LDL-C levels in older people: Cholesterol homeostasis and the free radical theory of ageing converge.

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Mc Auley, Mark T; Mooney, Kathleen M

2017-07-01

The cardiovascular disease (CVD) risk factor, low density lipoprotein cholesterol (LDL-C) increases with age, up until the midpoint of life in males and females. However, LDL-C can decrease with age in older men and women. Intriguingly, a recent systematic review also revealed an inverse association between LDL-C levels and cardiovascular mortality in older people; low levels of LDL-C were associated with reduced risk of mortality. Such findings are puzzling and require a biological explanation. In this paper a hypothesis is proposed to explain these observations. We hypothesize that the free radical theory of ageing (FRTA) together with disrupted cholesterol homeostasis can account for these observations. Based on this hypothesis, dysregulated hepatic cholesterol homeostasis in older people is characterised by two distinct metabolic states. The first state accounts for an older person who has elevated plasma LDL-C. This state is underpinned by the FRTA which suggests there is a decrease in cellular antioxidant capacity with age. This deficiency enables hepatic reactive oxidative species (ROS) to induce the total activation of HMG-CoA reductase, the key rate limiting enzyme in cholesterol biosynthesis. An increase in cholesterol synthesis elicits a corresponding rise in LDL-C, due to the downregulation of LDL receptor synthesis, and increased production of very low density lipoprotein cholesterol (VLDL-C). In the second state of dysregulation, ROS also trigger the total activation of HMG-CoA reductase. However, due to an age associated decrease in the activity of cholesterol-esterifying enzyme, acyl CoA: cholesterol acyltransferase, there is restricted conversion of excess free cholesterol (FC) to cholesterol esters. Consequently, the secretion of VLDL-C drops, and there is a corresponding decrease in LDL-C. As intracellular levels of FC accumulate, this state progresses to a pathophysiological condition akin to nonalcoholic fatty liver disease. It is our

Pitfalls in the detection of cholesterol in Huntington's disease models.

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Marullo, Manuela; Valenza, Marta; Leoni, Valerio; Caccia, Claudio; Scarlatti, Chiara; De Mario, Agnese; Zuccato, Chiara; Di Donato, Stefano; Carafoli, Ernesto; Cattaneo, Elena

2012-10-11

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington's disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it.

Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

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Fabian Arenas

2017-11-01

Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

Advanced glycation end products affect cholesterol homeostasis by impairing ABCA1 expression on macrophages.

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Kamtchueng Simo, Olivier; Ikhlef, Souade; Berrougui, Hicham; Khalil, Abdelouahed

2017-08-01

Reverse cholesterol transport (RCT), which is intimately linked to high-density lipoproteins (HDLs), plays a key role in cholesterol homeostasis and the prevention of atherosclerosis. The goal of the present study was to investigate the effect of aging and advanced glycation end products (AGEs) on RCT as well as on other factors that may affect the antiatherogenic property of HDLs. The transfer of macrophage-derived cholesterol to the plasma and liver and then to the feces for elimination was significantly lower in aged mice than in young mice. Chronic injection of d -galactose (D-gal) or AGEs also significantly reduced RCT (65.3% reduction in [ 3 H]cholesterol levels in the plasma of D-gal-treated mice after 48 h compared with control mice, P cholesterol levels in the plasma, although the levels were lower than those of control mice. The in vitro incubation of HDLs with dicarbonyl compounds increased the carbonyl and conjugated diene content of HDLs and significantly reduced PON1 paraoxonase activity (87.4% lower than control HDLs, P cholesterol (69.1% decrease, P < 0.0001). Our results showed, for the first time, that RCT is altered with aging and that AGEs contribute significantly to this alteration.

Pitfalls in the detection of cholesterol in Huntington’s disease models

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Marullo, Manuela; Valenza, Marta; Leoni, Valerio; Caccia, Claudio; Scarlatti, Chiara; De Mario, Agnese; Zuccato, Chiara; Di Donato, Stefano; Carafoli, Ernesto; Cattaneo, Elena

2012-01-01

Background Abnormalities in brain cholesterol homeostasis have been reported in Huntington’s disease (HD), an adult-onset neurodegenerative disorder caused by an expansion in the number of CAG repeats in the huntingtin (HTT) gene. However, the results have been contradictory with respect to whether cholesterol levels increase or decrease in HD models. Biochemical and mass spectrometry methods show reduced levels of cholesterol precursors and cholesterol in HD cells and in the brains of several HD animal models. Abnormal brain cholesterol homeostasis was also inferred from studies in HD patients. In contrast, colorimetric and enzymatic methods indicate cholesterol accumulation in HD cells and tissues. Here we used several methods to investigate cholesterol levels in cultured cells in the presence or absence of mutant HTT protein. Results Colorimetric and enzymatic methods with low sensitivity gave variable results, whereas results from a sensitive analytical method, gas chromatography-mass spectrometry, were more reliable. Sample preparation, high cell density and cell clonality also influenced the detection of intracellular cholesterol. Conclusions Detection of cholesterol in HD samples by colorimetric and enzymatic assays should be supplemented by detection using more sensitive analytical methods. Care must be taken to prepare the sample appropriately. By evaluating lathosterol levels using isotopic dilution mass spectrometry, we confirmed reduced cholesterol biosynthesis in knock-in cells expressing the polyQ mutation in a constitutive or inducible manner. *Correspondence should be addressed to Elena Cattaneo: elena.cattaneo@unimi.it PMID:23145355

Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

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Hamm, Rebecca; Zeino, Maen [Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Frewert, Simon [Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken (Germany); Efferth, Thomas, E-mail: efferth@uni-mainz.de [Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany)

2014-11-15

Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H{sup +}-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation.

Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

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Hamm, Rebecca; Zeino, Maen; Frewert, Simon; Efferth, Thomas

2014-01-01

Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H + -ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation

The Role of Macrophage Lipophagy in Reverse Cholesterol Transport

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Se-Jin Jeong

2017-03-01

Full Text Available Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis.

Two-Compartment Model as a Teaching Tool for Cholesterol Homeostasis

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Wrona, Artur; Balbus, Joanna; Hrydziuszko, Olga; Kubica, Krystian

2015-01-01

Cholesterol is a vital structural and functional molecule in the human body that is only slightly soluble in water and therefore does not easily travels by itself in the bloodstream. To enable cholesterol's targeted delivery to cells and tissues, it is encapsulated by different fractions of lipoproteins, complex particles containing both proteins…

Lead nitrate-induced development of hypercholesterolemia in rats: sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis.

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Kojima, Misaki; Masui, Toshimitsu; Nemoto, Kiyomitsu; Degawa, Masakuni

2004-12-01

Changes in the gene expressions of hepatic enzymes responsible for cholesterol homeostasis were examined during the process of lead nitrate (LN)-induced development of hypercholesterolemia in male rats. Total cholesterol levels in the liver and serum were significantly increased at 3-72 h and 12-72 h, respectively, after LN-treatment (100 micromol/kg, i.v.). Despite the development of hypercholesterolemia, the genes for hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes (FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; CYP51, lanosterol 14alpha-demethylase) responsible for cholesterol biosynthesis were activated at 3-24 h and 12-18 h, respectively. On the other hand, the gene expression of cholesterol 7alpha-hydroxylase (CYP7A1), a catabolic enzyme of cholesterol, was remarkably suppressed at 3-72 h. The gene expression levels of cytokines interleukin-1beta (IL-1beta) and TNF-alpha, which activate the HMGR gene and suppress the CYP7A1 gene, were significantly increased at 1-3 h and 3-24 h, respectively. Furthermore, gene activation of SREBP-2, a gene activator of several cholesterogenic enzymes, occurred before the gene activations of FPPS, SQS and CYP51. This is the first report demonstrating sterol-independent gene regulation of hepatic enzymes responsible for cholesterol homeostasis in LN-treated male rats. The mechanisms for the altered-gene expressions of hepatic enzymes in LN-treated rats are discussed.

Rational Targeting of Cellular Cholesterol in Diffuse Large B-Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin.

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Rink, Jonathan S; Yang, Shuo; Cen, Osman; Taxter, Tim; McMahon, Kaylin M; Misener, Sol; Behdad, Amir; Longnecker, Richard; Gordon, Leo I; Thaxton, C Shad

2017-11-06

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

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Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

Relationship between plasma cholesterol levels and cholesterol esterification in isolated human mononuclear cells

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Dallongeville, J.; Davignon, J.; Lussier-Cacan, S.

1990-01-01

The authors studied the relationship between plasma lipoprotein concentrations and cholesterol esterification in freshly isolated human mononuclear cells from 27 normolipidemic and 32 hyperlipidemic individuals. Cells were either incubated for 5 hours with radiolabeled oleate immediately after isolation or were preincubated for 18 hours in the presence of exogenous cholesterol, and then incubated with [ 14 C]sodium-oleate-albumin complex. In the absence of exogenous cholesterol, control and hypercholesterolemic subjects had similarly low values of intracellular cholesterol esterification. In the presence of exogenous cholesterol, both hypertriglyceridemic and hypercholesterolemic subjects had higher cholesterol esterification than controls. There was a significant correlation between the rate of cholesterol esterification and plasma total cholesterol. These results suggest that plasma cholesterol levels may regulate mononuclear cell intra-cellular cholesterol esterification in humans

Aβ-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Aβ Secretion in Neural Cells.

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Najem, Dema; Bamji-Mirza, Michelle; Yang, Ze; Zhang, Wandong

2016-06-01

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) toxicity, tau pathology, insulin resistance, neuroinflammation, and dysregulation of cholesterol homeostasis, all of which play roles in neurodegeneration. Insulin has polytrophic effects on neurons and may be at the center of these pathophysiological changes. In this study, we investigated possible relationships among insulin signaling and cholesterol biosynthesis, along with the effects of Aβ42 on these pathways in vitro. We found that neuroblastoma 2a (N2a) cells transfected with the human gene encoding amyloid-β protein precursor (AβPP) (N2a-AβPP) produced Aβ and exhibited insulin resistance by reduced p-Akt and a suppressed cholesterol-synthesis pathway following insulin treatment, and by increased phosphorylation of insulin receptor subunit-1 at serine 612 (p-IRS-S612) as compared to parental N2a cells. Treatment of human neuroblastoma SH-SY5Y cells with Aβ42 also increased p-IRS-S612, suggesting that Aβ42 is responsible for insulin resistance. The insulin resistance was alleviated when N2a-AβPP cells were treated with higher insulin concentrations. Insulin increased Aβ release from N2a-AβPP cells, by which it may promote Aβ clearance. Insulin increased cholesterol-synthesis gene expression in SH-SY5Y and N2a cells, including 24-dehydrocholesterol reductase (DHCR24) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) through sterol-regulatory element-binding protein-2 (SREBP2). While Aβ42-treated SH-SY5Y cells exhibited increased HMGCR expression and c-Jun phosphorylation as pro-inflammatory responses, they also showed down-regulation of neuro-protective/anti-inflammatory DHCR24. These results suggest that Aβ42 may cause insulin resistance, activate JNK for c-Jun phosphorylation, and lead to dysregulation of cholesterol homeostasis, and that enhancing insulin signaling may relieve the insulin-resistant phenotype and the dysregulated cholesterol-synthesis pathway to promote A�

Synthesis of the oxysterol, 24(S, 25-epoxycholesterol, parallels cholesterol production and may protect against cellular accumulation of newly-synthesized cholesterol

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Brown Andrew J

2007-04-01

Full Text Available Abstract Aim The effects of 24(S,25-epoxycholesterol (24,25EC on aspects of cholesterol homeostasis is well-documented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most? Methods We used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-14C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays. Results We showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP

Intracellular transport of cholesterol in mammalian cells

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Brasaemle, D.L.

1989-01-01

The erythrocyte was selected as a simple cell for the study of transbilayer movement of cholesterol. Cholesterol oxidase was used to measure the distribution of [ 3 H]cholesterol across the erythrocyte membrane. Cholesterol oxidase was also used to estimate the rate of transport of low density lipoprotein (LDL) cholesterol to the plasma membrane of cultured Chinese hamster ovary (CHO) fibroblasts; the half-time of this process was 42 minutes. The rate of transport of LDL cholesterol to the plasma membrane was confirmed by a second procedure using amphotericin B. Amphotericin B was also used to estimate the rate of transport of endogenously synthesized cholesterol to the plasma membrane of CHO cells. New methodology was developed including improvements of the previously published cholesterol oxidase assay for plasma membrane cholesterol. A new method for detecting transport of cholesterol to the plasma membrane in cultured cells was developed using amphotericin B. Preliminary studies investigated the use of fluorescent polyenes, pimaricin and etruscomycin, as probes for plasma membrane cholesterol in transport studies. Finally, a modification of a previously published cell staining protocol yielded a simple, quantitative assay for cell growth

The influence of saponins on cell membrane cholesterol.

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Böttger, Stefan; Melzig, Matthias F

2013-11-15

We studied the influence of structurally different saponins on the cholesterol content of cellular membranes. Therefore a cell culture model using ECV-304 urinary bladder carcinoma cells was developed. To measure the cholesterol content we used radiolabeled (3)H-cholesterol which is chemically and physiologically identical to natural cholesterol. The cells were pre-incubated with (3)H-cholesterol and after a medium change, they were treated with saponins to assess a saponin-induced cholesterol liberation from the cell membrane. In another experiment the cells were pre-incubated with saponins and after a medium change, they were treated with (3)H-cholesterol to assess a saponin-induced inhibition of cholesterol uptake into the cell membrane. Furthermore, the membrane toxicity of all applied saponins was analyzed using extracellular LDH quantification and the general cytotoxicity was analyzed using a colorimetric MTT-assay and DNA quantification. Our results revealed a correlation between membrane toxicity and general cytotoxicity. We also compared the results from the experiments on the saponin-induced cholesterol liberation as well as the saponin-induced inhibition of cholesterol uptake with the membrane toxicity. A significant reduction in the cell membrane cholesterol content was noted for those saponins who showed membrane toxicity (IC50 saponins either liberated (3)H-cholesterol from intact cell membranes or blocked the integration of supplemented (3)H-cholesterol into the cell membrane. Saponins with little influence on the cell membrane (IC50 >100 μM) insignificantly altered the cell membrane cholesterol content. The results suggested that the general cytotoxicity of saponins is mainly dependent on their membrane toxicity and that the membrane toxicity might be caused by the loss of cholesterol from the cell membrane. We also analyzed the influence of a significantly membrane toxic saponin on the cholesterol content of intracellular membranes such as those

Normal Non-HDL Cholesterol, Low Total Cholesterol, and HDL Cholesterol Levels in Sickle Cell Disease Patients in the Steady State: A Case-Control Study of Tema Metropolis.

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Ephraim, Richard K D; Adu, Patrick; Ake, Edem; Agbodzakey, Hope; Adoba, Prince; Cudjoe, Obed; Agoni, Clement

2016-01-01

Background. Abnormal lipid homeostasis in sickle cell disease (SCD) is characterized by defects in plasma and erythrocyte lipids and may increase the risk of cardiovascular disease. This study assessed the lipid profile and non-HDL cholesterol level of SCD patients. Methods. A hospital-based cross-sectional study was conducted in 50 SCD patients, in the steady state, aged 8-28 years, attending the SCD clinic, and 50 healthy volunteers between the ages of 8-38 years. Serum lipids were determined by enzymatic methods and non-HDL cholesterol calculated by this formula: non-HDL-C = TC-HDL-C. Results. Total cholesterol (TC) ( p = 0.001) and high-density lipoprotein cholesterol (HDL-C) ( p < 0.0001) were significantly decreased in cases compared to controls. The levels of non-HDL-C, low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) were similar among the participants. The levels of decrease in TC and HDL were associated with whether a patient was SCD-SS or SCD-SC. Systolic blood pressure and diastolic blood pressure were each significantly associated with increased VLDL [SBP, p = 0.01, OR: 0.74 (CI: 0.6-0.93); DBP, p = 0.023, OR: 1.45 (CI: 1.05-2.0)]. Conclusion. Dyslipidemia is common among participants in this study. It was more pronounced in the SCD-SS than in SCD-SC. This dyslipidemia was associated with high VLDL as well as increased SBP and DBP.

Temozolomide, sirolimus and chloroquine is a new therapeutic combination that synergizes to disrupt lysosomal function and cholesterol homeostasis in GBM cells.

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Hsu, Sanford P C; Kuo, John S; Chiang, Hsin-Chien; Wang, Hsin-Ell; Wang, Yu-Shan; Huang, Cheng-Chung; Huang, Yi-Chun; Chi, Mau-Shin; Mehta, Minesh P; Chi, Kwan-Hwa

2018-01-23

Glioblastoma (GBM) cells are characterized by high phagocytosis, lipogenesis, exocytosis activities, low autophagy capacity and high lysosomal demand are necessary for survival and invasion. The lysosome stands at the cross roads of lipid biosynthesis, transporting, sorting between exogenous and endogenous cholesterol. We hypothesized that three already approved drugs, the autophagy inducer, sirolimus (rapamycin, Rapa), the autophagy inhibitor, chloroquine (CQ), and DNA alkylating chemotherapy, temozolomide (TMZ) could synergize against GBM. This repurposed triple therapy combination induced GBM apoptosis in vitro and inhibited GBM xenograft growth in vivo . Cytotoxicity is caused by induction of lysosomal membrane permeabilization and release of hydrolases, and may be rescued by cholesterol supplementation. Triple treatment inhibits lysosomal function, prevents cholesterol extraction from low density lipoprotein (LDL), and causes clumping of lysosome associated membrane protein-1 (LAMP-1) and lipid droplets (LD) accumulation. Co-treatment of the cell lines with inhibitor of caspases and cathepsin B only partially reverse of cytotoxicities, while N-acetyl cysteine (NAC) can be more effective. A combination of reactive oxygen species (ROS) generation from cholesterol depletion are the early event of underling mechanism. Cholesterol repletion abolished the ROS production and reversed the cytotoxicity from QRT treatment. The shortage of free cholesterol destabilizes lysosomal membranes converting aborted autophagy to apoptosis through either direct mitochondria damage or cathepsin B release. This promising anti-GBM triple therapy combination severely decreases mitochondrial function, induces lysosome-dependent apoptotic cell death, and is now poised for further clinical testing and validation.

Cholesterol Bilayer Domains in the Eye Lens Health: A Review.

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Widomska, Justyna; Subczynski, Witold K; Mainali, Laxman; Raguz, Marija

2017-12-01

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

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Jorgelina Buschiazzo

Full Text Available Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane and cholesteryl esters (stored in lipid droplets, revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs among seasons and a dynamic organizational structure

Agaricus brasiliensis (sun mushroom) affects the expression of genes related to cholesterol homeostasis.

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de Miranda, Aline Mayrink; Rossoni Júnior, Joamyr Victor; Souza E Silva, Lorena; Dos Santos, Rinaldo Cardoso; Silva, Marcelo Eustáquio; Pedrosa, Maria Lúcia

2017-06-01

The sun mushroom (Agaricus brasiliensis) is considered a major source of bioactive compounds with potential health benefits. Mushrooms typically act as lipid-lowering agents; however, little is known about the mechanisms of action of A. brasiliensis in biological systems. This study aimed to determine the underlying mechanism involved in the cholesterol-lowering effect of A. brasiliensis through the assessment of fecal and serum lipid profiles in addition to gene expression analysis of specific transcription factors, enzymes, and transporters involved in cholesterol homeostasis. Twenty-four albino Fischer rats approximately 90 days old, with an average weight of 205 g, were divided into four groups of 6 each and fed a standard AIN-93 M diet (C), hypercholesterolemic diet (H), hypercholesterolemic diet +1 % A. brasiliensis (HAb), or hypercholesterolemic diet +0.008 % simvastatin (HS) for 6 weeks. Simvastatin was used as a positive control, as it is a typical drug prescribed for lipid disorders. Subsequently, blood, liver, and feces samples were collected for lipid profile and quantitative real-time polymerase chain reaction gene expression analyses. Diet supplementation with A. brasiliensis significantly improved serum lipid profiles, comparable to the effect observed for simvastatin. In addition, A. brasiliensis dietary supplementation markedly promoted fecal cholesterol excretion. Increased expression of 7α-hydroxylase (CYP7A1), ATP-binding cassette subfamily G-transporters (ABCG5/G8), and low-density lipoprotein receptor (LDLR) was observed following A. brasiliensis administration. Our results suggest that consumption of A. brasiliensis improves the serum lipid profile in hypercholesterolemic rats by modulating the expression of key genes involved in hepatic cholesterol metabolism.

Can non-cholesterol sterols and lipoprotein subclasses distribution predict different patterns of cholesterol metabolism and statin therapy response?

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Gojkovic, Tamara; Vladimirov, Sandra; Spasojevic-Kalimanovska, Vesna; Zeljkovic, Aleksandra; Vekic, Jelena; Kalimanovska-Ostric, Dimitra; Djuricic, Ivana; Sobajic, Sladjana; Jelic-Ivanovic, Zorana

2017-03-01

Cholesterol homeostasis disorders may cause dyslipidemia, atherosclerosis progression and coronary artery disease (CAD) development. Evaluation of non-cholesterol sterols (NCSs) as synthesis and absorption markers, and lipoprotein particles quality may indicate the dyslipidemia early development. This study investigates associations of different cholesterol homeostasis patterns with low-density (LDL) and high-density lipoproteins (HDL) subclasses distribution in statin-treated and statin-untreated CAD patients, and potential use of aforementioned markers for CAD treatment optimization. The study included 78 CAD patients (47 statin-untreated and 31 statin-treated) and 31 controls (CG). NCSs concentrations were quantified using gas chromatography- flame ionization detection (GC-FID). Lipoprotein subclasses were separated by gradient gel electrophoresis. In patients, cholesterol-synthesis markers were significantly higher comparing to CG. Cholesterol-synthesis markers were inversely associated with LDL size in all groups. For cholesterol homeostasis estimation, each group was divided to good and/or poor synthetizers and/or absorbers according to desmosterol and β-sitosterol median values. In CG, participants with reduced cholesterol absorption, the relative proportion of small, dense LDL was higher in those with increased cholesterol synthesis compared to those with reduced synthesis (p<0.01). LDL I fraction was significantly higher in poor synthetizers/poor absorbers subgroup compared to poor synthetizers/good absorbers (p<0.01), and good synthetizers/poor absorbers (p<0.01). Statin-treated patients with increased cholesterol absorption had increased proportion of LDL IVB (p<0.05). The results suggest the existence of different lipoprotein abnormalities according to various patterns of cholesterol homeostasis. Desmosterol/β-sitosterol ratio could be used for estimating individual propensity toward dyslipidemia development and direct the future treatment.

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction.

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Ruan, Banzhan; Zhang, Bo; Chen, Ang; Yuan, Long; Liang, Jianqing; Wang, Manna; Zhang, Zhengrong; Fan, Jie; Yu, Xiaochen; Zhang, Xin; Niu, Zubiao; Zheng, You; Gu, Songzhi; Liu, Xiaoqing; Du, Hongli; Wang, Jufang; Hu, Xianwen; Gao, Lihua; Chen, Zhaolie; Huang, Hongyan; Wang, Xiaoning; Sun, Qiang

2018-01-01

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction. Copyright © 2017 Elsevier Inc. All rights reserved.

Osmotic homeostasis and NKLy lymphoma cells radiosensitivity

International Nuclear Information System (INIS)

Tishchenko, V.V.; Magda, I.N.

1992-01-01

In experiments with cells of ascites NKLy lymphoma differing in ploidy and position in the cell cycle, a study was made of the radiosensitivity, osmotic homeostasis peculiarities and thermoradiation changes in potassium content. It was shown that the resistance of osmotic homeostasis of NKLy cells to thermoradiation correlated with their radioresistance

Localization and role of NPC1L1 in cholesterol absorption in human intestine.

Science.gov (United States)

Sané, Alain Théophile; Sinnett, Daniel; Delvin, Edgard; Bendayan, Moise; Marcil, Valérie; Ménard, Daniel; Beaulieu, Jean-François; Levy, Emile

2006-10-01

Recent studies have documented the presence of Niemann-Pick C1-Like 1 (NPC1L1) in the small intestine and its capacity to transport cholesterol in mice and rats. The current investigation was undertaken to explore the localization and function of NPC1L1 in human enterocytes. Cell fractionation experiments revealed an NPC1L1 association with apical membrane of the enterocyte in human jejunum. Signal was also detected in lysosomes, endosomes, and mitochondria. Confirmation of cellular NPC1L1 distribution was obtained by immunocytochemistry. Knockdown of NPC1L1 caused a decline in the ability of Caco-2 cells to capture micellar [(14)C]free cholesterol. Furthermore, this NPC1L1 suppression resulted in increased and decreased mRNA levels and activity of HMG-CoA reductase, the rate-limiting step in cholesterol synthesis, and of ACAT, the key enzyme in cholesterol esterification, respectively. An increase was also noted in the transcriptional factor sterol-regulatory element binding protein that modulates cholesterol homeostasis. Efforts were devoted to define the impact of NPC1L1 knockdown on other mediators of cholesterol uptake. RT-PCR evidence is presented to show the significant decrease in the levels of scavenger receptor class B type I (SR-BI) with no changes in ABCA1, ABCG5, and cluster determinant 36 in NPC1L1-deficient Caco-2 cells. Together, our data suggest that NPC1L1 contributes to intestinal cholesterol homeostasis and possibly cooperates with SR-BI to mediate cholesterol absorption in humans.

Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake

Science.gov (United States)

Yang, Muhua; Liu, Weidong; Pellicane, Christina; Sahyoun, Christine; Joseph, Biny K.; Gallo-Ebert, Christina; Donigan, Melissa; Pandya, Devanshi; Giordano, Caroline; Bata, Adam; Nickels, Joseph T.

2014-01-01

Dysregulation of cholesterol homeostasis is associated with various metabolic diseases, including atherosclerosis and type 2 diabetes. The sterol response element binding protein (SREBP)-2 transcription factor induces the expression of genes involved in de novo cholesterol biosynthesis and low density lipoprotein (LDL) uptake, thus it plays a crucial role in maintaining cholesterol homeostasis. Here, we found that overexpressing microRNA (miR)-185 in HepG2 cells repressed SREBP-2 expression and protein level. miR-185-directed inhibition caused decreased SREBP-2-dependent gene expression, LDL uptake, and HMG-CoA reductase activity. In addition, we found that miR-185 expression was tightly regulated by SREBP-1c, through its binding to a single sterol response element in the miR-185 promoter. Moreover, we found that miR-185 expression levels were elevated in mice fed a high-fat diet, and this increase correlated with an increase in total cholesterol level and a decrease in SREBP-2 expression and protein. Finally, we found that individuals with high cholesterol had a 5-fold increase in serum miR-185 expression compared with control individuals. Thus, miR-185 controls cholesterol homeostasis through regulating SREBP-2 expression and activity. In turn, SREBP-1c regulates miR-185 expression through a complex cholesterol-responsive feedback loop. Thus, a novel axis regulating cholesterol homeostasis exists that exploits miR-185-dependent regulation of SREBP-2 and requires SREBP-1c for function. PMID:24296663

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

Science.gov (United States)

Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of doxazosin on cholesterol synthesis in cell culture

International Nuclear Information System (INIS)

D'Eletto, R.D.; Javitt, N.B.

1989-01-01

The effect of doxazosin on cholesterol synthesis was determined by measuring the content of deuterium-enriched cholesterol in rabbit fibroblasts with and without receptors for low-density lipoproteins (LDL) and in hepatoma (Hep G2 cells). Doxazosin, at concentrations of 5-20 mumol/L, increased LDL binding to hepatic cells in a dose-related manner. Also, in these hepatic cells, doxazosin produced dose-related decreases in both newly synthesized cholesterol and cholesterol ester. In rabbit fibroblasts that were LDL receptor negative, de novo cholesterol synthesis was markedly reduced by increasing concentrations of doxazosin. Taken together, these results suggest that doxazosin may have a direct inhibitory effect on cholesterol synthesis independent of the LDL receptor. The inhibition of cholesterol synthesis by doxazosin may cause cells to compensate by upregulating the LDL receptor, thereby increasing the importation of lipoprotein cholesterol and reducing LDL cholesterol in the medium. This hypothesis supports findings in the clinical setting whereby doxazosin has a beneficial effect on the lipid profile, and suggests a useful additional property for this antihypertensive agent

Macrophage heterogeneity and cholesterol homeostasis: classically-activated macrophages are associated with reduced cholesterol accumulation following treatment with oxidized LDL.

Science.gov (United States)

Chu, Eugene M; Tai, Daven C; Beer, Jennifer L; Hill, John S

2013-02-01

Macrophages are centrally involved during atherosclerosis development and are the predominant cell type that accumulates cholesterol in the plaque. Macrophages however, are heterogeneous in nature reflecting a variety of microenvironments and different phenotypes may be more prone to contribute towards atherosclerosis progression. Using primary human monocyte-derived macrophages, we sought to evaluate one aspect of atherogenic potential of different macrophage phenotypes by determining their propensity to associate with and accumulate oxidized low density lipoprotein (oxLDL). Classically-activated macrophages treated simultaneously with interferon γ (IFNγ) and tumor necrosis factor α (TNFα) associated with less oxLDL and accumulated less cholesterol compared to untreated controls. The combined treatment of IFNγ and TNFα reduced the mRNA expression of CD36 and the expression of both cell surface CD36 and macrophage scavenger receptor 1 (MSR1) protein. Under oxLDL loaded conditions, IFNγ and TNFα did not reduce macrophage protein expression of the transcription factor peroxisome proliferator-actived receptor γ (PPARγ) which is known to positively regulate CD36 expression. However, macrophages treated with IFNγ attenuated the ability of the PPARγ-specific agonist rosiglitazone from upregulating cell surface CD36 protein expression. Our results demonstrate that the observed reduction of cholesterol accumulation in macrophages treated with IFNγ and TNFα following oxLDL treatment was due at least in part to reduced cell surface CD36 and MSR1 protein expression. Copyright © 2012 Elsevier B.V. All rights reserved.

Tocotrienol Affects Oxidative Stress, Cholesterol Homeostasis and the Amyloidogenic Pathway in Neuroblastoma Cells: Consequences for Alzheimer’s Disease

Science.gov (United States)

Grimm, Marcus O. W.; Regner, Liesa; Mett, Janine; Stahlmann, Christoph P.; Schorr, Pascal; Nelke, Christopher; Streidenberger, Olga; Stoetzel, Hannah; Winkler, Jakob; Zaidan, Shatha R.; Thiel, Andrea; Endres, Kristina; Grimm, Heike S.; Volmer, Dietrich A.; Hartmann, Tobias

2016-01-01

One of the characteristics of Alzheimer´s disease (AD) is an increased amyloid load and an enhanced level of reactive oxidative species (ROS). Vitamin E has known beneficial neuroprotective effects, and previously, some studies suggested that vitamin E is associated with a reduced risk of AD due to its antioxidative properties. However, epidemiological studies and nutritional approaches of vitamin E treatment are controversial. Here, we investigate the effect of α-tocotrienol, which belongs to the group of vitamin E, on AD-relevant processes in neuronal cell lines. In line with the literature, α-tocotrienol reduced the ROS level in SH-SY5Y cells. In the presence of tocotrienols, cholesterol and cholesterol esters, which have been shown to be risk factors in AD, were decreased. Besides the unambiguous positive effects of tocotrienol, amyloid-β (Aβ) levels were increased accompanied by an increase in the activity of enzymes responsible for Aβ production. Proteins and gene expression of the secretases and their components remained unchanged, whereas tocotrienol accelerates enzyme activity in cell-free assays. Besides enhanced Aβ production, tocotrienols inhibited Aβ degradation in neuro 2a (N2a)-cells. Our results might help to understand the controversial findings of vitamin E studies and demonstrate that besides the known positive neuroprotective properties, tocotrienols also have negative characteristics with respect to AD. PMID:27801864

Tocotrienol Affects Oxidative Stress, Cholesterol Homeostasis and the Amyloidogenic Pathway in Neuroblastoma Cells: Consequences for Alzheimer’s Disease

Directory of Open Access Journals (Sweden)

Marcus O. W. Grimm

2016-10-01

Full Text Available One of the characteristics of Alzheimer´s disease (AD is an increased amyloid load and an enhanced level of reactive oxidative species (ROS. Vitamin E has known beneficial neuroprotective effects, and previously, some studies suggested that vitamin E is associated with a reduced risk of AD due to its antioxidative properties. However, epidemiological studies and nutritional approaches of vitamin E treatment are controversial. Here, we investigate the effect of α-tocotrienol, which belongs to the group of vitamin E, on AD-relevant processes in neuronal cell lines. In line with the literature, α-tocotrienol reduced the ROS level in SH-SY5Y cells. In the presence of tocotrienols, cholesterol and cholesterol esters, which have been shown to be risk factors in AD, were decreased. Besides the unambiguous positive effects of tocotrienol, amyloid-β (Aβ levels were increased accompanied by an increase in the activity of enzymes responsible for Aβ production. Proteins and gene expression of the secretases and their components remained unchanged, whereas tocotrienol accelerates enzyme activity in cell-free assays. Besides enhanced Aβ production, tocotrienols inhibited Aβ degradation in neuro 2a (N2a-cells. Our results might help to understand the controversial findings of vitamin E studies and demonstrate that besides the known positive neuroprotective properties, tocotrienols also have negative characteristics with respect to AD.

Cholesterol: the debate should be terminated.

Science.gov (United States)

Nathan, David G

2017-07-01

Here, I offer personal perspectives on cholesterol homeostasis that reflect my belief that certain aspects of the debate have been overstated.-Nathan, D. G. Cholesterol: the debate should be terminated. © FASEB.

MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

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Elisa Balboa

2017-08-01

Full Text Available MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

Science.gov (United States)

Hannaoui, Samia; Shim, Su Yeon; Cheng, Yo Ching; Corda, Erica; Gilch, Sabine

2014-01-01

Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD): whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD. PMID:25419621

Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

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Samia Hannaoui

2014-11-01

Full Text Available Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD: whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD.

Mga2 transcription factor regulates an oxygen-responsive lipid homeostasis pathway in fission yeast

DEFF Research Database (Denmark)

Burr, Risa; Stewart, Emerson V; Shao, Wei

2016-01-01

-binding protein (SREBP) transcription factors regulate lipid homeostasis. In mammals, SREBP-2 controls cholesterol biosynthesis, whereas SREBP-1 controls triacylglycerol and glycerophospholipid biosynthesis. In the fission yeast Schizosaccharomyces pombe, the SREBP-2 homolog Sre1 regulates sterol homeostasis....... In the absence of mga2, fission yeast exhibited growth defects under both normoxia and low oxygen conditions. Mga2 transcriptional targets were enriched for lipid metabolism genes, and mga2Δ cells showed disrupted triacylglycerol and glycerophospholipid homeostasis, most notably with an increase in fatty acid...

Changes in cholesterol homeostasis modify the response of F1B hamsters to dietary very long chain n-3 and n-6 polyunsaturated fatty acids

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Rader Daniel J

2011-10-01

Full Text Available Abstract Background The plasma lipoprotein response of F1B Golden-Syrian hamsters fed diets high in very long chain (VLC n-3 polyunsaturated fatty acids (PUFA is paradoxical to that observed in humans. This anomaly is attributed, in part, to low lipoprotein lipase activity and is dependent on cholesterol status. To further elucidate the mechanism(s for these responses, hamsters were fed diets containing supplemental fish oil (VLC n-3 PUFA or safflower oil (n-6 PUFA (both 10% [w/w] and either cholesterol-supplemented (0.1% cholesterol [w/w] or cholesterol-depleted (0.01% cholesterol [w/w] and 10 days prior to killing fed 0.15% lovastatin+2% cholestyramine [w/w]. Results Cholesterol-supplemented hamsters fed fish oil, relative to safflower oil, had higher non-high density lipoprotein (HDL cholesterol and triglyceride concentrations (P Conclusion These data suggest disturbing cholesterol homeostasis in F1B hamsters alters their response to dietary fatty acids, which is reflected in altered plasma lipoprotein patterns and regulation of genes associated with their metabolism.

Localization and movement of newly synthesized cholesterol in rat ovarian granulosa cells

International Nuclear Information System (INIS)

Lange, Y.; Schmit, V.M.; Schreiber, J.R.

1988-01-01

The distribution and movement of cholesterol were studied in granulosa cells from the ovaries of estrogen-stimulated hypophysectomized immature rats cultured in serum-free medium. Plasma membrane cholesterol was distinguished from intracellular cholesterol with cholesterol oxidase, an enzyme that converts cell surface cholesterol to cholestenone, leaving intracellular cholesterol untouched. Using this approach we showed that 82% of unesterified cholesterol was associated with the plasma membrane in granulosa cells cultured for 48 h in serum-free medium in both the presence and absence of added androstenedione and FSH. FSH and androstenedione stimulated a marked increase in steroid hormone (progestin) production. The movement of newly synthesized cholesterol to the plasma membrane also was followed using cholesterol oxidase. Newly synthesized cholesterol reached the plasma membrane too rapidly to be measured in unstimulated cells (t1/2 less than 20 min); however, in cells stimulated by FSH and androstenedione, this rate was considerably slower (t1/2 approximately 2h). Therefore, cholesterol movement to the plasma membrane appears to be regulated by gonadotropins in these cells. We tested whether steroid biosynthesis used all cell cholesterol pools equally. To this end we administered [3H]acetate and [14C]acetate at different times and determined their relative specific contents in various steroids after defined intervals. The relative ages of the steroids (youngest to oldest) were: lanosterol, progestins, intracellular cholesterol, and plasma membrane cholesterol. This finding suggests that progestins use newly synthesized intracellular cholesterol in preference to preexisting intracellular or cell surface cholesterol

Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis.

Science.gov (United States)

Baker, Natasha; Sohn, Jihee; Tuan, Rocky S

2015-12-01

Stem cells are considered an important resource for tissue repair and regeneration. Their utilization in regenerative medicine will be aided by mechanistic insight into their responsiveness to external stimuli. It is likely that, similar to all other cells, an initial determinant of stem cell responsiveness to external stimuli is the organization of signaling molecules in cell membrane rafts. The clustering of signaling molecules in these cholesterol-rich membrane microdomains can affect the activity, specificity, cross-talk and amplification of cell signaling. Membrane rafts fall into two broad categories, non-caveolar and caveolar, based on the absence or presence, respectively, of caveolin scaffolding proteins. We have recently demonstrated that caveolin-1 (Cav-1) expression increases during, and knockdown of Cav-1 expression enhances, osteogenic differentiation of human bone marrow derived mesenchymal stem cells (MSCs). The increase in Cav-1 expression observed during osteogenesis is likely a negative feedback mechanism. We hypothesize that focal adhesion signaling pathways such as PI3K/Akt signaling may be negatively regulated by Cav-1 during human MSC osteogenesis. Human bone marrow MSCs were isolated from femoral heads obtained after total hip arthroplasty. MSCs were incubated in standard growth medium alone or induced to osteogenically differentiate by the addition of supplements (β-glycerophosphate, ascorbic acid, dexamethasone, and 1,25-dihydroxyvitamin D3). The activation of and requirement for PI3K/Akt signaling in MSC osteogenesis were assessed by immunoblotting for phosphorylated Akt, and treatment with the PI3K inhibitor LY294002 and Akt siRNA, respectively. The influences of Cav-1 and cholesterol membrane rafts on PI3K/Akt signaling were investigated by treatment with Cav-1 siRNA, methyl-β-cyclodextrin, or cholesterol oxidase, followed by cellular sub-fractionation and/or immunoblotting for phosphorylated Akt. LY294002 and Akt siRNA inhibited MSC

Domains of apolipoprotein E contributing to triglyceride and cholesterol homeostasis in vivo. Carboxyl-terminal region 203-299 promotes hepatic very low density lipoprotein-triglyceride secretion

NARCIS (Netherlands)

Kypreos, K.E.; Dijk, K.W. van; Zee, A. van der; Havekes, L.M.; Zannis, V.I.

