells was determined by MTT assay. Apoptosis was observed by Annexin-Ⅴ-FIFC/PI double staining and flow cytometry (FCM). Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resvera-trol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

  14. [Kidney, adipose tissue, adipocytes--what's new?].

    Science.gov (United States)

    Lafontan, Max

    2011-04-01

    Increased evidence suggests that obesity-related glomerulopathy and chronic kidney diseases should be identified as isolated complications of obesity. It is questioned if the numerous adipose tissue productions could play a role in the initiation/maintenance of such kidney diseases. This review will provide a sum-up of recent advances on fat cell metabolism and adipose tissue physiology. The adipose tissue behaves as an endocrine organ with multiple activities. It is secreting hormones (leptin, adiponectin, apelin) and numerous factors with autocrine, paracrine and systemic effects. These secretions are coming from adipocytes themselves or from cells present in the stroma-vascular fraction of the adipose tissue. When expanding, the adipose tissue of the obese is infiltrated by immune cells such as macrophages and lymphocytes; the role of which is not fully clarified. An attempt will be done to delineate if alterations of lipid storage/fatty acid release or of the secretion potencies of adipose tissue could contribute to kidney lipotoxicity and other chronic kidney diseases described in the obese. Copyright © 2010 Association Société de néphrologie. Published by Elsevier SAS. All rights reserved.

  15. Sildenafil Effect on Nitric Oxide Secretion by Normal Human Endometrial Epithelial Cells Cultured In vitro

    Directory of Open Access Journals (Sweden)

    Farzaneh Chobsaz

    2011-01-01

    Full Text Available Background: Sildenafil is a selective inhibitor of cyclic-guanosine monphosphat-specificphosphodiesterase type 5. It increases intracellular nitric oxide (NO production in some cells.There are reports on its positive effect on uterine circulation, endometrial thickness, and infertilityimprovement. Endometrial epithelial cells (EEC play an important role in embryo attachment andimplantation. The present work investigates the effect of sildenafil on human EEC and their NOsecretion in vitro.Materials and Methods: In this experimental in vitro study, endometrial biopsies (n=10 werewashed in a phosphate buffered solution (PBS and digested with collagenase I (2 mg/ml in DMEM/F12 medium at 37°C for 90 minutes. Epithelial glands were collected by sequential filtrationthrough nylon meshes (70 and 40 μm pores, respectively. Epithelial glands were then treated withtrypsin to obtain individual cells. The cells were counted and divided into four groups: control and1, 10, and 20 μM sildenafil concentrations. Cells were cultured for 15 days at 37ºC and 5% CO2; themedia were changed every 3 days, and their supernatants were collected for the NO assay. NO wasmeasured by standard Greiss methods. Data were analyzed by one way ANOVA.Results: There was no significant difference between groups in cell count and NO secretion, but thelevel of NO increased slightly in the experimental groups. The 10 μM dose showed the highest cellcount. EEC morphology changed into long spindle cells in the case groups.Conclusion: Sildenafil (1, 10, and 20 μM showed a mild proliferative effect on human EECnumbers, but no significant change was seen in NO production.

  16. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease

    Directory of Open Access Journals (Sweden)

    Peter L. Lee

    2016-06-01

    Conclusions: mTORC1 activity in mature adipocytes is essential for maintaining normal adipose tissue growth and its selective loss in mature adipocytes leads to a progressive lipodystrophy disorder and systemic metabolic disease that shares many of the hallmarks of human congenital generalized lipodystrophy.

  17. Targeting AMP-activated protein kinase in adipocytes to modulate obesity-related adipokine production associated with insulin resistance and breast cancer cell proliferation

    Directory of Open Access Journals (Sweden)

    Grisouard Jean

    2011-07-01

    Full Text Available Abstract Background Adipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance. AMP-activated protein kinase (AMPK activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS link systemic inflammation to high fat diet-induced insulin resistance. Modulation of LPS-induced adipokine production by metformin and AMPK activation might represent an alternative way to treat both, insulin resistance and breast cancer. Methods Human preadipocytes obtained from surgical biopsies were expanded and differentiated in vitro into adipocytes, and incubated with siRNA targeting AMPKalpha1 (72 h, LPS (24 h, 100 μg/ml and/or metformin (24 h, 1 mM followed by mRNA extraction and analyses. Additionally, the supernatant of preadipocytes or derived-adipocytes in culture for 24 h was used as conditioned media to evaluate MCF-7 breast cancer cell proliferation. Results Conditioned media from preadipocyte-derived adipocytes, but not from undifferentiated preadipocytes, increased MCF-7 cell proliferation (p Conclusions Adipocyte-secreted factors enhance breast cancer cell proliferation, while AMPK and metformin improve the LPS-induced adipokine imbalance. Possibly, AMPK activation may provide a new way not only to improve the obesity-related adipokine profile and insulin resistance, but also to prevent obesity-related breast cancer development and progression.

  18. Genome-wide profiling of peroxisome proliferator-activated receptor γ in primary epididymal, inguinal, and brown adipocytes reveals depot-selective binding correlated with gene expression

    DEFF Research Database (Denmark)

    Siersbæk, Majken; Loft, Anne; Jørgensen, Mads Malik Aagaard

    2012-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation and function. We and others have previously mapped PPARγ binding at a genome-wide level in murine and human adipocyte cell lines and in primary human adipocytes. However, little is known about...... how binding patterns of PPARγ differ between brown and white adipocytes and among different types of white adipocytes. Here we have employed chromatin immunoprecipitation combined with deep sequencing to map and compare PPARγ binding in in vitro differentiated primary mouse adipocytes isolated from...... epididymal, inguinal, and brown adipose tissues. While these PPARγ binding profiles are overall similar, there are clear depot-selective binding sites. Most PPARγ binding sites previously mapped in 3T3-L1 adipocytes can also be detected in primary adipocytes, but there are a large number of PPARγ binding...

