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Sample records for sirt1 protein expression

  1. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

    International Nuclear Information System (INIS)

    Srisuttee, Ratakorn; Koh, Sang Seok; Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae; Jhun, Byung Hak; Horio, Yoshiyuki; Chung, Young-Hwa

    2012-01-01

    Highlights: ► Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. ► Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. ► Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. ► Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of β-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

  2. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Srisuttee, Ratakorn [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Moon, Jeong; Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Jhun, Byung Hak [Department of Applied Nanoscience, Pusan National University, Busan 609-735 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer Up-regulation of SIRT1 protein and activity sensitizes Hep3B-HBX cells to oxidative stress-induced apoptosis. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for oxidation-induced apoptosis. Black-Right-Pointing-Pointer Ectopic expression and enhanced activity of SIRT1 attenuate JNK phosphorylation. Black-Right-Pointing-Pointer Inhibition of SIRT1 activity restores resistance to oxidation-induced apoptosis through JNK activation. -- Abstract: We previously showed that SIRT1 deacetylase inhibits proliferation of hepatocellular carcinoma cells expressing hepatitis B virus (HBV) X protein (HBX), by destabilization of {beta}-catenin. Here, we report another role for SIRT1 in HBX-mediated resistance to oxidative stress. Ectopic expression and enhanced activity of SIRT1 sensitize Hep3B cells stably expressing HBX to oxidative stress-induced apoptosis. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for sensitization of oxidation-mediated apoptosis. Furthermore, ectopic expression of SIRT1 and treatment with resveratrol (a SIRT1 activator) attenuated JNK phosphorylation, which is a prerequisite for resistance to oxidative stress-induced apoptosis. Conversely, suppression of SIRT1 activity with nicotinamide inhibited the effect of resveratrol on JNK phosphorylation, leading to restoration of resistance to oxidation-induced apoptosis. Taken together, these results suggest that up-regulation of SIRT1 under oxidative stress may be a therapeutic strategy for treatment of hepatocellular carcinoma cells related to HBV through inhibition of JNK activation.

  3. Ethanol extracts of black pepper or turmeric down-regulated SIRT1 protein expression in Daudi culture cells.

    Science.gov (United States)

    Nishimura, Yuri; Kitagishi, Yasuko; Yoshida, Hitomi; Okumura, Naoko; Matsuda, Satoru

    2011-01-01

    SIRT1 is a mammalian candidate molecule involved in longevity and diverse metabolic processes. The present study aimed to determine the effects of certain herbs and spices on SIRT1 expression. Human cell lines Daudi, Jurkat, U937 and K562 were cultured in RPMI-1640. Herb and spice powders were prepared and the supernatants were collected. RT-PCR was used to quantify the expression level of the gene. Protein samples were then analyzed by Western blotting. Western blotting revealed the down-regulation of SIRT1 protein expression in Daudi cells treated with extracts of black pepper or turmeric. On the other hand, the effect on the SIRT1 gene expression examined by reverse transcription polymerase chain reaction was unaltered. In conclusion, component(s) of certain herbs and spices may induce the down-regulation of SIRT1 protein.

  4. Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX.

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    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-09-04

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD(+)-dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  5. Transcriptional activation of NAD+-dependent protein deacetylase SIRT1 by nuclear receptor TLX

    International Nuclear Information System (INIS)

    Iwahara, Naotoshi; Hisahara, Shin; Hayashi, Takashi; Horio, Yoshiyuki

    2009-01-01

    An orphan nuclear receptor TLX is a transcriptional repressor that promotes the proliferation and self-renewal of neural precursor cells (NPCs). SIRT1, an NAD + -dependent protein deacetylase, is highly expressed in the NPCs and participates in neurogenesis. Here, we found that TLX colocalized with SIRT1 and knockdown of TLX by small interfering RNAs decreased SIRT1 levels in NPCs. TLX increased the SIRT1 expression by binding to the newly identified TLX-activating element in the SIRT1 gene promoter in HEK293 cells. Thus, TLX is an inducer of SIRT1 and may contribute to neurogenesis both as a transactivator and as a repressor.

  6. Seminal SIRT1 expression in infertile oligoasthenoteratozoospermic men with varicocoele.

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    Mostafa, T; Nabil, N; Rashed, L; Makeen, K; El-Kasas, M A; Mohamaed, H A

    2018-03-01

    In a case-controlled study, we assessed the expressed seminal NAD-dependent protein deacetylase (SIRT1) expression in infertile oligoasthenoteratozoospermic (OAT) men associated with varicocoele. Our study involved 81 men, recruited from the University hospitals, after ethical approval and informed consent. They were allocated into fertile normozoospermic men (n = 23), infertile OAT men without varicocoele (n = 23) and infertile OAT men with varicocoele (n = 35). Inclusion criteria consisted of confirmation of abnormal semen parameters and normal female partners whereas exclusion criteria were leukocytospermia, tobacco smoking, hormonal therapy, immunological disorders, dyslipidemia, hypogonadism, cardiovascular disorders, morbid obesity, and hepatic or renal failures. All participants had an interview to assess clinical history, clinical examination, semen analysis, and estimation of seminal SIRT1 expression. Seminal SIRT1 expression was significantly lower in infertile OAT men than fertile men. Among infertile OAT men, seminal SIRT1 expression was significantly lower in those with varicocoele than in those without. Additionally, seminal SIRT1 expression was significantly lower in varicocoele grade III cases compared with other grades. Seminal SIRT1 expression was positively correlated with sperm concentration (r = 0.327, p = 0.001), total sperm motility (r = 0.532, p = 0.001), and sperm normal forms (r = 0.469, p = 0.001). Our results suggest that seminal SIRT1 expression has a role of male infertility being significantly decreased in infertile OAT men in general and in infertile OAT men associated with varicocoele in particular. © 2018 American Society of Andrology and European Academy of Andrology.

  7. SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

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    Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

    2018-05-01

    SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

  8. Photobiomodulation on Bax and Bcl-2 Proteins and SIRT1/PGC-1α Axis mRNA Expression Levels of Aging Rat Skeletal Muscle

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    Fang-Hui Li

    2014-01-01

    Full Text Available Objective. This study aimed to analyze the effects of low level laser irradiation (LLLI on Bax and IGF-1 and Bcl-2 protein contents and SIRT1/PGC-1α axis mRNA expression levels to prevent sarcopenia in aged rats. Material and Methods. Twenty female Sprague Dawley rats (18 months old were randomly divided into two groups (n=10 per group: control (CON and LLLI groups. The gallium-aluminum-arsenium (GaAlAs laser irradiation at 810 nm was used in the single point contact mode (3.75 J/cm2; 0.4 cm2; 125 mW/cm2; 30 s. Bax, Bcl-2, and IGF-1 proteins and SIRT1/PGC-1α axis mRNA expression were assessed 24 h after LLLI on gastrocnemius in aged rat. Results. Gastrocnemius muscle weights, gastrocnemius mass/body mass, Bcl-2/BAX ratio, Bcl-2 protein, IGF-1 protein, and the mRNA contents in SIRT1, PGC-1α, NRF1, TMF, and SOD2 were significantly (P<0.05 increased by LLLI compared to CON group without LLLI. However, levels of BAX protein and caspase 3 mRNA were significantly attenuated by LLLI compared to CON group (P<0.05. Conclusion. LLLI at 810 nm inhibits sarcopenia associated with upregulation of Bcl-2/BAX ratio and IGF-1 and SIRT1/PGC-1α axis mRNA expression in aged rats. This indicates that LLLI has potential to decrease progression of myocyte apoptosis in sarcopenic muscles.

  9. NK cells of the oldest seniors represent constant and resistant to stimulation high expression of cellular protective proteins SIRT1 and HSP70.