2001-01-01

Apolipoprotein (apo) E has been implicated in cholesterol and triglyceride homeostasis in humans. At physiological concentration apoE promotes efficient clearance of apoE-containing lipoprotein remnants. However, high apoE plasma levels correlate with high plasma triglyceride levels. We have used

The role of serum non-cholesterol sterols as surrogate markers of absolute cholesterol synthesis and absorption.

Science.gov (United States)

Miettinen, T A; Gylling, H; Nissinen, M J

2011-10-01

To study the whole-body cholesterol metabolism in man, cholesterol synthesis and absorption need to be measured. Because of the complicated methods of the measurements, new approaches were developed including the analysis of serum non-cholesterol sterols. In current lipidologic papers and even in intervention studies, serum non-cholesterol sterols are frequently used as surrogate markers of cholesterol metabolism without any validation to the absolute metabolic variables. The present review compares serum non-cholesterol sterols with absolute measurements of cholesterol synthesis and absorption in published papers to find out whether the serum markers are valid indicators of cholesterol metabolism in various conditions. During statin treatment, during interventions of dietary fat, and in type 2 diabetes the relative and absolute variables of cholesterol synthesis and absorption were frequently but not constantly correlated with each other. In some occasions, especially in subjects with apolipoprotein E3/4 and E4/4 phenotypes, the relative metabolic markers were even more sensitive than the absolute ones to reflect changes in cholesterol metabolism during dietary interventions. Even in general population at very high absorption the homeostasis of cholesterol metabolism is disturbed damaging the validity of the serum markers. It is worth using several instead of only one precursor and absorption sterol marker for making conclusions of altered synthesis or absorption of cholesterol, and even then the presence of at least some absolute measurement is valuable. During consumption of plant sterol-enriched diets and in situations of interfered cholesterol homeostasis the relative markers do not adequately reflect cholesterol metabolism. Accordingly, the validity of the relative markers of cholesterol metabolism should not be considered as self-evident. Copyright © 2011 Elsevier B.V. All rights reserved.

Intestinal Farnesoid X Receptor Controls Transintestinal Cholesterol Excretion in Mice

NARCIS (Netherlands)

Boer, J.F. de; Schonewille, M.; Boesjes, M.; Wolters, H.; Bloks, V.W.; Bos, T.; Dijk, T.H. van; Jurdzinski, A.; Boverhof, R.; Wolters, J.C.; Kuivenhoven, J.A.; Deursen, J.M.A. van; Elferink, R.P.; Moschetta, A.; Kremoser, C.; Verkade, H.J.; Kuipers, F.; Groen, A.K.

2017-01-01

BACKGROUND & AIMS: The role of the intestine in the maintenance of cholesterol homeostasis increasingly is recognized. Fecal excretion of cholesterol is the last step in the atheroprotective reverse cholesterol transport pathway, to which biliary and transintestinal cholesterol excretion (TICE)

The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology.

Science.gov (United States)

Ayuyan, Artem G; Cohen, Fredric S

2018-02-27

Cholesterol is abundant in plasma membranes and exhibits a variety of interactions throughout the membrane. Chemical potential accounts for thermodynamic consequences of molecular interactions, and quantifies the effective concentration (i.e., activity) of any substance participating in a process. We have developed, to our knowledge, the first method to measure cholesterol chemical potential in plasma membranes. This was accomplished by complexing methyl-β-cyclodextrin with cholesterol in an aqueous solution and equilibrating it with an organic solvent containing dissolved cholesterol. The chemical potential of cholesterol was thereby equalized in the two phases. Because cholesterol is dilute in the organic phase, here activity and concentration were equivalent. This equivalence allowed the amount of cholesterol bound to methyl-β-cyclodextrin to be converted to cholesterol chemical potential. Our method was used to determine the chemical potential of cholesterol in erythrocytes and in plasma membranes of nucleated cells in culture. For erythrocytes, the chemical potential did not vary when the concentration was below a critical value. Above this value, the chemical potential progressively increased with concentration. We used standard cancer lines to characterize cholesterol chemical potential in plasma membranes of nucleated cells. This chemical potential was significantly greater for highly metastatic breast cancer cells than for nonmetastatic breast cancer cells. Chemical potential depended on density of the cancer cells. A method to alter and fix the cholesterol chemical potential to any value (i.e., a cholesterol chemical potential clamp) was also developed. Cholesterol content did not change when cells were clamped for 24-48 h. It was found that the level of activation of the transcription factor STAT3 increased with increasing cholesterol chemical potential. The cholesterol chemical potential may regulate signaling pathways. Copyright © 2018. Published by

Mitotic spindle defects and chromosome mis-segregation induced by LDL/cholesterol-implications for Niemann-Pick C1, Alzheimer's disease, and atherosclerosis.

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Antoneta Granic

Full Text Available Elevated low-density lipoprotein (LDL-cholesterol is a risk factor for both Alzheimer's disease (AD and Atherosclerosis (CVD, suggesting a common lipid-sensitive step in their pathogenesis. Previous results show that AD and CVD also share a cell cycle defect: chromosome instability and up to 30% aneuploidy-in neurons and other cells in AD and in smooth muscle cells in atherosclerotic plaques in CVD. Indeed, specific degeneration of aneuploid neurons accounts for 90% of neuronal loss in AD brain, indicating that aneuploidy underlies AD neurodegeneration. Cell/mouse models of AD develop similar aneuploidy through amyloid-beta (Aß inhibition of specific microtubule motors and consequent disruption of mitotic spindles. Here we tested the hypothesis that, like upregulated Aß, elevated LDL/cholesterol and altered intracellular cholesterol homeostasis also causes chromosomal instability. Specifically we found that: 1 high dietary cholesterol induces aneuploidy in mice, satisfying the hypothesis' first prediction, 2 Niemann-Pick C1 patients accumulate aneuploid fibroblasts, neurons, and glia, demonstrating a similar aneugenic effect of intracellular cholesterol accumulation in humans 3 oxidized LDL, LDL, and cholesterol, but not high-density lipoprotein (HDL, induce chromosome mis-segregation and aneuploidy in cultured cells, including neuronal precursors, indicating that LDL/cholesterol directly affects the cell cycle, 4 LDL-induced aneuploidy requires the LDL receptor, but not Aß, showing that LDL works differently than Aß, with the same end result, 5 cholesterol treatment disrupts the structure of the mitotic spindle, providing a cell biological mechanism for its aneugenic activity, and 6 ethanol or calcium chelation attenuates lipoprotein-induced chromosome mis-segregation, providing molecular insights into cholesterol's aneugenic mechanism, specifically through its rigidifying effect on the cell membrane, and potentially explaining why ethanol

Cholesterol Down-Regulates BK Channels Stably Expressed in HEK 293 Cells

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Deng, Xiu-Ling; Sun, Hai-Ying; Li, Gui-Rong

2013-01-01

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells and is involved in the regulation of a number of ion channels. The present study investigates how large conductance Ca2+-activated K+ (BK) channels are regulated by membrane cholesterol in BK-HEK 293 cells expressing both the α-subunit hKCa1.1 and the auxiliary β1-subunit or in hKCa1.1-HEK 293 cells expressing only the α-subunit hKCa1.1 using approaches of electrophysiology, molecular biology, and immunocytochemistry. Membrane cholesterol was depleted in these cells with methyl-β-cyclodextrin (MβCD), and enriched with cholesterol-saturated MβCD (MβCD-cholesterol) or low-density lipoprotein (LDL). We found that BK current density was decreased by cholesterol enrichment in BK-HEK 293 cells, with a reduced expression of KCa1.1 protein, but not the β1-subunit protein. This effect was fully countered by the proteasome inhibitor lactacystin or the lysosome function inhibitor bafilomycin A1. Interestingly, in hKCa1.1-HEK 293 cells, the current density was not affected by cholesterol enrichment, but directly decreased by MβCD, suggesting that the down-regulation of BK channels by cholesterol depends on the auxiliary β1-subunit. The reduced KCa1.1 channel protein expression was also observed in cultured human coronary artery smooth muscle cells with cholesterol enrichment using MβCD-cholesterol or LDL. These results demonstrate the novel information that cholesterol down-regulates BK channels by reducing KCa1.1 protein expression via increasing the channel protein degradation, and the effect is dependent on the auxiliary β1-subunit. PMID:24260325

Cholesterol metabolism in blood cells of irradiated rats

International Nuclear Information System (INIS)

Novoselova, E.G.; Kulagina, T.P.; Potekhina, N.I.

1985-01-01

Cholesterol metabolism in blood erythrocytes and lymphocytes of irradiated rats has been investigated. It has been found that at all terms and doses of irradiation, a suppression of the synthesis of erythrocyte cholesterol is observed. The increase of cholesterol quantiy in erythrocytes upon total gamma irradiation in the 10 Gr dose possibly is the result of growth of cholesterol transfer from plasma into erythrocyte cells. The study of the cholesterol synthesis in suspension of lymphocytes elminated from peripheral blood of control and irradiated rats has shown that at irradiation doses of 4 and 10 Gr in an hour acivation of cholesterol synthesis in vitro takes places

Increased membrane cholesterol in lymphocytes diverts T-cells toward an inflammatory response.

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Jacqueline Surls

Full Text Available Cell signaling for T-cell growth, differentiation, and apoptosis is initiated in the cholesterol-rich microdomains of the plasma membrane known as lipid rafts. Herein, we investigated whether enrichment of membrane cholesterol in lipid rafts affects antigen-specific CD4 T-helper cell functions. Enrichment of membrane cholesterol by 40-50% following squalene administration in mice was paralleled by an increased number of resting CD4 T helper cells in periphery. We also observed sensitization of the Th1 differentiation machinery through co-localization of IL-2Rα, IL-4Rα, and IL-12Rβ2 subunits with GM1 positive lipid rafts, and increased STAT-4 and STAT-5 phosphorylation following membrane cholesterol enrichment. Antigen stimulation or CD3/CD28 polyclonal stimulation of membrane cholesterol-enriched, resting CD4 T-cells followed a path of Th1 differentiation, which was more vigorous in the presence of increased IL-12 secretion by APCs enriched in membrane cholesterol. Enrichment of membrane cholesterol in antigen-specific, autoimmune Th1 cells fostered their organ-specific reactivity, as confirmed in an autoimmune mouse model for diabetes. However, membrane cholesterol enrichment in CD4(+Foxp3(+ T-reg cells did not alter their suppressogenic function. These findings revealed a differential regulatory effect of membrane cholesterol on the function of CD4 T-cell subsets. This first suggests that membrane cholesterol could be a new therapeutic target to modulate the immune functions, and second that increased membrane cholesterol in various physiopathological conditions may bias the immune system toward an inflammatory Th1 type response.

Changes in cholesterol homeostasis modify the response of F1B hamsters to dietary very long chain n-3 and n-6 polyunsaturated fatty acids.

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Lecker, Jaime L; Matthan, Nirupa R; Billheimer, Jeffrey T; Rader, Daniel J; Lichtenstein, Alice H

2011-10-21

The plasma lipoprotein response of F1B Golden-Syrian hamsters fed diets high in very long chain (VLC) n-3 polyunsaturated fatty acids (PUFA) is paradoxical to that observed in humans. This anomaly is attributed, in part, to low lipoprotein lipase activity and is dependent on cholesterol status. To further elucidate the mechanism(s) for these responses, hamsters were fed diets containing supplemental fish oil (VLC n-3 PUFA) or safflower oil (n-6 PUFA) (both 10% [w/w]) and either cholesterol-supplemented (0.1% cholesterol [w/w]) or cholesterol-depleted (0.01% cholesterol [w/w] and 10 days prior to killing fed 0.15% lovastatin+2% cholestyramine [w/w]). Cholesterol-supplemented hamsters fed fish oil, relative to safflower oil, had higher non-high density lipoprotein (HDL) cholesterol and triglyceride concentrations (P safflower oil, had lower non-HDL cholesterol and triglyceride concentrations (P < 0.001) which were associated with lower hepatic SREBP-1c (p < 0.05) but not apo B-100, apo E or ACAT-2 mRNA or protein levels. Independent of cholesterol status, fish oil fed hamsters had lower HDL cholesterol concentrations (p < 0.001), which were associated with lower hepatic apoA-I protein levels (p < 0.05). These data suggest disturbing cholesterol homeostasis in F1B hamsters alters their response to dietary fatty acids, which is reflected in altered plasma lipoprotein patterns and regulation of genes associated with their metabolism.

The intracellular cholesterol landscape: dynamic integrator of the immune response

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Fessler, Michael B.

2016-01-01

Cholesterol has typically been considered an exogenous, disease-related factor in immunity; however, recent literature suggests that a paradigm shift is in order. Sterols are now recognized to ligate several immune receptors. Altered flux through the mevalonic acid synthesis pathway also appears to be a required event in the antiviral interferon response of macrophages and in the activation, proliferation, and differentiation of T cells. In this review, evidence is discussed that suggests an intrinsic, ‘professional’ role for sterols and oxysterols in macrophage and T cell immunity. Host defense may have been the original selection pressure behind the development of mechanisms for intracellular cholesterol homeostasis. Functional coupling between sterol metabolism and immunity has fundamental implications for health and disease. PMID:27692616

Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation

International Nuclear Information System (INIS)

Fernandez, Carlos; Lobo, Maria del Val T.; Gomez-Coronado, Diego; Lasuncion, Miguel A.

2004-01-01

As an essential component of mammalian cell membranes, cells require cholesterol for proliferation, which is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-CoA. In addition to cholesterol, other non-sterol mevalonate derivatives are necessary for DNA synthesis, such as the phosphorylated forms of isopentane, farnesol, geranylgeraniol, and dolichol. The aim of the present study was to elucidate the role of cholesterol in mitosis. For this, human leukemia cells (HL-60) were incubated in a cholesterol-free medium and treated with SKF 104976, which inhibits cholesterol biosynthesis by blocking sterol 14α-demethylase, and the expression of relevant cyclins in the different phases of the cell cycle was analyzed by flow cytometry. Prolonged cholesterol starvation induced the inhibition of cytokinesis and the formation of polyploid cells, which were multinucleated and had mitotic aberrations. Supplementing the medium with cholesterol completely abolished these effects, demonstrating they were specifically due to cholesterol deficiency. This is the first evidence that cholesterol is essential for mitosis completion and that, in the absence of cholesterol, the cells fail to undergo cytokinesis, entered G1 phase at higher DNA ploidy (tetraploidy), and then progressed through S (rereplication) into G2, generating polyploid cells

Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

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Martinakova, Z.; Horilova, J.; Lajdova, I.; Marcek Chorvatova, A.

2014-12-01

Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

Emerging roles of the intestine in control of cholesterol metabolism

NARCIS (Netherlands)

Kruit, Janine-K.; Groen, Albert K.; van Berkel, Theo J.; Kuipers, Folkert

2006-01-01

The liver is considered the major "control center" for maintenance of whole body cholesterol homeostasis. This organ is the main site for de novo cholesterol synthesis, clears cholesterol-containing chylomicron remnants and low density lipoprotein particles from plasma and is the major contributor

Cholesterol as a modifying agent of the neurovascular unit structure and function under physiological and pathological conditions.

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Czuba, Ewelina; Steliga, Aleksandra; Lietzau, Grażyna; Kowiański, Przemysław

2017-08-01

The brain, demanding constant level of cholesterol, precisely controls its synthesis and homeostasis. The brain cholesterol pool is almost completely separated from the rest of the body by the functional blood-brain barrier (BBB). Only a part of cholesterol pool can be exchanged with the blood circulation in the form of the oxysterol metabolites such, as 27-hydroxycholesterol (27-OHC) and 24S-hydroxycholesterol (24S-OHC). Not only neurons but also blood vessels and neuroglia, constituting neurovascular unit (NVU), are crucial for the brain cholesterol metabolism and undergo precise regulation by numerous modulators, metabolites and signal molecules. In physiological conditions maintaining the optimal cholesterol concentration is important for the energetic metabolism, composition of cell membranes and myelination. However, a growing body of evidence indicates the consequences of the cholesterol homeostasis dysregulation in several pathophysiological processes. There is a causal relationship between hypercholesterolemia and 1) development of type 2 diabetes due to long-term high-fat diet consumption, 2) significance of the oxidative stress consequences for cerebral amyloid angiopathy and neurodegenerative diseases, 3) insulin resistance on progression of the neurodegenerative brain diseases. In this review, we summarize the current state of knowledge concerning the cholesterol influence upon functioning of the NVU under physiological and pathological conditions.

Carbohydrate restriction and dietary cholesterol modulate the expression of HMG-CoA reductase and the LDL receptor in mononuclear cells from adult men

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Volek Jeff S

2007-11-01

Full Text Available Abstract The liver is responsible for controlling cholesterol homeostasis in the body. HMG-CoA reductase and the LDL receptor (LDL-r are involved in this regulation and are also ubiquitously expressed in all major tissues. We have previously shown in guinea pigs that there is a correlation in gene expression of HMG-CoA reductase and the LDL-r between liver and mononuclear cells. The present study evaluated human mononuclear cells as a surrogate for hepatic expression of these genes. The purpose was to evaluate the effect of dietary carbohydrate restriction with low and high cholesterol content on HMG-CoA reductase and LDL-r mRNA expression in mononuclear cells. All subjects were counseled to consume a carbohydrate restricted diet with 10–15% energy from carbohydrate, 30–35% energy from protein and 55–60% energy from fat. Subjects were randomly assigned to either EGG (640 mg/d additional dietary cholesterol or SUB groups [equivalent amount of egg substitute (0 dietary cholesterol contributions per day] for 12 weeks. At the end of the intervention, there were no changes in plasma total or LDL cholesterol (LDL-C compared to baseline (P > 0.10 or differences in plasma total or LDL-C between groups. The mRNA abundance for HMG-CoA reductase and LDL-r were measured in mononuclear cells using real time PCR. The EGG group showed a significant decrease in HMG-CoA reductase mRNA (1.98 ± 1.26 to 1.32 ± 0.92 arbitrary units P

Role of low density lipoprotein-bound cholesterol esters in acute lymphoblastic leukemia cells

International Nuclear Information System (INIS)

Cutts, J.L.; Madden, E.A.; Melnykovych, G.

1986-01-01

The glucocorticoid sensitive CEM-C7 T-cell line was derived from human acute lymphoblastic leukemia cells by Norman and Thompson. Madden et al. have demonstrated that this growth inhibitory effect is due in part to a glucocorticoid-mediated inhibition of cholesterol synthesis and can be partially reversed by cholesterol dispersions. To further delineate the role of cholesterol in this growth inhibition, they have examined the ability of low density lipoprotein (LDL)-bound [ 3 H]cholesterol linoleate to reverse the growth inhibitory effect of 1 μM dexamethasone (Dex) on the CEM-C7 cells. LDL-bound cholesterol linoleate was unable to reverse the Dex-mediated growth inhibition, although incorporation of [ 14 C] acetate into free cholesterol was inhibited by 29%, following the Brown and Goldstein model. The presence of Dex further inhibited acetate incorporation into free cholesterol in the LDL-treated cells. Under all conditions, more than 99% of the acetate incorporated into cholesterol was present as free cholesterol, while over 87% of the LDL-bound cholesterol linoleate taken up remained in the ester compartment. These results indicate that CEM-C7 cells are unable to utilize LDL-bound cholesterol esters as a source of free cholesterol and rely on endogenous synthesis for their free cholesterol requirements

Cholesterol as a co-solvent and a ligand for membrane proteins

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Song, Yuanli; Kenworthy, Anne K; Sanders, Charles R

2014-01-01

As of mid 2013 a Medline search on “cholesterol” yielded over 200,000 hits, reflecting the prominence of this lipid in numerous aspects of animal cell biology and physiology under conditions of health and disease. Aberrations in cholesterol homeostasis underlie both a number of rare genetic disorders and contribute to common sporadic and complex disorders including heart disease, stroke, type II diabetes, and Alzheimer's disease. The corresponding author of this review and his lab stumbled only recently into the sprawling area of cholesterol research when they discovered that the amyloid precursor protein (APP) binds cholesterol, a topic covered by the Hans Neurath Award lecture at the 2013 Protein Society Meeting. Here, we first provide a brief overview of cholesterol-protein interactions and then offer our perspective on how and why binding of cholesterol to APP and its C99 domain (β-CTF) promotes the amyloidogenic pathway, which is closely related to the etiology of Alzheimer's disease. PMID:24155031

LDL Receptor-Related Protein-1 (LRP1 Regulates Cholesterol Accumulation in Macrophages.

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Anna P Lillis

Full Text Available Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1 to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR-deficient background (macLRP1-/-. After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.

The non-psychoactive plant cannabinoid, cannabidiol affects cholesterol metabolism-related genes in microglial cells.

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Rimmerman, Neta; Juknat, Ana; Kozela, Ewa; Levy, Rivka; Bradshaw, Heather B; Vogel, Zvi

2011-08-01

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that is clinically used in a 1:1 mixture with the psychoactive cannabinoid Δ(9)-tetrahydrocannabinol (THC) for the treatment of neuropathic pain and spasticity in multiple sclerosis. Our group previously reported that CBD exerts anti-inflammatory effects on microglial cells. In addition, we found that CBD treatment increases the accumulation of the endocannabinoid N-arachidonoyl ethanolamine (AEA), thus enhancing endocannabinoid signaling. Here we proceeded to investigate the effects of CBD on the modulation of lipid-related genes in microglial cells. Cell viability was tested using FACS analysis, AEA levels were measured using LC/MS/MS, gene array analysis was validated with real-time qPCR, and cytokine release was measured using ELISA. We report that CBD significantly upregulated the mRNAs of the enzymes sterol-O-acyl transferase (Soat2), which synthesizes cholesteryl esters, and of sterol 27-hydroxylase (Cyp27a1). In addition, CBD increased the mRNA of the lipid droplet-associated protein, perilipin2 (Plin2). Moreover, we found that pretreatment of the cells with the cholesterol chelating agent, methyl-β-cyclodextrin (MBCD), reversed the CBD-induced increase in Soat2 mRNA but not in Plin2 mRNA. Incubation with AEA increased the level of Plin2, but not of Soat2 mRNA. Furthermore, MBCD treatment did not affect the reduction by CBD of the LPS-induced release of the proinflammatory cytokine IL-1β. CBD treatment modulates cholesterol homeostasis in microglial cells, and pretreatment with MBCD reverses this effect without interfering with CBD's anti-inflammatory effects. The effects of the CBD-induced increase in AEA accumulation on lipid-gene expression are discussed.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis

DEFF Research Database (Denmark)

Luda, Katarzyna M.; Joeris, Thorsten; Persson, Emma K.

2016-01-01

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence...... dependent DCs in the maintenance of intestinal T cell homeostasis....

Control of Immune Cell Homeostasis and Function by lncRNAs.

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Mowel, Walter K; Kotzin, Jonathan J; McCright, Sam J; Neal, Vanessa D; Henao-Mejia, Jorge

2018-01-01

The immune system is composed of diverse cell types that coordinate responses to infection and maintain tissue homeostasis. In each of these cells, extracellular cues determine highly specific epigenetic landscapes and transcriptional profiles to promote immunity while maintaining homeostasis. New evidence indicates that long non-coding RNAs (lncRNAs) play crucial roles in epigenetic and transcriptional regulation in mammals. Thus, lncRNAs have emerged as key regulatory molecules of immune cell gene expression programs in response to microbial and tissue-derived cues. We review here how lncRNAs control the function and homeostasis of cell populations during immune responses, emphasizing the diverse molecular mechanisms by which lncRNAs tune highly contextualized transcriptional programs. In addition, we discuss the new challenges faced in interrogating lncRNA mechanisms and function in the immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

LXR regulates cholesterol uptake through Idol-dependent ubiquitination of the LDL receptor

NARCIS (Netherlands)

Zelcer, Noam; Hong, Cynthia; Boyadjian, Rima; Tontonoz, Peter

2009-01-01

Cellular cholesterol levels reflect a balance between uptake, efflux, and endogenous synthesis. Here we show that the sterol-responsive nuclear liver X receptor (LXR) helps maintain cholesterol homeostasis, not only through promotion of cholesterol efflux but also through suppression of low-density

Interlink between cholesterol & cell cycle in prostate carcinoma

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Govind Singh

2017-01-01

Interpretation & conclusions: The present findings along with increased expression of cell cycle protein cyclin E in the cell nucleus of the tumour tissue suggested the possibility of an intriguing role of cholesterol in the mechanism of cell cycle process of prostate cell proliferation.

Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1.

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Fumin Dong

Full Text Available ATP-binding cassette transporter A1 (ABCA1 plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I, a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.

Impact of high cholesterol and endoplasmic reticulum stress on metabolic diseases: An updated mini-review

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Erdi Sozen

2017-08-01

Full Text Available Endoplasmic reticulum (ER is the major site of protein folding and calcium storage. Beside the role of ER in protein homeostasis, it controls the cholesterol production and lipid-membrane biosynthesis as well as surviving and cell death signaling mechanisms in the cell. It is well-documented that elevated plasma cholesterol induces adverse effects in cardiovascular diseases (CVDs, liver disorders, such as non-alcoholic fatty liver disease (NAFLD, non-alcoholic steatosis hepatitis (NASH, and metabolic diseases which are associated with oxidative and ER stress. Recent animal model and human studies have showed high cholesterol and ER stress as an emerging factors involved in the development of many metabolic diseases. In this review, we will summarize the crucial effects of hypercholesterolemia and ER stress response in the pathogenesis of CVDs, NAFLD/NASH, diabetes and obesity which are major health problems in western countries. Keywords: Endoplasmic reticulum stress, High cholesterol, Cardiovascular diseases, Non-alcoholic fatty liver disease, Non-alcoholic steatosis hepatitis

Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis.

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Bevins, Charles L; Salzman, Nita H

2011-05-01

Building and maintaining a homeostatic relationship between a host and its colonizing microbiota entails ongoing complex interactions between the host and the microorganisms. The mucosal immune system, including epithelial cells, plays an essential part in negotiating this equilibrium. Paneth cells (specialized cells in the epithelium of the small intestine) are an important source of antimicrobial peptides in the intestine. These cells have become the focus of investigations that explore the mechanisms of host-microorganism homeostasis in the small intestine and its collapse in the processes of infection and chronic inflammation. In this Review, we provide an overview of the intestinal microbiota and describe the cell biology of Paneth cells, emphasizing the composition of their secretions and the roles of these cells in intestinal host defence and homeostasis. We also highlight the implications of Paneth cell dysfunction in susceptibility to chronic inflammatory bowel disease.

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux.

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Soazig Le Lay

Full Text Available Caveolin-1 (Cav1, a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs derived from wild type (WT or Cav1-deficient (Cav1(-/- animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/- cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/- MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/- cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

Ets transcription factor GABP controls T cell homeostasis and immunity.

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Luo, Chong T; Osmanbeyoglu, Hatice U; Do, Mytrang H; Bivona, Michael R; Toure, Ahmed; Kang, Davina; Xie, Yuchen; Leslie, Christina S; Li, Ming O

2017-10-20

Peripheral T cells are maintained in the absence of vigorous stimuli, and respond to antigenic stimulation by initiating cell cycle progression and functional differentiation. Here we show that depletion of the Ets family transcription factor GA-binding protein (GABP) in T cells impairs T-cell homeostasis. In addition, GABP is critically required for antigen-stimulated T-cell responses in vitro and in vivo. Transcriptome and genome-wide GABP-binding site analyses identify GABP direct targets encoding proteins involved in cellular redox balance and DNA replication, including the Mcm replicative helicases. These findings show that GABP has a nonredundant role in the control of T-cell homeostasis and immunity.

Reduced absorption and enhanced synthesis of cholesterol in patients with cystic fibrosis: a preliminary study of plasma sterols.

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Gelzo, Monica; Sica, Concetta; Elce, Ausilia; Dello Russo, Antonio; Iacotucci, Paola; Carnovale, Vincenzo; Raia, Valeria; Salvatore, Donatello; Corso, Gaetano; Castaldo, Giuseppe

2016-09-01

Low cholesterol is typically observed in the plasma of patients with cystic fibrosis (CF) contrasting with the subcellular accumulation of cholesterol demonstrated in CF cells and in mice models. However, the homeostasis of cholesterol has not been well investigated in patients with CF. We studied the plasma of 26 patients with CF and 33 unaffected controls campesterol and β-sitosterol as markers of intestinal absorption and lathosterol as a marker of de novo cholesterol biosynthesis by gas chromatography (GC-FID and GC-MS). Plasma campesterol and β-sitosterol results were significantly (p=0.01) lower while plasma lathosterol was significantly higher (p=0.001) in patients with CF as compared to control subjects. Plasma cholesterol results were significantly lower (p=0.01) in CF patients. Our data suggest that the impaired intestinal absorption of exogenous sterols in patients with CF stimulates the endogenous synthesis of cholesterol, but the levels of total cholesterol in plasma remain lower. This may be due to the CFTR dysfunction that reduces cholesterol blood excretion causing the accumulation of cholesterol in liver cells and in other tissues contributing to trigger CF chronic inflammation.

Calcium homeostasis in fly photoreceptor cells

NARCIS (Netherlands)

Oberwinkler, J

2002-01-01

In fly photoreceptor cells, two processes dominate the Ca2+ homeostasis: light-induced Ca2+ influx through members of the TRP family of ion channels, and Ca2+ extrusion by Na+/Ca2+ exchange.Ca2+ release from intracellular stores is quantitatively insignificant. Both, the light-activated channels and

Role of UBIAD1 in Intracellular Cholesterol Metabolism and Vascular Cell Calcification.

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Sha Liu

Full Text Available Vascular calcification is an important risk factor associated with mortality among patients with chronic kidney disease. Intracellular cholesterol metabolism is involved in the process of vascular cell calcification. In this study, we investigated the role of UbiA prenyltransferase domain containing 1 (UBIAD1 in intracellular cholesterol metabolism and vascular cell calcification, and identified its subcellular location. Primary human umbilical vein smooth muscle cells (HUVSMCs were incubated with either growth medium (1.4 mmol/L Pi or calcification medium (CM (3.0 mmol/L Pi. Under treatment with CM, HUVSMCs were further incubated with exogenous cholesterol, or menaquinone-4, a product of UBIAD1. The plasmid and small interfering RNA were transfected in HUVSMCs to alter the expression of UBIAD1. Matrix calcium quantitation, alkaline phosphatase activity, intracellular cholesterol level and menaquinone-4 level were measured. The expression of several genes involved in cholesterol metabolism were analyzed. Using an anti-UBIAD1 antibody, an endoplasmic reticulum marker and a Golgi marker, the subcellular location of UBIAD1 in HUVSMCs was analyzed. CM increased matrix calcium, alkaline phosphatase activity and intracellular cholesterol level, and reduced UBIAD1 expression and menaquinone-4 level. Addition of cholesterol contributed to increased matrix calcification and alkaline phosphatase activity in a dose-dependent manner. Elevated expression of UBIAD1 or menaquinone-4 in HUVSMCs treated with CM significantly reduced intracellular cholesterol level, matrix calcification and alkaline phosphatase activity, but increased menaquinone-4 level. Elevated expression of UBIAD1 or menaquinone-4 reduced the gene expression of sterol regulatory element-binding protein-2, and increased gene expression of ATP binding cassette transporters A1, which are in charge of cholesterol synthesis and efflux. UBIAD1 co-localized with the endoplasmic reticulum marker and

Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity.

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Westerterp, Marit; Gautier, Emmanuel L; Ganda, Anjali; Molusky, Matthew M; Wang, Wei; Fotakis, Panagiotis; Wang, Nan; Randolph, Gwendalyn J; D'Agati, Vivette D; Yvan-Charvet, Laurent; Tall, Alan R

2017-06-06

Autoimmune diseases such as systemic lupus erythematosus (SLE) are associated with increased cardiovascular disease and reduced plasma high-density lipoprotein (HDL) levels. HDL mediates cholesterol efflux from immune cells via the ATP binding cassette transporters A1 and G1 (ABCA1/G1). The significance of impaired cholesterol efflux pathways in autoimmunity is unknown. We observed that Abca1/g1-deficient mice develop enlarged lymph nodes (LNs) and glomerulonephritis suggestive of SLE. This lupus-like phenotype was recapitulated in mice with knockouts of Abca1/g1 in dendritic cells (DCs), but not in macrophages or T cells. DC-Abca1/g1 deficiency increased LN and splenic CD11b + DCs, which displayed cholesterol accumulation and inflammasome activation, increased cell surface levels of the granulocyte macrophage-colony stimulating factor receptor, and enhanced inflammatory cytokine secretion. Consequently, DC-Abca1/g1 deficiency enhanced T cell activation and T h 1 and T h 17 cell polarization. Nlrp3 inflammasome deficiency diminished the enlarged LNs and enhanced T h 1 cell polarization. These findings identify an essential role of DC cholesterol efflux pathways in maintaining immune tolerance. Copyright © 2017 Elsevier Inc. All rights reserved.

Integrating physiological regulation with stem cell and tissue homeostasis

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Nakada, Daisuke; Levi, Boaz P.; Morrison, Sean J.

2015-01-01

Summary Stem cells are uniquely able to self-renew, to undergo multilineage differentiation, and to persist throughout life in a number of tissues. Stem cells are regulated by a combination of shared and tissue-specific mechanisms and are distinguished from restricted progenitors by differences in transcriptional and epigenetic regulation. Emerging evidence suggests that other aspects of cellular physiology, including mitosis, signal transduction, and metabolic regulation also differ between stem cells and their progeny. These differences may allow stem cells to be regulated independently of differentiated cells in response to circadian rhythms, changes in metabolism, diet, exercise, mating, aging, infection, and disease. This allows stem cells to sustain homeostasis or to remodel relevant tissues in response to physiological change. Stem cells are therefore not only regulated by short-range signals that maintain homeostasis within their tissue of origin, but also by long-range signals that integrate stem cell function with systemic physiology. PMID:21609826

Cholesterol negatively regulates IL-9-producing CD8+ T cell differentiation and antitumor activity.

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Ma, Xingzhe; Bi, Enguang; Huang, Chunjian; Lu, Yong; Xue, Gang; Guo, Xing; Wang, Aibo; Yang, Maojie; Qian, Jianfei; Dong, Chen; Yi, Qing

2018-05-09

CD8 + T cells can be polarized into IL-9-secreting (Tc9) cells. We previously showed that adoptive therapy using tumor-specific Tc9 cells generated stronger antitumor responses in mouse melanoma than classical Tc1 cells. To understand why Tc9 cells exert stronger antitumor responses, we used gene profiling to compare Tc9 and Tc1 cells. Tc9 cells expressed different levels of cholesterol synthesis and efflux genes and possessed significantly lower cholesterol content than Tc1 cells. Unique to Tc9, but not other CD8 + or CD4 + T cell subsets, manipulating cholesterol content in polarizing Tc9 cells significantly affected IL-9 expression and Tc9 differentiation and antitumor response in vivo. Mechanistic studies showed that IL-9 was indispensable for Tc9 cell persistence and antitumor effects, and cholesterol or its derivatives inhibited IL-9 expression by activating liver X receptors (LXRs), leading to LXR Sumoylation and reduced p65 binding to Il9 promoter. Our study identifies cholesterol as a critical regulator of Tc9 cell differentiation and function. © 2018 Ma et al.

ILDR2: an endoplasmic reticulum resident molecule mediating hepatic lipid homeostasis.

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Kazuhisa Watanabe

Full Text Available Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. We used adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting "relief" of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, we investigated the possible participation of ILDR2 in ER stress responses. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. We conclude that manipulation of Ildr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time

Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

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Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

2018-01-01

Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

C282Y-HFE gene variant affects cholesterol metabolism in human neuroblastoma cells.

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Ali-Rahmani, Fatima; Huang, Michael A; Schengrund, C-L; Connor, James R; Lee, Sang Y

2014-01-01

Although disruptions in the maintenance of iron and cholesterol metabolism have been implicated in several cancers, the association between variants in the HFE gene that is associated with cellular iron uptake and cholesterol metabolism has not been studied. The C282Y-HFE variant is a risk factor for different cancers, is known to affect sphingolipid metabolism, and to result in increased cellular iron uptake. The effect of this variant on cholesterol metabolism and its possible relevance to cancer phenotype was investigated using wild type (WT) and C282Y-HFE transfected human neuroblastoma SH-SY5Y cells. Expression of C282Y-HFE in SH-SY5Y cells resulted in a significant increase in total cholesterol as well as increased transcription of a number of genes involved in its metabolism compared to cells expressing WT-HFE. The marked increase in expression of NPC1L1 relative to that of most other genes, was accompanied by a significant increase in expression of NPC1, a protein that functions in cholesterol uptake by cells. Because inhibitors of cholesterol metabolism have been proposed to be beneficial for treating certain cancers, their effect on the viability of C282Y-HFE neuroblastoma cells was ascertained. C282Y-HFE cells were significantly more sensitive than WT-HFE cells to U18666A, an inhibitor of desmosterol Δ24-reductase the enzyme catalyzing the last step in cholesterol biosynthesis. This was not seen for simvastatin, ezetimibe, or a sphingosine kinase inhibitor. These studies indicate that cancers presenting in carriers of the C282Y-HFE allele might be responsive to treatment designed to selectively reduce cholesterol content in their tumor cells.

The mevalonate pathway in neurons: It's not just about cholesterol.

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Moutinho, Miguel; Nunes, Maria João; Rodrigues, Elsa

2017-11-01

Cholesterol homeostasis greatly impacts neuronal function due to the essential role of this sterol in the brain. The mevalonate (MVA) pathway leads to the synthesis of cholesterol, but also supplies cells with many other intermediary molecules crucial for neuronal function. Compelling evidence point to a model in which neurons shutdown cholesterol synthesis, and rely on a shuttle derived from astrocytes to meet their cholesterol needs. Nevertheless, several reports suggest that neurons maintain the MVA pathway active, even with sustained cholesterol supply by astrocytes. Hence, in this review we focus not on cholesterol production, but rather on the role of the MVA pathway in the synthesis of particular intermediaries, namely isoprenoids, and on their role on neuronal function. Isoprenoids act as anchors for membrane association, after being covalently bound to proteins, such as most of the small guanosine triphosphate-binding proteins, which are critical to neuronal cell function. Based on literature, on our own results, and on the analysis of public transcriptomics databases, we raise the idea that in neurons there is a shift of the MVA pathway towards the non-sterol branch, responsible for isoprenoid synthesis, in detriment to post-squalene branch, and that this is ultimately essential for synaptic activity. Nevertheless new tools that facilitate imaging and the biochemical characterization and quantification of the prenylome in neurons and astrocytes are needed to understand the regulation of isoprenoid production and protein prenylation in the brain, and to analyze its differences on diverse physiological or pathological conditions, such as aging and neurodegenerative states. Copyright © 2017 Elsevier Inc. All rights reserved.

Leucine supplementation improves adiponectin and total cholesterol concentrations despite the lack of changes in adiposity or glucose homeostasis in rats previously exposed to a high-fat diet

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Donato Jose

2011-09-01

Full Text Available Abstract Background Studies suggest that leucine supplementation (LS has a therapeutic potential to prevent obesity and to promote glucose homeostasis. Furthermore, regular physical exercise is a widely accepted strategy for body weight maintenance and also for the prevention of obesity. The aim of this study was to determine the effect of chronic LS alone or combined with endurance training (ET as potential approaches for reversing the insulin resistance and obesity induced by a high-fat diet (HFD in rats. Methods Forty-seven rats were randomly divided into two groups. Animals were fed a control diet-low fat (n = 10 or HFD (n = 37. After 15 weeks on HFD, all rats received the control diet-low fat and were randomly divided according to treatment: reference (REF, LS, ET, and LS+ET (n = 7-8 rats per group. After 6 weeks of treatment, the animals were sacrificed and body composition, fat cell volume, and serum concentrations of total cholesterol, HDL-cholesterol, triacylglycerol, glucose, adiponectin, leptin and tumor necrosis factor-alpha (TNF-α were analyzed. Results At the end of the sixth week of treatment, there was no significant difference in body weight between the REF, LS, ET and LS+ET groups. However, ET increased lean body mass in rats (P = 0.019. In addition, ET was more effective than LS in reducing adiposity (P = 0.019, serum insulin (P = 0.022 and TNF-α (P = 0.044. Conversely, LS increased serum adiponectin (P = 0.021 levels and reduced serum total cholesterol concentration (P = 0.042. Conclusions The results showed that LS had no beneficial effects on insulin sensitivity or adiposity in previously obese rats. On the other hand, LS was effective in increasing adiponectin levels and in reducing total cholesterol concentration.