  19. Moderate Hypoxia Down-Regulates Interleukin-6 Secretion and TLR4 Expression in Human Sw.71 Placental Cells

    Directory of Open Access Journals (Sweden)

    Koumei Shirasuna

    2015-07-01

    Full Text Available Background/Aims: The placenta is a vital organ for pregnancy. Many in vitro placental experiments are conducted under 21% O2; however, O2 tension could influence cellular functions, including cytokine secretion. We investigated the effects of oxygen tension between moderate hypoxia (5% O2 and normoxia (21% O2 by testing the hypothesis that moderate hypoxia regulates cellular phenotypes differently from normoxia in human trophoblast cells. Methods and Results: Sw.71 trophoblast cells were incubated under normoxic or moderately hypoxic conditions. Cells were also treated with lipopolysaccharide (LPS as a Toll-like receptor 4 (TLR4 ligand inducing inflammation. Interleukin-6 (IL-6 as an inflammatory cytokine was determined, and TLR4, hypoxia-induced factor-1α (HIF1α, and reactive oxygen species (ROS production were detected. Moderate hypoxia increased HIF1α expression and cell proliferation and acted by two different mechanisms to decrease IL-6 secretion compared with normoxia: it limits the TLR4 expression and ROS production. Treatment with cobalt chloride as an HIF1 activator inhibited IL-6 secretion and TLR4 expression; this effect was reversed on treatment with PX-12 as an HIF1 suppressor. Conclusion: IL-6 secretion, TLR4 expression, and ROS production, classical markers of inflammation, are down-regulated by moderate hypoxia, and HIF1α and ROS have a potential to regulate these responses in human trophoblast cells.

  20. Quercetin Impacts Expression of Metabolism- and Obesity-Associated Genes in SGBS Adipocytes

    Directory of Open Access Journals (Sweden)

    Andreas Leiherer

    2016-05-01

    Full Text Available Obesity is characterized by the rapid expansion of visceral adipose tissue, resulting in a hypoxic environment in adipose tissue which leads to a profound change of gene expression in adipocytes. As a consequence, there is a dysregulation of metabolism and adipokine secretion in adipose tissue leading to the development of systemic inflammation and finally resulting in the onset of metabolic diseases. The flavonoid quercetin as well as other secondary plant metabolites also referred to as phytochemicals have anti-oxidant, anti-inflammatory, and anti-diabetic effects known to be protective in view of obesity-related-diseases. Nevertheless, its underlying molecular mechanism is still obscure and thus the focus of this study was to explore the influence of quercetin on human SGBS (Simpson Golabi Behmel Syndrome adipocytes’ gene expression. We revealed for the first time that quercetin significantly changed expression of adipokine (Angptl4, adipsin, irisin and PAI-1 and glycolysis-involved (ENO2, PFKP and PFKFB4 genes, and that this effect not only antagonized but in part even overcompensated the effect mediated by hypoxia in adipocytes. Thus, these results are explained by the recently proposed hypothesis that the protective effect of quercetin is not solely due to its free radical-scavenging activity but also to a direct effect on mitochondrial processes, and they demonstrate that quercetin might have the potential to counteract the development of obesity-associated complications.

  1. Adipocytes properties and crosstalk with immune system in obesity-related inflammation.

    Science.gov (United States)

    Maurizi, Giulia; Della Guardia, Lucio; Maurizi, Angela; Poloni, Antonella

    2018-01-01

    Obesity is a condition likely associated with several dysmetabolic conditions or worsening of cardiovascular and other chronic disturbances. A key role in this mechanism seem to be played by the onset of low-grade systemic inflammation, highlighting the importance of the interplay between adipocytes and immune system cells. Adipocytes express a complex and highly adaptive biological profile being capable to selectively activate different metabolic pathways in order to respond to environmental stimuli. It has been demonstrated how adipocytes, under appropriate stimulation, can easily differentiate and de-differentiate thereby converting themselves into different phenotypes according to metabolic necessities. Although underlying mechanisms are not fully understood, growing in adipocyte size and the inability of storing triglycerides under overfeeding conditions seem to be crucial for the switching to a dysfunctional metabolic profile, which is characterized by inflammatory and apoptotic pathways activation, and by the shifting to pro-inflammatory adipokines secretion. In obesity, changes in adipokines secretion along with adipocyte deregulation and fatty acids release into circulation contribute to maintain immune cells activation as well as their infiltration into regulatory organs. Over the well-established role of macrophages, recent findings suggest the involvement of new classes of immune cells such as T regulatory lymphocytes and neutrophils in the development inflammation and multi systemic worsening. Deeply understanding the pathways of adipocyte regulation and the de-differentiation process could be extremely useful for developing novel strategies aimed at curbing obesity-related inflammation and related metabolic disorders. © 2017 Wiley Periodicals, Inc.

  2. Identification of plant extracts with potential antidiabetic properties: effect on human peroxisome proliferator-activated receptor (PPAR), adipocyte differentiation and insulin-stimulated glucose uptake.

    Science.gov (United States)

    Christensen, Kathrine B; Minet, Ariane; Svenstrup, Henrik; Grevsen, Kai; Zhang, Hongbin; Schrader, Eva; Rimbach, Gerald; Wein, Silvia; Wolffram, Siegfried; Kristiansen, Karsten; Christensen, Lars P

    2009-09-01

    Thiazolidinediones (TZDs) are insulin sensitizing drugs used to treat type 2 diabetes