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    Kaszubowska, Lucyna; Foerster, Jerzy; Kaczor, Jan Jacek; Schetz, Daria; Ślebioda, Tomasz Jerzy; Kmieć, Zbigniew

    2018-01-01

    Natural killer cells (NK cells) are cytotoxic lymphocytes of innate immunity that reveal some immunoregulatory properties, however, their role in the process of ageing is not completely understood. The study aimed to analyze the expression of proteins involved in cellular stress response: sirtuin 1 (SIRT1), heat shock protein 70 (HSP70) and manganese superoxide dismutase (SOD2) in human NK cells with reference to the process of ageing. Non-stimulated and stimulated with IL-2, LPS or PMA with ionomycin cells originated from peripheral blood samples of: seniors aged over 85 ('the oldest'; n  = 25; 88.5 ± 0.5 years, mean ± SEM), seniors aged under 85 ('the old'; n  = 30; 75.6 ± 0.9 years) and the young ( n  = 31; 20.9 ± 0.3 years). The relationships between the levels of expression of cellular protective proteins in the studied population were also analyzed. The concentrations of carbonyl groups and 8-isoprostanes, markers of oxidative stress, in both stimulated and non-stimulated cultured NK cells were measured to assess the level of the oxidative stress in the cells. The oldest seniors varied from the other age groups by significantly higher expression of SIRT1 and HSP70 both in non-stimulated and stimulated NK cells. These cells also appeared to be resistant to further stimulations with IL-2, LPS or PMA with ionomycin. Highly positive correlations between SIRT1 and intracellular HSP70 in both stimulated and non-stimulated NK cells were observed. SOD2 presented low expression in non-stimulated cells, whereas its sensitivity to stimulation increased with age of donors. High positive correlations between SOD2 and surface HSP70 were observed. We found that the markers of oxidative stress in NK cells did not change with ageing. The oldest seniors revealed well developed adaptive stress response in NK cells with increased, constant levels of SIRT1 and intracellular HSP70. They presented also very high positive correlations between

  10. Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

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    Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

    2018-05-01

    Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Sirt1 protects against oxidative stress-induced renal tubular cell apoptosis by the bidirectional regulation of catalase expression

    International Nuclear Information System (INIS)

    Hasegawa, Kazuhiro; Wakino, Shu; Yoshioka, Kyoko; Tatematsu, Satoru; Hara, Yoshikazu; Minakuchi, Hitoshi; Washida, Naoki; Tokuyama, Hirobumi; Hayashi, Koichi; Itoh, Hiroshi

    2008-01-01

    NAD + -dependent protein deacetylase Sirt1 regulates cellular apoptosis. We examined the role of Sirt1 in renal tubular cell apoptosis by using HK-2 cells, proximal tubular cell lines with or without reactive oxygen species (ROS), H 2 O 2 . Without any ROS, Sirt1 inhibitors enhanced apoptosis and the expression of ROS scavenger, catalase, and Sirt1 overexpression downregulated catalase. When apoptosis was induced with H 2 O 2 , Sirt1 was upregulated with the concomitant increase in catalase expression. Sirt1 overexpression rescued H 2 O 2 -induced apoptosis through the upregulation of catalase. H 2 O 2 induced the nuclear accumulation of forkhead transcription factor, FoxO3a and the gene silencing of FoxO3a enhanced H 2 O 2 -induced apoptosis. In conclusion, endogenous Sirt1 maintains cell survival by regulating catalase expression and by preventing the depletion of ROS required for cell survival. In contrast, excess ROS upregulates Sirt1, which activates FoxO3a and catalase leading to rescuing apoptosis. Thus, Sirt1 constitutes a determinant of renal tubular cell apoptosis by regulating cellular ROS levels

  12. Resveratrol promotes expression of SIRT1 and StAR in rat ovarian granulosa cells: an implicative role of SIRT1 in the ovary

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    Morita Yoshihiro

    2012-02-01

    Full Text Available Abstract Background Resveratrol is a natural polyphenolic compound known for its beneficial effects on energy homeostasis, and it also has multiple properties, including anti-oxidant, anti-inflammatory, and anti-tumor activities. Recently, silent information regulator genes (Sirtuins have been identified as targets of resveratrol. Sirtuin 1 (SIRT1, originally found as an NAD+-dependent histone deacetylase, is a principal modulator of pathways downstream of calorie restriction, and the activation of SIRT1 ameliorates glucose homeostasis and insulin sensitivity. To date, the presence and physiological role of SIRT1 in the ovary are not known. Here we found that SIRT1 was localized in granulosa cells of the human ovary. Methods The physiological roles of resveratrol and SIRT1 in the ovary were analyzed. Immunohistochemistry was performed to localize the SIRT1 expression. SIRT1 protein expression of cultured cells and luteinized human granulosa cells was investigated by Western blot. Rat granulosa cells were obtained from diethylstilbestrol treated rats. The cells were treated with increasing doses of resveratrol, and subsequently harvested to determine mRNA levels and protein levels. Cell viability was tested by MTS assay. Cellular apoptosis was analyzed by caspase 3/7 activity test and Hoechst 33342 staining. Results SIRT1 protein was expressed in the human ovarian tissues and human luteinized granulosa cells. We demonstrated that resveratrol exhibited a potent concentration-dependent inhibition of rat granulosa cells viability. However, resveratrol-induced inhibition of rat granulosa cells viability is independent of apoptosis signal. Resveratrol increased mRNA levels of SIRT1, LH receptor, StAR, and P450 aromatase, while mRNA levels of FSH receptor remained unchanged. Western blot analysis was consistent with the results of quantitative real-time RT-PCR assay. In addition, progesterone secretion was induced by the treatment of resveratrol

  13. Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein

    DEFF Research Database (Denmark)

    Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro

    2011-01-01

    those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-¿B pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1...... cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching...

  14. [Rhein promotes the expression of SIRT1 in kidney tissues of type 2 diabetic rat].

    Science.gov (United States)

    Chen, Weidong; Chang, Baochao; Zhang, Yan; Yang, Ping; Liu, Lei

    2015-05-01

    To observe the effect of rhein on the expression of SIRT1(Sirtuin 1) in kidney of diabetic rats, and to explore the role of rhein in protecting rat kidney against diabetic nephropathy and possible mechanism. The type 2 diabetic rats were induced by high-glucose and high-fat diet combined with streptozotocin (35 mg/kg body mass). Seventy-five eight-week-old male SD rats were randomly divided into 6 groups: normal group, diabetic group, low-, medium- and high-dose (50, 100, 150 mg/kg) rhein treatment groups and 10 mg/kg pioglitazone treatment group. The rats were given corresponding substances intragastrically once a day. At the end of the 16th week, the fasting plasma glucose (FPG), fasting insulin (FINS), triglycerides (TG), total cholesterol (TC), serum creatinine (Scr) and 24 hours urine protein (24 h U-PRO) were determined. The renal hypertrophy index (KM/BM), insulin resistance index (HOMA-IR) were calculated. The pathological changes in renal tissues were examined by PAS staining under a light microscopy. The mean glomerular area (MGA) and mean glomerular volume (MGV) were measured by pathological image analysis system. Western blotting and real-time quantitative PCR were used to determine the expression of SIRT1 in renal tissues at protein and mRNA levels, respectively. The expression of SIRT1 was down-regulated in the kidney of diabetic rats. The levels of FPG, FINS, HOMA-IR, TG, TC, Scr, 24 h U-PRO, KM/BM, MGA and MGV significantly decreased and the histopathology of renal tissues were significantly improved in all treatment groups compared with diabetic group. The expression of SIRT1 mRNA and protein markedly increased in rhein treatment groups and pioglitazone treatment group compared with diabetic group. The indicators in high-dose rhein treatment group were improved more significantly than those in the other groups. Correlation analysis showed that the expression of SIRT1 was negatively correlated with 24 h U-PRO and MGV. The expression of SIRT1 was

  15. Effects of SIRT1 gene knock-out via activation of SREBP2 protein-mediated PI3K/AKT signaling on osteoarthritis in mice.

    Science.gov (United States)

    Yu, Fei; Zeng, Hui; Lei, Ming; Xiao, De-Ming; Li, Wei; Yuan, Hao; Lin, Jian-Jing

    2016-10-01

    This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1 +/+ control group (group A, n=6); SIRT1 +/+ osteoarthritis group (group B, n=6); SIRT1 -/- control group (group C, n=6); SIRT1 -/- osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1 -/- osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1 +/+ osteoarthritis group and SIRT1 -/- control group, SIRT1 protein expression was not obviously changed in the SIRT1 -/- osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (Pknock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.

  16. Expression of SIRT1 and oxidative stress in diabetic dry eye.

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    Liu, Hao; Sheng, Minjie; Liu, Yu; Wang, Peng; Chen, Yihui; Chen, Li; Wang, Weifang; Li, Bing

    2015-01-01

    To explore the expression of SIRT1 with oxidative stress and observe physiological and pathological changes in the corneas as well as the association between SIRT1 and oxidative stress of diabetic dry eyes in mice. Forty-eight C57BL/6Jdb/db mice at eight weeks of age were divided randomly into two groups: the diabetic dry eye group and the diabetic group. An additional forty-eight C57BL/6J mice at eight weeks of age were divided randomly into two groups: the dry eye group and the control group. Every mouse in the dry eye groups (diabetic and normal) was injected with scopolamine hydrobromide three times daily, combined with low humidity to establish a dry eye model. After the intervention, phenol red cotton string tests and corneal fluorescein staining were performed. In addition, HE staining and immunofluorescence were done. Expression of SIRT1 in the cornea was examined by real-time PCR and Western Blot and expression of FOXO3 and MnSOD proteins was detected by Western Blot. At one, four, and eight weeks post intervention, all of the groups except the controls showed significant decreases in tear production and increases in the corneal fluorescein stain (Pdry eye group had the least tear production and the highest corneal fluorescein stain score (Pdry eye group. In the 1(st) and 4(th) week, the expression of SIRT1, FOXO3, and MnSOD were significantly higher in the diabetic DE and DM groups but lower in the DE group compared to the controls (Pdry eye, tear production declined markedly coupled with seriously wounded corneal epithelium. Oxidative stress in the cornea was enhanced significantly and the expression of SIRT1 was decreased.