Dynamic thiol/disulphide homeostasis in patients with basal cell carcinoma.

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Demirseren, Duriye Deniz; Cicek, Cagla; Alisik, Murat; Demirseren, Mustafa Erol; Aktaş, Akın; Erel, Ozcan

2017-09-01

The aim of this study is to measure and compare the dynamic thiol/disulphide homeostasis of patients with basal cell carcinoma and healthy subjects with a newly developed and original method. Thirty four patients attending our outpatient clinic and clinically and histopathologically diagnosed as nodular basal cell carcinoma, and age and gender matched 30 healthy individuals have been involved in the study. Thiol/disulphide homeostasis tests have been measured with a novel automatic spectrophotometric method developed and the results have been compared statistically. Serum native thiol and disulphide levels in the patient and control group show a considerable variance statistically (p = 0.028, 0.039, respectively). Total thiol levels do not reveal a considerable variation (p = 0.094). Disulphide/native thiol ratios and native thiol/total thiol ratios also show a considerable variance statistically (p = 0.012, 0.013, 0.010, respectively). Thiol disulphide homeostasis in patients with basal cell carcinoma alters in the way that disulphide gets lower and thiols get higher. Thiol/disulphide level is likely to have a role in basal cell carcinoma pathogenesis.

Redox Homeostasis in Pancreatic beta Cells

Czech Academy of Sciences Publication Activity Database

Ježek, Petr; Dlasková, Andrea; Plecitá-Hlavatá, Lydie

2012-01-01

Roč. 2012, č. 2012 (2012), s. 932838 ISSN 1942-0900 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA ČR(CZ) GPP304/10/P204 Institutional support: RVO:67985823 Keywords : beta cells * reactive oxygen species homeostasis * mitochondria Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 3.393, year: 2012

Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells[S

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Huang, Huan; McIntosh, Avery L.; Atshaves, Barbara P.; Ohno-Iwashita, Yoshiko; Kier, Ann B.; Schroeder, Friedhelm

2010-01-01

While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains. PMID:20008119

Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype.

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Ngo, Kevin; Patil, Prashanti; McGowan, Sara J; Niedernhofer, Laura J; Robbins, Paul D; Kang, James; Sowa, Gwendolyn; Vo, Nam

2017-09-01

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1 -/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss. Published by Elsevier B.V.

Reactive Oxygen Species and Mitochondrial Homeostasis as Regulators of Stem Cell Fate and Function.

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Tan, Darren Q; Suda, Toshio

2018-07-10

The precise role and impact of reactive oxygen species (ROS) in stem cells, which are essential for lifelong tissue homeostasis and regeneration, remain of significant interest to the field. The long-term regenerative potential of a stem cell compartment is determined by the delicate balance between quiescence, self-renewal, and differentiation, all of which can be influenced by ROS levels. Recent Advances: The past decade has seen a growing appreciation for the importance of ROS and redox homeostasis in various stem cell compartments, particularly those of hematopoietic, neural, and muscle tissues. In recent years, the importance of proteostasis and mitochondria in relation to stem cell biology and redox homeostasis has garnered considerable interest. Here, we explore the reciprocal relationship between ROS and stem cells, with significant emphasis on mitochondria as a core component of redox homeostasis. We discuss how redox signaling, involving cell-fate determining protein kinases and transcription factors, can control stem cell function and fate. We also address the impact of oxidative stress on stem cells, especially oxidative damage of lipids, proteins, and nucleic acids. We further discuss ROS management in stem cells, and present recent evidence supporting the importance of mitochondrial activity and its modulation (via mitochondrial clearance, biogenesis, dynamics, and distribution [i.e., segregation and transfer]) in stem cell redox homeostasis. Therefore, elucidating the intricate links between mitochondria, cellular metabolism, and redox homeostasis is envisioned to be critical for our understanding of ROS in stem cell biology and its therapeutic relevance in regenerative medicine. Antioxid. Redox Signal. 00, 000-000.

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells.

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Erin K Purcell

Full Text Available The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%, ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.

Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

DEFF Research Database (Denmark)

Neuvonen, M.; Manna, M.; Mokkila, S.

2014-01-01

of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human...... fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either...... similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone....

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

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Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

The effects of membrane cholesterol and simvastatin on red blood cell deformability and ATP release.

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Forsyth, Alison M; Braunmüller, Susanne; Wan, Jiandi; Franke, Thomas; Stone, Howard A

2012-05-01

It is known that deformation of red blood cells (RBCs) is linked to ATP release from the cells. Further, membrane cholesterol has been shown to alter properties of the cell membrane such as fluidity and bending stiffness. Membrane cholesterol content is increased in some cardiovascular diseases, for example, in individuals with acute coronary syndromes and chronic stable angina, and therefore, because of the potential clinical relevance, we investigated the influence of altered RBC membrane cholesterol levels on ATP release. Because of the correlation between statins and reduced membrane cholesterol in vivo, we also investigated the effects of simvastatin on RBC deformation and ATP release. We found that reducing membrane cholesterol increases cell deformability and ATP release. We also found that simvastatin increases deformability by acting directly on the membrane in the absence of the liver, and that ATP release was increased for cells with enriched cholesterol after treatment with simvastatin. Copyright © 2012 Elsevier Inc. All rights reserved.

Microenvironmental regulation of stem cells in intestinal homeostasis and cancer

NARCIS (Netherlands)

Medema, Jan Paul; Vermeulen, Louis

2011-01-01

The identification of intestinal stem cells as well as their malignant counterparts, colon cancer stem cells, has undergone rapid development in recent years. Under physiological conditions, intestinal homeostasis is a carefully balanced and efficient interplay between stem cells, their progeny and

Cell Adhesion Molecule CD166/ALCAM Functions Within the Crypt to Orchestrate Murine Intestinal Stem Cell HomeostasisSummary

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Nicholas R. Smith

2017-05-01

Full Text Available Background & Aims: Intestinal epithelial homeostasis is maintained by active-cycling and slow-cycling stem cells confined within an instructive crypt-based niche. Exquisite regulating of these stem cell populations along the proliferation-to-differentiation axis maintains a homeostatic balance to prevent hyperproliferation and cancer. Although recent studies focus on how secreted ligands from mesenchymal and epithelial populations regulate intestinal stem cells (ISCs, it remains unclear what role cell adhesion plays in shaping the regulatory niche. Previously we have shown that the cell adhesion molecule and cancer stem cell marker, CD166/ALCAM (activated leukocyte cell adhesion molecule, is highly expressed by both active-cycling Lgr5+ ISCs and adjacent Paneth cells within the crypt base, supporting the hypothesis that CD166 functions to mediate ISC maintenance and signal coordination. Methods: Here we tested this hypothesis by analyzing a CD166–/– mouse combined with immunohistochemical, flow cytometry, gene expression, and enteroid culture. Results: We found that animals lacking CD166 expression harbored fewer active-cycling Lgr5+ ISCs. Homeostasis was maintained by expansion of the transit-amplifying compartment and not by slow-cycling Bmi1+ ISC stimulation. Loss of active-cycling ISCs was coupled with deregulated Paneth cell homeostasis, manifested as increased numbers of immature Paneth progenitors due to decreased terminal differentiation, linked to defective Wnt signaling. CD166–/– Paneth cells expressed reduced Wnt3 ligand expression and depleted nuclear β-catenin. Conclusions: These data support a function for CD166 as an important cell adhesion molecule that shapes the signaling microenvironment by mediating ISC–niche cell interactions. Furthermore, loss of CD166 expression results in decreased ISC and Paneth cell homeostasis and an altered Wnt microenvironment. Keywords: Intestinal Stem Cell, Homeostasis

Introducing inducible fluorescent split cholesterol oxidase to mammalian cells.

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Chernov, Konstantin G; Neuvonen, Maarit; Brock, Ivonne; Ikonen, Elina; Verkhusha, Vladislav V

2017-05-26

Cholesterol oxidase (COase) is a bacterial enzyme catalyzing the first step in the biodegradation of cholesterol. COase is an important biotechnological tool for clinical diagnostics and production of steroid drugs and insecticides. It is also used for tracking intracellular cholesterol; however, its utility is limited by the lack of an efficient temporal control of its activity. To overcome this we have developed a regulatable fragment complementation system for COase cloned from Chromobacterium sp. The enzyme was split into two moieties that were fused to FKBP (FK506-binding protein) and FRB (rapamycin-binding domain) pair and split GFP fragments. The addition of rapamycin reconstituted a fluorescent enzyme, termed split GFP-COase, the fluorescence level of which correlated with its oxidation activity. A rapid decrease of cellular cholesterol induced by intracellular expression of the split GFP-COase promoted the dissociation of a cholesterol biosensor D4H from the plasma membrane. The process was reversible as upon rapamycin removal, the split GFP-COase fluorescence was lost, and cellular cholesterol levels returned to normal. These data demonstrate that the split GFP-COase provides a novel tool to manipulate cholesterol in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

FLIM studies of 22- and 25-NBD-cholesterol in living HEK293 cells: Plasma membrane change induced by cholesterol depletion

Czech Academy of Sciences Publication Activity Database

Ostašov, Pavel; Sýkora, Jan; Brejchová, Jana; Olžyńska, Agnieszka; Hof, Martin; Svoboda, Petr

167-168, FEB-MAR (2013), s. 62-69 ISSN 0009-3084 R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 ; RVO:61388955 Keywords : cholesterol depletion * beta-Cyclodextrin * 22-NBD-cholesterol * 25-NBD-cholesterol * FLIM studies * intact HEK293 cells Subject RIV: CE - Biochemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 2.593, year: 2013

MicroRNA-27a decreases the level and efficiency of the LDL receptor and contributes to the dysregulation of cholesterol homeostasis.

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Alvarez, M Lucrecia; Khosroheidari, Mahdieh; Eddy, Elena; Done, Stefania C

2015-10-01

A strong risk factor for atherosclerosis- the leading cause of heart attacks and strokes- is the elevation of low-density lipoprotein cholesterol (LDL-C) in blood. The LDL receptor (LDLR) is the primary pathway for LDL-C removal from circulation, and their levels are increased by statins -the main treatment for high blood LDL-C. However, statins have low efficiency because they also increase PCSK9 which targets LDLR for degradation. Since microRNAs have recently emerged as key regulators of cholesterol homeostasis, our aim was to identify potential microRNA-based therapeutics to decrease blood LDL-C and prevent atherosclerosis. We over expressed and knocked down miR-27a in HepG2 cells to assess its effect on the expression of key players in the LDLR pathway using PCR Arrays, Elisas, and Western blots. We found that miR-27a decreases LDLR levels by 40% not only through a direct binding to its 3' untranslated region but also indirectly by inducing a 3-fold increase in PCSK9, which enhances LDLR degradation. Interestingly, miR-27a also directly decreases LRP6 and LDLRAP1, two other key players in the LDLR pathway that are required for efficient endocytosis of the LDLR-LDL-C complex in the liver. The inhibition of miR-27a using lock nucleic acids induced a 70% increase in LDLR levels and, therefore, it would be a more efficient treatment for hypercholesterolemia because of its desirable effects not only on LDLR but also on PCSK9. The results presented here provide evidence supporting the potential of miR-27a as a novel therapeutic target for the prevention of atherosclerosis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Trafficking of cholesterol from cell bodies to distal axons in Niemann Pick C1-deficient neurons.

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Karten, Barbara; Vance, Dennis E; Campenot, Robert B; Vance, Jean E

2003-02-07

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disorder. In cells lacking functional NPC1 protein, endocytosed cholesterol accumulates in late endosomes/lysosomes. We utilized primary neuronal cultures in which cell bodies and distal axons reside in separate compartments to investigate the requirement of NPC1 protein for transport of cholesterol from cell bodies to distal axons. We have recently observed that in NPC1-deficient neurons compared with wild-type neurons, cholesterol accumulates in cell bodies but is reduced in distal axons (Karten, B., Vance, D. E., Campenot, R. B., and Vance, J. E. (2002) J. Neurochem. 83, 1154-1163). We now show that NPC1 protein is expressed in both cell bodies and distal axons. In NPC1-deficient neurons, cholesterol delivered to cell bodies from low density lipoproteins (LDLs), high density lipoproteins, or cyclodextrin complexes was transported into axons in normal amounts, whereas transport of endogenously synthesized cholesterol was impaired. Inhibition of cholesterol synthesis with pravastatin in wild-type and NPC1-deficient neurons reduced axonal growth. However, LDLs restored a normal rate of growth to wild-type but not NPC1-deficient neurons treated with pravastatin. Thus, although LDL cholesterol is transported into axons of NPC1-deficient neurons, this source of cholesterol does not sustain normal axonal growth. Over the lifespan of NPC1-deficient neurons, these defects in cholesterol transport might be responsible for the observed altered distribution of cholesterol between cell bodies and axons and, consequently, might contribute to the neurological dysfunction in NPC disease.

Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

Science.gov (United States)

Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

2015-07-07

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine.

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Klunder, Leon J; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C D

2017-07-05

Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we highlight recent advances with regard to the molecular mechanisms of cell polarity-controlled epithelial homeostasis and immunity in the human intestine. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Cell-size distribution in epithelial tissue formation and homeostasis.

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Puliafito, Alberto; Primo, Luca; Celani, Antonio

2017-03-01

How cell growth and proliferation are orchestrated in living tissues to achieve a given biological function is a central problem in biology. During development, tissue regeneration and homeostasis, cell proliferation must be coordinated by spatial cues in order for cells to attain the correct size and shape. Biological tissues also feature a notable homogeneity of cell size, which, in specific cases, represents a physiological need. Here, we study the temporal evolution of the cell-size distribution by applying the theory of kinetic fragmentation to tissue development and homeostasis. Our theory predicts self-similar probability density function (PDF) of cell size and explains how division times and redistribution ensure cell size homogeneity across the tissue. Theoretical predictions and numerical simulations of confluent non-homeostatic tissue cultures show that cell size distribution is self-similar. Our experimental data confirm predictions and reveal that, as assumed in the theory, cell division times scale like a power-law of the cell size. We find that in homeostatic conditions there is a stationary distribution with lognormal tails, consistently with our experimental data. Our theoretical predictions and numerical simulations show that the shape of the PDF depends on how the space inherited by apoptotic cells is redistributed and that apoptotic cell rates might also depend on size. © 2017 The Author(s).

Innate lymphoid cells in tissue homeostasis and diseases.

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Ignacio, Aline; Breda, Cristiane Naffah Souza; Camara, Niels Olsen Saraiva

2017-08-18

Innate lymphoid cells (ILCs) are the most recently discovered family of innate immune cells. They are a part of the innate immune system, but develop from the lymphoid lineage. They lack pattern-recognition receptors and rearranged receptors, and therefore cannot directly mediate antigen specific responses. The progenitors specifically associated with the ILCs lineage have been uncovered, enabling the distinction between ILCs and natural killer cells. Based on the requirement of specific transcription factors and their patterns of cytokine production, ILCs are categorized into three subsets (ILC1, ILC2 and ILC3). First observed in mucosal surfaces, these cell populations interact with hematopoietic and non-hematopoietic cells throughout the body during homeostasis and diseases, promoting immunity, commensal microbiota tolerance, tissue repair and inflammation. Over the last 8 years, ILCs came into the spotlight as an essential cell type able to integrate diverse host immune responses. Recently, it became known that ILC subsets play a key role in immune responses at barrier surfaces, interacting with the microbiota, nutrients and metabolites. Since the liver receives the venous blood directly from the intestinal vein, the intestine and liver are essential to maintain tolerance and can rapidly respond to infections or tissue damage. Therefore, in this review, we discuss recent findings regarding ILC functions in homeostasis and disease, with a focus on the intestine and liver.

Quorum sensing in CD4+ T cell homeostasis: a hypothesis and a model.

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Afonso R.M. Almeida

2012-05-01

Full Text Available Homeostasis of lymphocyte numbers is believed to be due to competition between cellular populations for a common niche of restricted size, defined by the combination of interactions and trophic factors required for cell survival. Here we propose a new mechanism: homeostasis of lymphocyte numbers could also be achieved by the ability of lymphocytes to perceive the density of their own populations. Such a mechanism would be reminiscent of the primordial quorum sensing systems used by bacteria, in which some bacteria sense the accumulation of bacterial metabolites secreted by other elements of the population, allowing them to count the number of cells present and adapt their growth accordingly. We propose that homeostasis of CD4+ T cell numbers may occur via a quorum-sensing-like mechanism, where IL-2 is produced by activated CD4+ T cells and sensed by a population of CD4+ Treg cells that expresses the high-affinity IL-2Rα-chain and can regulate the number of activated IL-2-producing CD4+ T cells and the total CD4+T cell population. In other words, CD4+ T cell populations can restrain their growth by monitoring the number of activated cells, thus preventing uncontrolled lymphocyte proliferation during immune responses. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled T cell activation and autoimmunity. Finally, we present a mathematical model that describes the role of IL-2 and quorum-sensing mechanisms in CD4+ T cell homeostasis during an immune response.

MILD CHOLESTEROL DEPLETION REDUCES AMYLOID-β PRODUCTION BY IMPAIRING APP TRAFFICKING TO THE CELL SURFACE

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Guardia-Laguarta, Cristina; Coma, Mireia; Pera, Marta; Clarimón, Jordi; Sereno, Lidia; Agulló, José M.; Molina-Porcel, Laura; Gallardo, Eduard; Deng, Amy; Berezovska, Oksana; Hyman, Bradley T.; Blesa, Rafael; Gómez-Isla, Teresa; Lleó, Alberto

2009-01-01

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ40 and Aβ42 in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts. PMID:19457132

Sensibilization of escherichia coli cells by cholesterol incorporated into their membrane

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Breslev, S.E.; Rozenberg, O.A.; Noskin, L.A.; Stepanova, I.M.; Beketova, A.G.; Loshakova, L.V.; Kovaleva, I.G.

1984-01-01

It has been established earlier that a level of cell radiosensitivity is defined by membrane viscosity changing in a wide temperature range. Therefore in epsilon coli cells of a natural type lethal doses of gamma rays are increased approximately a 3.5 times at 45 deg C, as compared to 4 deg C. Cholesterol changing a phase state of membrane lipids was used as a modifying factor. Liposomes were used with the goal of effective bacteria transfer to a membrane. It is established that liposomes without cholesterol do not affect their radioresistance and an increase of its content leads to resistance decrease. The effect is attained only at a sufficient long time of incubation of cells with liposomes (10-16 h). At 4 deg C lipids of E. coli membrane are in a solid-crystalline state independently on pholesterol presence, because of this, radiosensitivity does not change. Temperature increase up to 45 deg C transfer a part of lipids to a liquid-crystalline state, thus decreasing membrane viscosity. In this case cholesterol manifests itself. The authors explain viscosity increase with a violation in functioning of those enzyme systems, which activity is connected with membrane structural state, including enzymes of DNA repair. The authors assume that the radiosensibilization effect of cholesterol introduction into a bacterial membrane in high-temperature cell irradiation is explained by this phenomenon

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

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Schäfer, Balázs; Orbán, Erika; Kele, Zoltán; Tömböly, Csaba

2015-01-01

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3β-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions. Copyright © 2015 John Wiley & Sons, Ltd.

Role of Membrane Cholesterol Levels in Activation of Lyn upon Cell Detachment

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Takao Morinaga

2018-06-01

Full Text Available Cholesterol, a major component of the plasma membrane, determines the physicalproperties of biological membranes and plays a critical role in the assembly of membranemicrodomains. Enrichment or deprivation of membrane cholesterol affects the activities of manysignaling molecules at the plasma membrane. Cell detachment changes the structure of the plasmamembrane and influences the localizations of lipids, including cholesterol. Recent studies showedthat cell detachment changes the activities of a variety of signaling molecules. We previously reportedthat the localization and the function of the Src-family kinase Lyn are critically regulated by itsmembrane anchorage through lipid modifications. More recently, we found that the localization andthe activity of Lyn were changed upon cell detachment, although the manners of which vary betweencell types. In this review, we highlight the changes in the localization of Lyn and a role of cholesterolin the regulation of Lyn’s activation following cell detachment.

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes.

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Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D

2016-03-08

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.

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Imjeti, Naga Salaija; Lebreton, Stéphanie; Paladino, Simona; de la Fuente, Erwin; Gonzalez, Alfonso; Zurzolo, Chiara

2011-12-01

Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis

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Jones, Steven; Boisvert, Annie; Naghi, Andrada; Hullin-Matsuda, Françoise; Greimel, Peter; Kobayashi, Toshihide; Papadopoulos, Vassilios; Culty, Martine

2016-01-01

Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10 μM GEN + 10 μM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN + MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10 μM combined GEN + MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRα agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN + MEHP. We propose a working model for GEN + MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally

The homeostasis of Plasmodium falciparum-infected red blood cells.

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Jakob M A Mauritz

2009-04-01

Full Text Available The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis. However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

Pitavastatin Differentially Modulates MicroRNA-Associated Cholesterol Transport Proteins in Macrophages.

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Haijun Zhang

Full Text Available There is emerging evidence identifying microRNAs (miRNAs as mediators of statin-induced cholesterol efflux, notably through the ATP-binding cassette transporter A1 (ABCA1 in macrophages. The objective of this study was to assess the impact of an HMG-CoA reductase inhibitor, pitavastatin, on macrophage miRNAs in the presence and absence of oxidized-LDL, a hallmark of a pro-atherogenic milieu. Treatment of human THP-1 cells with pitavastatin prevented the oxLDL-mediated suppression of miR-33a, -33b and -758 mRNA in these cells, an effect which was not uniquely attributable to induction of SREBP2. Induction of ABCA1 mRNA and protein by oxLDL was inhibited (30% by pitavastatin, while oxLDL or pitavastatin alone significantly induced and repressed ABCA1 expression, respectively. These findings are consistent with previous reports in macrophages. miRNA profiling was also performed using a miRNA array. We identified specific miRNAs which were up-regulated (122 and down-regulated (107 in THP-1 cells treated with oxLDL plus pitavastatin versus oxLDL alone, indicating distinct regulatory networks in these cells. Moreover, several of the differentially expressed miRNAs identified are functionally associated with cholesterol trafficking (six miRNAs in cells treated with oxLDL versus oxLDL plus pitavastatin. Our findings indicate that pitavastatin can differentially modulate miRNA in the presence of oxLDL; and, our results provide evidence that the net effect on cholesterol homeostasis is mediated by a network of miRNAs.

Mechanisms of foam cell formation in atherosclerosis.

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Chistiakov, Dimitry A; Melnichenko, Alexandra A; Myasoedova, Veronika A; Grechko, Andrey V; Orekhov, Alexander N

2017-11-01

Low-density lipoprotein (LDL) and cholesterol homeostasis in the peripheral blood is maintained by specialized cells, such as macrophages. Macrophages express a variety of scavenger receptors (SR) that interact with lipoproteins, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1). These cells also have several cholesterol transporters, including ATP-binding cassette transporter ABCA1, ABCG1, and SR-BI, that are involved in reverse cholesterol transport. Lipids internalized by phagocytosis are transported to late endosomes/lysosomes, where lysosomal acid lipase (LAL) digests cholesteryl esters releasing free cholesterol. Free cholesterol in turn is processed by acetyl-CoA acetyltransferase (ACAT1), an enzyme that transforms cholesterol to cholesteryl esters. The endoplasmic reticulum serves as a depot for maintaining newly synthesized cholesteryl esters that can be processed by neutral cholesterol ester hydrolase (NCEH), which generates free cholesterol that can exit via cholesterol transporters. In atherosclerosis, pro-inflammatory stimuli upregulate expression of scavenger receptors, especially LOX-1, and downregulate expression of cholesterol transporters. ACAT1 is also increased, while NCEH expression is reduced. This results in deposition of free and esterified cholesterol in macrophages and generation of foam cells. Moreover, other cell types, such as endothelial (ECs) and vascular smooth muscle cells (VSMCs), can also become foam cells. In this review, we discuss known pathways of foam cell formation in atherosclerosis.

Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

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Lee, Waisin; Xu, Mingjing; Li, Yue; Gu, Yong; Chen, Jianping; Wong, Derek; Fung, Peter C W; Shen, Jiangang

2011-10-01

Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death. Copyright © 2011 Elsevier Inc. All rights reserved.

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

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Duan Rui-Dong

2010-04-01

Full Text Available Abstract Background Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1 protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. Methods Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. Results We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. Conclusion

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression.

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Feng, Dan; Ohlsson, Lena; Duan, Rui-Dong

2010-04-19

Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells. Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification. We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 muM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 muM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively. Curcumin inhibits cholesterol uptake through suppression of NPC1L1

Effects of dietary beef tallow and soy oil on glucose and cholesterol homeostasis in normal and diabetic pigs

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Woollett, L.A.

1987-01-01

Toe valuate whether dietary fats of different degrees of unsaturation alter glucose and very low density lipoprotein-cholesterol (VLDL-CH) homeostasis, normal and alloxan-diabetic pigs were fed diets containing either beef tallow or soy oil as the primary source of fat for 6 weeks. After intra-arterial and oral doses of glucose, pigs fed soy oil had similar glucose and greater insulin concentrations in plasma when compared with pigs fed beef tallow. Beef tallow-fed pigs additionally were 40% more glucose effective than were soy oil-fed pigs. Disappearance of injected autologous 14 C-VLDL-CH was analyzed in pigs using a two-pool model. Diabetes resulted in a twofold increase in half-lives and a 60-fold increase in pool sizes of the primary and secondary components of VLDL-CH disappearance when compared with those of normal pigs. In normal pigs, feeding beef tallow resulted in longer half-lives of both components of VLDL-CH disappearance and no effect in pool size of both components of VLDL-CH disappearance than did feeding soy oil. In comparison, diabetic pigs fed beef tallow had a similar half-life of the primary component, a twofold shorter half-life of the secondary component, and threefold larger pool size of the primary component, and a similar pool size of the secondary component of VLDL-CH disappearance than did diabetic pigs fed soy oil. Thus, dietary fat seems to play an important role in regulation of glucose and VLDL-CH homeostasis in normal and diabetic animals

Cholesterol transfer at endosomal-organelle membrane contact sites.

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Ridgway, Neale D; Zhao, Kexin

2018-06-01

Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

Potentiating the antitumour response of CD8+ T cells by modulating cholesterol metabolism

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Yang, Wei; Bai, Yibing; Xiong, Ying; Zhang, Jin; Chen, Shuokai; Zheng, Xiaojun; Meng, Xiangbo; Li, Lunyi; Wang, Jing; Xu, Chenguang; Yan, Chengsong; Wang, Lijuan; Chang, Catharine C. Y.; Chang, Ta-Yuan; Zhang, Ti; Zhou, Penghui; Song, Bao-Liang; Liu, Wanli; Sun, Shao-cong; Liu, Xiaolong; Li, Bo-liang; Xu, Chenqi

2016-01-01

CD8+ T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment1–4. Reactivating the cytotoxicity of CD8+ T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8+ T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme5, led to potentiated effector function and enhanced proliferation of CD8+ but not CD4+ T cells. This is due to the increase in the plasma membrane cholesterol level of CD8+ T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8+ T cells were better than wild-type CD8+ T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile6,7, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy. PMID:26982734

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis.

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Luda, Katarzyna M; Joeris, Thorsten; Persson, Emma K; Rivollier, Aymeric; Demiri, Mimoza; Sitnik, Katarzyna M; Pool, Lieneke; Holm, Jacob B; Melo-Gonzalez, Felipe; Richter, Lisa; Lambrecht, Bart N; Kristiansen, Karsten; Travis, Mark A; Svensson-Frej, Marcus; Kotarsky, Knut; Agace, William W

2016-04-19

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αβ(+) and CD4(+)CD8αα(+) T cells; the latter requiring β8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis.

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Klose, Christoph S N; Artis, David

2016-06-21

Research over the last 7 years has led to the formal identification of innate lymphoid cells (ILCs), increased the understanding of their tissue distribution and has established essential functions of ILCs in diverse physiological processes. These include resistance to pathogens, the regulation of autoimmune inflammation, tissue remodeling, cancer and metabolic homeostasis. Notably, many ILC functions appear to be regulated by mechanisms distinct from those of other innate and adaptive immune cells. In this Review, we focus on how group 2 ILC (ILC2) and group 3 ILC (ILC3) responses are regulated and how these cells interact with other immune and non-immune cells to mediate their functions. We highlight experimental evidence from mouse models and patient-based studies that have elucidated the effects of ILCs on the maintenance of tissue homeostasis and the consequences for health and disease.

Redox homeostasis: the linchpin in stem cell self-renewal and differentiation.

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Wang, Kui; Zhang, Tao; Dong, Qiang; Nice, Edouard Collins; Huang, Canhua; Wei, Yuquan

2013-03-14

Stem cells are characterized by their unique ability of self-renewal to maintain the so-called stem cell pool. Over the past decades, reactive oxygen species (ROS) have been recognized as toxic aerobic metabolism byproducts that are harmful to stem cells, leading to DNA damage, senescence or cell death. Recently, a growing body of literature has shown that stem cells reside in redox niches with low ROS levels. The balance of Redox homeostasis facilitates stem cell self-renewal by an intricate network. Thus, to fully decipher the underlying molecular mechanisms involved in the maintenance of stem cell self-renewal, it is critical to address the important role of redox homeostasis in the regulation of self-renewal and differentiation of stem cells. In this regard, we will discuss the regulatory mechanisms involved in the subtly orchestrated balance of redox status in stem cells by scavenger antioxidant enzyme systems that are well monitored by the hypoxia niches and crucial redox regulators including forkhead homeobox type O family (FoxOs), apurinic/apyrimidinic (AP) endonuclease1/redox factor-1 (APE1/Ref-1), nuclear factor erythroid-2-related factor 2 (Nrf2) and ataxia telangiectasia mutated (ATM). We will also introduce several pivotal ROS-sensitive molecules, such as hypoxia-inducible factors, p38 mitogen-activated protein kinase (p38) and p53, involved in the redox-regulated stem cell self-renewal. Specifically, all the aforementioned molecules can act as 'redox sensors' by virtue of redox modifications of their cysteine residues, which are critically important in the control of protein function. Given the importance of redox homeostasis in the regulation of stem cell self-renewal, understanding the underlying molecular mechanisms involved will provide important new insights into stem cell biology.

Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine.

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McFarlane, Matthew R; Liang, Guosheng; Engelking, Luke J

2014-01-24

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes.

Insig Proteins Mediate Feedback Inhibition of Cholesterol Synthesis in the Intestine*

Science.gov (United States)

McFarlane, Matthew R.; Liang, Guosheng; Engelking, Luke J.

2014-01-01

Enterocytes are the only cell type that must balance the de novo synthesis and absorption of cholesterol, although the coordinate regulation of these processes is not well understood. Our previous studies demonstrated that enterocytes respond to the pharmacological blockade of cholesterol absorption by ramping up de novo sterol synthesis through activation of sterol regulatory element-binding protein-2 (SREBP-2). Here, we genetically disrupt both Insig1 and Insig2 in the intestine, two closely related proteins that are required for the feedback inhibition of SREBP and HMG-CoA reductase (HMGR). This double knock-out was achieved by generating mice with an intestine-specific deletion of Insig1 using Villin-Cre in combination with a germ line deletion of Insig2. Deficiency of both Insigs in enterocytes resulted in constitutive activation of SREBP and HMGR, leading to an 11-fold increase in sterol synthesis in the small intestine and producing lipidosis of the intestinal crypts. The intestine-derived cholesterol accumulated in plasma and liver, leading to secondary feedback inhibition of hepatic SREBP2 activity. Pharmacological blockade of cholesterol absorption was unable to further induce the already elevated activities of SREBP-2 or HMGR in Insig-deficient enterocytes. These studies confirm the essential role of Insig proteins in the sterol homeostasis of enterocytes. PMID:24337570

Cholesterol can modulate mitochondrial aquaporin-8 expression in human hepatic cells.

Science.gov (United States)

Danielli, Mauro; Capiglioni, Alejo M; Marrone, Julieta; Calamita, Giuseppe; Marinelli, Raúl A

2017-05-01

Hepatocyte mitochondrial aquaporin-8 (mtAQP8) works as a multifunctional membrane channel protein that facilitates the uptake of ammonia for its detoxification to urea as well as the mitochondrial release of hydrogen peroxide. Since early oligonucleotide microarray studies in liver of cholesterol-fed mice showed an AQP8 downregulation, we tested whether alterations of cholesterol content per se modulate mtAQP8 expression in human hepatocyte-derived Huh-7 cells. Cholesterol loading with methyl-β-cyclodextrin (mβCD):cholesterol complexes downregulated the proteolytic activation of cholesterol-responsive sterol regulatory element-binding protein (SREBP) transcriptions factors 1 and 2, and the expression of the target gene 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Under such conditions, mtAQP8 mRNA and protein expressions were significantly reduced. In contrast, cholesterol depletion using mβCD alone increased SREBP-1 and 2 activation and upregulated HMGCR and mtAQP8 mRNA and protein expressions. The results suggest that cholesterol can regulate transcriptionally human hepatocyte mtAQP8 expression likely via SREBPs. The functional implications of our findings are discussed. © 2017 IUBMB Life, 69(5):341-346, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Cholesterol Depletion from a Ceramide/Cholesterol Mixed Monolayer: A Brewster Angle Microscope Study

KAUST Repository

Mandal, Pritam

2016-06-01

Cholesterol is crucial to the mechanical properties of cell membranes that are important to cells’ behavior. Its depletion from the cell membranes could be dramatic. Among cyclodextrins (CDs), methyl beta cyclodextrin (MβCD) is the most efficient to deplete cholesterol (Chol) from biomembranes. Here, we focus on the depletion of cholesterol from a C16 ceramide/cholesterol (C16-Cer/Chol) mixed monolayer using MβCD. While the removal of cholesterol by MβCD depends on the cholesterol concentration in most mixed lipid monolayers, it does not depend very much on the concentration of cholesterol in C16-Cer/Chol monolayers. The surface pressure decay during depletion were described by a stretched exponential that suggested that the cholesterol molecules are unable to diffuse laterally and behave like static traps for the MβCD molecules. Cholesterol depletion causes morphology changes of domains but these disrupted monolayers domains seem to reform even when cholesterol level was low.

Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apoA-I from murine RAW 264.7 macrophages

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Allen Anne Marie

2012-12-01

Full Text Available Abstract Background Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function. Methods Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett’s or Bonferroni post t-tests, as appropriate. Results The positive control, resveratrol (24 h, significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; ppAbca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1 and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2, indicating profound dysregulation of cholesterol homeostasis. Conclusions Acute loss of mitochondrial function, and in particular Δψm, reduces

The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

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Cao, Wei; Kayama, Hisako; Chen, Mei Lan; Delmas, Amber; Sun, Amy; Kim, Sang Yong; Rangarajan, Erumbi S; McKevitt, Kelly; Beck, Amanda P; Jackson, Cody B; Crynen, Gogce; Oikonomopoulos, Angelos; Lacey, Precious N; Martinez, Gustavo J; Izard, Tina; Lorenz, Robin G; Rodriguez-Palacios, Alex; Cominelli, Fabio; Abreu, Maria T; Hommes, Daniel W; Koralov, Sergei B; Takeda, Kiyoshi; Sundrud, Mark S

2017-12-19

CD4 + T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4 + T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1 -/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1 -/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

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Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-01-01

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

LDL cholesterol counteracts the antitumour effect of tyrosine kinase inhibitors against renal cell carcinoma.

Science.gov (United States)

Naito, Sei; Makhov, Peter; Astsaturov, Igor; Golovine, Konstantin; Tulin, Alexei; Kutikov, Alexander; Uzzo, Robert G; Kolenko, Vladimir M

2017-04-25

Treatment with tyrosine kinase inhibitors (TKIs) significantly improves survival of patients with renal cell carcinoma (RCC). However, about one-quarter of the RCC patients are primarily refractory to treatment with TKIs. We examined viability of RCC and endothelial cells treated with low-density lipoprotein (LDL) and/or TKIs. Next, we validated the potential role of PI3K/AKT signalling in LDL-mediated TKI resistance. Finally, we examined the effect of a high-fat/high-cholesterol diet on the response of RCC xenograft tumours to sunitinib. The addition of LDL cholesterol increases activation of PI3K/AKT signalling and compromises the antitumour efficacy of TKIs against RCC and endothelial cells. Furthermore, RCC xenograft tumours resist TKIs in mice fed a high-fat/high-cholesterol diet. The ability of renal tumours to maintain their cholesterol homoeostasis may be a critical component of TKI resistance in RCC patients.

Biogenesis of plasma membrane cholesterol

International Nuclear Information System (INIS)

Lange, Y.

1986-01-01

A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from [ 3 H] acetate moved to the plasma membrane with a half-time of 1 h at 37 0 C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells

Science.gov (United States)

Asalla, Suman; Girada, Shravan Babu; Kuna, Ramya S.; Chowdhury, Debabrata; Kandagatla, Bhaskar; Oruganti, Srinivas; Bhadra, Utpal; Bhadra, Manika Pal; Kalivendi, Shasi Vardhan; Rao, Swetha Pavani; Row, Anupama; Ibrahim, A.; Ghosh, Partha Pratim; Mitra, Prasenjit

2016-06-01

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

NKT cell self-reactivity: evolutionary master key of immune homeostasis?

Science.gov (United States)

Issazadeh-Navikas, Shohreh

2012-04-01

Complex immune responses have evolved to protect multicellular organisms against the invasion of pathogens. This has exerted strong developmental pressure for specialized functions that can also limit damage to self-tissue. Two arms of immunity, the innate and adaptive immune systems, have evolved for quick, non-specific immune responses to pathogens and more efficient, long-lasting ones upon specific recognition of recurrent pathogens. Specialized cells have arisen as the sentinels of these functions, including macrophages, natural killer (NK), and T and B-lymphocytes. Interestingly, a population of immune cells that can exert both of these complex functions, NKT cells, not only share common functions but also exhibit shared cell surface markers of cells of both arms of the immune system. These features, in combination with sophisticated maintenance of immune homeostasis, will be discussed. The recent finding of self-peptide reactivity of NKT cells in the context of CD1d, with capacity to regulate multiple autoimmune and inflammatory conditions, motivates the current proposal that self-reactive NKT cells might be the ancestral link between present NK and T cells. Their parallel selection through evolution by higher vertebrates could be related to their central function as master regulators of immune homeostasis that in part is shared with regulatory T cells. Hypothetical views on how self-reactive NKT cells secure such a central role will also be proposed.

STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

DEFF Research Database (Denmark)

Garbarino, J.; Pan, M. H.; Chin, H. F.

2012-01-01

small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...

Cholesterol pathways affected by small molecules that decrease sterol levels in Niemann-Pick type C mutant cells.

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Madalina Rujoi

2010-09-01

Full Text Available Niemann-Pick type C (NPC disease is a genetically inherited multi-lipid storage disorder with impaired efflux of cholesterol from lysosomal storage organelles.The effect of screen-selected cholesterol lowering compounds on the major sterol pathways was studied in CT60 mutant CHO cells lacking NPC1 protein. Each of the selected chemicals decreases cholesterol in the lysosomal storage organelles of NPC1 mutant cells through one or more of the following mechanisms: increased cholesterol efflux from the cell, decreased uptake of low-density lipoproteins, and/or increased levels of cholesteryl esters. Several chemicals promote efflux of cholesterol to extracellular acceptors in both non-NPC and NPC1 mutant cells. The uptake of low-density lipoprotein-derived cholesterol is inhibited by some of the studied compounds.Results herein provide the information for prioritized further studies in identifying molecular targets of the chemicals. This approach proved successful in the identification of seven chemicals as novel inhibitors of lysosomal acid lipase (Rosenbaum et al, Biochim. Biophys. Acta. 2009, 1791:1155-1165.

Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

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Bárbara Hissa

Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

Regulatory T Cells in Post-stroke Immune Homeostasis.

Science.gov (United States)

Liesz, Arthur; Kleinschnitz, Christoph

2016-08-01

The secondary neuroinflammatory response has come into focus of experimental stroke research. Immunological mechanisms after acute stroke are being investigated in the hope to identify novel and druggable pathways that contribute to secondary infarct growth after stroke. Among a variety of neuroimmunological events after acute brain ischemia, including microglial activation, brain leukocyte invasion, and secretion of pro-inflammatory factors, lymphocytes have been identified as the key leukocyte subpopulation driving the neuroinflammatory response and contributing to stroke outcome. Several studies have shown that pro-inflammatory lymphocyte subpopulations worsen stroke outcome and that inhibiting their invasion to the injured brain is neuroprotective. In contrast to the effector functions of pro-inflammatory lymphocytes, regulatory T cells (Treg) are critically involved in maintaining immune homeostasis and have been characterized as disease-limiting protective cells in several inflammatory conditions, particularly in primary inflammatory diseases of the central nervous system (CNS). However, due to the complex function of regulatory cells in immune homeostasis and disease, divergent findings have been described for the role of Treg in stroke models. Emerging evidence suggests that this discrepancy arises from potentially differing functions of Treg depending on the predominant site of action within the neurovascular unit and the surrounding inflammatory milieu. This article will provide a comprehensive review of current findings on Treg in brain ischemia models and discuss potential reasons for the observed discrepancies.

Treg cell-IgA axis in maintenance of host immune homeostasis with microbiota

OpenAIRE

Feng, Ting; Elson, Charles O.; Cong, Yingzi

2010-01-01

The intestine is the home to a vast diversity of microbiota and a complex of mucosal immune system. Multiple regulatory mechanisms control host immune responses to microbiota and maintain intestinal immune homeostasis. This mini review will provide evidence indicating a Treg cell-IgA axis and such axis playing a major role in maintenance of intestinal homeostasis.

Intestinal stromal cells in mucosal immunity and homeostasis.

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Owens, B M J; Simmons, A

2013-03-01

A growing body of evidence suggests that non-hematopoietic stromal cells of the intestine have multiple roles in immune responses and inflammation at this mucosal site. Despite this, many still consider gut stromal cells as passive structural entities, with past research focused heavily on their roles in fibrosis, tumor progression, and wound healing, rather than their contributions to immune function. In this review, we discuss our current knowledge of stromal cells in intestinal immunity, highlighting the many immunological axes in which stromal cells have a functional role. We also consider emerging data that broaden the potential scope of their contribution to immunity in the gut and argue that these so-called "non-immune" cells are reclassified in light of their diverse contributions to intestinal innate immunity and the maintenance of mucosal homeostasis.

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth.

Science.gov (United States)

Lyu, Junfang; Yang, Eun Ju; Head, Sarah A; Ai, Nana; Zhang, Baoyuan; Wu, Changjie; Li, Ruo-Jing; Liu, Yifan; Yang, Chen; Dang, Yongjun; Kwon, Ho Jeong; Ge, Wei; Liu, Jun O; Shim, Joong Sup

2017-11-28

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Cholesterol in the retina: the best is yet to come

Science.gov (United States)

Pikuleva, Irina A.; Curcio, Christine A.

2014-01-01

Historically understudied, cholesterol in the retina is receiving more attention now because of genetic studies showing that several cholesterol-related genes are risk factors for age-related macular degeneration (AMD) and because eye pathology studies showing high cholesterol content of drusen, aging Bruch's membrane, and newly found subretinal lesions. The challenge before us is determining how the cholesterol-AMD link is realized. Meeting this challenge will require an excellent understanding these genes’ roles in retinal physiology and how chorioretinal cholesterol is maintained. In the first half of this review, we will succinctly summarize physico-chemical properties of cholesterol, its distribution in the human body, general principles of maintenance and metabolism, and differences in cholesterol handling in human and mouse that impact on experimental approaches. This information will provide a backdrop to the second part of the review focusing on unique aspects of chorioretinal cholesterol homeostasis, aging in Bruch's membrane, cholesterol in AMD lesions, a model for lesion biogenesis, a model for macular vulnerability based on vascular biology, and alignment of AMD-related genes and pathobiology using cholesterol and an atherosclerosis-like progression as unifying features. We conclude with recommendations for the most important research steps we can take towards delineating the cholesterol-AMD link. PMID:24704580

Cholesterol-lowing effect of taurine in HepG2 cell.

Science.gov (United States)

Guo, Junxia; Gao, Ya; Cao, Xuelian; Zhang, Jing; Chen, Wen

2017-03-16

A number of studies indicate that taurine promotes cholesterol conversion to bile acids by upregulating CYP7A1 gene expression. Few in vitro studies are concerned the concentration change of cholesterol and its product of bile acids, and the molecular mechanism of CYP7A1 induction by taurine. The levels of intracellular total cholesterol (TC), free cholesterol (FC), cholesterol ester (EC), total bile acids (TBA) and medium TBA were determined after HepG2 cells were cultured for 24/48 h in DMEM supplemented with taurine at the final concentrations of 1/10/20 mM respectively. The protein expressions of CYP7A1, MEK1/2, c-Jun, p-c-Jun and HNF-4α were detected. Taurine significantly reduced cellular TC and FC in dose -and time-dependent ways, and obviously increased intracellular/medium TBA and CYP7A1 expressions. There was no change in c-Jun expression, but the protein expressions of MEK1/2 and p-c-Jun were increased at 24 h and inhibited at 48 h by 20 mM taurine while HNF4α was induced after both of the 24 h and 48 h treatment. Taurine could enhance CYP7A1 expression by inducing HNF4α and inhibiting MEK1/2 and p-c-Jun expressions to promote intracellular cholesterol metabolism.

Effect of the combinations between pea proteins and soluble fibres on cholesterolaemia and cholesterol metabolism in rats.

Science.gov (United States)

Parolini, Cinzia; Manzini, Stefano; Busnelli, Marco; Rigamonti, Elena; Marchesi, Marta; Diani, Erika; Sirtori, Cesare R; Chiesa, Giulia

2013-10-01

Many functional foods and dietary supplements have been reported to be beneficial for the management of dyslipidaemia, one of the major risk factors for CVD. Soluble fibres and legume proteins are known to be a safe and practical approach for cholesterol reduction. The present study aimed at investigating the hypocholesterolaemic effect of the combinations of these bioactive vegetable ingredients and their possible effects on the expression of genes regulating cholesterol homeostasis. A total of six groups of twelve rats each were fed, for 28 d, Nath's hypercholesterolaemic diets, differing in protein and fibre sources, being, respectively, casein and cellulose (control), pea proteins and cellulose (pea), casein and oat fibres (oat), casein and apple pectin (pectin), pea proteins and oat fibres (pea+oat) and pea proteins and apple pectin (pea+pectin). Administration of each vegetable-containing diet was associated with lower total cholesterol concentrations compared with the control. The combinations (pea+oat and pea+pectin) were more efficacious than fibres alone in modulating cholesterolaemia ( - 53 and - 54%, respectively, at 28 d; Ppea proteins, a lower hepatic cholesterol content (Ppea proteins and oat fibres or apple pectin are extremely effective in lowering plasma cholesterol concentrations in rats and affect cellular cholesterol homeostasis by up-regulating genes involved in hepatic cholesterol turnover.

STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC.

Science.gov (United States)

Garbarino, Jeanne; Pan, Meihui; Chin, Harvey F; Lund, Frederik W; Maxfield, Frederick R; Breslow, Jan L

2012-12-01

STARD4, a member of the evolutionarily conserved START gene family, has been implicated in the nonvesicular intracellular transport of cholesterol. However, the direction of transport and the membranes with which this protein interacts are not clear. We present studies of STARD4 function using small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein receptor (LDLR) levels were increased and decreased, respectively. We also observed a decrease in NPC1 protein expression, suggesting the induction of compensatory pathways to maintain cholesterol balance. These data indicate a role for STARD4 in nonvesicular transport of cholesterol from the plasma membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well.

2-heptyl-formononetin increases cholesterol and induces hepatic steatosis in mice

DEFF Research Database (Denmark)

Andersen, Charlotte; Schjoldager, Janne Gram; Tortzen, Christian

2013-01-01

Consumption of isoflavones may prevent adiposity, hepatic steatosis, and dyslipidaemia. However, studies in the area are few and primarily with genistein. This study investigated the effects of formononetin and its synthetic analogue, 2-heptyl-formononetin (C7F), on lipid and cholesterol metabolism...... in C57BL/6J mice. The mice were fed a cholesterol-enriched diet for five weeks to induce hypercholesterolemia and were then fed either the cholesterol-enriched diet or the cholesterol-enriched diet-supplemented formononetin or C7F for three weeks. Body weight and composition, glucose homeostasis......, and plasma lipids were compared. In another experiment, mice were fed the above diets for five weeks, and hepatic triglyceride accumulation and gene expression and histology of adipose tissue and liver were examined. Supplementation with C7F increased plasma HDL-cholesterol thereby increasing the plasma...

γδ T cells in homeostasis and host defence of epithelial barrier tissues.

Science.gov (United States)

Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

2017-12-01

Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

Loss of niche-satellite cell interactions in syndecan-3 null mice alters muscle progenitor cell homeostasis improving muscle regeneration.

Science.gov (United States)

Pisconti, Addolorata; Banks, Glen B; Babaeijandaghi, Farshad; Betta, Nicole Dalla; Rossi, Fabio M V; Chamberlain, Jeffrey S; Olwin, Bradley B

2016-01-01

The skeletal muscle stem cell niche provides an environment that maintains quiescent satellite cells, required for skeletal muscle homeostasis and regeneration. Syndecan-3, a transmembrane proteoglycan expressed in satellite cells, supports communication with the niche, providing cell interactions and signals to maintain quiescent satellite cells. Syndecan-3 ablation unexpectedly improves regeneration in repeatedly injured muscle and in dystrophic mice, accompanied by the persistence of sublaminar and interstitial, proliferating myoblasts. Additionally, muscle aging is improved in syndecan-3 null mice. Since syndecan-3 null myofiber-associated satellite cells downregulate Pax7 and migrate away from the niche more readily than wild type cells, syxndecan-3 appears to regulate satellite cell homeostasis and satellite cell homing to the niche. Manipulating syndecan-3 provides a promising target for development of therapies to enhance muscle regeneration in muscular dystrophies and in aged muscle.

Gravity and positional homeostasis of the cell

Science.gov (United States)

Nace, G. W.

1983-01-01

The effect of gravity upon cytoplasmic aggregates of the size present in eggs and upon cells is investigated. An expression is developed to describe the tendency of torque to rotate the egg and reorganize its constituents. This expression provides the net torque resulting from buoyancy and gravity acting upon a dumbbell-shaped cell, with heavy and light masses at either end and floating in a medium. Torques of approximately 2.5 x 10 to the -13th to 0.85 dyne-cm are found to act upon cells ranging from 6.4 microns to 31 mm (chicken egg). It is noted that cells must expend energy to maintain positional homeostasis against gravity, as demonstrated by results from Skylab 3, where tissue cultures used 58 percent more glucose on earth than in space. The implications for developmental biology, physiology, genetics, and evolution are discussed. It is argued that at the cellular and tissue levels the concept of gravity receptors may be unnecessary.

Cholesterol accumulation in Niemann Pick type C (NPC) model cells causes a shift in APP localization to lipid rafts

International Nuclear Information System (INIS)

Kosicek, Marko; Malnar, Martina; Goate, Alison; Hecimovic, Silva

2010-01-01

It has been suggested that cholesterol may modulate amyloid-β (Aβ) formation, a causative factor of Alzheimer's disease (AD), by regulating distribution of the three key proteins in the pathogenesis of AD (β-amyloid precursor protein (APP), β-secretase (BACE1) and/or presenilin 1 (PS1)) within lipid rafts. In this work we tested whether cholesterol accumulation upon NPC1 dysfunction, which causes Niemann Pick type C disease (NPC), causes increased partitioning of APP into lipid rafts leading to increased CTF/Aβ formation in these cholesterol-rich membrane microdomains. To test this we used CHO NPC1 -/- cells (NPC cells) and parental CHOwt cells. By sucrose density gradient centrifugation we observed a shift in fl-APP/CTF compartmentalization into lipid raft fractions upon cholesterol accumulation in NPC vs. wt cells. Furthermore, γ-secretase inhibitor treatment significantly increased fl-APP/CTF distribution in raft fractions in NPC vs. wt cells, suggesting that upon cholesterol accumulation in NPC1-null cells increased formation of APP-CTF and its increased processing towards Aβ occurs in lipid rafts. Our results support that cholesterol overload, such as in NPC disease, leads to increased partitioning of APP/CTF into lipid rafts resulting in increased amyloidogenic processing of APP in these cholesterol-rich membranes. This work adds to the mechanism of the cholesterol-effect on APP processing and the pathogenesis of Alzheimer's disease and supports the role of lipid rafts in these processes.

Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein

International Nuclear Information System (INIS)

Castro, G.R.; Fielding, C.J.

1988-01-01

Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [ 3 H]cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-beta-migrating apo A-I lipoprotein fraction with a molecular weight of ca. 70,000. Label was enriched about 30-fold in this fraction relative to its content of apo A-I (1-2% of total apo A-I). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-beta apo A-I species, to low-density lipoprotein, and to the alpha-migrating apo A-I that made up the bulk (96%) of total apo A-I in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-beta-migrating lipoprotein A-I species as part of a cholesterol transport transfer sequence in plasma

Lycopene reduces cholesterol absorption through the downregulation of Niemann-Pick C1-like 1 in Caco-2 cells.

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Zou, Jun; Feng, Dan

2015-11-01

Elevated blood cholesterol is an important risk factor associated with atherosclerosis and coronary heart disease. Tomato lycopene has been found to have a hypocholesterolemic effect, and the effect was considered to be related to inhibition of cholesterol synthesis. However, since plasma cholesterol levels are also influenced by the absorption of cholesterol in the gut, the present study is to investigate whether lycopene affects cholesterol absorption in the intestinal Caco-2 cells. The Caco-2 cells were pretreated with lycopene at different concentrations for 24 h and then incubated with radioactive micellar cholesterol for 2 h. The absorption of radioactive cholesterol was quantified by liquid scintillation. The expression of Niemann-Pick C1-like 1 (NPC1L1) and liver X receptor α (LXRα) was analyzed by Western blot and qPCR. We found that lycopene dose dependently inhibited cholesterol absorption and the expression of NPC1L1 protein and NPC1L1 mRNA. The inhibitory effects of lycopene on cholesterol absorption and NPC1L1 expression could be prevented by blockade of the LXRα pathway. This study provides the first evidence that lycopene inhibits cholesterol absorption in the intestinal cells and this inhibitory effect of lycopene is mediated, at least in part, by LXRα-NPC1L1 signaling pathway. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tissues Use Resident Dendritic Cells and Macrophages to Maintain Homeostasis and to Regain Homeostasis upon Tissue Injury: The Immunoregulatory Role of Changing Tissue Environments

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Lech, Maciej; Gröbmayr, Regina; Weidenbusch, Marc; Anders, Hans-Joachim

2012-01-01

Most tissues harbor resident mononuclear phagocytes, that is, dendritic cells and macrophages. A classification that sufficiently covers their phenotypic heterogeneity and plasticity during homeostasis and disease does not yet exist because cell culture-based phenotypes often do not match those found in vivo. The plasticity of mononuclear phagocytes becomes obvious during dynamic or complex disease processes. Different data interpretation also originates from different conceptual perspectives. An immune-centric view assumes that a particular priming of phagocytes then causes a particular type of pathology in target tissues, conceptually similar to antigen-specific T-cell priming. A tissue-centric view assumes that changing tissue microenvironments shape the phenotypes of their resident and infiltrating mononuclear phagocytes to fulfill the tissue's need to maintain or regain homeostasis. Here we discuss the latter concept, for example, why different organs host different types of mononuclear phagocytes during homeostasis. We further discuss how injuries alter tissue environments and how this primes mononuclear phagocytes to enforce this particular environment, for example, to support host defense and pathogen clearance, to support the resolution of inflammation, to support epithelial and mesenchymal healing, and to support the resolution of fibrosis to the smallest possible scar. Thus, organ- and disease phase-specific microenvironments determine macrophage and dendritic cell heterogeneity in a temporal and spatial manner, which assures their support to maintain and regain homeostasis in whatever condition. Mononuclear phagocytes contributions to tissue pathologies relate to their central roles in orchestrating all stages of host defense and wound healing, which often become maladaptive processes, especially in sterile and/or diffuse tissue injuries. PMID:23251037

Steryl ester synthesis, storage and hydrolysis: A contribution to sterol homeostasis.

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Korber, Martina; Klein, Isabella; Daum, Günther

2017-12-01

Sterols are essential lipids of all eukaryotic cells, appearing either as free sterols or steryl esters. Besides other regulatory mechanisms, esterification of sterols and hydrolysis of steryl esters serve to buffer both an excess and a lack of free sterols. In this review, the esterification process, the storage of steryl esters and their mobilization will be described. Several model organisms are discussed but the focus was set on mammals and the yeast Saccharomyces cerevisiae. The contribution of imbalanced cholesterol homeostasis to several human diseases, namely Wolman disease, cholesteryl ester storage disease, atherosclerosis and Alzheimer's disease, Niemann-Pick type C and Tangier disease is described. Copyright © 2017 Elsevier B.V. All rights reserved.

Cholesterol Transporters ABCA1 and ABCG1 Gene Expression in Peripheral Blood Mononuclear Cells in Patients with Metabolic Syndrome

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Zahra Tavoosi

2015-01-01

Full Text Available ABCA1 and ABCG1 genes encode the cholesterol transporter proteins that play a key role in cholesterol and phospholipids homeostasis. This study was aimed at evaluating and comparing ABCA1 and ABCG1 genes expression in metabolic syndrome patients and healthy individuals. This case-control study was performed on 36 patients with metabolic syndrome and the same number of healthy individuals in Hamadan (west of Iran during 2013-2014. Total RNA was extracted from mononuclear cells and purified using RNeasy Mini Kit column. The expression of ABCA1 and ABCG1 genes was performed by qRT-PCR. Lipid profile and fasting blood glucose were measured using colorimetric procedures. ABCG1 expression in metabolic syndrome patients was significantly lower (about 75% compared to that of control group, while for ABCA1 expression, there was no significant difference between the two studied groups. Comparison of other parameters such as HDL-C, FBS, BMI, waist circumference, and systolic and diastolic blood pressure between metabolic syndrome patients and healthy individuals showed significant differences (P<0.05. Decrease in ABCG1 expression in metabolic syndrome patients compared to healthy individuals suggests that hyperglycemia, related metabolites, and hyperlipidemia over the transporter capacity resulted in decreased expression of ABCG1. Absence of a significant change in ABCA1 gene expression between two groups can indicate a different regulation mechanism for ABCA1 expression.

The cholesterol-raising factor from coffee beans, cafestol, as an agonist ligand for the farnesoid and pregnane X receptors

NARCIS (Netherlands)

Ricketts, Marie-Louise; Boekschoten, Mark V.; Kreeft, Arja J.; Hooiveld, Guido J. E. J.; Moen, Corina J. A.; Mueller, Michael; Frants, Rune R.; Kasanmoentalib, Soemini; Post, Sabine M.; Princen, Hans M. G.; Porter, J. Gordon; Katan, Martijn B.; Hofker, Marten H.; Moore, David D.

Cafestol, a diterpene present in unfiltered coffee brews such as Scandinavian boiled, Turkish, and cafetiere coffee, is the most potent cholesterol-elevating compound-knownin the human diet. Several genes involved in cholesterol homeostasis have previously been shown to be targets of cafestol,

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

NARCIS (Netherlands)

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Oude Elferink, Ronald P. J.; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary

The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells.

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Martins, Ana Sofia; Alves, Inês; Helguero, Luisa; Domingues, Maria Rosário; Neves, Bruno Miguel

2016-11-01

The endoplasmic reticulum (ER) plays important roles in eukaryotic protein folding and lipid biosynthesis. Several exogenous and endogenous cellular sources of stress can perturb ER homeostasis leading to the accumulation of unfolded proteins in the lumen. Unfolded protein accumulation triggers a signal-transduction cascade known as the unfolded protein response (UPR), an adaptive mechanism which aims to protect cells from protein aggregates and to restore ER functions. Further to this protective mechanism, in immune cells, UPR molecular effectors have been shown to participate in a wide range of biological processes such as cell differentiation, survival and immunoglobulin and cytokine production. Recent findings also highlight the involvement of the UPR machinery in the maturational program and antigen presentation capacities of dendritic cells. UPR is therefore a key element in immune system homeostasis with direct implications on both adaptive and innate immune responses. The present review summarizes the knowledge on the emerging roles of UPR signaling cascades in mammalian immune cells as well as the consequences of their dysregulation in relation to the pathogenesis of several diseases.

Cholesterol metabolism: use of D2O for determination of synthesis rate in cell culture

International Nuclear Information System (INIS)

Esterman, A.L.; Cohen, B.I.; Javitt, N.B.

1985-01-01

Cholesterol synthesis in cell culture in the presence of D 2 O yields a spectrum of enriched molecules having a relative abundance that indicates random substitution of deuterium for hydrogen. Quantitation of the absolute rate of cholesterol synthesis is obtained by isotope ratio mass spectrometry. Mevinolin and 26-hydroxycholesterol both decrease cholesterol synthesis rate but have a discordant effect on HMG-CoA reductase activity

Andrographolide Inhibits Oxidized LDL-Induced Cholesterol Accumulation and Foam Cell Formation in Macrophages.

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Lin, Hung-Chih; Lii, Chong-Kuei; Chen, Hui-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Chen, Haw-Wen

2018-01-01

oxLDL is involved in the pathogenesis of atherosclerotic lesions through cholesterol accumulation in macrophage foam cells. Andrographolide, the bioactive component of Andrographis paniculata, possesses several biological activities such as anti-inflammatory, anti-oxidant, and anticancer functions. Scavenger receptors (SRs), including class A SR (SR-A) and CD36, are responsible for the internalization of oxLDL. In contrast, receptors for reverse cholesterol transport, including ABCA1 and ABCG1, mediate the efflux of cholesterol from macrophage foam cells. Transcription factor liver X receptor [Formula: see text] (LXR[Formula: see text] plays a key role in lipid metabolism and inflammation as well as in the regulation of ABCA1 and ABCG1 expression. Because of the contribution of inflammation to macrophage foam cell formation and the potent anti-inflammatory activity of andrographolide, we hypothesized that andrographolide might inhibit oxLDL-induced macrophage foam cell formation. The results showed that andrographolide reduced oxLDL-induced lipid accumulation in macrophage foam cells. Andrographolide decreased the mRNA and protein expression of CD36 by inducing the degradation of CD36 mRNA; however, andrographolide had no effect on SR-A expression. In contrast, andrographolide increased the mRNA and protein expression of ABCA1 and ABCG1, which were dependent on LXR[Formula: see text]. Andrographolide enhanced LXR[Formula: see text] nuclear translocation and DNA binding activity. Treatment with the LXR[Formula: see text] antagonist GGPP and transfection with LXR[Formula: see text] siRNA reversed the ability of andrographolide to stimulate ABCA1 and ABCG1 protein expression. In conclusion, inhibition of CD36-mediated oxLDL uptake and induction of ABCA1- and ABCG1-dependent cholesterol efflux are two working mechanisms by which andrographolide inhibits macrophage foam cell formation, which suggests that andrographolide could be a potential candidate to prevent

Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

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Hissa, Barbara; Pontes, Bruno; Roma, Paula Magda S; Alves, Ana Paula; Rocha, Carolina D; Valverde, Thalita M; Aguiar, Pedro Henrique N; Almeida, Fernando P; Guimarães, Allan J; Guatimosim, Cristina; Silva, Aristóbolo M; Fernandes, Maria C; Andrews, Norma W; Viana, Nathan B; Mesquita, Oscar N; Agero, Ubirajara; Andrade, Luciana O

2013-01-01

In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal.

Evolution of Cell Size Homeostasis and Growth Rate Diversity during Initial Surface Colonization of Shewanella oneidensis.

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Lee, Calvin K; Kim, Alexander J; Santos, Giancarlo S; Lai, Peter Y; Lee, Stella Y; Qiao, David F; Anda, Jaime De; Young, Thomas D; Chen, Yujie; Rowe, Annette R; Nealson, Kenneth H; Weiss, Paul S; Wong, Gerard C L

2016-09-06

Cell size control and homeostasis are fundamental features of bacterial metabolism. Recent work suggests that cells add a constant size between birth and division ("adder" model). However, it is not known how cell size homeostasis is influenced by the existence of heterogeneous microenvironments, such as those during biofilm formation. Shewanella oneidensis MR-1 can use diverse energy sources on a range of surfaces via extracellular electron transport (EET), which can impact growth, metabolism, and size diversity. Here, we track bacterial surface communities at single-cell resolution to show that not only do bacterial motility appendages influence the transition from two- to three-dimensional biofilm growth and control postdivisional cell fates, they strongly impact cell size homeostasis. For every generation, we find that the average growth rate for cells that stay on the surface and continue to divide (nondetaching population) and that for cells that detach before their next division (detaching population) are roughly constant. However, the growth rate distribution is narrow for the nondetaching population, but broad for the detaching population in each generation. Interestingly, the appendage deletion mutants (ΔpilA, ΔmshA-D, Δflg) have significantly broader growth rate distributions than that of the wild type for both detaching and nondetaching populations, which suggests that Shewanella appendages are important for sensing and integrating environmental inputs that contribute to size homeostasis. Moreover, our results suggest multiplexing of appendages for sensing and motility functions contributes to cell size dysregulation. These results can potentially provide a framework for generating metabolic diversity in S. oneidensis populations to optimize EET in heterogeneous environments.

Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Wang, Jiying [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Ohno-Matsui, Kyoko, E-mail: k.ohno.oph@tmd.ac.jp [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Morita, Ikuo [Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)

2012-08-10

Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

NARCIS (Netherlands)

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Elferink, Ronald P. J. Oude; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPAR delta) is involved in regulation of energy homeostasis. Activation of PPAR delta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased

COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.

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Fang, Jingye; Liu, Ming; Zhang, Xuebao; Sakamoto, Takeshi; Taatjes, Douglas J; Jena, Bhanu P; Sun, Fei; Woods, James; Bryson, Tim; Kowluru, Anjaneyulu; Zhang, Kezhong; Chen, Xuequn

2015-08-01

Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.

The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts.

Science.gov (United States)

Krause, Martin R; Regen, Steven L

2014-12-16

CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been

IMB2026791, a Xanthone, Stimulates Cholesterol Efflux by Increasing the Binding of Apolipoprotein A-I to ATP-Binding Cassette Transporter A1

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Zijian Xie

2012-03-01

Full Text Available It is known that the ATP-binding cassette transporter A1 (ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL metabolism. Several laboratories have demonstrated that ABCA1 binding to lipid-poor apolipoprotein A-I (apoA-I will mediate the assembly of nascent HDL and cellular cholesterol efflux, which suggests a possible receptor-ligand interaction between ABCA1 and apoA-I. In this study, a cell-based-ELISA-like high-throughput screening (HTS method was developed to identify the synthetic and natural compounds that can regulate binding activity of ABCA1 to apoA-I. The cell-based-ELISA-like high-throughput screen was conducted in a 96-well format using Chinese hamster ovary (CHO cells stably transfected with ABCA1 pIRE2-EGFP (Enhanced Green Fluorecence Protein expression vector and the known ABCA1 inhibitor glibenclamide as the antagonist control. From 2,600 compounds, a xanthone compound (IMB 2026791 was selected using this HTS assay, and it was proved as an apoA-I binding agonist to ABCA1 by a flow cytometry assay and western blot analysis. The [3H] cholesterol efflux assay of IMB2026791 treated ABCA1-CHO cells and PMA induced THP-1 macrophages (human acute monocytic leukemia cell further confirmed the compound as an accelerator of cholesterol efflux in a dose-dependent manner with an EC50 of 25.23 μM.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Science.gov (United States)

Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

2017-10-01

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

Ablation of cholesterol biosynthesis in neural stem cells increases their VEGF expression and angiogenesis but causes neuron apoptosis.

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Saito, Kanako; Dubreuil, Veronique; Arai, Yoko; Wilsch-Bräuninger, Michaela; Schwudke, Dominik; Saher, Gesine; Miyata, Takaki; Breier, Georg; Thiele, Christoph; Shevchenko, Andrej; Nave, Klaus-Armin; Huttner, Wieland B

2009-05-19

Although sufficient cholesterol supply is known to be crucial for neurons in the developing mammalian brain, the cholesterol requirement of neural stem and progenitor cells in the embryonic central nervous system has not been addressed. Here we have conditionally ablated the activity of squalene synthase (SQS), a key enzyme for endogenous cholesterol production, in the neural stem and progenitor cells of the ventricular zone (VZ) of the embryonic mouse brain. Mutant embryos exhibited a reduced brain size due to the atrophy of the neuronal layers, and died at birth. Analyses of the E11.5-E15.5 dorsal telencephalon and diencephalon revealed that this atrophy was due to massive apoptosis of newborn neurons, implying that this progeny of the SQS-ablated neural stem and progenitor cells was dependent on endogenous cholesterol biosynthesis for survival. Interestingly, the neural stem and progenitor cells of the VZ, the primary target of SQS inactivation, did not undergo significant apoptosis. Instead, vascular endothelial growth factor (VEGF) expression in these cells was strongly upregulated via a hypoxia-inducible factor-1-independent pathway, and angiogenesis in the VZ was increased. Consistent with an increased supply of lipoproteins to these cells, the level of lipid droplets containing triacylglycerides with unsaturated fatty acyl chains was found to be elevated. Our study establishes a direct link between intracellular cholesterol levels, VEGF expression, and angiogenesis. Moreover, our data reveal a hitherto unknown compensatory process by which the neural stem and progenitor cells of the developing mammalian brain evade the detrimental consequences of impaired endogenous cholesterol biosynthesis.

Cutting edge: CD95 maintains effector T cell homeostasis in chronic immune activation

NARCIS (Netherlands)

Arens, Ramon; Baars, Paul A.; Jak, Margot; Tesselaar, Kiki; van der Valk, Martin; van Oers, Marinus H. J.; van Lier, René A. W.

2005-01-01

The elimination of activated T cells is important to maintain homeostasis and avoid immunopathology. CD95 (Fas/APO-1) has been identified as a death mediator for activated T cells in vitro but the function of CD95 in death of mature T cells in vivo is still controversial. Here we show that

Mucosal Ecological Network of Epithelium and Immune Cells for Gut Homeostasis and Tissue Healing.

Science.gov (United States)

Kurashima, Yosuke; Kiyono, Hiroshi

2017-04-26

The intestinal epithelial barrier includes columnar epithelial, Paneth, goblet, enteroendocrine, and tuft cells as well as other cell populations, all of which contribute properties essential for gastrointestinal homeostasis. The intestinal mucosa is covered by mucin, which contains antimicrobial peptides and secretory IgA and prevents luminal bacteria, fungi, and viruses from stimulating intestinal immune responses. Conversely, the transport of luminal microorganisms-mediated by M, dendritic, and goblet cells-into intestinal tissues facilitates the harmonization of active and quiescent mucosal immune responses. The bacterial population within gut-associated lymphoid tissues creates the intratissue cohabitations for harmonized mucosal immunity. Intermolecular and intercellular communication among epithelial, immune, and mesenchymal cells creates an environment conducive for epithelial regeneration and mucosal healing. This review summarizes the so-called intestinal mucosal ecological network-the complex but vital molecular and cellular interactions of epithelial mesenchymal cells, immune cells, and commensal microbiota that achieve intestinal homeostasis, regeneration, and healing.

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

Directory of Open Access Journals (Sweden)

Dhritiman Samanta

2017-05-01

Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

Phytosterols Differentially Influence ABC transporter Expression, Cholesterol Efflux and Inflammatory Cytokine Secretion in Macrophage Foam Cells

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Sabeva, Nadezhda S; McPhaul, Christopher M; Li, Xiangan; Cory, Theodore J.; Feola, David J.; Graf, Gregory A

2010-01-01

Phytosterol supplements lower low density lipoprotein (LDL) cholesterol, but accumulate in vascular lesions of patients and limit the anti-atherosclerotic effects of LDL lowering in apolipoprotein E deficient mice, suggesting that the cholesterol lowering benefit of phytosterol supplementation may not be fully realized. Individual phytosterols have cell-type specific effects that may either be beneficial or deleterious with respect to atherosclerosis, but little is known concerning their effects on macrophage function. The effects of phytosterols on ABCA1 and ABCG1 abundance, cholesterol efflux, and inflammatory cytokine secretion were determined in cultured macrophage foam cells. Among the commonly consumed phytosterols, stigmasterol increased expression of ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein (Apo) AI and high density lipoprotein (HDL). Campesterol and sitosterol had no effect on ABCA1 or ABCG1 levels. Sitosterol had no effect of cholesterol efflux to Apo AI or HDL, whereas campesterol had a modest, but significant reduction in cholesterol efflux to HDL in THP-1 macrophages. Whereas stigmasterol blunted aggregated LDL-induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β secretion, sitosterol exacerbated these effects. The presence of campesterol had no effect on agLDL-induced inflammatory cytokine secretion from THP-1 macrophages. In conclusion, the presence of stigmasterol in modified lipoproteins promoted cholesterol efflux and suppressed inflammatory cytokine secretion in response to lipid loading in macrophage foam cells. While campesterol was largely inert, the presence of sitosterol increased the proinflammatory cytokine secretion. PMID:21111593

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

International Nuclear Information System (INIS)

Doi, Keiko; Fujimoto, Takahiro; Okamura, Tadashi; Ogawa, Masahiro; Tanaka, Yoko; Mototani, Yasumasa; Goto, Motohito; Ota, Takeharu; Matsuzaki, Hiroshi; Kuroki, Masahide; Tsunoda, Toshiyuki; Sasazuki, Takehiko; Shirasawa, Senji

2012-01-01

Highlights: ► We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. ► Zfat-deficiency leads to reduction in the number of the peripheral T cells. ► Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. ► Decreased expression of IL-7Rα, IL-2Rα and IL-2 in Zfat-deficient peripheral T cells. ► Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7Rα and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2Rα expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

Sulforaphane Protects against High Cholesterol-Induced Mitochondrial Bioenergetics Impairments, Inflammation, and Oxidative Stress and Preserves Pancreatic β-Cells Function

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Catalina Carrasco-Pozo

2017-01-01

Full Text Available Cholesterol plays an important role in inducing pancreatic β-cell dysfunction, leading to an impaired insulin secretory response to glucose. This study aimed to determine the protective effects of sulforaphane, a natural isothiocyanate Nrf2-inducer, against cholesterol-induced pancreatic β-cells dysfunction, through molecular and cellular mechanisms involving mitochondrial bioenergetics. Sulforaphane prevented cholesterol-induced alterations in the coupling efficiency of mitochondrial respiration, improving ATP turnover and spare capacity, and averted the impairment of the electron flow at complexes I, II, and IV. Sulforaphane also attenuated the cholesterol-induced activation of the NFκB pathway, normalizing the expression of pro- and anti-inflammatory cytokines. In addition, it also inhibited the decrease in sirtuin 1 expression and greatly increased Pgc-1α expression in Min6 cells. Sulforaphane increased the expression of antioxidant enzymes downstream of the Nrf2 pathway and prevented lipid peroxidation induced by cholesterol. The antioxidant and anti-inflammatory properties of sulforaphane and its ability to protect and improve mitochondrial bioenergetic function contribute to its protective action against cholesterol-induced pancreatic β-cell dysfunction. Our data provide a scientifically tested foundation upon which sulforaphane can be developed as nutraceutical to preserve β-cell function and eventually control hyperglycemia.

Regulation of NKT Cell Localization in Homeostasis and Infection

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Slauenwhite, Drew; Johnston, Brent

2015-01-01

Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection. PMID:26074921

Regulation of NKT Cell Localization in Homeostasis and Infection.

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Slauenwhite, Drew; Johnston, Brent

2015-01-01

Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection.

Comparative effect of amidated pectin and psyllium on cholesterol homeostasis in rats

Czech Academy of Sciences Publication Activity Database

Marounek, Milan; Volek, Z.; Skřivanová, E.; Tůma, J.; Dušková, D.

2010-01-01

Roč. 5, č. 3 (2010), s. 299-303 ISSN 1895-104X Institutional research plan: CEZ:AV0Z50450515 Keywords : Cholesterol homeostazis * Pectin * Psyllium Subject RIV: GH - Livestock Nutrition Impact factor: 0.685, year: 2010

Epigenetic Control of Stem Cell Potential During Homeostasis, Aging, and Disease

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Beerman, Isabel; Rossi, Derrick J.

2015-01-01

Stem cell decline is an important cellular driver of aging-associated pathophysiology in multiple tissues. Epigenetic regulation is central to establishing and maintaining stem cell function, and emerging evidence indicates that epigenetic dysregulation contributes to the altered potential of stem cells during aging. Unlike terminally differentiated cells, the impact of epigenetic dysregulation in stem cells is propagated beyond self; alterations can be heritably transmitted to differentiated progeny, in addition to being perpetuated and amplified within the stem cell pool through self-renewal divisions. This review focuses on recent studies examining epigenetic regulation of tissue-specific stem cells in homeostasis, aging, and aging-related disease. PMID:26046761

Membrane Cholesterol Modulates Superwarfarin Toxicity

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Marangoni, M. Natalia; Martynowycz, Michael W.; Kuzmenko, Ivan; Braun, David; Polak, Paul E.; Weinberg, Guy; Rubinstein, Israel; Gidalevitz, David; Feinstein, Douglas L.

2016-04-26

Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

Gly[14]-humanin inhibits ox-LDL uptake and stimulates cholesterol efflux in macrophage-derived foam cells.