  17. Expression of SIRT1 in the ovaries of rats with polycystic ovary syndrome before and after therapeutic intervention with exenatide.

    Science.gov (United States)

    Tao, Xin; Zhang, Xiao; Ge, Shu-Qi; Zhang, Er-Hong; Zhang, Bin

    2015-01-01

    To investigate the expression of silent information regulator 1 (SIRT1) in rats with polycystic ovary syndrome (PCOS) and its alteration after exenatide treatment. PCOS rat model was established by dehydroepiandrosterone induction. The animals were randomly divided into exenatide treatment group (EX group, n = 10), metformin treatment group (MF group, n = 10), PCOS group (PCOS group, n = 9) and normal control group (NC group, n = 10). Histological changes of the ovarian tissues were examined by HE staining. SIRT1 expression in the ovarian tissue was detected by RT-PCR and immunohistochemistry. Rats in the PCOS group lost their estrous cycle. Histological observation of the ovary showed saccular dilatation of the follicle, decreased number of corpora lutea, fewer layers of granulosa cells aligned loosely, and thickened layer of theca cells. The changes in reproductive hormones and the development of insulin resistance suggested the successful establishment of the animal models. Immunohistochemistry and Q-PCR detected the mRNA and protein expressions of SIRT1 in the ovary tissues of rats in the normal control group. The SIRT1 expression was significantly lower in PCOS group than in control group (P < 0.05); after drug intervention, the SIRT1 expression significantly increased in EX and MF groups (compared with the PCOS group), whereas no significant difference was noted between the EX group and MF group. The SIRT1 expression in the ovary tissue decreases in PCOS rats (compare with the normal rats) but can be up-regulated after Ex or MF treatment. These drugs may affect the process and development of PCOS by regulating the SIRT1 expression. Exenatide may be therapeutic for PCOS by up-regulating the SITR1 expression.

  18. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of β-catenin

    International Nuclear Information System (INIS)

    Cho, Il-Rae; Koh, Sang Seok; Malilas, Waraporn; Srisuttee, Ratakorn; Moon, Jeong; Choi, Young-Whan; Horio, Yoshiyuki; Oh, Sangtaek; Chung, Young-Hwa

    2012-01-01

    Highlights: ► SIRT1 inhibits protein levels of β-catenin and its transcriptional activity. ► Nuclear localization of SIRT1 is not required for the decrease of β-catenin expression. ► SIRT1-mediated degradation of β-catenin is not required for GSK-3β and Siah-1 but for proteosome. ► SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of β-catenin, we postulated that β-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target β-catenin in a colon cancer model, suppresses β-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of β-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced β-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of β-catenin. Treatment with MG132, a proteasomal inhibitor, restored β-catenin protein levels, suggesting that SIRT1-mediated degradation of β-catenin requires proteasomal activity. It was reported that inhibition of GSK-3β or Siah-1 stabilizes β-catenin in colon cancer cells, but suppression of GSK-3β or Siah-1 using siRNA in the presence of resveratrol instead diminished β-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3β and Siah-1 are not involved in SIRT1-mediated degradation of β-catenin in the cells. Finally, activation of SIRT1 inhibited the proliferation of Panc-PAUF cells by down-regulation of cyclin-D1, a target

  19. SIRT1 inhibits proliferation of pancreatic cancer cells expressing pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by suppression of {beta}-catenin

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Il-Rae [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Koh, Sang Seok [Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology, Daejeon 305-333 (Korea, Republic of); Malilas, Waraporn; Srisuttee, Ratakorn; Moon, Jeong [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of); Choi, Young-Whan [Department of Horticultural Bioscience, Pusan National University, Miryang 627-706 (Korea, Republic of); Horio, Yoshiyuki [Department of Pharmacology, Sapporo Medical University, Sapporo 060-8556 (Japan); Oh, Sangtaek [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of); Chung, Young-Hwa, E-mail: younghc@pusan.ac.kr [WCU, Department of Cogno-Mechatronics Engineering, Pusan National University, Busan 609-735 (Korea, Republic of)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer SIRT1 inhibits protein levels of {beta}-catenin and its transcriptional activity. Black-Right-Pointing-Pointer Nuclear localization of SIRT1 is not required for the decrease of {beta}-catenin expression. Black-Right-Pointing-Pointer SIRT1-mediated degradation of {beta}-catenin is not required for GSK-3{beta} and Siah-1 but for proteosome. Black-Right-Pointing-Pointer SIRT1 activation inhibits proliferation of pancreatic cancer cells expressing PAUF. -- Abstract: Because we found in a recent study that pancreatic adenocarcinoma up-regulated factor (PAUF), a novel oncogene, induces a rapid proliferation of pancreatic cells by up-regulation of {beta}-catenin, we postulated that {beta}-catenin might be a target molecule for pancreatic cancer treatment. We thus speculated whether SIRT1, known to target {beta}-catenin in a colon cancer model, suppresses {beta}-catenin in those pancreatic cancer cells that express PAUF (Panc-PAUF). We further evaluated whether such suppression would lead to inhibition of the proliferation of these cells. The ectopic expression of either SIRT1 or resveratrol (an activator of SIRT1) suppressed levels of {beta}-catenin protein and its transcriptional activity in Panc-PAUF cells. Conversely, suppression of SIRT1 expression by siRNA enhanced {beta}-catenin expression and transcriptional activity. SIRT1 mutant analysis showed that nuclear localization of SIRT1 is not required for reduction of {beta}-catenin. Treatment with MG132, a proteasomal inhibitor, restored {beta}-catenin protein levels, suggesting that SIRT1-mediated degradation of {beta}-catenin requires proteasomal activity. It was reported that inhibition of GSK-3{beta} or Siah-1 stabilizes {beta}-catenin in colon cancer cells, but suppression of GSK-3{beta} or Siah-1 using siRNA in the presence of resveratrol instead diminished {beta}-catenin protein levels in Panc-PAUF cells. This suggests that GSK-3{beta} and Siah-1 are not involved in SIRT1

  20. Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation.

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    Kalous, Kelsey S; Wynia-Smith, Sarah L; Olp, Michael D; Smith, Brian C

    2016-12-02

    The sirtuin family of proteins catalyze the NAD + -dependent deacylation of acyl-lysine residues. Humans encode seven sirtuins (Sirt1-7), and recent studies have suggested that post-translational modification of Sirt1 by cysteine S-nitrosation correlates with increased acetylation of Sirt1 deacetylase substrates. However, the mechanism of Sirt1 inhibition by S-nitrosation was unknown. Here, we show that Sirt1 is transnitrosated and inhibited by the physiologically relevant nitrosothiol S-nitrosoglutathione. Steady-state kinetic analyses and binding assays were consistent with Sirt1 S-nitrosation inhibiting binding of both the NAD + and acetyl-lysine substrates. Sirt1 S-nitrosation correlated with Zn 2+ release from the conserved sirtuin Zn 2+ -tetrathiolate and a loss of α-helical structure without overall thermal destabilization of the enzyme. Molecular dynamics simulations suggested that Zn 2+ loss due to Sirt1 S-nitrosation results in repositioning of the tetrathiolate subdomain away from the rest of the catalytic domain, thereby disrupting the NAD + and acetyl-lysine-binding sites. Sirt1 S-nitrosation was reversed upon exposure to the thiol-based reducing agents, including physiologically relevant concentrations of the cellular reducing agent glutathione. Reversal of S-nitrosation resulted in full restoration of Sirt1 activity only in the presence of Zn 2+ , consistent with S-nitrosation of the Zn 2+ -tetrathiolate as the primary source of Sirt1 inhibition upon S-nitrosoglutathione treatment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. SIRT1 attenuates palmitate-induced endoplasmic reticulum stress and insulin resistance in HepG2 cells via induction of oxygen-regulated protein 150

    Science.gov (United States)

    Jung, T.W.; Lee, K.T.; Lee, M.W.; Ka, K.H.

    2012-01-01

    Endoplasmic reticulum (ER) stress has been implicated in the pathology of type 2 diabetes mellitus (T2DM). Although SIRT1 has a therapeutic effect on T2DM, the mechanisms by which SIRT1 ameliorates insulin resistance (IR) remain unclear. In this study, we investigated the impact of SIRT1 on palmitate-induced ER stress in HepG2 cells and its underlying signal pathway. Treatment with resveratrol, a SIRT1 activator significantly inhibited palmitate-induced ER stress, leading to the protection against palmitate-induced ER stress and insulin resistance. Resveratrol and SIRT1 overexpression induced the expression of oxygen-regulated protein (ORP) 150 in HepG2 cells. Forkhead box O1 (FOXO1) was involved in the regulation of ORP150 expression because suppression of FOXO1 inhibited the induction of ORP150 by SIRT1. Our results indicate a novel mechanism by which SIRT1 regulates ER stress by overexpression of ORP150, and suggest that SIRT1 ameliorates palmitate-induced insulin resistance in HepG2 cells via regulation of ER stress.

  2. SIRT1 expression is associated with poor prognosis of lung adenocarcinoma

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    Li C

    2015-04-01

    Full Text Available Chong Li,1,2,* Lingling Wang,3,* Liang Zheng,4 Xianghong Zhan,4 Bin Xu,1,2 Jingting Jiang,1,2 Changping Wu1,2 1Department of Tumor Biological Treatment, the Third Affiliated Hospital, Soochow University, Changzhou, 2Cancer Immunotherapy Engineering Research Center of Jiangsu Province, Changzhou, 3Department of Medical Education, Jinling Hospital, Medical School of Nanjing University, Nanjing, 4Department of Thoracic Surgery, the Third Affiliated Hospital, Soochow University, Changzhou, Jiangsu, People’s Republic of China *These authors contributed equally to this work Abstract: Several studies have reported that the overexpression of Sirtuin 1 (SIRT1 was associated with poor prognosis in various human cancers. However, little is known regarding the prognostic value of SIRT1 in lung adenocarcinoma. Therefore, the aim of this study is to evaluate the role of SIRT1 in the prognosis of lung adenocarcinoma patients. Using a tissue microarray, we detected SIRT1 expression by immunohistochemistry in lung adenocarcinoma tissue, as well as in corresponding noncancerous tissues (NCTs. A high expression level of SIRT1 was observed in 74.7% (56/75 of patients with lung adenocarcinoma and 6.7% (5/75 of NCTs (P<0.001. SIRT1 expression was significantly associated with high pathological stage. Importantly, we found that SIRT1 expression was associated with worse overall survival in these lung adenocarcinoma patients (67.0 months vs 104.5 months; P=0.005. In addition, anaplastic lymphoma kinase, epidermal growth factor receptor, vascular endothelial growth factor (VEGF, and Survivin expression were evaluated by fluorescent in situ hybridization or immunohistochemistry, respectively. We found that VEGF and Survivin were both highly expressed in the lung adenocarcinoma tissues, as compared to NCTs. Moreover, the SIRT1 and VEGF expression statuses were significantly positively correlated (r=0.238, P=0.039, while SIRT1 and Survivin expression status were not

  3. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

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    Kim, Sang Chon; Kim, Yoo Hoon; Son, Sung Wook; Moon, Eun-Yi; Pyo, Suhkneung; Um, Sung Hee

    2015-11-27

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. SIRT1 promotes N-Myc oncogenesis through a positive feedback loop involving the effects of MKP3 and ERK on N-Myc protein stability.

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    Glenn M Marshall

    2011-06-01

    Full Text Available The N-Myc oncoprotein is a critical factor in neuroblastoma tumorigenesis which requires additional mechanisms converting a low-level to a high-level N-Myc expression. N-Myc protein is stabilized when phosphorylated at Serine 62 by phosphorylated ERK protein. Here we describe a novel positive feedback loop whereby N-Myc directly induced the transcription of the class III histone deacetylase SIRT1, which in turn increased N-Myc protein stability. SIRT1 binds to Myc Box I domain of N-Myc protein to form a novel transcriptional repressor complex at gene promoter of mitogen-activated protein kinase phosphatase 3 (MKP3, leading to transcriptional repression of MKP3, ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, and N-Myc protein stabilization. Importantly, SIRT1 was up-regulated, MKP3 down-regulated, in pre-cancerous cells, and preventative treatment with the SIRT1 inhibitor Cambinol reduced tumorigenesis in TH-MYCN transgenic mice. Our data demonstrate the important roles of SIRT1 in N-Myc oncogenesis and SIRT1 inhibitors in the prevention and therapy of N-Myc-induced neuroblastoma.

  5. The Impact of Ramadan Fasting on SIRT1 mRNA Expression in Peripheral Blood Mononuclear Cells

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    Mostafa Haji Molahoseini

    2016-11-01

    Full Text Available Background:The aim of this study was to evaluate the effect of Ramadan fasting on SIRT1 mRNA expression in healthy men.Islamic Ramadan fasting is a holy religious ceremony that has many spiritual benefits. Additionally, it can be considered as the equivalent of calorie restriction that may affect physical health. The results of previous studies revealed that calorie restriction increases the lifespan in laboratory rodents via increasing the expression of a histone deacetylase named SIRT1. Additionally, SIRT1 is known for its anti-inflammatory properties. Materials and Methods: Overall, 43 men volunteered for participating in this one-group before and after (self-controlled study. Two mL blood samples were taken prior to fasting and at the end of the 30th day of fasting. Routine biochemical tests and SIRT1 mRNA expression analysis were performed. Results: Cholesterol and low-density lipoproteins increase, however, high-density lipoproteins level decreased after Ramadan fasting. The analysis of real-time PCR results revealed that SIRT1 mRNA expression in human peripheral blood mononuclear cells increased 4.63 fold in fasting state in comparison with non-fasting state. Conclusion: Ramadan fasting has a significant effect on SIRT1 gene expression. Considering the immunosuppressive and anti-inflammatory properties of SIRT1, further studies are needed to evaluate the effects of SIRT1 up-regulation on the autoimmune and inflammatory diseases during Ramadan fasting.

  6. Prognostic role of sirt1 in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Song, S.; Zhang, H.

    2014-01-01

    To determine the clinical significance of silent mating type information regulation 2 homolog 1 (SIRT1) expression in Hepatocellular Carcinoma (HCC) and its association with P53 and Yes-associated protein 2 (YAP2) expression. Study Design: Observational study. Place and Duration of Study: Department of General Surgery, China-Japan Union Hospital of Jilin University, Changchun, China, from January 2000 to January 2010. Methodology: Tissue microarray technique and immunohistochemistry were conducted to detect the expression of SIRT1, P53 and YAP2 proteins in 300 self-paired HCC samples. Associations with clinicopathologic manifestations were analyzed, overall survival analysis and multivariate analysis were performed. Results: By tissue microarray technique and immunohistochemistry on 300 self-paired HCC samples, it was found that SIRT1, P53 and YAP2 were significantly overexpressed in HCC tumor tissues compared with adjacent non-tumor tissues. SIRT1 immunostaining localized both in the nucleus (145/300, 48.3%) and the cytoplasm (70/300, 23.3%), and the overexpression of nuclear SIRT1 was positively related to the overexpression of P53 and YAP2. Survival analysis showed that nuclear SIRT1, P53 and YAP2 overexpression predicted poor overall survival while cytoplasmic SIRT1 overexpression predicted longer overall survival. Multivariate analysis showed nuclear SIRT1 and P53 overexpression as independent tumor promoters while cytoplasmic SIRT1 overexpression as an independent tumor suppressor. Conclusion: SIRT1 was overexpressed in HCC and the expression was positively related to P53 and YAP2 expression. As the nuclear SIRT1 functions as a tumor promoter and cytoplasmic SIRT1 functions as a tumor suppressor, the role of SIRT1 in HCC should be reconsidered. (author)

  7. The Impact of Ramadan Fasting on SIRT1 mRNA Expression in Peripheral Blood Mononuclear Cells

    OpenAIRE

    Mostafa Haji Molahoseini; kanaan Gorjipour; Farshid Yeganeh

    2016-01-01

    Background:The aim of this study was to evaluate the effect of Ramadan fasting on SIRT1 mRNA expression in healthy men.Islamic Ramadan fasting is a holy religious ceremony that has many spiritual benefits. Additionally, it can be considered as the equivalent of calorie restriction that may affect physical health. The results of previous studies revealed that calorie restriction increases the lifespan in laboratory rodents via increasing the expression of a histone deacetylase named SIRT1. Add...

  8. Epigenetic-based combinatorial resveratrol and pterostilbene alters DNA damage response by affecting SIRT1 and DNMT enzyme expression, including SIRT1-dependent γ-H2AX and telomerase regulation in triple-negative breast cancer

    International Nuclear Information System (INIS)

    Kala, Rishabh; Shah, Harsh N.; Martin, Samantha L.; Tollefsbol, Trygve O.