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Zhu, Wa-Wa; Wang, Shu-Rong; Liu, Zhi-Hua; Cao, Yong-Jun; Wang, Fen; Wang, Jing; Liu, Chun-Feng; Xie, Ying; Xie, Ying; Zhang, Yan-Lin

2017-01-01

Foam cell formation, which is caused by imbalanced cholesterol influx and efflux by macrophages, plays a vital role in the occurrence and development of atherosclerosis. Humanin (HN), a mitochondria-derived peptide, can prevent the production of reactive oxygen species and death of human aortic endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL) and has a protective effect on patients with in early atherosclerosis. However, the effects of HN on the regulation of cholesterol metabolism in RAW 264.7 macrophages are still unknown. This study was designed to investigate the role of [Gly14]-humanin (HNG) in lipid uptake and cholesterol efflux in RAW 264.7 macrophages. Flow cytometry and live cell imaging results showed that HNG reduced Dil-ox-LDL accumulation in the RAW 264.7 macrophages. A similar result was obtained for lipid accumulation by measuring cellular cholesterol content. Western blot analysis showed that ox-LDL treatment upregulated not only the protein expression of CD36 and LOX-1, which mediate ox-LDL endocytosis, but also ATP-binding cassette (ABC) transporter A1 and ABCG1, which mediate ox-LDL exflux. HNG pretreatment inhibited the upregulation of CD36 and LOX-1 levels, prompting the upregulation of ABCA1 and ABCG1 levels induced by ox-LDL. Therefore we concluded that HNG could inhibit ox-LDL-induced macrophage-derived foam cell formation, which occurs because of a decrease in lipid uptake and an increase in cholesterol efflux from macrophage cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Current Views on Genetics and Epigenetics of Cholesterol Gallstone Disease

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Agostino Di Ciaula

2013-01-01

Full Text Available Cholesterol gallstone disease, one of the commonest digestive diseases in western countries, is induced by an imbalance in cholesterol metabolism, which involves intestinal absorption, hepatic biosynthesis, and biliary output of cholesterol, and its conversion to bile acids. Several components of the metabolic syndrome (e.g., obesity, type 2 diabetes, dyslipidemia, and hyperinsulinemia are also well-known risk factors for gallstones, suggesting the existence of interplay between common pathophysiological pathways influenced by insulin resistance, genetic, epigenetic, and environmental factors. Cholesterol gallstones may be enhanced, at least in part, by the abnormal expression of a set of the genes that affect cholesterol homeostasis and lead to insulin resistance. Additionally, epigenetic mechanisms (mainly DNA methylation, histone acetylation/deacetylation, and noncoding microRNAs may modify gene expression in the absence of an altered DNA sequence, in response to different lithogenic environmental stimuli, such as diet, lifestyle, pollutants, also occurring in utero before birth. In this review, we will comment on various steps of the pathogenesis of cholesterol gallstones and interaction between environmental and genetic factors. The epigenomic approach may offer new options for therapy of gallstones and better possibilities for primary prevention in subjects at risk.

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft.

Science.gov (United States)

Yamamoto, Yutaro; Tomiyama, Arata; Sasaki, Nobuyoshi; Yamaguchi, Hideki; Shirakihara, Takuya; Nakashima, Katsuhiko; Kumagai, Kosuke; Takeuchi, Satoru; Toyooka, Terushige; Otani, Naoki; Wada, Kojiro; Narita, Yoshitaka; Ichimura, Koichi; Sakai, Ryuichi; Namba, Hiroki; Mori, Kentaro

2018-01-01

Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism. Copyright © 2017 Elsevier Inc. All rights reserved.

Cholesterol synthesis by human fetal hepatocytes: effect of lipoproteins

International Nuclear Information System (INIS)

Carr, B.R.; Simpson, E.R.

1984-01-01

The purpose of the present investigation was to determine the effect of various lipoproteins on the rate of cholesterol synthesis of human fetal liver cells maintained in culture. This was accomplished by measuring the rate of incorporation of tritium from tritiated water or carbon 14-labeled acetate into cholesterol in human fetal liver cells. Optimal conditions for each assay were determined. When human fetal liver cells were maintained in the presence of low-density lipoprotein, cholesterol synthesis was inhibited in a concentration-dependent fashion. Intermediate--density lipoprotein and very-low-density lipoprotein also suppressed cholesterol synthesis in human fetal liver cells. In contrast, high-density lipoprotein stimulated cholesterol synthesis in human fetal liver cells. The results of the present as well as our previous investigations suggest that multiple interrelationships exist between fetal liver cholesterol synthesis and lipoprotein-cholesterol utilization by the human fetal adrenal gland and that these processes serve to regulate the lipoprotein-cholesterol levels in fetal plasma

CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis

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McNally, Alice; Hill, Geoffrey R.; Sparwasser, Tim; Thomas, Ranjeny; Steptoe, Raymond J.

2011-01-01

CD4+CD25+ regulatory T cells (Treg) play a crucial role in the regulation of immune responses. Although many mechanisms of Treg suppression in vitro have been described, the mechanisms by which Treg modulate CD8+ T cell differentiation and effector function in vivo are more poorly defined. It has been proposed, in many instances, that modulation of cytokine homeostasis could be an important mechanism by which Treg regulate adaptive immunity; however, direct experimental evidence is sparse. Here we demonstrate that CD4+CD25+ Treg, by critically regulating IL-2 homeostasis, modulate CD8+ T-cell effector differentiation. Expansion and effector differentiation of CD8+ T cells is promoted by autocrine IL-2 but, by competing for IL-2, Treg limit CD8+ effector differentiation. Furthermore, a regulatory loop exists between Treg and CD8+ effector T cells, where IL-2 produced during CD8+ T-cell effector differentiation promotes Treg expansion. PMID:21502514

Cholesterol and phytosterols differentially regulate the expression of caveolin 1 and a downstream prostate cell growth-suppressor gene

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Ifere, Godwin O.; Equan, Anita; Gordon, Kereen; Nagappan, Peri; Igietseme, Joseph U.; Ananaba, Godwin A.

2010-01-01

Background The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream regulated gene1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor. Methods PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72 h, followed by trypan blue dye exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members. Results Physiological doses (16 µM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol enrichment promoted cell growth (Pphytosterols significantly induced growth-suppression (Pphytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1(cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis. Conclusions Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor-suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling

Thyroid hormone regulation of adult intestinal stem cells: Implications on intestinal development and homeostasis.

Science.gov (United States)

Sun, Guihong; Roediger, Julia; Shi, Yun-Bo

2016-12-01

Organ-specific adult stem cells are essential for organ homeostasis, tissue repair and regeneration. The formation of such stem cells often takes place during postembryonic development, a period around birth in mammals when plasma thyroid hormone concentration is high. The life-long self-renewal of the intestinal epithelium has made mammalian intestine a valuable model to study the function and regulation and adult stem cells. On the other hand, much less is known about how the adult intestinal stem cells are formed during vertebrate development. Here, we will review some recent progresses on this subject, focusing mainly on the formation of the adult intestine during Xenopus metamorphosis. We will discuss the role of thyroid hormone signaling pathway in the process and potential molecular conservations between amphibians and mammals as well as the implications in organ homeostasis and human diseases.

Chlordecone, a mixed pregnane X receptor (PXR) and estrogen receptor alpha (ERα) agonist, alters cholesterol homeostasis and lipoprotein metabolism in C57BL/6 mice

International Nuclear Information System (INIS)

Lee, Junga; Scheri, Richard C.; Zhang Yuan; Curtis, Lawrence R.

2008-01-01

Chlordecone (CD) is one of many banned organochlorine (OC) insecticides that are widespread persistent organic pollutants. OC insecticides alter lipid homeostasis in rodents at doses that are not neurotoxic or carcinogenic. Pretreatment of mice or rats with CD altered tissue distribution of a subsequent dose of [ 14 C]CD or [ 14 C]cholesterol (CH). Nuclear receptors regulate expression of genes important in the homeostasis of CH and other lipids. In this study, we report that CD suppresses in vitro reporter systems for human liver X receptors (LXRs) and activates those for human farnesoid X receptor (FXR), pregnane X receptor (PXR) and estrogen receptor α (ERα) in a concentration-dependent manner (0-50 μM). Consistent with human PXR activation in vitro, three days after a single dose of CD (15 mg/kg) hepatic microsomal CYP3A11 protein increases in C57BL/6 mice. CD decreases hepatic CH ester content without altering total CH concentration. Apolipoprotein A-I (apoA-I) contents of hepatic lipoprotein-rich and microsomal fractions of CD-treated mice are higher than controls. There is a significant reduction in non-high density lipoprotein CH but not apolipoprotein B-48/100 (apoB-48/100) in plasma from CD-treated mice after a 4 h fast. At 14 days after 15 mg CD/kg apoA-I and apoB-100 proteins but not CYP3A11 protein in hepatic microsomes are similar to controls. This work indicates that altered CH homeostasis is a mode of OC insecticide action of relevance after a single dose. This at least partially explains altered CH tissue distribution in CD-pretreated mice

Cholesterol in unusual places

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Kucerka, N; Nieh, M P; Marquardt, D; Harroun, T A; Wassail, S R; Katsaras, J, E-mail: John.Katsaras@nrc.gc.ca, E-mail: Norbert.Kucerka@nrc.gc.ca

2010-11-01

Cholesterol is an essential component of mammalian cells, and is required for building and maintaining cell membranes, regulating their fluidity, and possibly acting as an antioxidant. Cholesterol has also been implicated in cell signaling processes, where it has been suggested that it triggers the formation of lipid rafts in the plasma membrane. Aside from cholesterol's physiological roles, what is also becoming clear is its poor affinity for lipids with unsaturated fatty acids as opposed to saturated lipids, such as sphingomyelin with which it forms rafts. We previously reported the location of cholesterol in membranes with varying degrees of acyl chain unsaturation as determined by neutron diffraction studies (Harroun et al 2006 Biochemistry 45, 1227; Harroun et al 2008 Biochemistry 47, 7090). In bilayers composed of phosphatidylcholine (PC) molecules with a saturated acyl chain at the sn-1 position or a monounsaturated acyl chain at both sn-1 and sn-2 positions, cholesterol was found in its much-accepted 'upright' position. However, in dipolyunsaturated 1,2-diarachidonyl phosphatidylcholine (20:4-20:4PC) membranes the molecule was found sequestered in the center of the bilayers. In further experiments, mixing l-palmitoyl-2-oleoyl phosphatidylcholine (16:0-18:1 PC) with 20:4-20:4PC resulted in cholesterol reverting to its upright orientation at approximately 40 mol% 16:0-18:1 PC. Interestingly, the same effect was achieved with only 5 mol% 1,2-dimyristoyl phosphatidylchoile (14:0-14:0PC).

Cholesterol in unusual places

International Nuclear Information System (INIS)

Kucerka, N; Nieh, M P; Marquardt, D; Harroun, T A; Wassail, S R; Katsaras, J

2010-01-01

Cholesterol is an essential component of mammalian cells, and is required for building and maintaining cell membranes, regulating their fluidity, and possibly acting as an antioxidant. Cholesterol has also been implicated in cell signaling processes, where it has been suggested that it triggers the formation of lipid rafts in the plasma membrane. Aside from cholesterol's physiological roles, what is also becoming clear is its poor affinity for lipids with unsaturated fatty acids as opposed to saturated lipids, such as sphingomyelin with which it forms rafts. We previously reported the location of cholesterol in membranes with varying degrees of acyl chain unsaturation as determined by neutron diffraction studies (Harroun et al 2006 Biochemistry 45, 1227; Harroun et al 2008 Biochemistry 47, 7090). In bilayers composed of phosphatidylcholine (PC) molecules with a saturated acyl chain at the sn-1 position or a monounsaturated acyl chain at both sn-1 and sn-2 positions, cholesterol was found in its much-accepted 'upright' position. However, in dipolyunsaturated 1,2-diarachidonyl phosphatidylcholine (20:4-20:4PC) membranes the molecule was found sequestered in the center of the bilayers. In further experiments, mixing l-palmitoyl-2-oleoyl phosphatidylcholine (16:0-18:1 PC) with 20:4-20:4PC resulted in cholesterol reverting to its upright orientation at approximately 40 mol% 16:0-18:1 PC. Interestingly, the same effect was achieved with only 5 mol% 1,2-dimyristoyl phosphatidylchoile (14:0-14:0PC).

The effect of 24S-hydroxycholesterol on cholesterol homeostasis in neurons: quantitative changes to the cortical neuron proteome.

Science.gov (United States)

Wang, Yuqin; Muneton, Sabina; Sjövall, Jan; Jovanovic, Jasmina N; Griffiths, William J

2008-04-01

In humans, the brain represents only about 2% of the body's mass but contains about one-quarter of the body's free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 2.3.3.10), diphosphomevalonate decarboxylase (EC 4.1.1.33), isopentenyl-diphosphate delta isomerase (EC 5.3.3.2), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 2.5.1.10), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 2.5.1.21) and methylsterol monooxygenase (EC 1.14.13.72). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not

Regulation of CD4+ T-Cell Function by Membrane Cholesterol

Science.gov (United States)

2012-03-13

and intracellular synthesis [Lehoux et al 1985]. Early studies using in vivo administration of radio-labeled squalene, a late cholesterol...mice expressing the HA of PR8/A/34 influenza virus in the pancreatic -cells (RAG2 KO, RIP-PR8/HA Tg mice) leads to fulminate autoimmune diabetes within...transgenic mouse model in which infusion of influenza PR8/HA-specific T-effector cells (from a TCR- PR8/HA Tg mouse) induces fulminate diabetes, we found

Age-dependent transition from cell-level to population-level control in murine intestinal homeostasis revealed by coalescence analysis.

Directory of Open Access Journals (Sweden)

Zheng Hu

Full Text Available In multi-cellular organisms, tissue homeostasis is maintained by an exquisite balance between stem cell proliferation and differentiation. This equilibrium can be achieved either at the single cell level (a.k.a. cell asymmetry, where stem cells follow strict asymmetric divisions, or the population level (a.k.a. population asymmetry, where gains and losses in individual stem cell lineages are randomly distributed, but the net effect is homeostasis. In the mature mouse intestinal crypt, previous evidence has revealed a pattern of population asymmetry through predominantly symmetric divisions of stem cells. In this work, using population genetic theory together with previously published crypt single-cell data obtained at different mouse life stages, we reveal a strikingly dynamic pattern of stem cell homeostatic control. We find that single-cell asymmetric divisions are gradually replaced by stochastic population-level asymmetry as the mouse matures to adulthood. This lifelong process has important developmental and evolutionary implications in understanding how adult tissues maintain their homeostasis integrating the trade-off between intrinsic and extrinsic regulations.

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington's disease.

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Valenza, M; Marullo, M; Di Paolo, E; Cesana, E; Zuccato, C; Biella, G; Cattaneo, E

2015-04-01

In the adult brain, neurons require local cholesterol production, which is supplied by astrocytes through apoE-containing lipoproteins. In Huntington's disease (HD), such cholesterol biosynthesis in the brain is severely reduced. Here we show that this defect, occurring in astrocytes, is detrimental for HD neurons. Astrocytes bearing the huntingtin protein containing increasing CAG repeats secreted less apoE-lipoprotein-bound cholesterol in the medium. Conditioned media from HD astrocytes and lipoprotein-depleted conditioned media from wild-type (wt) astrocytes were equally detrimental in a neurite outgrowth assay and did not support synaptic activity in HD neurons, compared with conditions of cholesterol supplementation or conditioned media from wt astrocytes. Molecular perturbation of cholesterol biosynthesis and efflux in astrocytes caused similarly altered astrocyte-neuron cross talk, whereas enhancement of glial SREBP2 and ABCA1 function reversed the aspects of neuronal dysfunction in HD. These findings indicate that astrocyte-mediated cholesterol homeostasis could be a potential therapeutic target to ameliorate neuronal dysfunction in HD.

ZFAT plays critical roles in peripheral T cell homeostasis and its T cell receptor-mediated response

Energy Technology Data Exchange (ETDEWEB)

Doi, Keiko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute of Life Sciences for the Next Generation of Women Scientists, Fukuoka University, Fukuoka (Japan); Fujimoto, Takahiro [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Okamura, Tadashi [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ogawa, Masahiro [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tanaka, Yoko [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Mototani, Yasumasa; Goto, Motohito [Division of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Ota, Takeharu; Matsuzaki, Hiroshi [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Kuroki, Masahide [Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Tsunoda, Toshiyuki [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan); Sasazuki, Takehiko [Institute for Advanced Study, Kyushu University, Fukuoka (Japan); Shirasawa, Senji, E-mail: sshirasa@fukuoka-u.ac.jp [Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka (Japan); Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka (Japan)

2012-08-17

Highlights: Black-Right-Pointing-Pointer We generated Cd4-Cre-mediated T cell-specific Zfat-deficient mice. Black-Right-Pointing-Pointer Zfat-deficiency leads to reduction in the number of the peripheral T cells. Black-Right-Pointing-Pointer Impaired T cell receptor-mediated response in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Decreased expression of IL-7R{alpha}, IL-2R{alpha} and IL-2 in Zfat-deficient peripheral T cells. Black-Right-Pointing-Pointer Zfat plays critical roles in peripheral T cell homeostasis. -- Abstract: ZFAT, originally identified as a candidate susceptibility gene for autoimmune thyroid disease, has been reported to be involved in apoptosis, development and primitive hematopoiesis. Zfat is highly expressed in T- and B-cells in the lymphoid tissues, however, its physiological function in the immune system remains totally unknown. Here, we generated the T cell-specific Zfat-deficient mice and demonstrated that Zfat-deficiency leads to a remarkable reduction in the number of the peripheral T cells. Intriguingly, a reduced expression of IL-7R{alpha} and the impaired responsiveness to IL-7 for the survival were observed in the Zfat-deficient T cells. Furthermore, a severe defect in proliferation and increased apoptosis in the Zfat-deficient T cells following T cell receptor (TCR) stimulation was observed with a reduced IL-2R{alpha} expression as well as a reduced IL-2 production. Thus, our findings reveal that Zfat is a critical regulator in peripheral T cell homeostasis and its TCR-mediated response.

Stomach Organ and Cell Lineage Differentiation: From?Embryogenesis to Adult Homeostasis

OpenAIRE

Willet, Spencer G.; Mills, Jason C.

2016-01-01

Gastric diseases cause considerable worldwide burden. However, the stomach is still poorly understood in terms of the molecularâcellular processes that govern its development and homeostasis. In particular, the complex relationship between the differentiated cell types located within the stomach and the stem and progenitor cells that give rise to them is significantly understudied relative to other organs. In this review, we highlight the current state of the literature relating to specificat...

Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

NARCIS (Netherlands)

Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

DEFF Research Database (Denmark)

Wustner, D.; Solanko, L.; Sokol, Olena

2011-01-01

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehythoergosterol (DHE), a well-established marker for cholesterol, by introducing simultan......Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehythoergosterol (DHE), a well-established marker for cholesterol, by introducing...... and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference...

Diet and Age Interactions with Regards to Cholesterol Regulation and Brain Pathogenesis

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Romina M. Uranga

2010-01-01

Full Text Available Cholesterol is an essential molecule for brain homeostasis; yet, hypercholesterolemia and its numerous complications are believed to play a role in promoting multiple aspects of brain pathogenesis. An ever increasing number of individuals in modern Western Society are regularly consuming diets high in fat which promote the development of hypercholesterolemia. Additionally, modern societies are becoming increasingly aged, causing a collision between increased hypercholesterolemia and increased aging, which will likely lead to the development of increased pathological conditions due to hypercholesterolemia, thereby promoting deleterious neurochemical and behavioral changes in the brain. Lastly, while beneficial in controlling cholesterol levels, the long-term use of statins itself may potentially promote adverse effects on brain homeostasis, although specifics on this remain largely unknown. This review will focus on linking the current understanding of diet-induced hypercholesterolemia (as well as statin use to the development of oxidative stress, neurochemical alterations, and cognitive disturbances in the aging brain.

Impaired Autophagy and Defective T Cell Homeostasis in Mice with T Cell-Specific Deletion of Receptor for Activated C Kinase 1

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Guihua Qiu

2017-05-01

Full Text Available Autophagy plays a central role in maintaining T cell homeostasis. Our previous study has shown that hepatocyte-specific deficiency of receptor for activated C kinase 1 (RACK1 leads to lipid accumulation in the liver, accompanied by impaired autophagy, but its in vivo role in T cells remains unclear. Here, we report that mice with T cell-specific deletion of RACK1 exhibit normal intrathymic development of conventional T cells and regulatory T (Treg cells but reduced numbers of peripheral CD4+ and CD8+ T cells. Such defects are cell intrinsic with impaired mitochondrial clearance, increased sensitivity to cell death, and decreased proliferation that could be explained by impaired autophagy. Furthermore, RACK1 is essential for invariant natural T cell development. In vivo, T cell-specific loss of RACK1 dampens concanavalin A-induced acute liver injury. Our data suggest that RACK1 is a key regulator of T cell homeostasis.

Stem/progenitor cells in pituitary organ homeostasis and tumourigenesis

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Manshaei, Saba

2018-01-01

Evidence for the presence of pituitary gland stem cells has been provided over the last decade using a combination of approaches including in vitro clonogenicity assays, flow cytometric side population analysis, immunohistochemical analysis and genetic approaches. These cells have been demonstrated to be able to self-renew and undergo multipotent differentiation to give rise to all hormonal lineages of the anterior pituitary. Furthermore, evidence exists for their contribution to regeneration of the organ and plastic responses to changing physiological demand. Recently, stem-like cells have been isolated from pituitary neoplasms raising the possibility that a cytological hierarchy exists, in keeping with the cancer stem cell paradigm. In this manuscript, we review the evidence for the existence of pituitary stem cells, their role in maintaining organ homeostasis and the regulation of their differentiation. Furthermore, we explore the emerging concept of stem cells in pituitary tumours and their potential roles in these diseases. PMID:28855316

High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review.

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Subczynski, Witold K; Pasenkiewicz-Gierula, Marta; Widomska, Justyna; Mainali, Laxman; Raguz, Marija

2017-12-01

Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells

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Liang Y

2016-05-01

Full Text Available Yayun Liang,1 Benford Mafuvadze,1 Johannes D Aebi,2 Salman M Hyder1 1Dalton Cardiovascular Research Center and Department of Biomedical Sciences, University of Missouri-Columbia, Columbia, MO, USA; 2Medicinal Chemistry, Roche Pharma Research and Early Development (pRED, Roche Innovation Center Basel, F Hoffmann-La Roche Ltd., Basel, Switzerland Abstract: Standard treatment for primary prostate cancer includes systemic exposure to chemotherapeutic drugs that target androgen receptor or antihormone therapy (chemical castration; however, drug-resistant cancer cells generally emerge during treatment, limiting the continued use of systemic chemotherapy. Patients are then treated with more toxic standard therapies. Therefore, there is an urgent need for novel and more effective treatments for prostate cancer. The cholesterol biosynthetic pathway is an attractive therapeutic target for treating endocrine-dependent cancers because cholesterol is an essential structural and functional component of cell membranes as well as the metabolic precursor of endogenous steroid hormones. In this study, we have examined the effects of RO 48-8071 (4'-[6-(allylmethylaminohexyloxy]-4-bromo-2'-fluorobenzophenone fumarate; Roche Pharmaceuticals internal reference: RO0488071 (RO, which is an inhibitor of 2, 3-oxidosqualene cyclase (a key enzyme in the cholesterol biosynthetic pathway, on prostate cancer cells. Exposure of both hormone-dependent and castration-resistant human prostate cancer cells to RO reduced prostate cancer cell viability and induced apoptosis in vitro. RO treatment reduced androgen receptor protein expression in hormone-dependent prostate cancer cells and increased estrogen receptor β (ERβ protein expression in both hormone-dependent and castration-resistant prostate cancer cell lines. Combining RO with an ERβ agonist increased its ability to reduce castration-resistant prostate cancer cell viability. In addition, RO effectively suppressed the

Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

International Nuclear Information System (INIS)

Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

1987-01-01

Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in 3 H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture

The role of cholesterol metabolism and cholesterol transport in carcinogenesis; A review of scientific findings, relevant to future cancer therapeutics.

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Pedro Miguel Cruz

2013-09-01

Full Text Available While the unique metabolic activities of malignant tissues as potential targets for cancer therapeutics has been the subject of several recent reviews, the role of cholesterol metabolism in this context is yet to be fully explored. Cholesterol is an essential component of mammalian cell membranes as well as a precursor of bile acids and steroid hormones. The hypothesis that cancer cells need excess cholesterol and intermediates of the cholesterol biosynthesis pathway to maintain a high level of proliferation is well accepted, however the mechanisms by which malignant cells and tissues reprogram cholesterol synthesis, uptake and efflux are yet to be fully elucidated as potential therapeutic targets. High and low density plasma lipoproteins, area the likely major suppliers of cholesterol to cancer cells and tumors, potentially via receptor mediated mechanisms. This review is primarily focused on the role(s of lipoproteins in carcinogenesis, and their future roles as drug delivery vehicles for targeted cancer chemotherapy.

Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells.

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He, Chao; Chen, Ying; Liu, Chun; Cao, Ming; Fan, Yu-jin; Guo, Xiao-mei

2013-04-01

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (PLDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (Pcholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.

A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization

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Melanie L. Hutton

2017-06-01

Full Text Available The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as “lipid rafts.” Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248 with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01. HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05, and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05. Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.

Science.gov (United States)

Baar, Marjolein P; Brandt, Renata M C; Putavet, Diana A; Klein, Julian D D; Derks, Kasper W J; Bourgeois, Benjamin R M; Stryeck, Sarah; Rijksen, Yvonne; van Willigenburg, Hester; Feijtel, Danny A; van der Pluijm, Ingrid; Essers, Jeroen; van Cappellen, Wiggert A; van IJcken, Wilfred F; Houtsmuller, Adriaan B; Pothof, Joris; de Bruin, Ron W F; Madl, Tobias; Hoeijmakers, Jan H J; Campisi, Judith; de Keizer, Peter L J

2017-03-23

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging Xpd TTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored. Copyright © 2017 Elsevier Inc. All rights reserved.

IRF8 dependent classical dendritic cells are essential for intestinal T cell homeostasis

DEFF Research Database (Denmark)

Luda, K.; Joeris, Thorsten; Persson, E. K.

2016-01-01

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 dependent DCs have reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8ab+ andCD4+CD8......aa+ T cells; the latter requiring b8 integrin expression by migratory IRF8 dependent CD103+CD11b- DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI derived MLN DCs......, and inefficient T cell localization to the SI. Finally, mice with a DC deletion in IRF8 lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8...

The scavenger endothelial cell: a new player in homeostasis and immunity.

Science.gov (United States)

Sørensen, Karen Kristine; McCourt, Peter; Berg, Trond; Crossley, Clive; Le Couteur, David; Wake, Kenjiro; Smedsrød, Bård

2012-12-15

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 μm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

Survival of adult neurons lacking cholesterol synthesis in vivo.

Science.gov (United States)

Fünfschilling, Ursula; Saher, Gesine; Xiao, Le; Möbius, Wiebke; Nave, Klaus-Armin

2007-01-02

Cholesterol, an essential component of all mammalian plasma membranes, is highly enriched in the brain. Both during development and in the adult, brain cholesterol is derived from local cholesterol synthesis and not taken up from the circulation. However, the contribution of neurons and glial cells to total brain cholesterol metabolism is unknown. Using conditional gene inactivation in the mouse, we disrupted the squalene synthase gene (fdft1), which is critical for cholesterol synthesis, in cerebellar granule cells and some precerebellar nuclei. Mutant mice showed no histological signs of neuronal degeneration, displayed ultrastructurally normal synapses, and exhibited normal motor coordination. This revealed that these adult neurons do not require cell-autonomous cholesterol synthesis for survival or function. We conclude that at least some adult neurons no longer require endogenous cholesterol synthesis and can fully meet their cholesterol needs by uptake from their surrounding. Glia are a likely source of cholesterol in the central nervous system.

Survival of adult neurons lacking cholesterol synthesis in vivo

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Möbius Wiebke

2007-01-01

Full Text Available Abstract Background Cholesterol, an essential component of all mammalian plasma membranes, is highly enriched in the brain. Both during development and in the adult, brain cholesterol is derived from local cholesterol synthesis and not taken up from the circulation. However, the contribution of neurons and glial cells to total brain cholesterol metabolism is unknown. Results Using conditional gene inactivation in the mouse, we disrupted the squalene synthase gene (fdft1, which is critical for cholesterol synthesis, in cerebellar granule cells and some precerebellar nuclei. Mutant mice showed no histological signs of neuronal degeneration, displayed ultrastructurally normal synapses, and exhibited normal motor coordination. This revealed that these adult neurons do not require cell-autonomous cholesterol synthesis for survival or function. Conclusion We conclude that at least some adult neurons no longer require endogenous cholesterol synthesis and can fully meet their cholesterol needs by uptake from their surrounding. Glia are a likely source of cholesterol in the central nervous system.

GM-CSF Controls Nonlymphoid Tissue Dendritic Cell Homeostasis but Is Dispensable for the Differentiation of Inflammatory Dendritic Cells

Science.gov (United States)

Greter, Melanie; Helft, Julie; Chow, Andrew; Hashimoto, Daigo; Mortha, Arthur; Agudo-Cantero, Judith; Bogunovic, Milena; Gautier, Emmanuel L.; Miller, Jennifer; Leboeuf, Marylene; Lu, Geming; Aloman, Costica; Brown, Brian D.; Pollard, Jeffrey W.; Xiong, Huabao; Randolph, Gwendalyn J.; Chipuk, Jerry E.; Frenette, Paul S.; Merad, Miriam

2012-01-01

SUMMARY GM-CSF (Csf-2) is a critical cytokine for the in vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103+ DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103+ and CD11b+ DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8+ T cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8+ T cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in vivo. PMID:22749353

Longitudinal Trajectories of Cholesterol from Midlife through Late Life according to Apolipoprotein E Allele Status

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Brian Downer

2014-10-01

Full Text Available Background: Previous research indicates that total cholesterol levels increase with age during young adulthood and middle age and decline with age later in life. This is attributed to changes in diet, body composition, medication use, physical activity, and hormone levels. In the current study we utilized data from the Framingham Heart Study Original Cohort to determine if variations in apolipoprotein E (APOE, a gene involved in regulating cholesterol homeostasis, influence trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio from midlife through late life. Methods: Cholesterol trajectories from midlife through late life were modeled using generalized additive mixed models and mixed-effects regression models. Results: APOE e2+ subjects had lower total cholesterol levels, higher HDL cholesterol levels, and lower total: HDL cholesterol ratios from midlife to late life compared to APOE e3 and APOE e4+ subjects. Statistically significant differences in life span cholesterol trajectories according to gender and use of cholesterol-lowering medications were also detected. Conclusion: The findings from this research provide evidence that variations in APOE modify trajectories of serum cholesterol from midlife to late life. In order to efficiently modify cholesterol through the life span, it is important to take into account APOE allele status.

The effect of plant sterol-enriched turkey meat on cholesterol bio-accessibility during in vitro digestion and Caco-2 cell uptake.

Science.gov (United States)

Grasso, S; Harrison, S M; Monahan, F J; Brayden, D; Brunton, N P

2018-03-01

This study evaluated the effect of a plant sterol-enriched turkey product on cholesterol bio-accessibility during in vitro digestion and cholesterol uptake by Caco-2 monolayers. Turkey products, one plant sterol-enriched (PS) and one plant sterol-free (C), were produced in an industrial pilot plant. Before simulated digestion, matrices were spiked with cholesterol (1:5 weight ratio of cholesterol to plant sterol). Plant sterols were included at a concentration equivalent to the minimum daily intake recommended by the European Food Safety Authority (EFSA) for cholesterol lowering. After simulated digestion, the percentage of cholesterol micellarization and uptake by Caco-2 cells in the presence of PS meat were measured. Compared to C meat, PS meat significantly inhibited cholesterol micellarization on average by 24% and Caco-2 cell accumulation by 10%. This study suggests that plant sterols in meat can reduce cholesterol uptake by intestinal epithelia and it encourages efforts to make new PS-based functional foods.

Dendrogenin A arises from cholesterol and histamine metabolism and shows cell differentiation and anti-tumour properties.

Science.gov (United States)

de Medina, Philippe; Paillasse, Michael R; Segala, Gregory; Voisin, Maud; Mhamdi, Loubna; Dalenc, Florence; Lacroix-Triki, Magali; Filleron, Thomas; Pont, Frederic; Saati, Talal Al; Morisseau, Christophe; Hammock, Bruce D; Silvente-Poirot, Sandrine; Poirot, Marc

2013-01-01

We previously synthesized dendrogenin A and hypothesized that it could be a natural metabolite occurring in mammals. Here we explore this hypothesis and report the discovery of dendrogenin A in mammalian tissues and normal cells as an enzymatic product of the conjugation of 5,6α-epoxy-cholesterol and histamine. Dendrogenin A was not detected in cancer cell lines and was fivefold lower in human breast tumours compared with normal tissues, suggesting a deregulation of dendrogenin A metabolism during carcinogenesis. We established that dendrogenin A is a selective inhibitor of cholesterol epoxide hydrolase and it triggered tumour re-differentiation and growth control in mice and improved animal survival. The properties of dendrogenin A and its decreased level in tumours suggest a physiological function in maintaining cell integrity and differentiation. The discovery of dendrogenin A reveals a new metabolic pathway at the crossroads of cholesterol and histamine metabolism and the existence of steroidal alkaloids in mammals.

What Are High Blood Cholesterol and Triglycerides?

Science.gov (United States)

... Reduction Cholesterol What Are High Blood Cholesterol and Triglycerides? Cholesterol travels to the body’s cells through the ... doctor about medicines that can help. What are triglycerides? Triglycerides are the most common type of fat ...

Salisphere derived c-Kit+ cell transplantation restores tissue homeostasis in irradiated salivary gland

International Nuclear Information System (INIS)

Nanduri, Lalitha S.Y.; Lombaert, Isabelle M.A.; Zwaag, Marianne van der; Faber, Hette; Brunsting, Jeanette F.; Os, Ronald P. van; Coppes, Robert P.

2013-01-01

Introduction: During radiotherapy salivary glands of head and neck cancer patients are unavoidably co-irradiated, potentially resulting in life-long impairment. Recently we showed that transplantation of salisphere-derived c-Kit expressing cells can functionally regenerate irradiated salivary glands. This study aims to select a more potent subpopulation of c-Kit + cells, co-expressing stem cell markers and to investigate whether long-term tissue homeostasis is restored after stem cell transplantation. Methods and results: Salisphere derived c-Kit + cells that co-expressed CD24 and/or CD49f markers, were intra-glandularly injected into 15 Gy irradiated submandibular glands of mice. Particularly, c-Kit + /CD24 + /CD49f + cell transplanted mice improved saliva production (54.59 ± 11.1%) versus the irradiated control group (21.5 ± 8.7%). Increase in expression of cells with differentiated duct cell markers like, cytokeratins (CK8, 18, 7 and 14) indicated functional recovery of this compartment. Moreover, ductal stem cell marker expression like c-Kit, CD133, CD24 and CD49f reappeared after transplantation indicating long-term functional maintenance potential of the gland. Furthermore, a normalization of vascularization as indicated by CD31 expression and reduction of fibrosis was observed, indicative of normalization of the microenvironment. Conclusions: Our results show that stem cell transplantation not only rescues hypo-salivation, but also restores tissue homeostasis of the irradiated gland, necessary for long-term maintenance of adult tissue

Effects of cholesterol on progesterone production by goat luteal cell subpopulations at two different stages of the luteal phase.

Science.gov (United States)

Arikan, Ş; Kalender, H; Simsek, O

2010-12-01

The aim of the present study was to evaluate the effects of cholesterol on progesterone production during long-term culturing of luteal cell subpopulations at early and late luteal stages of the goat corpora lutea. Corpora lutea were collected from Angora goats on days 5 and 15 of the oestrous cycle. Luteal cells were isolated by collagenase digestion. The cells were separated into two distinct subpopulations by Percoll density-gradient centrifugation. Both subpopulations of luteal cells staining positively for 3β-HSD activities (5 × 10(4)  cell/well) were cultured with or without 22(R)-hydroxycholesterol (22R-HC) in serum-free culture medium for periods of up to 7 days. Cells were incubated with serum (10%) for the first 18 h of incubation followed by serum-free medium. Cell treatment (10 and 20 μg/ml) was performed on days 1, 3 and 5. Treatment of cells with both concentrations of 22R-HC resulted in significant (p  0.05) on progesterone production in both fractions of cells throughout 7 days of incubation. Treatment of the cells with cholesterol resulted in 2.5- and 9.0-fold increases in progesterone accumulation on day 3 of incubation. Steroid production was maintained throughout the incubations when cells are incubated in serum-free media treated with cholesterol and ITS premix. Cells collected from higher density of percoll layers produced 2.82 and 2.32 times more progesterone, in comparison to the lover density percoll layer, on days 5 and 15 of the oestrous cycle in untreated cell groups, respectively. Progesterone accumulation was decreased as incubation time advanced in all groups of untreated cells. These results demonstrated that goat luteal cell subpopulations secrete substantial amounts of progesterone in response to cholesterol treatment at least for 7 days, and cholesterol is required as progesterone precursor for maintaining a high-level steroidogenesis during long-life culturing of both cell subpopulations. © 2010 Blackwell

Use of quantitative optical imaging to examine the role of cholesterol-rich lipid raft microdomains in the migration of breast cancer cells

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You, Minghai; Chen, Jianling; Wang, Shaobing; Dong, Shiqing; Wang, Yuhua; Xie, Shusen; Wang, Zhengchao; Yang, Hongqin

2018-04-01

Lipid rafts have been extensively studied and shown to be involved in many cancers, including breast cancer. However, the exact role of lipid rafts in the migration of breast cancer cells remains unclear. This study was designed to examine lipid rafts (cholesterol) in the plasma membrane of breast cancer cells (MDA-MB-231 and MCF-7) and normal breast epithelial cells (MCF-10A) through generalized polarization values, and further investigate the role of cholesterol-rich lipid rafts in the migration of breast cancer cells. The results showed that the plasma membrane in breast cancer cells was more orderly than that in normal epithelial cells; this was correlated with expression changes of matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR), the markers of cancer cell migration. Moreover, the breast cancer cells were more sensitive to the reagent that induced cholesterol depletion than the normal breast epithelial cells, while the capacity of cancer cells to migrate decreased significantly according to changes in MMP-9 and uPAR expression. To our best knowledge, this is the first demonstration of the relationship between cholesterol-rich lipid rafts and the migration of breast cancer cells; it could be useful for the prevention of breast cancer and early treatment through reduction of the level of cholesterol in the plasma membrane of the cells.

Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits

International Nuclear Information System (INIS)

Cavallero, C.; Tondo, U. di; Mingazinni, P.L.; Nicosia, R.; Pericoli, M.N.; Sarti, P.; Spagnoli, L.G.; Villaschi, S.

1976-01-01

Tritiated thymidine radioautography was employed to study the effect of cortisol and other glucocorticoids on cellular proliferation in the aorta and pulmonary artery of rabbits with cholesterol atherosclerosis. Labelled cell counts showed that glucocorticoids, even after one day and at a relatively low dose, decrease sharply the deoxyribonucleic acid synthesis in the intimal plaques. The hormonal influence on [ 3 H] thymidine uptake seems to be a dose-dependent process. The relative potency of these steroids in inhibiting DNA synthesis in the plaques parallels closely their anti-inflammatory effectiveness. Conversely mineralocorticoids, including aldosterone and deoxycorticosterone, increase the rate of DNA synthesis in the plaques. It is concluded that the anti-atherogenic effect of glucocorticoids on cholesterol-fed rabbits may be due, at least partly, to the inhibitory effect of these steroids on the DNA synthesis of the cellular components of the intimal plaques

Trapping crystal nucleation of cholesterol monohydrate

DEFF Research Database (Denmark)

Solomonov, I.; Weygand, M.J.; Kjær, K.

2005-01-01

Crystalline nucleation of cholesterol at the air-water interface has been studied via grazing incidence x-ray diffraction using synchrotron radiation. The various stages of cholesterol molecular assembly from monolayer to three bilayers incorporating interleaving hydrogen-bonded water layers......, at least initially, an intralayer cholesterol rearrangement in a single-crystal-to-single-crystal transition. The preferred nucleation of the monoclinic phase of cholesterol . H2O followed by transformation to the stable monohydrate phase may be associated with an energetically more stable cholesterol...... bilayer arrangement of the former and a more favorable hydrogen-bonding arrangement of the latter. The relevance of this nucleation process of cholesterol monohydrate to pathological crystallization of cholesterol from cell biomembranes is discussed....

Innate lymphoid cells: models of plasticity for immune homeostasis and rapid responsiveness in protection.