    2015-01-01

    Nutrition is believed to be a primary contributor in regulating gene expression by affecting epigenetic pathways such as DNA methylation and histone modification. Resveratrol and pterostilbene are phytoalexins produced by plants as part of their defense system. These two bioactive compounds when used alone have been shown to alter genetic and epigenetic profiles of tumor cells, but the concentrations employed in various studies often far exceed physiologically achievable doses. Triple-negative breast cancer (TNBC) is an often fatal condition that may be prevented or treated through novel dietary-based approaches. HCC1806 and MDA-MB-157 breast cancer cells were used as TNBC cell lines in this study. MCF10A cells were used as control breast epithelial cells to determine the safety of this dietary regimen. CompuSyn software was used to determine the combination index (CI) for drug combinations. Combinatorial resveratrol and pterostilbene administered at close to physiologically relevant doses resulted in synergistic (CI <1) growth inhibition of TNBCs. SIRT1, a type III histone deacetylase (HDAC), was down-regulated in response to this combinatorial treatment. We further explored the effects of this novel combinatorial approach on DNA damage response by monitoring γ-H2AX and telomerase expression. With combination of these two compounds there was a significant decrease in these two proteins which might further resulted in significant growth inhibition, apoptosis and cell cycle arrest in HCC1806 and MDA-MB-157 breast cancer cells, while there was no significant effect on cellular viability, colony forming potential, morphology or apoptosis in control MCF10A breast epithelial cells. SIRT1 knockdown reproduced the effects of combinatorial resveratrol and pterostilbene-induced SIRT1 down-regulation through inhibition of both telomerase activity and γ-H2AX expression in HCC1806 breast cancer cells. As a part of the repair mechanisms and role of SIRT1 in recruiting DNMTs

  9. Frizzled 7 expression is positively regulated by SIRT1 and β-catenin in breast cancer cells.

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    Glenn E Simmons

    Full Text Available The Wnt signaling pathway is often chronically activated in diverse human tumors, and the Frizzled (FZD family of receptors for Wnt ligands, are central to propagating oncogenic signals in a β-catenin-dependent and independent manner. SIRT1 is a class III histone deacetylase (HDAC that deacetylates histone and non-histone proteins to regulate gene transcription and protein function. We previously demonstrated that SIRT1 loss of function led to a significant decrease in the levels of Dishevelled (Dvl proteins. To further explore this connection between the sirtuins and components of the Wnt pathway, we analyzed sirtuin-mediated regulation of FZD proteins. Here we explore the contribution of sirtuin deacetylases in promoting constitutive Wnt pathway activation in breast cancer cells. We demonstrate that the use of small molecule inhibitors of SIRT1 and SIRT2, and siRNA specific to SIRT1, all reduce the levels of FZD7 mRNA. We further demonstrate that pharmacologic inhibition of SIRT1/2 causes a marked reduction in FZD7 protein levels. Additionally, we show that β-catenin and c-Jun occupy the 7 kb region upstream of the transcription start site of the FZD7 gene, and SIRT1 inhibition leads to a reduction in the occupancy of both β-catenin and c-Jun at points along this region. This work uncovers a new mechanism for the regulation of FZD7 and provides a critical new link between the sirtuins and FZD7, one of the earliest nodal points from which oncogenic Wnt signaling emanates. This study shows that inhibition of specific sirtuins may provide a unique strategy for inhibiting the constitutively active Wnt pathway at the level of the receptor.

  10. Inhibition of transcriptional activity of c-JUN by SIRT1

    International Nuclear Information System (INIS)

    Gao Zhanguo; Ye Jianping

    2008-01-01

    c-JUN is a major component of heterodimer transcription factor AP-1 (Activator Protein-1) that activates gene transcription in cell proliferation, inflammation and stress responses. SIRT1 (Sirtuin 1) is a histone deacetylase that controls gene transcription through modification of chromatin structure. However, it is not clear if SIRT1 regulates c-JUN activity in the control of gene transcription. Here, we show that SIRT1 associated with c-JUN in co-immunoprecipitation of whole cell lysate, and inhibited the transcriptional activity of c-JUN in the mammalian two hybridization system. SIRT1 was found in the AP-1 response element in the matrix metalloproteinase-9 (MMP9) promoter DNA leading to inhibition of histone 3 acetylation as shown in a ChIP assay. The SIRT1 signal was reduced by the AP-1 activator PMA, and induced by the SIRT1 activator Resveratrol in the promoter DNA. SIRT1-mediaetd inhibition of AP-1 was demonstrated in the MMP9 gene expression at the gene promoter, mRNA and protein levels. In mouse embryonic fibroblast (MEF) with SIRT1 deficiency (SIRT1 -/- ), mRNA and protein of MMP9 were increased in the basal condition, and the inhibitory activity of Resveratrol was significantly attenuated. Glucose-induced MMP9 expression was also inhibited by SIRT1 in response to Resveratrol. These data consistently suggest that SIRT1 directly inhibits the transcriptional activity of AP-1 by targeting c-JUN

  11. Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

    Science.gov (United States)

    Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

    2016-11-01

    Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

  12. SIRT1 and FOXO1 mRNA expression in PBMC correlates to physical activity in COPD patients

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    Taka C

    2017-11-01

    Full Text Available Chihiro Taka, Ryuji Hayashi, Kazuki Shimokawa, Kotaro Tokui, Seisuke Okazawa, Kenta Kambara, Minehiko Inomata, Toru Yamada, Shoko Matsui, Kazuyuki Tobe First Department of Internal Medicine, Faculty of Medicine, University of Toyama, Sugitani, Toyama, Toyama, Japan Background: Physical activity (PA is considered as one of the most important prognostic predictors in chronic obstructive pulmonary disease (COPD patients. Longevity gene, SIRT1, is reported to be involved in the pathogenesis of COPD by regulating the signaling pathways of oxidative stress, inflammation, and aging. We hypothesize that SIRT1 and related genes are also associated with the benefits of PA in COPD patients.Methods: Eighteen COPD outpatients were enrolled in this study, and their PA level was assessed with an accelerometer. We assessed the SIRT1 and related genes mRNA expression levels in the peripheral blood mononuclear cells (PBMCs of the subjects. We carried out respiratory function testing, blood gas analysis, the 6-minute walk test, and measurement of the cross-sectional area of the erector spinae muscles (ESMCSA by chest computed tomography. We analyzed the association of PA with the results of each of the examinations.Results: The mean age was 72±9 years, and the mean forced expiratory volume in 1 second was 1.4±0.56 L (52%±19% predicted. Our findings revealed a correlation between the daily PA and ESMCSA. The SIRT1 and Forkhead box O (FOXO1 mRNA expression levels in PBMCs were positively correlated with moderate-PA time (r=0.60, p=0.008 for SIRT1 and r=0.59, p=0.01 for FOXO1. Keywords: COPD, accelerometer, mRNA, walking, sedentary, moderate

  13. SIRT1 overexpression is an independent prognosticator for patients with esophageal squamous cell carcinoma.

    Science.gov (United States)

    Ma, Ming-Chun; Chiu, Tai-Jan; Lu, Hung-I; Huang, Wan-Ting; Lo, Chien-Ming; Tien, Wan-Yu; Lan, Ya-Chun; Chen, Yen-Yang; Chen, Chang-Han; Li, Shau-Hsuan

    2018-04-10

    Sirtuin 1 (SIRT1) regulates DNA repair and metabolism by deacetylating target proteins. SIRT1 may be oncogenic because its overexpression has been detected in many cancers. The aim of the present study was to clarify the prognostic role of SIRT1 in patients with esophageal squamous cell carcinoma (ESCC) and evaluate the effect of SIRT1 inhibitor in vitro. The expression of SIRT1 was evaluated immunohistochemically in 155 surgically resected ESCC and the staining results were evaluated semiquantitatively by the Immunoreactive Scoring System. The clinical features and treatment outcome were analyzed. The effect of SIRT1 inhibitor, SIRT 1 inhibitor IV, (S)-35, was investigated in vitro on ESCC cell lines. The expression of SIRT1 on ESCC did not correlate with age, gender, tumor location, stage, T classification, N classification, surgical margin or histology. Univariate analysis showed that SIRT1 overexpression was associated with inferior overall survival (P = 0.004) and disease-free survival (P = 0.004). In multivariate comparison, SIRT1 overexpression remained independently associated with worse overall survival (P = 0.009, hazard ratio = 1.776) and disease-free survival (P = 0.017, hazard ratio = 1.642). In cell lines, SIRT1 inhibitor inhibited ESCC growth. Our study suggests that SIRT1 overexpression is an independent prognosticator for patients with ESCC and the SIRT1 inhibitor suppressed cell proliferation of ESCC cell lines. Our findings suggest that inhibition of SIRT1 signaling may be a promising novel target for ESCC.

  14. Effect of telmisartan and insulin on endoplasmic reticulum stress and the expression of Sirt1 in testis of type 1 diabetic rats

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    Yan HUANG

    2016-10-01

    Full Text Available Objective  To observe the regulation effect of insulin and telmisartan on the endoplasmic reticulum stress (ERS and the expression of silent information regulator 1 (Sirt1 in the testes of type 1 diabetic rats, and explore the protective effect and mechanism of insulin and telmisartan on the testicular tissue of diabetic rats. Methods  Male Sprague-Dawley (SD rats were randomly divided into normal control group (group A, n=8, diabetic control group (group B, n=8, diabetic treated with insulin group (group C, n=8 and diabetic treated with telmisartan group (group D, n=8. Diabetic rats were induced by a single intraperitoneal injection of streptozotocin. Rats in group C were subcutaneously injected with protamine-zinc insulin once a day, and in group D were given telmisartan once a day by gavage. Only at the end of the eighth week can specimens be taken. And then some tasks were performed such as calculating the testicular weight and testis index, accounting the quantity and activity rate of sperm, detecting the testosterone levels and mRNA expression levels of CCAAT/enhancer binding protein homologous protein (CHOP, glucose regulated protein (GRP-78, cysteine aspartic acid specific protease 12 (caspase-12 and Sirt1. Results  The levels of testosterone, testes weight, sperm counts and motility significantly decreased in group B than in group A (P0.05. The mRNA expression levels of testicular CHOP, GRP-78 and caspase-12 significantly decreased (P0.05. Conclusion  Insulin and telmisartan may play a protective role on testicular tissues by down-regulating the expression of endoplasmic reticulum stress and up-regulating the expression of Sirt1. DOI: 10.11855/j.issn.0577-7402.2016.10.03

  15. Phosphorylation regulates SIRT1 function.

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    Tsutomu Sasaki

    Full Text Available BACKGROUND: SIR2 is an NAD(+-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540 disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify

  16. PRMT1 and PRMT4 Regulate Oxidative Stress-Induced Retinal Pigment Epithelial Cell Damage in SIRT1-Dependent and SIRT1-Independent Manners

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    Dong-Il Kim

    2015-01-01

    Full Text Available Oxidative stress-induced retinal pigment epithelial (RPE cell damage is involved in the progression of diabetic retinopathy. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs has emerged as an important histone modification involved in diverse diseases. Sirtuin (SIRT1 is a protein deacetylase implicated in the onset of metabolic diseases. Therefore, we examined the roles of type I PRMTs and their relationship with SIRT1 in human RPE cells under H2O2-induced oxidative stress. H2O2 treatment increased PRMT1 and PRMT4 expression but decreased SIRT1 expression. Similar to H2O2 treatment, PRMT1 or PRMT4 overexpression increased RPE cell damage. Moreover, the H2O2-induced RPE cell damage was attenuated by PRMT1 or PRMT4 knockdown and SIRT1 overexpression. In this study, we revealed that SIRT1 expression was regulated by PRMT1 but not by PRMT4. Finally, we found that PRMT1 and PRMT4 expression is increased in the RPE layer of streptozotocin-treated rats. Taken together, we demonstrated that oxidative stress induces apoptosis both via PRMT1 in a SIRT1-dependent manner and via PRMT4 in a SIRT1-independent manner. The inhibition of the expression of type I PRMTs, especially PRMT1 and PRMT4, and increased SIRT1 could be therapeutic approaches for diabetic retinopathy.

  17. SIRT1 Functions as an Important Regulator of Estrogen-Mediated Cardiomyocyte Protection in Angiotensin II-Induced Heart Hypertrophy

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    Tao Shen

    2014-01-01

    Full Text Available Background. Sirtuin 1 (SIRT1 is a member of the sirtuin family, which could activate cell survival machinery and has been shown to be protective in regulation of heart function. Here, we determined the mechanism by which SIRT1 regulates Angiotensin II- (AngII- induced cardiac hypertrophy and injury in vivo and in vitro. Methods. We analyzed SIRT1 expression in the hearts of control and AngII-induced mouse hypertrophy. Female C57BL/6 mice were ovariectomized and pretreated with 17β-estradiol to measure SIRT1 expression. Protein synthesis, cardiomyocyte surface area analysis, qRT-PCR, TUNEL staining, and Western blot were performed on AngII-induced mouse heart hypertrophy samples and cultured neonatal rat ventricular myocytes (NRVMs to investigate the function of SIRT1. Results. SIRT1 expression was slightly upregulated in AngII-induced mouse heart hypertrophy in vivo and in vitro, accompanied by elevated cardiomyocyte apoptosis. SIRT1 overexpression relieves AngII-induced cardiomyocyte hypertrophy and apoptosis. 17β-Estradiol was able to protect cardiomyocytes from AngII-induced injury with a profound upregulation of SIRT1 and activation of AMPK. Moreover, estrogen receptor inhibitor ICI 182,780 and SIRT1 inhibitor niacinamide could block SIRT1’s protective effect. Conclusions. These results indicate that SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection during AngII-induced heart hypertrophy and injury.

  18. Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

    International Nuclear Information System (INIS)

    Chang, C.-J.; Hsu, C.-C.; Yung, M.-C.; Chen, K.-Y.; Tzao Ching; Wu, W.-F.; Chou, H.-Y.; Lee, Y.-Y.; Lu, K.-H.; Chiou, S.-H.; Ma, H.-I

    2009-01-01

    CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133 + ) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133 - cells. To evaluate the role of SirT1 in GBM-CD133 + , we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133 + . Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133 + to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133 + . Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133 + mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.

  19. SIRT1 Regulates the Chemoresistance and Invasiveness of Ovarian Carcinoma Cells

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    David Hamisi Mvunta

    2017-08-01

    Full Text Available BACKGROUND: SIRT1 is a longevity gene that forestalls aging and age-related diseases including cancer, and has recently attracted widespread attention due to its overexpression in some cancers. We previously identified the overexpression of SIRT1 in ovarian carcinoma (OvCa as a poor prognostic factor. However, mechanistic insights into the function of SIRT1 in OvCa have yet to be elucidated. METHODS: Quantitative real-time reverse PCR (qRT-PCR and Western blotting were employed to examine the expression of SIRT1 in a panel of human OvCa cell lines. si-RNA or sh-RNA and cDNA technologies were utilized to knockdown or overexpress SIRT1, respectively. The effects of SIRT1 on proliferation and chemoresistance were examined using a WST-1 assay, and the underlying mechanisms were confirmed using an apoptotic assay, and the quantification of glutathione (GSH, and reactive oxygen species (ROS. The aggressiveness of SIRT1 was analyzed using in vitro invasion and migration assays. RESULTS: SIRT1 was more strongly expressed in OvCa cell lines than in the immortalized ovarian epithelium at the gene and protein levels. Stress up-regulated the expression of SIRT1 in dose- and time-dependent manners. SIRT1 significantly enhanced the proliferation (P < .05, chemoresistance (P < .05, and aggressiveness of OvCa cells by up-regulating multiple antioxidant pathways to inhibit oxidative stress. Further study into the overexpression of SIRT1 demonstrated the up-regulation of several stemness-associated genes and enrichment of CD44v9 via an as-yet-unidentified pathway. CONCLUSIONS: Our results suggest that SIRT1 plays a role in the acquisition of aggressiveness and chemoresistance by OvCa, and has potential as a therapeutic target for OvCa.

  20. SIRT1 induces resistance to apoptosis in human granulosa cells by activating the ERK pathway and inhibiting NF-κB signaling with anti-inflammatory functions.

    Science.gov (United States)

    Han, Ying; Luo, Haining; Wang, Hui; Cai, Jun; Zhang, Yunshan

    2017-10-01

    SIRT1, a member of the sirtuin family, has recently emerged as a vital molecule in controlling ovarian function. The aims of the present study were to investigate SIRT1 expression and analyze SIRT1-mediated apoptosis in human granulosa cells (GCs). Human ovarian tissues were subjected to immunohistochemistry for localization of SIRT1 expression. SIRT1 knockdown in a human ovarian GC tumor line (COV434) was achieved by small interfering RNA, and the relationship between apoptosis and SIRT1 was assessed by quantitative reverse transcription polymerase chain reaction and western blotting. We further detected SIRT1 expression in human luteinized GCs. Associations among SIRT1 knockdown, SIRT1 stimulation (resveratrol) and expression of ERK1/2 and apoptotic regulatory proteins were analyzed in cell lines and luteinized GCs. Resveratrol downregulated the levels of nuclear factor (NF)-κB/p65, but this inhibitory effect was attenuated by suppressing SIRT1 activity. The NF-κB/p65 inhibitor pyrrolidine dithiocarbamate achieved similar anti-apoptosis effects. These results suggest that SIRT1 might play an anti-apoptotic role in apoptosis processes in GCs, possibly by sensing and regulating the ERK1/2 pathway, which has important clinical implications. Thus, our study provides a mechanistic link, whereby activation of SIRT1 function might help to sustain human reproduction by maintaining GCs as well as oocytes, offering a novel approach for developing a new class of therapeutic anti-inflammatory agents.