Science.gov (United States)

Almeida, F F; Belz, G T

2016-09-01

Innate lymphoid cells (ILCs) have stormed onto the immune landscape as "newly discovered" cell types. These tissue-resident sentinels are enriched at mucosal surfaces and engage in complex cross talk with elements of the adaptive immune system and microenvironment to orchestrate immune homeostasis. Many parallels exist between innate cells and T cells leading to the initial partitioning of ILCs into rather rigid subsets that reflect their "adaptive-like" effector cytokines profiles. ILCs themselves, however, have unique attributes that are only just beginning to be elucidated. These features result in complementarity with, rather than complete duplication of, functions of the adaptive immune system. Key transcription factors determine the pathway of differentiation of progenitors towards an ILC1, ILC2, or ILC3 subset. Once formed, flexibility in the responses of these subsets to stimuli unexpectedly allows transdifferentation between the different subsets and the acquisition of altered phenotypes and function. This provides a mechanism for rapid innate immune responsiveness. Here, we discuss the models of differentiation for maintenance and activation of tissue-resident ILCs in maintaining immune homeostasis and protection.

Anatomical localization of commensal bacteria in immune cell homeostasis and disease.

Science.gov (United States)

Fung, Thomas C; Artis, David; Sonnenberg, Gregory F

2014-07-01

The mammalian gastrointestinal (GI) tract is colonized by trillions of beneficial commensal bacteria that are essential for promoting normal intestinal physiology. While the majority of commensal bacteria are found in the intestinal lumen, many species have also adapted to colonize different anatomical locations in the intestine, including the surface of intestinal epithelial cells (IECs) and the interior of gut-associated lymphoid tissues. These distinct tissue localization patterns permit unique interactions with the mammalian immune system and collectively influence intestinal immune cell homeostasis. Conversely, dysregulated localization of commensal bacteria can lead to inappropriate activation of the immune system and is associated with numerous chronic infectious, inflammatory, and metabolic diseases. Therefore, regulatory mechanisms that control proper anatomical containment of commensal bacteria are essential to maintain tissue homeostasis and limit pathology. In this review, we propose that commensal bacteria associated with the mammalian GI tract can be anatomically defined as (i) luminal, (ii) epithelial-associated, or (iii) lymphoid tissue-resident, and we discuss the role and regulation of these microbial populations in health and disease. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Novel Role for C5a in B-1 Cell Homeostasis

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Katharina Bröker

2018-02-01

Full Text Available B-1 cells constitute a unique subpopulation of lymphocytes residing mainly in body cavities like the peritoneal cavity (PerC but are also found in spleen and bone marrow (BM. As innate-like B cells, they mediate first line immune defense through low-affinity natural IgM (nIgM antibodies. PerC B-1 cells can egress to the spleen and differentiate into nIgM antibody-secreting plasma cells that recognize conserved exogenous and endogenous cellular structures. Homing to and homeostasis within the PerC are regulated by the chemokine CXCL13 released by PerC macrophages and stroma cells. However, the exact mechanisms underlying the regulation of CXCL13 and B-1 homeostasis are not fully explored. B-1 cells play important roles in the inflammatory response to infection, autoimmunity, ischemia/reperfusion injury, obesity, and atherosclerosis. Remarkably, this list of inflammatory entities has a strong overlap with diseases that are regulated by complement suggesting a link between B-1 cells and the complement system. Interestingly, up to now, no data exist regarding the role of complement in B-1 cell biology. Here, we demonstrate for the first time that C5a regulates B-1 cell steady-state dynamics within the peritoneum, the spleen, and the BM. We found decreased B-1a cell numbers in the peritoneum and the spleen of C5aR1−/− mice associated with increased B1-a and B1-b numbers in the spleen and high serum titers of nIgM antibodies directed against phosphorylcholine and several pneumococcal polysaccharides. Similarly, peritoneal B-1a cells were decreased in the peritoneum and splenic B-1a and B-1b cells were increased in C5aR2−/− mice. The decrease in peritoneal B-1 cell numbers was associated with decreased peritoneal CXCL13 levels in C5aR1−/− and C5aR2−/− mice. In search for mechanisms, we found that combined TLR2 and IL-10 receptor activation in PerC macrophages induced strong CXCL13 production, which was significantly reduced in cells

NAC selectively inhibit cancer telomerase activity: A higher redox homeostasis threshold exists in cancer cells

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Pengying Li

2016-08-01

Full Text Available Telomerase activity controls telomere length, and this plays an important role in stem cells, aging and tumors. Antioxidant was shown to protect telomerase activity in normal cells but inhibit that in cancer cells, but the underlying mechanism is elusive. Here we found that 7721 hepatoma cells held a higher redox homeostasis threshold than L02 normal liver cells which caused 7721 cells to have a higher demand for ROS; MnSOD over-expression in 7721 decreased endogenous reactive oxygen species (ROS and inhibited telomerase activity; Akt phosphorylation inhibitor and NAC both inhibited 7721 telomerase activity. The over-elimination of ROS by NAC resulted in the inhibition of Akt pathway. Our results suggest that ROS is involved in the regulation of cancer telomerase activity through Akt pathway. The different intracellular redox homeostasis and antioxidant system in normal cells and tumor cells may be the cause of the opposite effect on telomerase activity in response to NAC treatment. Our results provide a theoretical base of using antioxidants selectively inhibit cancer telomerase activity. Findings of the present study may provide insights into novel approaches for cancer treatment.

Response of normal stem cells to ionizing radiation: A balance between homeostasis and genomic stability

International Nuclear Information System (INIS)

Harfouche, G.; Martin, M.T.

2010-01-01

Stem cells have been described in most adult tissues, where they play a key role in maintaining tissue homeostasis. As they self-renew throughout life, accumulating genetic anomalies can compromise their genomic integrity and potentially give rise to cancer. Stem cells (SCs) may thus be a major target of radiation carcinogenesis. In addition, unrepaired genotoxic damage may cause cell death and stem cell pool depletion, impairing lineage functionality and accelerating aging. Developments in SC biology enabled the characterization of the responses of stem cells to genotoxic stress and their role in tissue damage. We here examine how these cells react to ionizing radiation (IR), and more specifically their radiosensitivity, stress signaling and DNA repair. We first review embryonic SCs, as a paradigm of primitive pluri-potent cells, then three adult tissues, bone marrow, skin and intestine, capable of long-term regeneration and at high risk for acute radiation syndromes and long-term carcinogenesis. We discuss IR disruption of the fine balance between maintenance of tissue homeostasis and genomic stability. We show that stem cell radiosensitivity does not follow a unique model, but differs notably according to the turnover rates of the tissues. (authors)

TAK1 modulates satellite stem cell homeostasis and skeletal muscle repair

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Ogura, Yuji; Hindi, Sajedah M.; Sato, Shuichi; Xiong, Guangyan; Akira, Shizuo; Kumar, Ashok

2015-01-01

Satellite cells are resident adult stem cells that are required for regeneration of skeletal muscle. However, signalling mechanisms that regulate satellite cell function are less understood. Here we demonstrate that transforming growth factor-β-activated kinase 1 (TAK1) is important in satellite stem cell homeostasis and function. Inactivation of TAK1 in satellite cells inhibits muscle regeneration in adult mice. TAK1 is essential for satellite cell proliferation and its inactivation causes precocious differentiation. Moreover, TAK1-deficient satellite cells exhibit increased oxidative stress and undergo spontaneous cell death, primarily through necroptosis. TAK1 is required for the activation of NF-κB and JNK in satellite cells. Forced activation of NF-κB improves survival and proliferation of TAK1-deficient satellite cells. Furthermore, TAK1-mediated activation of JNK is essential to prevent oxidative stress and precocious differentiation of satellite cells. Collectively, our study suggests that TAK1 is required for maintaining the pool of satellite stem cells and for regenerative myogenesis. PMID:26648529

Prion protein misfolding affects calcium homeostasis and sensitizes cells to endoplasmic reticulum stress.

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Mauricio Torres

2010-12-01

Full Text Available Prion-related disorders (PrDs are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrP(RES. Altered endoplasmic reticulum (ER homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrP(RES. Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrP(RES and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.

Angiogenin activates phospholipase C and elicits a rapid incorporation of fatty acid into cholesterol esters in vascular smooth muscle cells

International Nuclear Information System (INIS)

Moore, F.; Riordan, J.F.

1990-01-01

Angiogenin activates the phosphoinositide-specific phospholipase C (PLC) in cultured rat aortic smooth muscle cells to yield a transient (30 s) peak of 1,2-diacylglycerol (DG) and inositol trisphosphate. Within 1 min, the DG level falls below that of the control and remains so for at least 20 min. A transient increase in monoacylglycerol indicates that depletion of DG may be the consequence of hydrolysis by DG lipase. In addition to these changes in second messengers, a rapid increase in incorporating of radiolabeled tracer into cellular cholesterol esters is observed. Stimulated cholesterol ester labeling is inhibited by preincubation with either the DG lipase inhibitor RHC 80267 or the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58035. Cells prelabeled with [ 3 H]arachidonate show a sustained increase in labeling of cholesterol esters following exposure to angiogenin. In contrast, cells prelabeled with [ 3 H]oleate show only a transient elevation that returns to the basal level by 5 min. This suggests initial cholesterol esterification by oleate followed by arachidonate that is released by stimulation of the PLC/DG lipase pathway

Effect of methanolic extract of Piper sarmentosum leaves on neointimal foam cell infiltration in rabbits fed with high cholesterol diet

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Amran, Adel A.; Zakaria, Zaiton; Othman, Faizah; Das, Srijit; Al-Mekhlafi, Hesham M.; Raj, Santhana; Nordin, Nor-Anita MM

2012-01-01

Previous research has shown the beneficial effects of aqueous extract of Piper sarmentosum (P.s) on atherosclerosis. The first stage in atherosclerosis is the formation of foam cell. The aim of this study was to investigate the effect of the methanol extract of P.s on fatty streaks by calculating neointimal foam cell infiltration in rabbits fed with high cholesterol diet. Thirty six male New Zealand white rabbits were divided equally into six groups: (i) C: control group fed normal rabbit chow; (ii) CH: cholesterol diet (1 % cholesterol); (iii) PM1: 1 % cholesterol with methanol extract of P.s (62.5 mg/kg); (iv) PM2: 1 % cholesterol with methanol extract of P.s (125 mg/kg); (v) PM3: 1 % cholesterol with methanol extract of P.s (250 mg/kg); (vi) SMV group fed 1 % cholesterol supplemented with Simvistatin drug (1.2 mg/kg). All animals were treated for 10 weeks. At the end of the treatment, the rabbits were fasted and sacrificed and the aortic tissues were collected for histological studies to measure the area of the neointimal foam cell infiltration using software. The thickening of intima ratio of atherosclerosis and morphological changes by scanning electron microscope were measured. The results showed that the atherosclerotic group had significantly bigger area of fatty streak compared to the control group. The area of fatty streak in the abdominal aorta was significantly reduced in the treatment groups which were similar with the SMV group. Similarly, there was a reduction in the number of foam cell in the treatment groups compared to the atherosclerotic group as seen under scanning microscope. In conclusion, histological study demonstrated that the methanol extract of the P.s could reduce the neointimal foam cell infiltration in the lumen of the aorta and the atherosclerotic lesion. PMID:27366140

Sex Hormones and Their Receptors Regulate Liver Energy Homeostasis

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Minqian Shen

2015-01-01

Full Text Available The liver is one of the most essential organs involved in the regulation of energy homeostasis. Hepatic steatosis, a major manifestation of metabolic syndrome, is associated with imbalance between lipid formation and breakdown, glucose production and catabolism, and cholesterol synthesis and secretion. Epidemiological studies show sex difference in the prevalence in fatty liver disease and suggest that sex hormones may play vital roles in regulating hepatic steatosis. In this review, we summarize current literature and discuss the role of estrogens and androgens and the mechanisms through which estrogen receptors and androgen receptors regulate lipid and glucose metabolism in the liver. In females, estradiol regulates liver metabolism via estrogen receptors by decreasing lipogenesis, gluconeogenesis, and fatty acid uptake, while enhancing lipolysis, cholesterol secretion, and glucose catabolism. In males, testosterone works via androgen receptors to increase insulin receptor expression and glycogen synthesis, decrease glucose uptake and lipogenesis, and promote cholesterol storage in the liver. These recent integrated concepts suggest that sex hormone receptors could be potential promising targets for the prevention of hepatic steatosis.

Cellular Cholesterol Facilitates the Postentry Replication Cycle of Herpes Simplex Virus 1.

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Wudiri, George A; Nicola, Anthony V

2017-07-15

Cholesterol is an essential component of cell membranes and is required for herpes simplex virus 1 (HSV-1) entry (1-3). Treatment of HSV-1-infected Vero cells with methyl beta-cyclodextrin from 2 to 9 h postentry reduced plaque numbers. Transport of incoming viral capsids to the nuclear periphery was unaffected by the cholesterol reduction, suggesting that cell cholesterol is important for the HSV-1 replicative cycle at a stage(s) beyond entry, after the arrival of capsids at the nucleus. The synthesis and release of infectious HSV-1 and cell-to-cell spread of infection were all impaired in cholesterol-reduced cells. Propagation of HSV-1 on DHCR24 -/- fibroblasts, which lack the desmosterol-to-cholesterol conversion enzyme, resulted in the generation of infectious extracellular virions (HSV des ) that lack cholesterol and likely contain desmosterol. The specific infectivities (PFU per viral genome) of HSV chol and HSV des were similar, suggesting cholesterol and desmosterol in the HSV envelope support similar levels of infectivity. However, infected DHCR24 -/- fibroblasts released ∼1 log less infectious HSV des and ∼1.5 log fewer particles than release of cholesterol-containing particles (HSV chol ) from parental fibroblasts, suggesting that the hydrocarbon tail of cholesterol facilitates viral synthesis. Together, the results suggest multiple roles for cholesterol in the HSV-1 replicative cycle. IMPORTANCE HSV-1 infections are associated with a wide range of clinical manifestations that are of public health importance. Cholesterol is a key player in the complex interaction between viral and cellular factors that allows HSV-1 to enter host cells and establish infection. Previous reports have demonstrated a role for cellular cholesterol in the entry of HSV-1 into target cells. Here, we employed both chemical treatment and cells that were genetically defined to synthesize only desmosterol to demonstrate that cholesterol is important at stages following the

Increased expression of RXRα in dementia: an early harbinger for the cholesterol dyshomeostasis?

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Katsel Pavel

2010-09-01

Full Text Available Abstract Background Cholesterol content of cerebral membranes is tightly regulated by elaborate mechanisms that balance the level of cholesterol synthesis, uptake and efflux. Among the conventional regulatory elements, a recent research focus has been nuclear receptors, a superfamily of ligand-activated transcription factors providing an indispensable regulatory framework in controlling cholesterol metabolism pathway genes. The mechanism of transcriptional regulation by nuclear receptors such as LXRs involves formation of heterodimers with RXRs. LXR/RXR functions as a sensor of cellular cholesterol concentration and mediates cholesterol efflux by inducing the transcription of key cholesterol shuffling vehicles namely, ATP-binding cassette transporter A1 (ABCA1 and ApoE. Results In the absence of quantitative data from humans, the relevance of expression of nuclear receptors and their involvement in cerebral cholesterol homeostasis has remained elusive. In this work, new evidence is provided from direct analysis of human postmortem brain gene and protein expression suggesting that RXRα, a key regulator of cholesterol metabolism is differentially expressed in individuals with dementia. Importantly, RXRα expression showed strong association with ABCA1 and ApoE gene expression, particularly in AD vulnerable regions. Conclusions These findings suggest that LXR/RXR-induced upregulation of ABCA1 and ApoE levels may be the molecular determinants of cholesterol dyshomeostasis and of the accompanying dementia observed in AD.

Cellular copper homeostasis: current concepts on its interplay with glutathione homeostasis and its implication in physiology and human diseases.

Science.gov (United States)

Bhattacharjee, Ashima; Chakraborty, Kaustav; Shukla, Aditya

2017-10-18

Copper is a trace element essential for almost all living organisms. But the level of intracellular copper needs to be tightly regulated. Dysregulation of cellular copper homeostasis leading to various diseases demonstrates the importance of this tight regulation. Copper homeostasis is regulated not only within the cell but also within individual intracellular compartments. Inactivation of export machinery results in excess copper being redistributed into various intracellular organelles. Recent evidence suggests the involvement of glutathione in playing an important role in regulating copper entry and intracellular copper homeostasis. Therefore interplay of both homeostases might play an important role within the cell. Similar to copper, glutathione balance is tightly regulated within individual cellular compartments. This review explores the existing literature on the role of glutathione in regulating cellular copper homeostasis. On the one hand, interplay of glutathione and copper homeostasis performs an important role in normal physiological processes, for example neuronal differentiation. On the other hand, perturbation of the interplay might play a key role in the pathogenesis of copper homeostasis disorders.

Impact of heme oxygenase-1 on cholesterol synthesis, cholesterol efflux and oxysterol formation in cultured astroglia.

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Hascalovici, Jacob R; Song, Wei; Vaya, Jacob; Khatib, Soliman; Fuhrman, Bianca; Aviram, Michael; Schipper, Hyman M

2009-01-01

Up-regulation of heme oxygenase-1 (HO-1) and altered cholesterol (CH) metabolism are characteristic of Alzheimer-diseased neural tissues. The liver X receptor (LXR) is a molecular sensor of CH homeostasis. In the current study, we determined the effects of HO-1 over-expression and its byproducts iron (Fe(2+)), carbon monoxide (CO) and bilirubin on CH biosynthesis, CH efflux and oxysterol formation in cultured astroglia. HO-1/LXR interactions were also investigated in the context of CH efflux. hHO-1 over-expression for 3 days ( approximately 2-3-fold increase) resulted in a 30% increase in CH biosynthesis and a two-fold rise in CH efflux. Both effects were abrogated by the competitive HO inhibitor, tin mesoporphyrin. CO, released from administered CORM-3, significantly enhanced CH biosynthesis; a combination of CO and iron stimulated CH efflux. Free iron increased oxysterol formation three-fold. Co-treatment with LXR antagonists implicated LXR activation in the modulation of CH homeostasis by heme degradation products. In Alzheimer's disease and other neuropathological states, glial HO-1 induction may transduce ambient noxious stimuli (e.g. beta-amyloid) into altered patterns of glial CH homeostasis. As the latter may impact synaptic plasticity and neuronal repair, modulation of glial HO-1 expression (by pharmacological or other means) may confer neuroprotection in patients with degenerative brain disorders.

Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes

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Habchi, Johnny; Chia, Sean; Galvagnion, Céline; Michaels, Thomas C. T.; Bellaiche, Mathias M. J.; Ruggeri, Francesco Simone; Sanguanini, Michele; Idini, Ilaria; Kumita, Janet R.; Sparr, Emma; Linse, Sara; Dobson, Christopher M.; Knowles, Tuomas P. J.; Vendruscolo, Michele

2018-06-01

Alzheimer's disease is a neurodegenerative disorder associated with the aberrant aggregation of the amyloid-β peptide. Although increasing evidence implicates cholesterol in the pathogenesis of Alzheimer's disease, the detailed mechanistic link between this lipid molecule and the disease process remains to be fully established. To address this problem, we adopt a kinetics-based strategy that reveals a specific catalytic role of cholesterol in the aggregation of Aβ42 (the 42-residue form of the amyloid-β peptide). More specifically, we demonstrate that lipid membranes containing cholesterol promote Aβ42 aggregation by enhancing its primary nucleation rate by up to 20-fold through a heterogeneous nucleation pathway. We further show that this process occurs as a result of cooperativity in the interaction of multiple cholesterol molecules with Aβ42. These results identify a specific microscopic pathway by which cholesterol dramatically enhances the onset of Aβ42 aggregation, thereby helping rationalize the link between Alzheimer's disease and the impairment of cholesterol homeostasis.

Conditional ablation of CD205+ conventional dendritic cells impacts the regulation of T-cell immunity and homeostasis in vivo.

Science.gov (United States)

Fukaya, Tomohiro; Murakami, Ryuichi; Takagi, Hideaki; Sato, Kaori; Sato, Yumiko; Otsuka, Haruna; Ohno, Michiko; Hijikata, Atsushi; Ohara, Osamu; Hikida, Masaki; Malissen, Bernard; Sato, Katsuaki

2012-07-10

Dendritic cells (DCs) are composed of multiple subsets that play a dual role in inducing immunity and tolerance. However, it is unclear how CD205(+) conventional DCs (cDCs) control immune responses in vivo. Here we generated knock-in mice with the selective conditional ablation of CD205(+) cDCs. CD205(+) cDCs contributed to antigen-specific priming of CD4(+) T cells under steady-state conditions, whereas they were dispensable for antigen-specific CD4(+) T-cell responses under inflammatory conditions. In contrast, CD205(+) cDCs were required for antigen-specific priming of CD8(+) T cells to generate cytotoxic T lymphocytes (CTLs) mediated through cross-presentation. Although CD205(+) cDCs were involved in the thymic generation of CD4(+) regulatory T cells (Tregs), they maintained the homeostasis of CD4(+) Tregs and CD4(+) effector T cells in peripheral and mucosal tissues. On the other hand, CD205(+) cDCs were involved in the inflammation triggered by Toll-like receptor ligand as well as bacterial and viral infections. Upon microbial infections, CD205(+) cDCs contributed to the cross-priming of CD8(+) T cells for generating antimicrobial CTLs to efficiently eliminate pathogens, whereas they suppressed antimicrobial CD4(+) T-cell responses. Thus, these findings reveal a critical role for CD205(+) cDCs in the regulation of T-cell immunity and homeostasis in vivo.

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

Science.gov (United States)

Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

2009-11-15

A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells

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Dorothea Maetzel

2014-06-01

Full Text Available Niemann-Pick type C (NPC disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs using transcription activator-like effector nucleases (TALENs. We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.

Propiconazole-enhanced hepatic cell proliferation is associated with dysregulation of the cholesterol biosynthesis pathway leading to activation of Erk1/2 through Ras farnesylation

Energy Technology Data Exchange (ETDEWEB)

Murphy, Lynea A.; Moore, Tanya; Nesnow, Stephen, E-mail: nesnow.stephen@epa.gov

2012-04-15

Propiconazole is a mouse hepatotumorigenic fungicide designed to inhibit CYP51, a key enzyme in the biosynthesis of ergosterol in fungi and is widely used in agriculture to prevent fungal growth. Metabolomic studies in mice revealed that propiconazole increased levels of hepatic cholesterol metabolites and bile acids, and transcriptomic studies revealed that genes within the cholesterol biosynthesis, cholesterol metabolism and bile acid biosyntheses pathways were up-regulated. Hepatic cell proliferation was also increased by propiconazole. AML12 immortalized hepatocytes were used to study propiconazole's effects on cell proliferation focusing on the dysregulation of cholesterol biosynthesis and resulting effects on Ras farnesylation and Erk1/2 activation as a primary pathway. Mevalonate, a key intermediate in the cholesterol biosynthesis pathway, increases cell proliferation in several cancer cell lines and tumors in vivo and serves as the precursor for isoprenoids (e.g. farnesyl pyrophosphate) which are crucial in the farnesylation of the Ras protein by farnesyl transferase. Farnesylation targets Ras to the cell membrane where it is involved in signal transduction, including the mitogen-activated protein kinase (MAPK) pathway. In our studies, mevalonic acid lactone (MVAL), a source of mevalonic acid, increased cell proliferation in AML12 cells which was reduced by farnesyl transferase inhibitors (L-744,832 or manumycin) or simvastatin, an HMG-CoA reductase inhibitor, indicating that this cell system responded to alterations in the cholesterol biosynthesis pathway. Cell proliferation in AML12 cells was increased by propiconazole which was reversed by co-incubation with L-744,832 or simvastatin. Increasing concentrations of exogenous cholesterol muted the proliferative effects of propiconazole and the inhibitory effects of L-733,832, results ascribed to reduced stimulation of the endogenous cholesterol biosynthesis pathway. Western blot analysis of subcellular

Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin(1A) receptor in the plasma membrane of living cells.

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Pucadyil, Thomas J; Chattopadhyay, Amitabha

2007-03-01

Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin(1A) receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin(1A) receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin(1A) receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin(1A) receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.

Membrane cholesterol mediates the cellular effects of monolayer graphene substrates.

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Kitko, Kristina E; Hong, Tu; Lazarenko, Roman M; Ying, Da; Xu, Ya-Qiong; Zhang, Qi

2018-02-23

Graphene possesses extraordinary properties that promise great potential in biomedicine. However, fully leveraging these properties requires close contact with the cell surface, raising the concern of unexpected biological consequences. Computational models have demonstrated that graphene preferentially interacts with cholesterol, a multifunctional lipid unique to eukaryotic membranes. Here we demonstrate an interaction between graphene and cholesterol. We find that graphene increases cell membrane cholesterol and potentiates neurotransmission, which is mediated by increases in the number, release probability, and recycling rate of synaptic vesicles. In fibroblasts grown on graphene, we also find an increase in cholesterol, which promotes the activation of P2Y receptors, a family of receptor regulated by cholesterol. In both cases, direct manipulation of cholesterol levels elucidates that a graphene-induced cholesterol increase underlies the observed potentiation of each cell signaling pathway. These findings identify cholesterol as a mediator of graphene's cellular effects, providing insight into the biological impact of graphene.

Altered GPM6A/M6 dosage impairs cognition and causes phenotypes responsive to cholesterol in human and Drosophila.

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Gregor, Anne; Kramer, Jamie M; van der Voet, Monique; Schanze, Ina; Uebe, Steffen; Donders, Rogier; Reis, André; Schenck, Annette; Zweier, Christiane

2014-12-01

Glycoprotein M6A (GPM6A) is a neuronal transmembrane protein of the PLP/DM20 (proteolipid protein) family that associates with cholesterol-rich lipid rafts and promotes filopodia formation. We identified a de novo duplication of the GPM6A gene in a patient with learning disability and behavioral anomalies. Expression analysis in blood lymphocytes showed increased GPM6A levels. An increase of patient-derived lymphoblastoid cells carrying membrane protrusions supports a functional effect of this duplication. To study the consequences of GPM6A dosage alterations in an intact nervous system, we employed Drosophila melanogaster as a model organism. We found that knockdown of Drosophila M6, the sole member of the PLP family in flies, in the wing, and whole organism causes malformation and lethality, respectively. These phenotypes as well as the protrusions of patient-derived lymphoblastoid cells with increased GPM6A levels can be alleviated by cholesterol supplementation. Notably, overexpression as well as loss of M6 in neurons specifically compromises long-term memory in the courtship conditioning paradigm. Our findings thus indicate a critical role of correct GPM6A/M6 levels for cognitive function and support a role of the GPM6A duplication for the patient's phenotype. Together with other recent findings, this study highlights compromised cholesterol homeostasis as a recurrent feature in cognitive phenotypes. © 2014 WILEY PERIODICALS, INC.

Cell Extrusion: A Stress-Responsive Force for Good or Evil in Epithelial Homeostasis.

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Ohsawa, Shizue; Vaughen, John; Igaki, Tatsushi

2018-02-05

Epithelial tissues robustly respond to internal and external stressors via dynamic cellular rearrangements. Cell extrusion acts as a key regulator of epithelial homeostasis by removing apoptotic cells, orchestrating morphogenesis, and mediating competitive cellular battles during tumorigenesis. Here, we delineate the diverse functions of cell extrusion during development and disease. We emphasize the expanding role for apoptotic cell extrusion in exerting morphogenetic forces, as well as the strong intersection of cell extrusion with cell competition, a homeostatic mechanism that eliminates aberrant or unfit cells. While cell competition and extrusion can exert potent, tumor-suppressive effects, dysregulation of either critical homeostatic program can fuel cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Oxidation of cholesterol moiety of low density lipoprotein in the presence of human endothelial cells or Cu+2 ions: identification of major products and their effects.

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Bhadra, S; Arshad, M A; Rymaszewski, Z; Norman, E; Wherley, R; Subbiah, M T

1991-04-15

Oxidation of lipoproteins is believed to play a key role in atherogenesis. In this study, low density lipoproteins (LDL) was subjected to oxidation in the presence of either human umbilical vein endothelial cells or with Cu+2 ions and the major oxides formed were identified. While cholesterol-alpha-epoxide (C-alpha EP) was the major product of cholesterol peroxidation in the presence of endothelial cells, cholest-3,5-dien-7-one (CD) predominated in the presence of Cu+2 ion. Both steroids were identified by gas chromatography/mass spectrometry. HDL cholesterol was resistant to oxidation. When tested on human skin fibroblasts in culture C-alpha EP (10 micrograms/ml) caused marked stimulation of 14C-oleate incorporation into cholesterol esters, while CD stimulated cholesterol esterification only mildly. These studies show that a) C-alpha EP is the major peroxidation product of LDL cholesterol moiety in the presence of endothelial cells and b) it causes marked stimulation of cholesterol esterification in cells. C-alpha EP may play a key role in increasing cholesterol esterification noted in atherogenesis.

The Effect of 24S-Hydroxycholesterol on Cholesterol Homeostasis in Neurons: Quantitative Changes to the Cortical Neuron Proteome

OpenAIRE

Wang, Yuqin; Muneton, Sabina; Sjövall, Jan; Jovanovic, Jasmina N.; Griffiths, William J.

2008-01-01

In human the brain represents only about 2% of the body’s mass but contains about one quarter of the body’s free cholesterol. Cholesterol is synthesised de novo in brain, and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study we investigated the effect of 24S-hydroxycholesterol on the pr...

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

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Paiva, Aline Dias; de Oliveira, Michelle Dias; de Paula, Sérgio Oliveira; Baracat-Pereira, Maria Cristina; Breukink, Eefjan; Mantovani, Hilário Cuquetto

2012-11-01

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

Disturbance of copper homeostasis is a mechanism for homocysteine-induced vascular endothelial cell injury.

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Daoyin Dong

Full Text Available Elevation of serum homocysteine (Hcy levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell viability and an increase in necrotic cell death. Pretreatment of the cells with a final concentration of 5 µM Cu in cultures prevented the effects of Hcy. Hcy decreased intracellular Cu concentrations. HPLC-ICP-MS analysis revealed that Hcy caused alterations in the distribution of intracellular Cu; more Cu was redistributed to low molecular weight fractions. ESI-Q-TOF detected the formation of Cu-Hcy complexes. Hcy also decreased the protein levels of Cu chaperone COX17, which was accompanied by a decrease in the activity of cytochrome c oxidase (CCO and a collapse of mitochondrial membrane potential. These effects of Hcy were all preventable by Cu pretreatment. The study thus demonstrated that Hcy disturbs Cu homeostasis and limits the availability of Cu to critical molecules such as COX17 and CCO, leading to mitochondrial dysfunction and endothelial cell injury.

Cholesterol lowering effects of mono-lactose-appended β-cyclodextrin in Niemann–Pick type C disease-like HepG2 cells

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Keiichi Motoyama

2015-11-01

Full Text Available The Niemann–Pick type C disease (NPC is one of inherited lysosomal storage disorders, emerges the accumulation of unesterified cholesterol in endolysosomes. Currently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD has been applied for the treatment of NPC. HP-β-CyD improved hepatosplenomegaly in NPC patients, however, a high dose of HP-β-CyD was necessary. Therefore, the decrease in dose by actively targeted-β-CyD to hepatocytes is expected. In the present study, to deliver β-CyD selectively to hepatocytes, we newly fabricated mono-lactose-appended β-CyD (Lac-β-CyD and evaluated its cholesterol lowering effects in NPC-like HepG2 cells, cholesterol accumulated HepG2 cells induced by treatment with U18666A. Lac-β-CyD (degree of substitution of lactose (DSL 1 significantly decreased the intracellular cholesterol content in a concentration-dependent manner. TRITC-Lac-β-CyD was associated with NPC-like HepG2 cells higher than TRITC-β-CyD. In addition, TRITC-Lac-β-CyD was partially localized with endolysosomes after endocytosis. Thus, Lac-β-CyD entered NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR-mediated endocytosis and decreased the accumulation of intracellular cholesterol in NPC-like HepG2 cells. These results suggest that Lac-β-CyD may have the potential as a drug for the treatment of hepatosplenomegaly in NPC disease.

A mouse model of harlequin ichthyosis delineates a key role for Abca12 in lipid homeostasis.

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Ian Smyth

2008-09-01

Full Text Available Harlequin Ichthyosis (HI is a severe and often lethal hyperkeratotic skin disease caused by mutations in the ABCA12 transport protein. In keratinocytes, ABCA12 is thought to regulate the transfer of lipids into small intracellular trafficking vesicles known as lamellar bodies. However, the nature and scope of this regulation remains unclear. As part of an original recessive mouse ENU mutagenesis screen, we have identified and characterised an animal model of HI and showed that it displays many of the hallmarks of the disease including hyperkeratosis, loss of barrier function, and defects in lipid homeostasis. We have used this model to follow disease progression in utero and present evidence that loss of Abca12 function leads to premature differentiation of basal keratinocytes. A comprehensive analysis of lipid levels in mutant epidermis demonstrated profound defects in lipid homeostasis, illustrating for the first time the extent to which Abca12 plays a pivotal role in maintaining lipid balance in the skin. To further investigate the scope of Abca12's activity, we have utilised cells from the mutant mouse to ascribe direct transport functions to the protein and, in doing so, we demonstrate activities independent of its role in lamellar body function. These cells have severely impaired lipid efflux leading to intracellular accumulation of neutral lipids. Furthermore, we identify Abca12 as a mediator of Abca1-regulated cellular cholesterol efflux, a finding that may have significant implications for other diseases of lipid metabolism and homeostasis, including atherosclerosis.

γδ T cells in homeostasis and host defence of epithelial barrier tissues

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Nielsen, Morten M.; Witherden, Deborah A.; Havran, Wendy L.

2017-01-01

Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue...... homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body — namely, the epidermis and the intestine — and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity...

Lack of P2Y(13) in mice fed a high cholesterol diet results in decreased hepatic cholesterol content, biliary lipid secretion and reverse cholesterol transport

NARCIS (Netherlands)

Lichtenstein, Laeticia; Serhan, Nizar; Annema, Wijtske; Combes, Guillaume; Robaye, Bernard; Boeynaems, Jean-Marie; Perret, Bertrand; Tietge, Uwe J. F.; Laffargue, Muriel; Martinez, Laurent O.

2013-01-01

Background: The protective effect of HDL is mostly attributed to their metabolic function in reverse cholesterol transport (RCT), a process whereby excess cellular cholesterol is taken up from peripheral cells, processed in HDL particles, and later delivered to the liver for further metabolism and

Small interfering RNA against transcription factor STAT6 leads to increased cholesterol synthesis in lung cancer cell lines.

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Richa Dubey

Full Text Available STAT6 transcription factor has become a potential molecule for therapeutic intervention because it regulates broad range of cellular processes in a large variety of cell types. Although some target genes and interacting partners of STAT6 have been identified, its exact mechanism of action needs to be elucidated. In this study, we sought to further characterize the molecular interactions, networks, and functions of STAT6 by profiling the mRNA expression of STAT6 silenced human lung cells (NCI-H460 using microarrays. Our analysis revealed 273 differentially expressed genes after STAT6 silencing. Analysis of the gene expression data with Ingenuity Pathway Analysis (IPA software revealed Gene expression, Cell death, Lipid metabolism as the functions associated with highest rated network. Cholesterol biosynthesis was among the most enriched pathways in IPA as well as in PANTHER analysis. These results have been validated by real-time PCR and cholesterol assay using scrambled siRNA as a negative control. Similar findings were also observed with human type II pulmonary alveolar epithelial cells, A549. In the present study we have, for the first time, shown the inverse relationship of STAT6 with the cholesterol biosynthesis in lung cancer cells. The present findings are potentially significant to advance the understanding and design of therapeutics for the pathological conditions where both STAT6 and cholesterol biosynthesis are implicated viz. asthma, atherosclerosis etc.

TRAF2 regulates peripheral CD8(+) T-cell and NKT-cell homeostasis by modulating sensitivity to IL-15.

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Villanueva, Jeanette E; Malle, Elisabeth K; Gardam, Sandra; Silveira, Pablo A; Zammit, Nathan W; Walters, Stacey N; Brink, Robert; Grey, Shane T

2015-06-01

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8(+) T-cell and NKT-cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8(+) T-cell subsets. IL-15-dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8(+) CD44(hi) CD122(+) T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8(+) CD44(hi) T cells exhibited impaired dose-dependent proliferation to exogenous IL-15. In contrast, TRAF2TKO CD8(+) T cells proliferated normally to anti-CD3 and TRAF2TKO CD8(+) CD44(hi) T cells exhibited normal proliferation to exogenous IL-2. TRAF2TKO CD8(+) T cells expressed normal levels of IL-15-associated receptors and possessed functional IL-15-mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8(+) CD44(hi) CD122(+) and NKT cells was mechanistically linked to an inability to respond to IL-15. The reduced CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell populations in TRAF2TKO mice were rescued in the presence of high dose IL-15 by IL-15/IL-15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8(+) CD44(hi) CD122(+) T-cell and NKT-cell homeostasis by modulating sensitivity to T-cell intrinsic growth factors such as IL-15. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cholesterol in brain disease: sometimes determinant and frequently implicated

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Martín, Mauricio G; Pfrieger, Frank; Dotti, Carlos G

2014-01-01

Cholesterol is essential for neuronal physiology, both during development and in the adult life: as a major component of cell membranes and precursor of steroid hormones, it contributes to the regulation of ion permeability, cell shape, cell–cell interaction, and transmembrane signaling. Consistently, hereditary diseases with mutations in cholesterol-related genes result in impaired brain function during early life. In addition, defects in brain cholesterol metabolism may contribute to neurological syndromes, such as Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD), and even to the cognitive deficits typical of the old age. In these cases, brain cholesterol defects may be secondary to disease-causing elements and contribute to the functional deficits by altering synaptic functions. In the first part of this review, we will describe hereditary and non-hereditary causes of cholesterol dyshomeostasis and the relationship to brain diseases. In the second part, we will focus on the mechanisms by which perturbation of cholesterol metabolism can affect synaptic function. PMID:25223281

Interaction of pathogens with host cholesterol metabolism.

Science.gov (United States)

Sviridov, Dmitri; Bukrinsky, Michael

2014-10-01

Pathogens of different taxa, from prions to protozoa, target cellular cholesterol metabolism to advance their own development and to impair host immune responses, but also causing metabolic complications, for example, atherosclerosis. This review describes recent findings of how pathogens do it. A common theme in interaction between pathogens and host cholesterol metabolism is pathogens targeting lipid rafts of the host plasma membrane. Many intracellular pathogens use rafts as an entry gate, taking advantage of the endocytic machinery and high abundance of outward-looking molecules that can be used as receptors. At the same time, disruption of the rafts' functional capacity, achieved by the pathogens through a number of various means, impairs the ability of the host to generate immune response, thus helping pathogen to thrive. Pathogens cannot synthesize cholesterol, and salvaging host cholesterol helps pathogens build advanced cholesterol-containing membranes and assembly platforms. Impact on cholesterol metabolism is not limited to the infected cells; proteins and microRNAs secreted by infected cells affect lipid metabolism systemically. Given an essential role that host cholesterol metabolism plays in pathogen development, targeting this interaction may be a viable strategy to fight infections, as well as metabolic complications of the infections.

The hedgehog receptor patched is involved in cholesterol transport.

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Michel Bidet

Full Text Available Sonic hedgehog (Shh signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.

Critical time window of neuronal cholesterol synthesis during neurite outgrowth.