  1. Nicotinamide phosphoribosyltransferase delays cellular senescence by upregulating SIRT1 activity and antioxidant gene expression in mouse cells.

    Science.gov (United States)

    Khaidizar, Fiqri D; Nakahata, Yasukazu; Kume, Akira; Sumizawa, Kyosuke; Kohno, Kenji; Matsui, Takaaki; Bessho, Yasumasa

    2017-12-01

    Senescent cells accumulate in tissues of aged animals and deteriorate tissue functions. The elimination of senescent cells from aged mice not only attenuates progression of already established age-related disorders, but also extends median lifespan. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD + salvage pathway, has shown a protective effect on cellular senescence of human primary cells. However, it still remains unclear how NAMPT has a protective impact on aging in vitro and in vivo. In this study, we found that primary mouse embryonic fibroblast (MEF) cells undergo progressive decline of NAMPT and NAD + contents during serial passaging before becoming senescent. Furthermore, we showed that constitutive Nampt over-expression increases cellular NAD + content and delays cellular senescence of MEF cells in vitro. We further found that constitutive Nampt over-expression increases SIRT1 activity, increases the expression of antioxidant genes, superoxide dismutase 2 and catalase and promotes resistance against oxidative stress. These findings suggest that Nampt over-expression in MEF cells delays cellular senescence by the mitigation of oxidative stress via the upregulation of superoxide dismutase 2 and catalase gene expressions by SIRT1 activation. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  2. Physiological and pathophysiological functions of SIRT1.

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    Wojcik, M; Mac-Marcjanek, K; Wozniak, L A

    2009-03-01

    The human SIRT1 is a nuclear enzyme from the class III histone deacetylases (HDACs) which is widely distributed in mammalian tissues. A variety of SIRT1 substrates hints that this protein is involved in the regulation of diverse biological processes, including cell survival, apoptosis, gluconeogenesis, adipogenesis, lipolysis, stress resistance, muscle differentiation, and insulin secretion. This review emphasizes catalytic properties of SIRT1 and its role in apoptosis, insulin pathway, and neuron survival.

  3. Silent information regulator 1 (SIRT1) ameliorates liver fibrosis via promoting activated stellate cell apoptosis and reversion

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yuting, E-mail: wuyuting1302@sina.com; Liu, Xuejiao; Zhou, Qun; Huang, Cheng; Meng, Xiaoming; Xu, Fengyun; Li, Jun, E-mail: lj@ahmu.edu.cn

    2015-12-01

    SIRT1 (silent information regulator 1), a conserved NAD +-dependent histone deacetylase, is closely related with various biological processes. Moreover, the important role of SIRT1 in alcoholic liver disease, nonalcoholic fatty liver and HCC had been widely reported. Recently, a novel role of SIRT1 was uncovered in organ fibrosis diseases. Here, we investigated the inhibitory effect of SIRT1 in liver fibrogenesis. SIRT1 protein was dramatically decreased in CCl4-treated mice livers. Stimulation of LX-2 cells with TGF-β1 also resulted in a significant suppression of SIRT1 protein. Nevertheless, TGF-β1-induced LX-2 cell activation was inhibited by SIRT1 plasmid, and this was accompanied by up-regulation of cell apoptosis-related proteins. Overexpression of SIRT1 also attenuated TGF-β1-induced expression of myofibroblast markers α-SMA and COL1a. However, the important characteristic of the recovery of liver fibrosis is not only the apoptosis of activated stellate cells but also the reversal of the myofibroblast-like phenotype to a quiescent-like phenotype. Restoration of SIRT1 protein was observed in the in vivo spontaneously liver fibrosis reversion model and in vitro MDI (isobutylmethylxanthine, dexamethasone, and insulin)-induced reversed stellate cells, and forced expression of SIRT1 also promoted the reversal of activated stellate cells. Furthermore, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was increased in liver fibrosis. RNAi-mediated suppression of MALAT1 resulted in a decrease of myofibroblast markers and restoration of SIRT1 protein. These observations suggested that SIRT1 contributed to apoptosis and reversion of activated LX-2 cells and SIRT1 might be regulated by MALAT1 in liver fibrosis. Therefore, SIRT1 could be considered as a valuable therapeutic target for translational studies of liver fibrosis. - Highlights: • This is the first report of SIRT1 expression and function in liver fibrogenesis and reversion.

  4. SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease.

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    Singh, Preeti; Hanson, Peter S; Morris, Christopher M

    2017-06-02

    Sirtuins (SIRTs) are NAD + dependent lysine deacetylases which are conserved from bacteria to humans and have been associated with longevity and lifespan extension. SIRT1, the best studied mammalian SIRT is involved in many physiological and pathological processes and changes in SIRT1 have been implicated in neurodegenerative disorders, with SIRT1 having a suggested protective role in Parkinson's disease. In this study, we determined the effect of SIRT1 on cell survival and α-synuclein aggregate formation in SH-SY5Y cells following oxidative stress. Over-expression of SIRT1 protected SH-SY5Y cells from toxin induced cell death and the protection conferred by SIRT1 was partially independent of its deacetylase activity, which was associated with the repression of NF-кB and cPARP expression. SIRT1 reduced the formation of α-synuclein aggregates but showed minimal co-localisation with α-synuclein. In post-mortem brain tissue obtained from patients with Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies and Alzheimer's disease, the activity of SIRT1 was observed to be down-regulated. These findings suggests a negative effect of oxidative stress in neurodegenerative disorders and possibly explain the reduced activity of SIRT1 in neurodegenerative disorders. Our study shows that SIRT1 is a pro-survival protein that is downregulated under cellular stress.

  5. Effect of scuba diving on the oxidant/antioxidant status, SIRT1 and SIRT3 expression in recreational divers after a winter nondive period.

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    Perović, Antonija; Sobočanec, Sandra; Dabelić, Sanja; Balog, Tihomir; Dumić, Jerka

    2018-02-01

    The aim of this study was to examine the effects of scuba diving on oxidative damage markers in erythrocytes and plasma, antioxidant system in peripheral blood mononuclear cells (PBMCs), as well as sirtuin 1 (SIRT1) and sirtuin 3 (SIRT3) gene expressions in recreational divers after a winter nondive period (at least 5 months). For that purpose, 17 male recreational divers performed an immersion at a depth of 30 m for 30 min. Blood samples were collected immediately before and after diving, 3 and 6 h after diving. Erythrocyte lipid peroxidation measured by thiobarbituric-reactive substances (TBARS) method was significantly increased immediately after diving, but returned to the baseline 6 h after diving, while no significant change was found for plasma TBARS and protein carbonyl derivates in both plasma and erythrocytes. Diving-induced catalase (CAT), superoxide dismutase 2 (SOD2), and consequently total superoxide dismutase (SOD) activities in the PBMC samples (significantly increased immediately after diving, reached the maximum activities 3 h after diving, while 6 h after diving only CAT activity remained significantly increased). No significant change was observed for SOD1 activity and gene expression, as well as SOD2 expression, while CAT and SIRT1 expressions were slightly decreased immediately after diving and 3 h after diving. Interestingly, SIRT3 expression was significantly increased 6 h after diving. In conclusion, after the first dive to 30 m after a nondive season, activation of antioxidant defence was not sufficient to prevent oxidative damage, while SIRT3 upregulation could be a step towards an adaptive response to the diving.

  6. MicroRNA-133b inhibits hepatocellular carcinoma cell progression by targeting Sirt1

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    Tian, Zhijie [School of Biomedicine, Chengdu Medical College, Chengdu, Sichuan 610500 (China); Jiang, Hequn [The First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan 610041 (China); Liu, Ying; Huang, Yong [School of Biomedicine, Chengdu Medical College, Chengdu, Sichuan 610500 (China); Xiong, Xin [Laboratory Research Center, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016 (China); Wu, Hongwei, E-mail: hongweiwu2118@sina.com [The First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan 610041 (China); Dai, Xiaozhen, E-mail: xiaozhendai2012@163.com [School of Biomedicine, Chengdu Medical College, Chengdu, Sichuan 610500 (China); Chongqing University, Key Laboratory of Biorheological Science and Technology, Ministry of Education, Chongqing 400044 (China); Department of Pediatrics, University of Louisville School of Medicine, Louisville, KY (United States)