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Fünfschilling, Ursula; Jockusch, Wolf J; Sivakumar, Nandhini; Möbius, Wiebke; Corthals, Kristina; Li, Sai; Quintes, Susanne; Kim, Younghoon; Schaap, Iwan A T; Rhee, Jeong-Seop; Nave, Klaus-Armin; Saher, Gesine

2012-05-30

Cholesterol is an essential membrane component enriched in plasma membranes, growth cones, and synapses. The brain normally synthesizes all cholesterol locally, but the contribution of individual cell types to brain cholesterol metabolism is unknown. To investigate whether cortical projection neurons in vivo essentially require cholesterol biosynthesis and which cell types support neurons, we have conditionally ablated the cholesterol biosynthesis in these neurons in mice either embryonically or postnatally. We found that cortical projection neurons synthesize cholesterol during their entire lifetime. At all stages, they can also benefit from glial support. Adult neurons that lack cholesterol biosynthesis are mainly supported by astrocytes such that their functional integrity is preserved. In contrast, microglial cells support young neurons. However, compensatory efforts of microglia are only transient leading to layer-specific neuronal death and the reduction of cortical projections. Hence, during the phase of maximal membrane growth and maximal cholesterol demand, neuronal cholesterol biosynthesis is indispensable. Analysis of primary neurons revealed that neurons tolerate only slight alteration in the cholesterol content and plasma membrane tension. This quality control allows neurons to differentiate normally and adjusts the extent of neurite outgrowth, the number of functional growth cones and synapses to the available cholesterol. This study highlights both the flexibility and the limits of horizontal cholesterol transfer in vivo and may have implications for the understanding of neurodegenerative diseases.

Three-component homeostasis control

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Xu, Jin; Hong, Hyunsuk; Jo, Junghyo

2014-03-01

Two reciprocal components seem to be sufficient to maintain a control variable constant. However, pancreatic islets adapt three components to control glucose homeostasis. They are α (secreting glucagon), β (insulin), and δ (somatostatin) cells. Glucagon and insulin are the reciprocal hormones for increasing and decreasing blood glucose levels, while the role of somatostatin is unknown. However, it has been known how each hormone affects other cell types. Based on the pulsatile hormone secretion and the cellular interactions, this system can be described as coupled oscillators. In particular, we used the Landau-Stuart model to consider both amplitudes and phases of hormone oscillations. We found that the presence of the third component, δ cell, was effective to resist under glucose perturbations, and to quickly return to the normal glucose level once perturbed. Our analysis suggested that three components are necessary for advanced homeostasis control.

Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

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Meritxell Reverter

2014-05-01

Full Text Available Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN. Here, using Chinese hamster ovary (CHO Niemann-Pick type C1 (NPC1 mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6 accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs. This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome

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Zhao Y

2016-10-01

Full Text Available Yun-Chun Zhao,1 Li Zhang,2 Shi-Sen Feng,3 Lu Hong,3 Hai-Li Zheng,3 Li-Li Chen,4 Xiao-Ling Zheng,1 Yi-Qing Ye,1 Meng-Dan Zhao,1 Wen-Xi Wang,3 Cai-Hong Zheng1 1Pharmacy Department, Women’s Hospital, 2Pharmacy Department, The Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China; 3Department of Pharmaceutic Preparation, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 4Department of Gynecologic Oncology, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China Abstract: A novel cationic cholesterol derivative-based small interfering RNA (siRNA interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl-N, N-dimethylamine (DMAPA-chems liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells. Keywords: siRNA, cationic cholesterol derivative, Notch1, ovarian cancer cells

Zinc oxide nanoparticles decrease the expression and activity of plasma membrane calcium ATPase, disrupt the intracellular calcium homeostasis in rat retinal ganglion cells.

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Guo, Dadong; Bi, Hongsheng; Wang, Daoguang; Wu, Qiuxin

2013-08-01

Zinc oxide nanoparticle is one of the most important materials with diverse applications. However, it has been reported that zinc oxide nanoparticles are toxic to organisms, and that oxidative stress is often hypothesized to be an important factor in cytotoxicity mediated by zinc oxide nanoparticles. Nevertheless, the mechanism of toxicity of zinc oxide nanoparticles has not been completely understood. In this study, we investigated the cytotoxic effect of zinc oxide nanoparticles and the possible molecular mechanism involved in calcium homeostasis mediated by plasma membrane calcium ATPase in rat retinal ganglion cells. Real-time cell electronic sensing assay showed that zinc oxide nanoparticles could exert cytotoxic effect on rat retinal ganglion cells in a concentration-dependent manner; flow cytometric analysis indicated that zinc oxide nanoparticles could lead to cell damage by inducing the overproduction of reactive oxygen species. Furthermore, zinc oxide nanoparticles could also apparently decrease the expression level and their activity of plasma membrane calcium ATPase, which finally disrupt the intracellular calcium homeostasis and result in cell death. Taken together, zinc oxide nanoparticles could apparently decrease the plasma membrane calcium ATPase expression, inhibit their activity, cause the elevated intracellular calcium ion level and disrupt the intracellular calcium homeostasis. Further, the disrupted calcium homeostasis will trigger mitochondrial dysfunction, generate excessive reactive oxygen species, and finally initiate cell death. Thus, the disrupted calcium homeostasis is involved in the zinc oxide nanoparticle-induced rat retinal ganglion cell death. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cholesterol modulates the volume-regulated anion current in Ehrlich-Lettre ascites cells via effects on Rho and F-actin

DEFF Research Database (Denmark)

Klausen, Thomas Kjaer; Hougaard, Charlotte; Hoffmann, Else K

2006-01-01

swollen cells, this reduction was prevented by cholesterol depletion, which also increased isotonic Rho activity. Thrombin, which stimulates Rho and causes actin polymerization, potentiated VRAC in modestly swollen cells. VRAC activity was unaffected by inclusion of a water-soluble PtdIns(4,5)P(2......) analogue or a PtdIns(4,5)P(2)-blocking antibody in the pipette, or neomycin treatment to sequester PtdIns(4,5)P(2). It is suggested that in ELA cells, F-actin and Rho-Rho kinase modulate VRAC magnitude and activation rate, respectively, and that cholesterol depletion potentiates VRAC at least in part......The mechanisms controlling the volume-regulated anion current (VRAC) are incompletely elucidated. Here, we investigate the modulation of VRAC by cellular cholesterol and the potential involvement of F-actin, Rho, Rho kinase, and phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P(2...

In situ probing of cholesterol in astrocytes at the single-cell level using laser desorption ionization mass spectrometric imaging with colloidal silver.

Science.gov (United States)

Perdian, D C; Cha, Sangwon; Oh, Jisun; Sakaguchi, Donald S; Yeung, Edward S; Lee, Young Jin

2010-04-30

Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level. Published in 2010 by John Wiley & Sons, Ltd.

Engineering cholesterol-based fibers for antibody immobilization and cell capture

Science.gov (United States)

Cohn, Celine

In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

The response of the prostate to circulating cholesterol: activating transcription factor 3 (ATF3 as a prominent node in a cholesterol-sensing network.

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Jayoung Kim

Full Text Available Elevated circulating cholesterol is a systemic risk factor for cardiovascular disease and metabolic syndrome, however the manner in which the normal prostate responds to variations in cholesterol levels is poorly understood. In this study we addressed the molecular and cellular effects of elevated and suppressed levels of circulating cholesterol on the normal prostate. Integrated bioinformatic analysis was performed using DNA microarray data from two experimental formats: (1 ventral prostate from male mice with chronically elevated circulating cholesterol and (2 human prostate cells exposed acutely to cholesterol depletion. A cholesterol-sensitive gene expression network was constructed from these data and the transcription factor ATF3 was identified as a prominent node in the network. Validation experiments confirmed that elevated cholesterol reduced ATF3 expression and enhanced proliferation of prostate cells, while cholesterol depletion increased ATF3 levels and inhibited proliferation. Cholesterol reduction in vivo alleviated dense lymphomononuclear infiltrates in the periprostatic adipose tissue, which were closely associated with nerve tracts and blood vessels. These findings open new perspectives on the role of cholesterol in prostate health, and provide a novel role for ATF3, and associated proteins within a large signaling network, as a cholesterol-sensing mechanism.

Imaging appearances of cholesterol pneumonia

International Nuclear Information System (INIS)

Miao Yanwei; Zhang Jingwen; Wu Jianlin; Zhou Yong; Li Mingwu; Lei Zhen; Shi Lifu

2006-01-01

Objection: To analyze the imaging appearances of cholesterol pneumonia. Methods We retrospectively analyzed the X-ray and CT findings of 3 patients with cholesterol pneumonia confirmed pathologically and reviewed correlative literature. Results: Lesions similar to mass were found in X-ray and CT imaging of three cases. Two of them appeared cavity with fluid-level and one showed multiple ring enhancement after CT contrast. The course of disease was very. long and it had no respond to antibiotic therapy. Amounts of foam cells rich in cholesterol crystal were detected in pathological examination. Conclusions: Cholesterol pneumonia is a rare chronic pulmonary idiopathic disease, and the radiological findings can do some help to its diagnosis. (authors)

NKT cell self-reactivity: evolutionary master key of immune homeostasis?

DEFF Research Database (Denmark)

Navikas, Shohreh

2011-01-01

Complex immune responses have evolved to protect multicellular organisms against the invasion of pathogens. This has exerted strong developmental pressure for specialized functions that can also limit damage to self-tissue. Two arms of immunity, the innate and adaptive immune system, have evolved...... through evolution by higher vertebrates could be related to their central function as master regulators of immune homeostasis that in part is shared with regulatory T cells. Hypothetical views on how self-reactive NKT cells secure such a central role will also be proposed.......Complex immune responses have evolved to protect multicellular organisms against the invasion of pathogens. This has exerted strong developmental pressure for specialized functions that can also limit damage to self-tissue. Two arms of immunity, the innate and adaptive immune system, have evolved....... The recent finding of self-peptide reactivity of NKT cells in the context of CD1d, with capacity to regulate multiple autoimmune and inflammatory conditions, motivates the current proposal that self-reactive NKT cells might be the ancestral link between present NK and T cells. Their parallel selection...

The monocyte counts to HDL cholesterol ratio in obese and lean patients with polycystic ovary syndrome.

Science.gov (United States)

Usta, Akin; Avci, Eyup; Bulbul, Cagla Bahar; Kadi, Hasan; Adali, Ertan

2018-04-10

Women with polycystic ovary syndrome are more likely to suffer from obesity, insulin resistance, and chronic low-grade inflammation. In fact, the excessive activation of monocytes exacerbates oxidative stress and inflammation. However, high-density lipoprotein cholesterol neutralizes the pro-inflammatory and pro-oxidant effects of monocytes. The aim of this study is to investigate whether monocyte counts to high-density lipoprotein cholesterol ratio can predict the inflammatory condition in patients with polycystic ovary syndrome. In this cross-sectional study, a total of 124 women (61 of them with polycystic ovary syndrome and 63 age-matched healthy volunteers) were included in the study population. Obese polycystic ovary syndrome patients (n = 30) with a body mass index of ≥25 kg/m 2 and lean polycystic ovary syndrome patients (n = 31) with a body mass index of polycystic ovary syndrome were significantly higher than in control subjects (p = 0.0018). Moreover, a regression analysis revealed that body mass index, the homeostasis model assessment of insulin resistance and the high sensitivity C-reactive protein levels were confounding factors that affected the monocyte counts to high density lipoprotein cholesterol values. Additionally, a univariate and multivariate logistic regression analysis demonstrated that the increased monocyte counts to high density lipoprotein cholesterol values were more sensitive than the other known risk factors (such as increased body mass index, homeostasis model assessment of insulin resistance and high sensitive C-reactive protein levels) in the prediction of the inflammation in patients with polycystic ovary syndrome. The present study demonstrated that the monocyte count to high density lipoprotein cholesterol may be a novel and useful predictor of the presence of polycystic ovary syndrome.

CISH has no non-redundant functions in glucose homeostasis or beta cell proliferation during pregnancy in mice.

Science.gov (United States)

Jiao, Yang; Rieck, Sebastian; Le Lay, John; Kaestner, Klaus H

2013-11-01

Increased beta cell proliferation during pregnancy is mediated by the Janus kinase 2/signal transducer and activator of transcription 5 (JAK2/STAT5) signalling pathway in response to increased lactogen levels. Activation of the pathway leads to transcriptional upregulation of Cish (encoding cytokine-inducible SH2 domain-containing protein), a member of the suppressor of cytokine signalling (SOCS) family of genes, forming a negative-feedback loop. Here, we examined whether conditional gene ablation of Cish in the pancreas improves beta cell proliferation and beta cell function during pregnancy in mice. We derived mice with a novel, conditional loxP allele for Cish. Pancreas-specific ablation of Cish was achieved by crossing Cish (loxP/loxP) mice with Pdx1-Cre (Early) mice. Beta cell proliferation was quantified by BrdU labelling. Glucose homeostasis was examined with glucose tolerance tests and determination of plasma insulin levels. The expression of other Socs genes and target genes of p-STAT5 related to beta cell function and beta cell proliferation was determined by quantitative PCR. There was no difference in beta cell proliferation or glucose homeostasis between the Cish mutant group and the control group. The p-STAT5 protein level was the same in Cish mutant and control mice. Socs2 gene expression was higher in Cish mutant than control mice at pregnancy day 9.5. The expression of other Socs genes was the same between control and mutant mice. Our results show that CISH has no non-redundant functions in beta cell proliferation or glucose homeostasis during pregnancy in mice. Socs2 might compensate for the loss of Cish during pregnancy.

Regulation of T Cell Homeostasis and Responses by Pten

Directory of Open Access Journals (Sweden)

Ryan H. Newton

2012-06-01

Full Text Available The generation of lipid products catalyzed by PI3K is critical for normal T cell homeostasis and a productive immune response. PI3K can be activated in response to antigen receptor, costimulatory, cytokine and chemokine signals. Moreover, dysregulation of this pathway frequently occurs in T cell lymphomas and is implicated in lymphoproliferative autoimmune disease. Akt acts as a central mediator of PI3K signals, downstream of which is the mTOR pathway, controlling cell growth and metabolism. Members of the Foxo family of transcription factors are also regulated by Akt, thus linking control over homing and migration of T cells, as well cell cycle entry, apoptosis, and DNA damage and oxidative stress responses, to PI3K signaling. PTEN, first identified as a tumor suppressor gene, encodes a lipid phosphatase that, by catalyzing the reverse of the PI3K reaction, directly opposes PI3K signaling. However, PTEN may have other functions as well, and recent reports have suggested roles for PTEN as a tumor suppressor independent of its effects on PI3K signaling. Through the use of models in which Pten is deleted specifically in T cells, it is becoming increasingly clear that control over autoimmunity and lymphomagenesis by PTEN involves multi-faceted functions of this molecule at multiple stages of T cell development.

Impact of a chronic ingestion of radionuclides on cholesterol metabolism in the rat: example of depleted uranium and cesium 137

International Nuclear Information System (INIS)

Racine, Radjini

2009-01-01

Depleted uranium (DU) and cesium-137 ( 137 Cs) are radionuclides spread in the environment due to industrial activities, incidents or accidents. This pollution sets a risk of human exposure to low levels of radiations through contaminated foodstuff. The impact of a chronic ingestion of DU or 137 Cs on cholesterol metabolism in the liver and the brain has been studied. Indeed, cholesterol is crucial in physiology, being a component of cell membranes and a precursor to numerous molecules (bile acids...). Disruption of its metabolism is associated to many pathologies such as atherosclerosis or Alzheimer disease. Rats daily ingested a low level of DU or 137 Cs over 9 months. For each radionuclide, a reference model (rats contaminated since adulthood) and a more sensitive model (hypercholesterolemic or contaminated since fetal life) were studied. The effects mainly consist of changes in gene expression or enzymatic activity of various actors of cholesterol metabolism. DU mainly affects one catabolism enzyme in both models, as well as membrane transporters and regulation factors. 137 Cs mainly affects the storage enzyme in both models as well as catabolism enzymes, apolipoproteins, and regulation factors. No change in the plasma profile or in the tissue concentration of cholesterol (hepatic/cerebral) is recorded, whatever the model and the radionuclide. Thus, a chronic internal contamination with DU or 137 Cs induces molecular modifications in cholesterol metabolism in the rat, without affecting its homeostasis or the general health status in all of our experimental models. (author)

Stromal cell contributions to the homeostasis and functionality of the immune system.

Science.gov (United States)

Mueller, Scott N; Germain, Ronald N

2009-09-01

A defining characteristic of the immune system is the constant movement of many of its constituent cells through the secondary lymphoid tissues, mainly the spleen and lymph nodes, where crucial interactions that underlie homeostatic regulation, peripheral tolerance and the effective development of adaptive immune responses take place. What has only recently been recognized is the role that non-haematopoietic stromal elements have in many aspects of immune cell migration, activation and survival. In this Review, we summarize our current understanding of lymphoid compartment stromal cells, examine their possible heterogeneity, discuss how these cells contribute to immune homeostasis and the efficient initiation of adaptive immune responses, and highlight how targeting of these elements by some pathogens can influence the host immune response.

Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes.

Science.gov (United States)

Subasinghe, Supundi; Unabia, Sharon; Barrow, Colin J; Mok, Su San; Aguilar, Marie-Isabel; Small, David H

2003-02-01

Accumulation of beta amyloid (Abeta) in the brain is central to the pathogenesis of Alzheimer's disease. Abeta can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Abeta binding to membranes. Abeta peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Abeta peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Abeta peptides and their membrane binding. 'Ageing' the Abeta peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Abeta analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Abeta to purified plasma membrane preparations but also reduced Abeta toxicity. The results support the view that Abeta toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Abeta-membrane binding.

Analysis of Cholesterol Trafficking with Fluorescent Probes

DEFF Research Database (Denmark)

Maxfield, Frederick R.; Wustner, Daniel

2012-01-01

Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport...... that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy...... and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly....

What Can We Learn about Cholesterol's Transmembrane Distribution Based on Cholesterol-Induced Changes in Membrane Dipole Potential?

DEFF Research Database (Denmark)

Falkovich, Stanislav G.; Martinez-Seara, Hector; Nesterenko, Alexey M.

2016-01-01

Cholesterol is abundant in the plasma membranes of animal cells and is known to regulate a variety of membrane properties. Despite decades of research, the transmembrane distribution of cholesterol is still a matter of debate. Here we consider this outstanding issue through atomistic simulations ...

Homeostasis of peripheral CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T cell numbers

NARCIS (Netherlands)

Almeida, Afonso R. M.; Legrand, Nicolas; Papiernik, Martine; Freitas, António A.

2002-01-01

We show that the lymphoid hyperplasia observed in IL-2Ralpha- and IL-2-deficient mice is due to the lack of a population of regulatory cells essential for CD4 T cell homeostasis. In chimeras reconstituted with bone marrow cells from IL-2Ralpha-deficient donors, restitution of a population of

DIP1 modulates stem cell homeostasis in Drosophila through regulation of sisR-1.

Science.gov (United States)

Wong, Jing Ting; Akhbar, Farzanah; Ng, Amanda Yunn Ee; Tay, Mandy Li-Ian; Loi, Gladys Jing En; Pek, Jun Wei

2017-10-02

Stable intronic sequence RNAs (sisRNAs) are by-products of splicing and regulate gene expression. How sisRNAs are regulated is unclear. Here we report that a double-stranded RNA binding protein, Disco-interacting protein 1 (DIP1) regulates sisRNAs in Drosophila. DIP1 negatively regulates the abundance of sisR-1 and INE-1 sisRNAs. Fine-tuning of sisR-1 by DIP1 is important to maintain female germline stem cell homeostasis by modulating germline stem cell differentiation and niche adhesion. Drosophila DIP1 localizes to a nuclear body (satellite body) and associates with the fourth chromosome, which contains a very high density of INE-1 transposable element sequences that are processed into sisRNAs. DIP1 presumably acts outside the satellite bodies to regulate sisR-1, which is not on the fourth chromosome. Thus, our study identifies DIP1 as a sisRNA regulatory protein that controls germline stem cell self-renewal in Drosophila.Stable intronic sequence RNAs (sisRNAs) are by-products of splicing from introns with roles in embryonic development in Drosophila. Here, the authors show that the RNA binding protein DIP1 regulates sisRNAs in Drosophila, which is necessary for germline stem cell homeostasis.

γδ T cells producing interleukin-17A regulate adipose regulatory T cell homeostasis and thermogenesis.

Science.gov (United States)

Kohlgruber, Ayano C; Gal-Oz, Shani T; LaMarche, Nelson M; Shimazaki, Moto; Duquette, Danielle; Nguyen, Hung N; Mina, Amir I; Paras, Tyler; Tavakkoli, Ali; von Andrian, Ulrich; Banks, Alexander S; Shay, Tal; Brenner, Michael B; Lynch, Lydia

2018-05-01

γδ T cells are situated at barrier sites and guard the body from infection and damage. However, little is known about their roles outside of host defense in nonbarrier tissues. Here, we characterize a highly enriched tissue-resident population of γδ T cells in adipose tissue that regulate age-dependent regulatory T cell (T reg ) expansion and control core body temperature in response to environmental fluctuations. Mechanistically, innate PLZF + γδ T cells produced tumor necrosis factor and interleukin (IL) 17 A and determined PDGFRα + and Pdpn + stromal-cell production of IL-33 in adipose tissue. Mice lacking γδ T cells or IL-17A exhibited decreases in both ST2 + T reg cells and IL-33 abundance in visceral adipose tissue. Remarkably, these mice also lacked the ability to regulate core body temperature at thermoneutrality and after cold challenge. Together, these findings uncover important physiological roles for resident γδ T cells in adipose tissue immune homeostasis and body-temperature control.

Osteoclasts and CD8 T cells form a negative feedback loop that contributes to homeostasis of both the skeletal and immune systems.

Science.gov (United States)

Buchwald, Zachary S; Aurora, Rajeev

2013-01-01

There are a number of dynamic regulatory loops that maintain homeostasis of the immune and skeletal systems. In this review, we highlight a number of these regulatory interactions that contribute to maintaining homeostasis. In addition, we review data on a negative regulatory feedback loop between osteoclasts and CD8 T cells that contributes to homeostasis of both the skeletal and immune systems.

Identification of the C-terminal domain of Daxx acts as a potential regulator of intracellular cholesterol synthesis in HepG2 cells

International Nuclear Information System (INIS)

Sun, Shaowei; Wen, Juan; Qiu, Fei; Yin, Yufang; Xu, Guina; Li, Tianping; Nie, Juan; Xiong, Guozuo; Zhang, Caiping; Liao, Duangfang; Chen, Jianxiong; Tuo, Qinhui

2016-01-01

Daxx is a highly conserved nuclear transcriptional factor, which has been implicated in many nuclear processes including transcription and cell cycle regulation. Our previous study demonstrated Daxx also plays a role in regulation of intracellular cholesterol content. Daxx contains several domains that are essential for interaction with a growing number of proteins. To delineate the underlying mechanism of hypocholesterolemic activity of Daxx, we constructed a set of plasmids which can be used to overexpress different fragments of Daxx and transfected to HepG2 cells. We found that the C- terminal region Daxx626–740 clearly reduced intracellular cholesterol levels and inhibited the expression of SREBPs and SCAP. In GST pull-down experiments and Double immunofluorescence assays, Daxx626–740 was demonstrated to bind directly to androgen receptor (AR). Our findings suggest that the interaction of Daxx626-740 and AR abolishes the AR-mediated activation of SCAP/SREBPs pathway, which suppresses the de novo cholesterol synthesis. Thus, C-terminal domain of Daxx acts as a potential regulator of intracellular cholesterol content in HepG2 cells. - Highlights: • Daxx C-terminal domain reduces cholesterol levels. • Daxx C-terminal domain binds directly to AR. • The interaction of Daxx C-terminal domain and AR suppresses cholesterol synthesis.

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

Science.gov (United States)

Ioannou, George N; Subramanian, Savitha; Chait, Alan; Haigh, W Geoffrey; Yeh, Matthew M; Farrell, Geoffrey C; Lee, Sum P; Savard, Christopher

2017-06-01

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ≥0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha , NLRP3, and interleukin 1beta ( IL1β ) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.

Intracellular transport of low density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts

International Nuclear Information System (INIS)

Liscum, L.; Ruggiero, R.M.; Faust, J.R.

1989-01-01

Niemann-Pick disease type C (NPC) is characterized by substantial intracellular accumulation of unesterified cholesterol. The accumulation of unesterified cholesterol in NPC fibroblasts cultured with low density lipoprotein (LDL) appears to result from the inability of LDL to stimulate cholesterol esterification in addition to impaired LDL-mediated downregulation of LDL receptor activity and cellular cholesterol synthesis. Although a defect in cholesterol transport in NPC cells has been inferred from previous studies, no experiments have been reported that measure the intracellular movement of LDL-cholesterol specifically. We have used four approaches to assess intracellular cholesterol transport in normal and NPC cells and have determined the following: (a) mevinolin-inhibited NPC cells are defective in using LDL-cholesterol for growth. However, exogenously added mevalonate restores cell growth equally in normal and NPC cells; (b) the transport of LDL-derived [3H]cholesterol to the plasma membrane is slower in NPC cells, while the rate of appearance of [3H]acetate-derived, endogenously synthesized [3H]cholesterol at the plasma membrane is the same for normal and NPC cells; (c) in NPC cells, LDL-derived [3H]cholesterol accumulates in lysosomes to higher levels than normal, resulting in defective movement to other cell membranes; and (d) incubation of cells with LDL causes an increase in cholesterol content of NPC lysosomes that is threefold greater than that observed in normal lysosomes. Our results indicate that a cholesterol transport defect exists in NPC that is specific for LDL-derived cholesterol

Single-cell-based system to monitor carrier driven cellular auxin homeostasis

Science.gov (United States)

2013-01-01

Background Abundance and distribution of the plant hormone auxin play important roles in plant development. Besides other metabolic processes, various auxin carriers control the cellular level of active auxin and, hence, are major regulators of cellular auxin homeostasis. Despite the developmental importance of auxin transporters, a simple medium-to-high throughput approach to assess carrier activities is still missing. Here we show that carrier driven depletion of cellular auxin correlates with reduced nuclear auxin signaling in tobacco Bright Yellow-2 (BY-2) cell cultures. Results We developed an easy to use transient single-cell-based system to detect carrier activity. We use the relative changes in signaling output of the auxin responsive promoter element DR5 to indirectly visualize auxin carrier activity. The feasibility of the transient approach was demonstrated by pharmacological and genetic interference with auxin signaling and transport. As a proof of concept, we provide visual evidence that the prominent auxin transport proteins PIN-FORMED (PIN)2 and PIN5 regulate cellular auxin homeostasis at the plasma membrane and endoplasmic reticulum (ER), respectively. Our data suggest that PIN2 and PIN5 have different sensitivities to the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Also the putative PIN-LIKES (PILS) auxin carrier activity at the ER is insensitive to NPA in our system, indicating that NPA blocks intercellular, but not intracellular auxin transport. Conclusions This single-cell-based system is a useful tool by which the activity of putative auxin carriers, such as PINs, PILS and WALLS ARE THIN1 (WAT1), can be indirectly visualized in a medium-to-high throughput manner. Moreover, our single cell system might be useful to investigate also other hormonal signaling pathways, such as cytokinin. PMID:23379388

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

Science.gov (United States)

Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H; Nachtergaele, Sigrid; Sagner, Andreas; Byrne, Eamon FX; Covey, Douglas F; Siebold, Christian; Rohatgi, Rajat

2016-01-01

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. DOI: http://dx.doi.org/10.7554/eLife.20304.001 PMID:27705744

CD 4 + CD 25 + T cells maintain homeostasis by promoting TER - 119 cell development and inhibiting T cell activation

Directory of Open Access Journals (Sweden)

Muhaimin Rifa’i

2014-05-01

Full Text Available CD4+ CD25+ regulatory T cells involved in the regulation of self- tolerance and normality of homeostasis. CD122 deficient mice are model animals that have an abnormal immune system characteristically have a high number of activated T cells and TER-119 cell decreased. Here we showed evidence that the transfer of CD4+ CD25+ regulatory T cells derived from normal mice to CD122- defficient neonates prevent the development of activated memory T cells and elicit TER-119 differentiation. Bone marrow reconstitution derived from CD122-/- mice to normal mice resulting tolerance to individual that genetically different. Importantly, CD4+ CD25+ regulatory T cells derived from normal mice can replace CD4+ CD25+ cells derived from CD122-/- mice. The results of this experiment suggest that regulatory T cells from normal mice exert a critical role in maintaining peripheral tolerance and controlling hematopoietic disorder.

Cholesterol: Its Regulation and Role in Central Nervous System Disorders

Directory of Open Access Journals (Sweden)

Matthias Orth

2012-01-01

Full Text Available Cholesterol is a major constituent of the human brain, and the brain is the most cholesterol-rich organ. Numerous lipoprotein receptors and apolipoproteins are expressed in the brain. Cholesterol is tightly regulated between the major brain cells and is essential for normal brain development. The metabolism of brain cholesterol differs markedly from that of other tissues. Brain cholesterol is primarily derived by de novo synthesis and the blood brain barrier prevents the uptake of lipoprotein cholesterol from the circulation. Defects in cholesterol metabolism lead to structural and functional central nervous system diseases such as Smith-Lemli-Opitz syndrome, Niemann-Pick type C disease, and Alzheimer’s disease. These diseases affect different metabolic pathways (cholesterol biosynthesis, lipid transport and lipoprotein assembly, apolipoproteins, lipoprotein receptors, and signaling molecules. We review the metabolic pathways of cholesterol in the CNS and its cell-specific and microdomain-specific interaction with other pathways such as the amyloid precursor protein and discuss potential treatment strategies as well as the effects of the widespread use of LDL cholesterol-lowering drugs on brain functions.

Cholesterol oxidation products and their biological importance

DEFF Research Database (Denmark)

Kulig, Waldemar; Cwiklik, Lukasz; Jurkiewicz, Piotr

2016-01-01

The main biological cause of oxysterols is the oxidation of cholesterol. They differ from cholesterol by the presence of additional polar groups that are typically hydroxyl, keto, hydroperoxy, epoxy, or carboxyl moieties. Under typical conditions, oxysterol concentration is maintained at a very low...... and precisely regulated level, with an excess of cholesterol. Like cholesterol, many oxysterols are hydrophobic and hence confined to cell membranes. However, small chemical differences between the sterols can significantly affect how they interact with other membrane components, and this in turn can have...

Metal ion transporters and homeostasis.

OpenAIRE

Nelson, N

1999-01-01

Transition metals are essential for many metabolic processes and their homeostasis is crucial for life. Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discover...

Rational heterodoxy: cholesterol reformation of the amyloid doctrine.

Science.gov (United States)

Castello, Michael A; Soriano, Salvador

2013-01-01

According to the amyloid cascade hypothesis, accumulation of the amyloid peptide Aβ, derived by proteolytic processing from the amyloid precursor protein (APP), is the key pathogenic trigger in Alzheimer's disease (AD). This view has led researchers for more than two decades and continues to be the most influential model of neurodegeneration. Nevertheless, close scrutiny of the current evidence does not support a central pathogenic role for Aβ in late-onset AD. Furthermore, the amyloid cascade hypothesis lacks a theoretical foundation from which the physiological generation of Aβ can be understood, and therapeutic approaches based on its premises have failed. We present an alternative model of neurodegeneration, in which sustained cholesterol-associated neuronal distress is the most likely pathogenic trigger in late-onset AD, directly causing oxidative stress, inflammation and tau hyperphosphorylation. In this scenario, Aβ generation is part of an APP-driven adaptive response to the initial cholesterol distress, and its accumulation is neither central to, nor a requirement for, the initiation of the disease. Our model provides a theoretical framework that places APP as a regulator of cholesterol homeostasis, accounts for the generation of Aβ in both healthy and demented brains, and provides suitable targets for therapeutic intervention. Copyright © 2012 Elsevier B.V. All rights reserved.

Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.

Directory of Open Access Journals (Sweden)

Zahedi Mujawar

2006-10-01

Full Text Available Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings

Neuroimmune regulation during intestinal development and homeostasis.

Science.gov (United States)

Veiga-Fernandes, Henrique; Pachnis, Vassilis

2017-02-01

Interactions between the nervous system and immune system are required for organ function and homeostasis. Evidence suggests that enteric neurons and intestinal immune cells share common regulatory mechanisms and can coordinate their responses to developmental challenges and environmental aggressions. These discoveries shed light on the physiology of system interactions and open novel perspectives for therapy designs that target underappreciated neurological-immunological commonalities. Here we highlight findings that address the importance of neuroimmune cell units (NICUs) in intestinal development, homeostasis and disease.

Taste bud homeostasis in health, disease, and aging.

Science.gov (United States)

Feng, Pu; Huang, Liquan; Wang, Hong

2014-01-01

The mammalian taste bud is an onion-shaped epithelial structure with 50-100 tightly packed cells, including taste receptor cells, supporting cells, and basal cells. Taste receptor cells detect nutrients and toxins in the oral cavity and transmit the sensory information to gustatory nerve endings in the buds. Supporting cells may play a role in the clearance of excess neurotransmitters after their release from taste receptor cells. Basal cells are precursor cells that differentiate into mature taste cells. Similar to other epithelial cells, taste cells turn over continuously, with an average life span of about 8-12 days. To maintain structural homeostasis in taste buds, new cells are generated to replace dying cells. Several recent studies using genetic lineage tracing methods have identified populations of progenitor/stem cells for taste buds, although contributions of these progenitor/stem cell populations to taste bud homeostasis have yet to be fully determined. Some regulatory factors of taste cell differentiation and degeneration have been identified, but our understanding of these aspects of taste bud homoeostasis remains limited. Many patients with various diseases develop taste disorders, including taste loss and taste distortion. Decline in taste function also occurs during aging. Recent studies suggest that disruption or alteration of taste bud homeostasis may contribute to taste dysfunction associated with disease and aging.

Taste Bud Homeostasis in Health, Disease, and Aging

Science.gov (United States)

2014-01-01

The mammalian taste bud is an onion-shaped epithelial structure with 50–100 tightly packed cells, including taste receptor cells, supporting cells, and basal cells. Taste receptor cells detect nutrients and toxins in the oral cavity and transmit the sensory information to gustatory nerve endings in the buds. Supporting cells may play a role in the clearance of excess neurotransmitters after their release from taste receptor cells. Basal cells are precursor cells that differentiate into mature taste cells. Similar to other epithelial cells, taste cells turn over continuously, with an average life span of about 8–12 days. To maintain structural homeostasis in taste buds, new cells are generated to replace dying cells. Several recent studies using genetic lineage tracing methods have identified populations of progenitor/stem cells for taste buds, although contributions of these progenitor/stem cell populations to taste bud homeostasis have yet to be fully determined. Some regulatory factors of taste cell differentiation and degeneration have been identified, but our understanding of these aspects of taste bud homoeostasis remains limited. Many patients with various diseases develop taste disorders, including taste loss and taste distortion. Decline in taste function also occurs during aging. Recent studies suggest that disruption or alteration of taste bud homeostasis may contribute to taste dysfunction associated with disease and aging. PMID:24287552

Novel role of a triglyceride-synthesizing enzyme: DGAT1 at the crossroad between triglyceride and cholesterol metabolism.

Science.gov (United States)

Sachdev, Vinay; Leopold, Christina; Bauer, Raimund; Patankar, Jay V; Iqbal, Jahangir; Obrowsky, Sascha; Boverhof, Renze; Doktorova, Marcela; Scheicher, Bernhard; Goeritzer, Madeleine; Kolb, Dagmar; Turnbull, Andrew V; Zimmer, Andreas; Hoefler, Gerald; Hussain, M Mahmood; Groen, Albert K; Kratky, Dagmar

2016-09-01

Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1(-/-)) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1(-/-) and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Rorγt+ innate lymphoid cells in intestinal homeostasis and immunity.

Science.gov (United States)

Aparicio-Domingo, Patricia; Cupedo, Tom

2011-01-01

Innate lymphoid cells (ILC) combine innate and adaptive immune functions and are part of the first line of defense against mucosal infections. ILC are set apart from adaptive lymphocytes by their independence on RAG genes and the resulting absence of specific antigen receptors. In this review, we will discuss the biology and function of intestinal ILC that express the nuclear hormone receptor Rorγt (encoded by the Rorc gene) and highlight their role in intestinal homeostasis and immunity. Copyright © 2011 S. Karger AG, Basel.

How cholesterol interacts with proteins and lipids during its intracellular transport

DEFF Research Database (Denmark)

Wüstner, Daniel; Solanko, Katarzyna

2015-01-01

as well as by non-vesicular sterol exchange between organelles. In this article, we will review recent progress in elucidating sterol-lipid and sterol-protein interactions contributing to proper sterol transport in living cells. We outline recent biophysical models of cholesterol distribution and dynamics...... for characterization of sterol-protein interactions and for monitoring intracellular sterol transport. Finally, we review recent work on the molecular mechanisms underlying lipoprotein-mediated cholesterol import into mammalian cells and describe the process of cellular cholesterol efflux. Overall, we emphasize how......Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein-lipid and protein-protein interactions...

Molecular control of steady-state dendritic cell maturation and immune homeostasis.

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Hammer, Gianna Elena; Ma, Averil

2013-01-01

Dendritic cells (DCs) are specialized sentinels responsible for coordinating adaptive immunity. This function is dependent upon coupled sensitivity to environmental signs of inflammation and infection to cellular maturation-the programmed alteration of DC phenotype and function to enhance immune cell activation. Although DCs are thus well equipped to respond to pathogens, maturation triggers are not unique to infection. Given that immune cells are exquisitely sensitive to the biological functions of DCs, we now appreciate that multiple layers of suppression are required to restrict the environmental sensitivity, cellular maturation, and even life span of DCs to prevent aberrant immune activation during the steady state. At the same time, steady-state DCs are not quiescent but rather perform key functions that support homeostasis of numerous cell types. Here we review these functions and molecular mechanisms of suppression that control steady-state DC maturation. Corruption of these steady-state operatives has diverse immunological consequences and pinpoints DCs as potent drivers of autoimmune and inflammatory disease.

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux.

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Chen, Liang; Yao, Qiying; Xu, Siwei; Wang, Hongyan; Qu, Peng

2018-01-01

The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients

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Maria Di Bari

2016-11-01

Full Text Available Multiple sclerosis (MS is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE and butyrylcholinesterase (BuChE. Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD, is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis.

Delineating the regulation of energy homeostasis using hypothalamic cell models.

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Wellhauser, Leigh; Gojska, Nicole M; Belsham, Denise D

2015-01-01

Attesting to its intimate peripheral connections, hypothalamic neurons integrate nutritional and hormonal cues to effectively manage energy homeostasis according to the overall status of the system. Extensive progress in the identification of essential transcriptional and post-translational mechanisms regulating the controlled expression and actions of hypothalamic neuropeptides has been identified through the use of animal and cell models. This review will introduce the basic techniques of hypothalamic investigation both in vivo and in vitro and will briefly highlight the key advantages and challenges of their use. Further emphasis will be place on the use of immortalized models of hypothalamic neurons for in vitro study of feeding regulation, with a particular focus on cell lines proving themselves most fruitful in deciphering fundamental basics of NPY/AgRP, Proglucagon, and POMC neuropeptide function. Copyright © 2014 Elsevier Inc. All rights reserved.

Viral MicroRNAs Repress the Cholesterol Pathway, and 25-Hydroxycholesterol Inhibits Infection.

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Serquiña, Anna K P; Kambach, Diane M; Sarker, Ontara; Ziegelbauer, Joseph M

2017-07-11

From various screens, we found that Kaposi's sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR [a rate-limiting step in the mevalonate pathway]), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 [a committed step in the cholesterol branch]) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells [HUVECs]) is sufficient to reduce intracellular cholesterol levels; however, small interfering RNAs (siRNAs) targeting only HMGCS1 did not reduce cholesterol levels. This suggests that multiple targets are needed to perturb this tightly regulated pathway. We also report here that cholesterol levels were decreased in de novo -infected HUVECs after 7 days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type infections. We hypothesized that KSHV is downregulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo -infected HUVECs but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of 25HC. IMPORTANCE A subset of viruses express unique microRNAs (miRNAs), which act like cellular miRNAs to generally repress host gene

Lifespan extension by preserving proliferative homeostasis in Drosophila.

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Benoît Biteau

2010-10-01

Full Text Available Regenerative processes are critical to maintain tissue homeostasis in high-turnover tissues. At the same time, proliferation of stem and progenitor cells has to be carefully controlled to prevent hyper-proliferative diseases. Mechanisms that ensure this balance, thus promoting proliferative homeostasis, are expected to be critical for longevity in metazoans. The intestinal epithelium of Drosophila provides an accessible model in which to test this prediction. In aging flies, the intestinal epithelium degenerates due to over-proliferation of intestinal stem cells (ISCs and mis-differentiation of ISC daughter cells, resulting in intestinal dysplasia. Here we show that conditions that impair tissue renewal lead to lifespan shortening, whereas genetic manipulations that improve proliferative homeostasis extend lifespan. These include reduced Insulin/IGF or Jun-N-terminal Kinase (JNK signaling activities, as well as over-expression of stress-protective genes in somatic stem cell lineages. Interestingly, proliferative activity in aging intestinal epithelia correlates with longevity over a range of genotypes, with maximal lifespan when intestinal proliferation is reduced but not completely inhibited. Our results highlight the importance of the balance between regenerative processes and strategies to prevent hyperproliferative disorders and demonstrate that promoting proliferative homeostasis in aging metazoans is a viable strategy to extend lifespan.

Regulation of cholesterol 25-hydroxylase expression by vitamin D3 metabolites in human prostate stromal cells

International Nuclear Information System (INIS)

Wang, J.-H.; Tuohimaa, Pentti

2006-01-01

Vitamin D 3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D 3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1α,25(OH) 2 D 3 and 500 nM 25OHD 3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1α,25(OH) 2 D 3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1α,25(OH) 2 D 3 induced CH25H mRNA expression. 1α,25(OH) 2 D 3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D 3 treated cells. Our data suggest that ch25h could be a vitamin D 3 target gene and may partly mediate anti-proliferative action of vitamin D 3 in human primary prostate stromal cells

Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation.

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Kurashima, Yosuke; Goto, Yoshiyuki; Kiyono, Hiroshi

2013-12-01

Continuous exposure of intestinal mucosal surfaces to diverse microorganisms and their metabolites reflects the biological necessity for a multifaceted, integrated epithelial and immune cell-mediated regulatory system. The development and function of the host cells responsible for the barrier function of the intestinal surface (e.g., M cells, Paneth cells, goblet cells, and columnar epithelial cells) are strictly regulated through both positive and negative stimulation by the luminal microbiota. Stimulation by damage-associated molecular patterns and commensal bacteria-derived microbe-associated molecular patterns provokes the assembly of inflammasomes, which are involved in maintaining the integrity of the intestinal epithelium. Mucosal immune cells located beneath the epithelium play critical roles in regulating both the mucosal barrier and the relative composition of the luminal microbiota. Innate lymphoid cells and mast cells, in particular, orchestrate the mucosal regulatory system to create a mutually beneficial environment for both the host and the microbiota. Disruption of mucosal homeostasis causes intestinal inflammation such as that seen in inflammatory bowel disease. Here, we review the recent research on the biological interplay among the luminal microbiota, epithelial cells, and mucosal innate immune cells in both healthy and pathological conditions. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Transforming growth factor beta-activated kinase 1 (TAK1)-dependent checkpoint in the survival of dendritic cells promotes immune homeostasis and function.

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Wang, Yanyan; Huang, Gonghua; Vogel, Peter; Neale, Geoffrey; Reizis, Boris; Chi, Hongbo

2012-02-07

Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and -inducible deletion systems, we report that transforming growth factor beta-activated kinase 1 (TAK1) is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c(+) cells induced markedly elevated apoptosis, leading to the depletion of DC populations, especially the CD8(+) and CD103(+) DC subsets in lymphoid and nonlymphoid tissues, respectively. TAK1 also contributed to DC development by promoting the generation of DC precursors. Prosurvival signals from Toll-like receptors, CD40 and receptor activator of nuclear factor-κB (RANK) are integrated by TAK1 in DCs, which in turn mediated activation of downstream NF-κB and AKT-Foxo pathways and established a gene-expression program. TAK1 deficiency in DCs caused a myeloid proliferative disorder characterized by expansion of neutrophils and inflammatory monocytes, disrupted T-cell homeostasis, and prevented effective T-cell priming and generation of regulatory T cells. Moreover, TAK1 signaling in DCs was required to prevent myeloid proliferation even in the absence of lymphocytes, indicating a previously unappreciated regulatory mechanism of DC-mediated control of myeloid cell-dependent inflammation. Therefore, TAK1 orchestrates a prosurvival checkpoint in DCs that affects the homeostasis and function of the immune system.

Homeostasis and function of regulatory T cells (Tregs) in vivo: lessons from TCR-transgenic Tregs

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Attridge, Kesley; Walker, Lucy S K

2014-01-01

The identification of CD25 and subsequently Forkhead box protein 3 (Foxp3) as markers for regulatory T cells (Tregs) has revolutionized our ability to explore this population experimentally. In a similar vein, our understanding of antigen-specific Treg responses in vivo owes much to the fortuitous generation of T-cell receptor (TCR)-transgenic Tregs. This has permitted tracking of Tregs with a defined specificity in vivo, facilitating analysis of how encounter with cognate antigen shapes Treg homeostasis and function. Here, we review the key lessons learned from a decade of analysis of TCR-transgenic Tregs and set this in the broader context of general progress in the field. Use of TCR-transgenic Tregs has led to an appreciation that Tregs are a highly dynamic proliferative population in vivo, rather than an anergic population as they were initially portrayed. It is now clear that Treg homeostasis is positively regulated by encounter with self-antigen expressed on peripheral tissues, which is likely to be relevant to the phenomenon of peripheral repertoire reshaping that has been described for Tregs and the observation that the Treg TCR specificities vary by anatomical location. Substantial evidence has also accumulated to support the role of CD28 costimulation and interleukin-2 in Treg homeostasis. The availability of TCR-transgenic Tregs has enabled analysis of Treg populations that are sufficient or deficient in particular genes, without the comparison being confounded by repertoire alterations. This approach has yielded insights into genes required for Treg function in vivo, with particular progress being made on the role of ctla-4 in this context. As the prospect of manipulating Treg populations in the clinic becomes reality, a full appreciation of the rules governing their homeostasis will prove increasingly important. PMID:24712457

Cholesterol Metabolism and Weight Reduction in Subjects with Mild Obstructive Sleep Apnoea: A Randomised, Controlled Study

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Maarit Hallikainen

2013-01-01

Full Text Available To evaluate whether parameters of obstructive sleep apnoea (OSA associate with cholesterol metabolism before and after weight reduction, 42 middle-aged overweight subjects with mild OSA were randomised to intensive lifestyle intervention (N=23 or to control group (N=18 with routine lifestyle counselling only. Cholesterol metabolism was evaluated with serum noncholesterol sterol ratios to cholesterol, surrogate markers of cholesterol absorption (cholestanol and plant sterols and synthesis (cholestenol, desmosterol, and lathosterol at baseline and after 1-year intervention. At baseline, arterial oxygen saturation (SaO2 was associated with serum campesterol (P<0.05 and inversely with desmosterol ratios (P<0.001 independently of gender, BMI, and homeostasis model assessment index of insulin resistance (HOMA-IR. Apnoea-hypopnoea index (AHI was not associated with cholesterol metabolism. Weight reduction significantly increased SaO2and serum cholestanol and decreased AHI and serum cholestenol ratios. In the groups combined, the changes in AHI were inversely associated with changes of cholestanol and positively with cholestenol ratios independent of gender and the changes of BMI and HOMA-IR (P<0.05. In conclusion, mild OSA seemed to be associated with cholesterol metabolism independent of BMI and HOMA-IR. Weight reduction increased the markers of cholesterol absorption and decreased those of cholesterol synthesis in the overweight subjects with mild OSA.

T-cell homeostasis in chronic HCV-infected patients treated with interferon and ribavirin or an interferon-free regimen

DEFF Research Database (Denmark)

Hartling, Hans Jakob; Birch, Carsten; Gaardbo, Julie C

2015-01-01

Direct-acting antiviral has replaced pegylated interferon-α and ribavirin-based treatment in the treatment of chronic hepatitis C virus (HCV) infection. While interferon-α is immune modulating and causes lymphopenia, interferon-free regimens seem to be well-tolerated. This study aimed to compare T......-cell homeostasis before, during, and after HCV treatment with or without interferon-α in patients with chronic HCV infection. A total of 20 patients with chronic HCV infection were treated with pegylated interferon-α and ribavirin, and six patients were treated with an interferon-free regimen. All patients were...... compared to prior treatment values. Finally, a proportion of CD8+ effector memory was lower while proportion of apoptotic T cells was higher after sustained virologic response compared to prior treatment. Despite lymphopenia during interferon, alterations in T-cell homeostasis during treatment were...

Hypercholesterolemia Tunes Hematopoietic Stem/Progenitor Cells for Inflammation and Atherosclerosis.

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Ma, Xiaojuan; Feng, Yingmei

2016-07-19

As the pathological basis of cardiovascular disease (CVD), atherosclerosis is featured as a chronic inflammation. Hypercholesterolemia is an independent risk factor for CVD. Accumulated studies have shown that hypercholesterolemia is associated with myeloid cell expansion, which stimulates innate and adaptive immune responses, strengthens inflammation, and accelerates atherosclerosis progression. Hematopoietic stem/progenitor cells (HSPC) in bone marrow (BM) expresses a panel of lipoprotein receptors to control cholesterol homeostasis. Deficiency of these receptors abrogates cellular cholesterol efflux, resulting in HSPC proliferation and differentiation in hypercholesterolemic mice. Reduction of the cholesterol level in the lipid rafts by infusion of reconstituted high-density lipoprotein (HDL) or its major apolipoprotein, apoA-I, reverses hypercholesterolemia-induced HSPC expansion. Apart from impaired cholesterol metabolism, inhibition of reactive oxygen species production suppresses HSPC activation and leukocytosis. These data indicate that the mechanisms underlying the effects of hypercholesterolemia on HSPC proliferation and differentiation could be multifaceted. Furthermore, dyslipidemia also regulates HSPC-neighboring cells, resulting in HSPC mobilization. In the article, we review how hypercholesterolemia evokes HSPC activation and mobilization directly or via its modification of BM microenvironment. We hope this review will bring light to finding key molecules to control HSPC expansion, inflammation, and atherosclerosis for the treatment of CVD.

The membrane as the gatekeeper of infection: Cholesterol in host-pathogen interaction.

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Kumar, G Aditya; Jafurulla, Md; Chattopadhyay, Amitabha

2016-09-01

The cellular plasma membrane serves as a portal for the entry of intracellular pathogens. An essential step for an intracellular pathogen to gain entry into a host cell therefore is to be able to cross the cell membrane. In this review, we highlight the role of host membrane cholesterol in regulating the entry of intracellular pathogens using insights obtained from work on the interaction of Leishmania and Mycobacterium with host cells. The entry of these pathogens is known to be dependent on host membrane cholesterol. Importantly, pathogen entry is inhibited either upon depletion (or complexation), or enrichment of membrane cholesterol. In other words, an optimum level of host membrane cholesterol is necessary for efficient infection by pathogens. In this overall context, we propose a general mechanism, based on cholesterol-induced conformational changes, involving cholesterol binding sites in host cell surface receptors that are implicated in this process. A therapeutic strategy targeting modulation of membrane cholesterol would have the advantage of avoiding the commonly encountered problem of drug resistance in tackling infection by intracellular pathogens. Insights into the role of host membrane cholesterol in pathogen entry would be instrumental in the development of novel therapeutic strategies to effectively tackle intracellular pathogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

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Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Cholesterol: Its Regulation and Role in Central Nervous System Disorders

OpenAIRE

Matthias Orth; Stefano Bellosta

2012-01-01

Cholesterol is a major constituent of the human brain, and the brain is the most cholesterol-rich organ. Numerous lipoprotein receptors and apolipoproteins are expressed in the brain. Cholesterol is tightly regulated between the major brain cells and is essential for normal brain development. The metabolism of brain cholesterol differs markedly from that of other tissues. Brain cholesterol is primarily derived by de novo synthesis and the blood brain barrier prevents the uptake of lipoprotein...

Anthocyanin prevents CD40-activated proinflammatory signaling in endothelial cells by regulating cholesterol distribution.

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Xia, Min; Ling, Wenhua; Zhu, Huilian; Wang, Qing; Ma, Jing; Hou, Mengjun; Tang, Zhihong; Li, Lan; Ye, Qinyuan

2007-03-01

Intracellular tumor necrosis factor receptor-associated factors (TRAFs) translocation to lipid rafts is a key element in CD40-induced signaling. The purpose of this study was to investigate the influence of anthocyanin on CD40-mediated proinflammatory events in human endothelial cells and the underlying possible molecular mechanism. Treatment of endothelial cells with anthocyanin prevented from CD40-induced proinflammatory status, measured by production of IL-6, IL-8, and monocyte chemoattractant protein-1 through inhibiting CD40-induced nuclear factor-kappaB (NF-kappaB) activation. TRAF-2 played pivotal role in CD40-NF-kappaB pathway as TRAF-2 small interference RNA (siRNA) diminished CD40-induced NF-kappaB activation and inflammation. TRAF-2 overexpression increased CD40-mediated NF-kappaB activation. Moreover, TRAF-2 almost totally recruited to lipid rafts after stimulation by CD40 ligand and depletion of cholesterol diminished CD40-mediated NF-kappaB activation. Exposure to anthocyanin not only interrupted TRAF-2 recruitment to lipid rafts but also decreased cholesterol content in Triton X-100 insoluble lipid rafts. However, anthocyanin did not influence the interaction between CD40 ligand and CD40 receptor. Our findings suggest that anthocyanin protects from CD40-induced proinflammatory signaling by preventing TRAF-2 translocation to lipid rafts through regulation of cholesterol distribution, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin.

FEL induced molecular operation on cultured fibroblast and cholesterol ester

International Nuclear Information System (INIS)

Awazu, Kunio; Ogino, Seiji; Nishimura, Eiichi; Tomimasu, Takio; Yasumoto, Masato.

1997-01-01

Free Electron Lasers can be used to molecular operation such as the delivery of a number of molecules into cells or the separation of cholesterol ester. First, cultured NIH3T3 cells are exposed to high-intensity short pulse Free Electron Laser (FEL). The FEL is tuned to an absorption maximum wavelength, 6.1 μm, which was measured by microscopic FTIR. A fluorescence dye in the cell suspension is more absorbed into the cell with the FEL exposure due to the FEL-induced mechanical stress to the cell membrane. A quantitative fluorescence microscopy is used to determine the efficiency of delivery. Second, as a compound in a lipid cell, cholesterol ester was exposed to 5.75 μm FEL. FTIR measurement was done to evaluate the modification of the cholesterol ester. The result showed that the fluorescence intensity of sample cells were higher than that of control cells, and there was significant difference between the control and the sample group. Blebbing and the colony formation of the cells were observed for cells with mechanical stress. As for the cholesterol ester, it can be modified by the FEL irradiation. These results showed that FEL can be used as a molecular operational tool by photo-chemical and photo-mechanical interaction. (author)

Cholesterol: a novel regulatory role in myelin formation.

Science.gov (United States)

Saher, Gesine; Quintes, Susanne; Nave, Klaus-Armin

2011-02-01

Myelin consists of tightly compacted membranes that form an insulating sheath around axons. The function of myelin for rapid saltatory nerve conduction is dependent on its unique composition, highly enriched in glycosphingolipids and cholesterol. Cholesterol emerged as the only integral myelin component that is essential and rate limiting for the development of CNS and PNS myelin. Experiments with conditional mouse mutants that lack cholesterol biosynthesis in oligodendrocytes revealed that only minimal changes of the CNS myelin lipid composition are tolerated. In Schwann cells of the PNS, protein trafficking and myelin compaction depend on cholesterol. In this review, the authors summarize the role of cholesterol in myelin biogenesis and myelin disease.

Triglycerides, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol in rats exposed to premium motor spirit fumes.

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Aberare, Ogbevire L; Okuonghae, Patrick; Mukoro, Nathaniel; Dirisu, John O; Osazuwa, Favour; Odigie, Elvis; Omoregie, Richard

2011-06-01

Deliberate and regular exposure to premium motor spirit fumes is common and could be a risk factor for liver disease in those who are occupationally exposed. A possible association between premium motor spirit fumes and plasma levels of triglyceride, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol using a rodent model could provide new insights in the pathology of diseases where cellular dysfunction is an established risk factor. The aim of this study was to evaluate the possible effect of premium motor spirit fumes on lipids and lipoproteins in workers occupationally exposed to premium motor spirit fumes using rodent model. Twenty-five Wister albino rats (of both sexes) were used for this study between the 4(th) of August and 7(th) of September, 2010. The rats were divided into five groups of five rats each. Group 1 rats were not exposed to premium motor spirit fumes (control group), group 2 rats were exposed for 1 hour daily, group 3 for 3 hours daily, group 4 for 5 hours daily and group 5 for 7 hours daily. The experiment lasted for a period of 4 weeks. Blood samples obtained from all the groups after 4 weeks of exposure were used for the estimation of plasma levels of triglyceride, total cholesterol, high density lipoprotein- cholesterol and low density lipoprotein- cholesterol. Results showed significant increase in means of plasma total cholesterol and low density lipoprotein levels (P<0.05). The mean triglyceride and total body weight were significantly lower (P<0.05) in the exposed group when compared with the unexposed. The plasma level of high density lipoprotein, the ratio of low density lipoprotein to high density lipoprotein and the ratio of total cholesterol to high density lipoprotein did not differ significantly in exposed subjects when compared with the control group. These results showed that frequent exposure to petrol fumes may be highly deleterious to the liver cells.

Loss of Folliculin Disrupts Hematopoietic Stem Cell Quiescence and Homeostasis Resulting in Bone Marrow Failure.

Science.gov (United States)

Baba, Masaya; Toyama, Hirofumi; Sun, Lei; Takubo, Keiyo; Suh, Hyung-Chan; Hasumi, Hisashi; Nakamura-Ishizu, Ayako; Hasumi, Yukiko; Klarmann, Kimberly D; Nakagata, Naomi; Schmidt, Laura S; Linehan, W Marston; Suda, Toshio; Keller, Jonathan R

2016-04-01

Folliculin (FLCN) is an autosomal dominant tumor suppressor gene that modulates diverse signaling pathways required for growth, proliferation, metabolism, survival, motility, and adhesion. FLCN is an essential protein required for murine embryonic development, embryonic stem cell (ESC) commitment, and Drosophila germline stem cell maintenance, suggesting that Flcn may be required for adult stem cell homeostasis. Conditional inactivation of Flcn in adult hematopoietic stem/progenitor cells (HSPCs) drives hematopoietic stem cells (HSC) into proliferative exhaustion resulting in the rapid depletion of HSPC, loss of all hematopoietic cell lineages, acute bone marrow (BM) failure, and mortality after 40 days. HSC that lack Flcn fail to reconstitute the hematopoietic compartment in recipient mice, demonstrating a cell-autonomous requirement for Flcn in HSC maintenance. BM cells showed increased phosphorylation of Akt and mTorc1, and extramedullary hematopoiesis was significantly reduced by treating mice with rapamycin in vivo, suggesting that the mTorc1 pathway was activated by loss of Flcn expression in hematopoietic cells in vivo. Tfe3 was activated and preferentially localized to the nucleus of Flcn knockout (KO) HSPCs. Tfe3 overexpression in HSPCs impaired long-term hematopoietic reconstitution in vivo, recapitulating the Flcn KO phenotype, and supporting the notion that abnormal activation of Tfe3 contributes to the Flcn KO phenotype. Flcn KO mice develop an acute histiocytic hyperplasia in multiple organs, suggesting a novel function for Flcn in macrophage development. Thus, Flcn is intrinsically required to maintain adult HSC quiescence and homeostasis, and Flcn loss leads to BM failure and mortality in mice. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Gut commensal flora: tolerance and homeostasis

OpenAIRE

Rescigno, Maria

2009-01-01

Commensal microorganisms are not ignored by the intestinal immune system. Recent evidence shows that commensals actively participate in maintaining intestinal immune homeostasis by interacting with intestinal epithelial cells and delivering tolerogenic signals that are transmitted to the underlying cells of the immune system.

Regulation of intestinal homeostasis by innate and adaptive immunity.

Science.gov (United States)

Kayama, Hisako; Takeda, Kiyoshi

2012-11-01

The intestine is a unique tissue where an elaborate balance is maintained between tolerance and immune responses against a variety of environmental factors such as food and the microflora. In a healthy individual, the microflora stimulates innate and adaptive immune systems to maintain gut homeostasis. However, the interaction of environmental factors with particular genetic backgrounds can lead to dramatic changes in the composition of the microflora (i.e. dysbiosis). Many of the specific commensal-bacterial products and the signaling pathways they trigger have been characterized. The role of T(h)1, T(h)2 and T(h)17 cells in inflammatory bowel disease has been widely investigated, as has the contribution of epithelial cells and subsets of dendritic cells and macrophages. To date, multiple regulatory cells in adaptive immunity, such as regulatory T cells and regulatory B cells, have been shown to maintain gut homeostasis by preventing inappropriate innate and adaptive immune responses to commensal bacteria. Additionally, regulatory myeloid cells have recently been identified that prevent intestinal inflammation by inhibiting T-cell proliferation. An increasing body of evidence has shown that multiple regulatory mechanisms contribute to the maintenance of gut homeostasis.

Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.

Directory of Open Access Journals (Sweden)

Kathie Béland

Full Text Available The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C in presence or absence of activated liver-specific autoreactive CD8(+ T cells. Regardless of autoreactive CD8(+ T cells, mice injected with CpG and Poly(I:C showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C and autoreactive CD8(+T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8(+ T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8(+ T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

Liver restores immune homeostasis after local inflammation despite the presence of autoreactive T cells.

Science.gov (United States)

Béland, Kathie; Lapierre, Pascal; Djilali-Saiah, Idriss; Alvarez, Fernando

2012-01-01

The liver must keep equilibrium between immune tolerance and immunity in order to protect itself from pathogens while maintaining tolerance to food antigens. An imbalance between these two states could result in an inflammatory liver disease. The aims of this study were to identify factors responsible for a break of tolerance and characterize the subsequent restoration of liver immune homeostasis. A pro-inflammatory environment was created in the liver by the co-administration of TLR ligands CpG and Poly(I:C) in presence or absence of activated liver-specific autoreactive CD8(+) T cells. Regardless of autoreactive CD8(+) T cells, mice injected with CpG and Poly(I:C) showed elevated serum ALT levels and a transient liver inflammation. Both CpG/Poly(I:C) and autoreactive CD8(+)T cells induced expression of TLR9 and INF-γ by the liver, and an up-regulation of homing and adhesion molecules CXCL9, CXCL10, CXCL16, ICAM-1 and VCAM-1. Transferred CFSE-labeled autoreactive CD8(+) T cells, in presence of TLR3 and 9 ligands, were recruited by the liver and spleen and proliferated. This population then contracted by apoptosis through intrinsic and extrinsic pathways. Up-regulation of FasL and PD-L1 in the liver was observed. In conclusion, TLR-mediated activation of the innate immune system results in a pro-inflammatory environment that promotes the recruitment of lymphocytes resulting in bystander hepatitis. Despite this pro-inflammatory environment, the presence of autoreactive CD8(+) T cells is not sufficient to sustain an autoimmune response against the liver and immune homeostasis is rapidly restored through the apoptosis of T cells.

Single-Cell RNA Sequencing Reveals T Helper Cells Synthesizing Steroids De Novo to Contribute to Immune Homeostasis

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Bidesh Mahata

2014-05-01

Full Text Available T helper 2 (Th2 cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a “lymphosteroid,” a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.

Cognition, learning behaviour and hippocampal synaptic plasticity are not disrupted in mice over-expressing the cholesterol transporter ABCG1

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Eadie Brennan D

2009-02-01

Full Text Available Abstract Background Cognitive deficits are a hallmark feature of both Down Syndrome (DS and Alzheimer's Disease (AD. Extra copies of the genes on chromosome 21 may also play an important role in the accelerated onset of AD in DS individuals. Growing evidence suggests an important function for cholesterol in the pathogenesis of AD, particularly in APP metabolism and production of Aβ peptides. The ATP-Binding Cassette-G1 (ABCG1 transporter is located on chromosome 21, and participates in the maintenance of tissue cholesterol homeostasis. Results To assess the role of ABCG1 in DS-related cognition, we evaluated the cognitive performance of mice selectively over-expressing the ABCG1 gene from its endogenous regulatory signals. Both wild-type and ABCG1 transgenic mice performed equivalently on several behavioral tests, including measures of anxiety, as well as on reference and working memory tasks. No deficits in hippocampal CA1 synaptic plasticity as determined with electrophysiological studies were apparent in mice over-expressing ABCG1. Conclusion These findings indicate that although ABCG1 may play a role in maintaining cellular or tissue cholesterol homeostasis, it is unlikely that excess ABCG1 expression contributes to the cognitive deficits in DS individuals.

Circulating PCSK9 affects serum LDL and cholesterol levels more than SREBP-2 expression.

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Mohammadi, Asghar; Shabani, Mohamad; Naseri, Faezeh; Hosseni, Bita; Soltanmohammadi, Elham; Piran, Sadegh; Najafi, Mohammad

2017-07-01

Cholesterol homeostasis is dependent upon the sterol regulatory element binding protein 2 (SREBP-2) regulatory system and the functioning of plasma proprotein convertase subtilisin/kexin type 9 (PCSK9). Many studies have also reported that low density lipoprotein receptor (LDLR) levels in cellular membranes are related to the functioning of these proteins. The aim of this study was to investigate the association of lipid profiles with circulating PCSK9 protein values and SREBP-2 expression levels in normal subjects. The study involved 120 randomly chosen healthy subjects. Their lipid profiles were measured using routine laboratory techniques, and the plasma PCSK9 protein and SREBP-2 expression levels were determined by ELISA and real time quantitative PCR methods, respectively. A statistical analysis was carried out using a statistical software package. Linear regression analyses showed a significant correlation between total cholesterol and PCSK9 (3.54 ± 1.31 ng/mL), as well as between total cholesterol and SREBP-2 (0.1-35.38) (p = 0.002 and p = 0.02, respectively). Furthermore, multiple regression analyses showed strict correlations between PCSK9 and cholesterol-related parameters especially the total cholesterol/HDL-C ratio (β = 3.53, p = 0.001). There was no significant correlation between circulating PCSK9 and SREBP-2 expression levels (r = 1.2, p = 0.3). The study results revealed that serum cholesterol-related parameters are strictly associated with plasma PCSK9 values, suggesting that PCSK9 function has a greater effect on serum total cholesterol levels than SREBP-2 expression does. Furthermore, the total cholesterol/HDL-C ratio was a better indicator for evaluating PCSK9 level than total cholesterol.

Functions of innate immune cells and commensal bacteria in gut homeostasis.

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Kayama, Hisako; Takeda, Kiyoshi

2016-02-01

The intestinal immune system remains unresponsive to beneficial microbes and dietary antigens while activating pro-inflammatory responses against pathogens for host defence. In intestinal mucosa, abnormal activation of innate immunity, which directs adaptive immune responses, causes the onset and/or progression of inflammatory bowel diseases. Thus, innate immunity is finely regulated in the gut. Multiple innate immune cell subsets have been identified in both murine and human intestinal lamina propria. Some innate immune cells play a key role in the maintenance of gut homeostasis by preventing inappropriate adaptive immune responses while others are associated with the pathogenesis of intestinal inflammation through development of Th1 and Th17 cells. In addition, intestinal microbiota and their metabolites contribute to the regulation of innate/adaptive immune responses. Accordingly, perturbation of microbiota composition can trigger intestinal inflammation by driving inappropriate immune responses. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Transcription controls growth, cell kinetics and cholesterol supply to sustain ACTH responses

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Robert I Menzies

2017-09-01

Full Text Available Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes (P 4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu, were downregulated and apoptotic cells (Tunel staining were fewer (P < 0.001 and more widely distributed throughout the cortex. In summary, long-term steroidogenesis with ACTH excess is sustained by genes controlling cholesterol supply and adrenal mass. ACTH effects on adrenal morphology and genes controlling cell hypertrophy, proliferation and apoptosis suggest the involvement of different cell types and separate molecular pathways.

Transcription controls growth, cell kinetics and cholesterol supply to sustain ACTH responses.

Science.gov (United States)

Menzies, Robert I; Zhao, Xin; Mullins, Linda J; Mullins, John J; Cairns, Carolynn; Wrobel, Nicola; Dunbar, Donald R; Bailey, Matthew A; Kenyon, Christopher J

2017-10-01

Chronic ACTH exposure is associated with adrenal hypertrophy and steroidogenesis. The underlying molecular processes in mice have been analysed by microarray, histological and immunohistochemical techniques. Synacthen infused for 2 weeks markedly increased adrenal mass and plasma corticosterone levels. Microarray analysis found greater than 2-fold changes in expression of 928 genes ( P  4-fold and cross-sectional area of fasciculata cells was 2-fold greater. In contrast, genes associated with apoptosis (eg Casp12, Clu, ) were downregulated and apoptotic cells (Tunel staining) were fewer ( P  < 0.001) and more widely distributed throughout the cortex. In summary, long-term steroidogenesis with ACTH excess is sustained by genes controlling cholesterol supply and adrenal mass. ACTH effects on adrenal morphology and genes controlling cell hypertrophy, proliferation and apoptosis suggest the involvement of different cell types and separate molecular pathways. © 2017 The authors.

Mitochondrial damage and cholesterol storage in human hepatocellular carcinoma cells with silencing of UBIAD1 gene expression

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Carlos R. Morales

2014-01-01

Full Text Available Heterozygous mutations in the UBIAD1 gene cause Schnyder corneal dystrophy characterized by abnormal cholesterol and phospholipid deposits in the cornea. Ubiad1 protein was recently identified as Golgi prenyltransferase responsible for biosynthesis of vitamin K2 and CoQ10, a key protein in the mitochondrial electron transport chain. Our study shows that silencing UBIAD1 in cultured human hepatocellular carcinoma cells causes dramatic morphological changes and cholesterol storage in the mitochondria, emphasizing an important role of UBIAD1 in mitochondrial function.

Inhibition of Cholesterol Synthesis in HepG2 Cells by GINST-Decreasing HMG-CoA Reductase Expression Via AMP-Activated Protein Kinase.

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Han, Joon-Seung; Sung, Jong Hwan; Lee, Seung Kwon

2017-11-01

GINST, a hydrolyzed ginseng extract, has been reported to have antidiabetic effects and to reduce hyperglycemia and hyperlipidemia. Hypercholesterolemia is caused by diet or genetic factors and can lead to atherosclerosis and coronary heart disease. Thus, the purpose of this study is to determine whether GINST and the ginsenoside metabolite, IH-901 (compound K), reduce cholesterol synthesis in HepG2 cells and the signal transduction pathways involved. Concentrations of cholesterol were measured by using an enzymatic method. Expression levels of sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer-binding proteins α (C/EBPα), GAPDH, and phosphorylation of AMP-activated protein kinase α (AMPKα), protein kinase B (PKB, also known as Akt), and mechanistic target of rapamycin complex 1 (mTORC1) were measured using western blot. Total cholesterol concentration decreased after GINST treatment for 24 and 48 h. Expression of HMGCR decreased more with GINST than with the inhibitors, U18666A and atorvastatin, after 48 h in a dose-dependent manner. Phosphorylation of AMPKα increased 2.5x by GINST after 360 min of treatment, and phosphorylation of Akt decreased after 120 and 360 min. We separated compound K from GINST extracts flash chromatography. Compound K decreased cholesterol synthesis in HepG2 cells at 24 and 48 h. Therefore, we conclude that GINST inhibits cholesterol synthesis in HepG2 cells by decreasing HMGCR expression via AMPKα activation. GINST, a hydrolyzed ginseng extract, can inhibit cholesterol synthesis in liver cells via activation of AMPKα. IH-901 (compound K), which is the main component with bioactivity in GINST, also has anticholesterol effects. Thus, we suggest that GINST can be used to reduce hypercholesterolemia. © 2017 Institute of Food Technologists®.

Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

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Jijon, H B; Suarez-Lopez, L; Diaz, O E; Das, S; De Calisto, J; Yaffe, M B; Pittet, M J; Mora, J R; Belkaid, Y; Xavier, R J; Villablanca, E J

2018-05-01

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4 + goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.

Adipose Type One Innate Lymphoid Cells Regulate Macrophage Homeostasis through Targeted Cytotoxicity.

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Boulenouar, Selma; Michelet, Xavier; Duquette, Danielle; Alvarez, David; Hogan, Andrew E; Dold, Christina; O'Connor, Donal; Stutte, Suzanne; Tavakkoli, Ali; Winters, Desmond; Exley, Mark A; O'Shea, Donal; Brenner, Michael B; von Andrian, Ulrich; Lynch, Lydia

2017-02-21

Adipose tissue has a dynamic immune system that adapts to changes in diet and maintains homeostatic tissue remodeling. Adipose type 1 innate lymphoid cells (AT1-ILCs) promote pro-inflammatory macrophages in obesity, but little is known about their functions at steady state. Here we found that human and murine adipose tissue harbor heterogeneous populations of AT1-ILCs. Experiments using parabiotic mice fed a high-fat diet (HFD) showed differential trafficking of AT1-ILCs, particularly in response to short- and long-term HFD and diet restriction. At steady state, AT1-ILCs displayed cytotoxic activity toward adipose tissue macrophages (ATMs). Depletion of AT1-ILCs and perforin deficiency resulted in alterations in the ratio of inflammatory to anti-inflammatory ATMs, and adoptive transfer of AT1-ILCs exacerbated metabolic disorder. Diet-induced obesity impaired AT1-ILC killing ability. Our findings reveal a role for AT1-ILCs in regulating ATM homeostasis through cytotoxicity and suggest that this function is relevant in both homeostasis and metabolic disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

NARCIS (Netherlands)

Beulens, J.W.J.; Sierksma, A.; Tol, A. van; Fournier, N.; Gent, T. van; Paul, J.L.; Hendriks, H.F.J.

2004-01-01

Moderate alcohol consumption increases HDL cholesterol, which is involved in reverse cholesterol transport (RCT). The aim of this study was to investigate the effect of moderate alcohol consumption on cholesterol efflux, using J774 mouse macrophages and Fu5AH cells, and on other parameters in the

Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

International Nuclear Information System (INIS)

Luo, Di-xian; Xia, Cheng-lai; Li, Jun-mu; Xiong, Yan; Yuan, Hao-yu; TANG, Zhen-Wang; Zeng, Yixin; Liao, Duan-fang

2010-01-01

Research highlights: → Vertical static pressure accelerates ox-LDL-induced cholesterol accumulation in cultured vascular smooth muscle cells. → Static pressure induces SREBP-1 activation. → Static pressure downregulates the expressions of caveolin-1 by activating SREBP-1. → Static pressure also downregulates the transcription of ABCA1 by activating SREBP-1. → Static pressure increases ox-LDL-induced cholesterol accumulation by SREBP-1-mediated caveolin-1 downregulation in vascular smooth muscle cells cultured in vitro. -- Abstract: Objective: To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism. Methods: Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different static pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure. Results: Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 ± 2.8 mg/g, 31.8 ± 0.7 mg/g, 92.3 ± 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 ± 9.4 mg/g, 235.9 ± 3.0 mg/g, 386.7 ± 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were upregulated. Conclusion: Static

Static pressure accelerates ox-LDL-induced cholesterol accumulation via SREBP-1-mediated caveolin-1 downregulation in cultured vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Luo, Di-xian, E-mail: luodixian_2@163.com [Department of Pharmacology, School of Pharmaceutics, Central South University, Changsha 410083, Hunan (China); Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan (China); First People' s Hospital of Chenzhou City, Chenzhou 423000, Hunan (China); Xia, Cheng-lai [Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan (China); Department of Pharmacy, Third Affiliated Hospital Medical College of Guangzhou, Guangzhou 510150, Guangdong (China); Li, Jun-mu [Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan (China); Xiong, Yan [Department of Pharmacology, School of Pharmaceutics, Central South University, Changsha 410083, Hunan (China); Yuan, Hao-yu [Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan (China); Lusong Center for Disease Control and Prevention, Zhuzhou 412000, Hunan (China); TANG, Zhen-Wang; Zeng, Yixin [Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan (China); Liao, Duan-fang, E-mail: dfliao66@yahoo.com.cn [Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang 421001, Hunan (China); Department of Traditional Chinese Diagnostics, School of Pharmacy, Hunan University of Chinese Medicine, Changsha 420108, Hunan (China)

2010-12-03

Research highlights: {yields} Vertical static pressure accelerates ox-LDL-induced cholesterol accumulation in cultured vascular smooth muscle cells. {yields} Static pressure induces SREBP-1 activation. {yields} Static pressure downregulates the expressions of caveolin-1 by activating SREBP-1. {yields} Static pressure also downregulates the transcription of ABCA1 by activating SREBP-1. {yields} Static pressure increases ox-LDL-induced cholesterol accumulation by SREBP-1-mediated caveolin-1 downregulation in vascular smooth muscle cells cultured in vitro. -- Abstract: Objective: To investigate the effect of static pressure on cholesterol accumulation in vascular smooth muscle cells (VSMCs) and its mechanism. Methods: Rat-derived VSMC cell line A10 treated with 50 mg/L ox-LDL and different static pressures (0, 60, 90, 120, 150, 180 mm Hg) in a custom-made pressure incubator for 48 h. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC; The mRNA levels of caveolin-1 and ABCA1, the protein levels of caveolin-1 SREBP-1 and mature SREBP-1 were respectively detected by RT-PCR or western blot. ALLN, an inhibitor of SREBP metabolism, was used to elevate SREBP-1 protein level in VSMCs treated with static pressure. Results: Static pressures significantly not only increase intracellular lipid droplets in VSMCs, but also elevate cellular lipid content in a pressure-dependent manner. Intracellular free cholesterol (FC), cholesterol ester (CE), total cholesterol (TC) were respectively increased from 60.5 {+-} 2.8 mg/g, 31.8 {+-} 0.7 mg/g, 92.3 {+-} 2.1 mg/g at atmosphere pressure (ATM, 0 mm Hg) to 150.8 {+-} 9.4 mg/g, 235.9 {+-} 3.0 mg/g, 386.7 {+-} 6.4 mg/g at 180 mm Hg. At the same time, static pressures decrease the mRNA and protein levels of caveolin-1, and induce the activation and nuclear translocation of SREBP-1. ALLN increases the protein level of mature SREBP-1 and decreases caveolin-1 expression, so that cellular lipid levels were

Dietary and biliary cholesterol absorption in rats. Effect of dietary cholesterol level and cholesterol saturation of bile

International Nuclear Information System (INIS)

Wilson, M.D.

1985-01-01

The principal objective of this research was to determine if cholesterol introduced into the duodenum of rats in a micellar form as occurs with bile, is absorbed more efficiently than cholesterol presented in a nonmicellar form, as occurs with dietary cholesterol. Cholesterol absorption was measured during the constant intraduodenal infusion of liquid diets ([ 14 C] cholesterol) and artificial biles ([ 3 H] cholesterol) in thoracic lymph duct cannulated rats. Percentage absorption was calculated by dividing the rate of appearance of radiolabeled cholesterol in lymph by its rate of infusion when lymph cholesterol specific activity was constant. Results provide strong evidence that under certain conditions biliary cholesterol is more efficiently absorbed than is dietary cholesterol, and that this differential must be considered when evaluating the influence of diet or drug therapy on cholesterol absorption

Regulation of α1 Na/K-ATPase Expression by Cholesterol*

OpenAIRE

Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

2011-01-01

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol tr...