    2016-05-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as critical gene regulators by targeting mRNAs for translational repression or degradation. In this study, we showed that the expression level of miR-133b was decreased, while Sirt1 mRNA expression levels were increased in hepatocellular carcinoma (HCC) and cell lines, and we identified Sirt1 as a novel direct target of miR-133b. The over-expression of miR-133b suppressed Sirt1 expression. In addition, miR-133b over-expression resulted in attenuating HCC cell proliferation and invasion together with apoptosis increase in vitro. HepG2 cell transplantation revealed that up-regulation of miR-133b could inhibit HCC tumor genesis in vivo. Forced expression of Sirt1 partly rescued the effect of miR-133b in vitro. Furthermore, our study showed that miR-133b over-expression or Sirt1 down-regulation elevated E-cadherin expression, and repressed glypican-3 (GPC3) and the anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) expression. The inhibition of GPC3 expression repressed Bcl-2, Bcl-xL, and Mcl-1 expression, and elevated E-cadherin expression. Moreover, the Sirt1 up-regulation resulted in increases in HCC cell proliferation and invasion together with decreases apoptosis, and increases in the cytosolic accumulation and nuclear translocation of the transcription factor β-catenin in vitro. But the effect of Sirt1 up-regulation was partly reversed by GPC3 down-regulation in vitro. Taken together, these findings provide insight into the role and mechanism of miR-133b in regulating HCC cell proliferation, invasion and apoptosis via the miR-133b/Sirt1/GPC3/Wnt β-catenin axis, and miR-133b may serve as a potential therapeutic target in HCC in the future. - Highlights: • Sirt1 is a direct target of miR-133b in HCC. • miR-133b over-expression suppresses HCC progression in vitro and in vivo. • Sirt1 restoration reverses the effect of miR-133b over-expression on HCC cells. • GPC3 down-regulation reverses

  7. MicroRNA-133b inhibits hepatocellular carcinoma cell progression by targeting Sirt1

    International Nuclear Information System (INIS)

    Tian, Zhijie; Jiang, Hequn; Liu, Ying; Huang, Yong; Xiong, Xin; Wu, Hongwei; Dai, Xiaozhen

    2016-01-01

    MicroRNAs (miRNAs) are a class of small non-coding RNAs that function as critical gene regulators by targeting mRNAs for translational repression or degradation. In this study, we showed that the expression level of miR-133b was decreased, while Sirt1 mRNA expression levels were increased in hepatocellular carcinoma (HCC) and cell lines, and we identified Sirt1 as a novel direct target of miR-133b. The over-expression of miR-133b suppressed Sirt1 expression. In addition, miR-133b over-expression resulted in attenuating HCC cell proliferation and invasion together with apoptosis increase in vitro. HepG2 cell transplantation revealed that up-regulation of miR-133b could inhibit HCC tumor genesis in vivo. Forced expression of Sirt1 partly rescued the effect of miR-133b in vitro. Furthermore, our study showed that miR-133b over-expression or Sirt1 down-regulation elevated E-cadherin expression, and repressed glypican-3 (GPC3) and the anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) expression. The inhibition of GPC3 expression repressed Bcl-2, Bcl-xL, and Mcl-1 expression, and elevated E-cadherin expression. Moreover, the Sirt1 up-regulation resulted in increases in HCC cell proliferation and invasion together with decreases apoptosis, and increases in the cytosolic accumulation and nuclear translocation of the transcription factor β-catenin in vitro. But the effect of Sirt1 up-regulation was partly reversed by GPC3 down-regulation in vitro. Taken together, these findings provide insight into the role and mechanism of miR-133b in regulating HCC cell proliferation, invasion and apoptosis via the miR-133b/Sirt1/GPC3/Wnt β-catenin axis, and miR-133b may serve as a potential therapeutic target in HCC in the future. - Highlights: • Sirt1 is a direct target of miR-133b in HCC. • miR-133b over-expression suppresses HCC progression in vitro and in vivo. • Sirt1 restoration reverses the effect of miR-133b over-expression on HCC cells. • GPC3 down-regulation reverses

  8. SIRT1 suppresses the senescence-associated secretory phenotype through epigenetic gene regulation.

    Directory of Open Access Journals (Sweden)

    Tomohisa Hayakawa

    Full Text Available Senescent cells develop a pro-inflammatory response termed the senescence-associated secretory phenotype (SASP. As many SASP components affect surrounding cells and alter their microenvironment, SASP may be a key phenomenon in linking cellular senesence with individual aging and age-related diseases. We herein demonstrated that the expression of Sirtuin1 (SIRT1 was decreased and the expression of SASP components was reciprocally increased during cellular senescence. The mRNAs and proteins of SASP components, such as IL-6 and IL-8, quickly accumulated in SIRT1-depleted cells, and the levels of these factors were also higher than those in control cells, indicating that SIRT1 negatively regulated the expression of SASP factors at the transcriptional level. SIRT1 bound to the promoter regions of IL-8 and IL-6, but dissociated from them during cellular senescence. The acetylation of Histone H3 (K9 and H4 (K16 of the IL-8 and IL-6 promoter regions gradually increased during cellular senescence. In SIRT1-depleted cells, the acetylation levels of these regions were already higher than those in control cells in the pre-senescent stage. Moreover, these acetylation levels in SIRT1-depleted cells were significantly higher than those in control cells during cellular senescence. These results suggest that SIRT1 repressed the expression of SASP factors through the deacetylation of histones in their promoter regions.

  9. MiR-181b regulates steatosis in nonalcoholic fatty liver disease via targeting SIRT1.

    Science.gov (United States)

    Wang, Yunxia; Zhu, Kongxi; Yu, Weihua; Wang, Hongjuan; Liu, Lan; Wu, Qiong; Li, Shuai; Guo, Jianqiang

    2017-11-04

    Non-alcoholic fatty liver diseases (NAFLD) is one of the leading cause of chronic liver diseases in the world. However, the pathogenesis of NAFLD is still unclear. Emerging studies have demonstrated that microRNAs (miRs) are profoundly involved in NAFLD and related metabolic diseases. Here, we investigated the mechanisms by which miR-181b influences NAFLD via direct targeting SIRT1. The expression of miR181b was up-regulated while SIRT1 was down-regulated in both human NAFLD patients and high fat diet (HFD) induced NAFDL mice model. And palmitic acid (PA) treatment increased the miR-181b expression while decreased SIRT1 expression in HepG2 cells. Further, we identified that SIRT1 is a direct downstream target of miR-181b. Ectopic expression of miR-181b significantly repressed the 3'-UTR reporter activities of SIRT1 in a dose-dependent manner, while the effect of miR-181b was interrupted when the binding site of miR-181b within the SIRT1 3'-UTR was mutated. And overexpression of miR-181b reduced both the mRNA and protein levels of SIRT1 in HepG2 cells. We also found that inhibition of miR-181b expression alleviates hepatic steatosis both in vitro and in vivo. And the effect of miR-181b on steatosis was blocked by SIRT1 overexpression. Taken together, our data indicated that increased expression of miR-181b potentially contributes to altered lipid metabolism in NAFLD. Downregulation of miR-34a may be a therapeutic strategy against NAFLD by regulating its target SIRT1. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Sirt1 Is Required for Resveratrol-Mediated Chemopreventive Effects in Colorectal Cancer Cells

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    Constanze Buhrmann

    2016-03-01

    Full Text Available Sirt1 is a NAD+-dependent protein-modifying enzyme involved in regulating gene expression, DNA damage repair, metabolism and survival, as well as acts as an important subcellular target of resveratrol. The complex mechanisms underlying Sirt1 signaling during carcinogenesis remain controversial, as it can serve both as a tumor promoter and suppressor. Whether resveratrol-mediated chemopreventive effects are mediated via Sirt1 in CRC growth and metastasis remains unclear; which was the subject of this study. We found that resveratrol suppressed proliferation and invasion of two different human CRC cells in a dose-dependent manner, and interestingly, this was accompanied with a significant decrease in Ki-67 expression. By transient transfection of CRC cells with Sirt1-ASO, we demonstrated that the anti-tumor effects of resveratrol on cells was abolished, suggesting the essential role of this enzyme in the resveratrol signaling pathway. Moreover, resveratrol downregulated nuclear localization of NF-κB, NF-κB phosphorylation and its acetylation, causing attenuation of NF-κB-regulated gene products (MMP-9, CXCR4 involved in tumor-invasion and metastasis. Finally, Sirt1 was found to interact directly with NF-κB, and resveratrol did not suppress Sirt1-ASO-induced NF-κB phosphorylation, acetylation and NF-κB-regulated gene products. Overall, our results demonstrate that resveratrol can suppress tumorigenesis, at least in part by targeting Sirt1 and suppression of NF-κB activation.

  11. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells.

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    Laura Bordone

    2006-02-01

    Full Text Available Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic beta cells. Sirt1 represses the uncoupling protein (UCP gene UCP2 by binding directly to the UCP2 promoter. In beta cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in beta cells to affect insulin secretion.

  12. Arctigenin attenuates ischemic stroke via SIRT1-dependent inhibition of NLRP3 inflammasome.

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    Zhang, Shimeng; Jiang, Liangjun; Che, Fengyuan; Lu, Yucheng; Xie, Zhongxiang; Wang, Hao

    2017-11-04

    Arctigenin (ARC), a phenylpropanoid dibenzylbutyrolactone lignan derived from Arctium lappa L, has been reported to protect against cerebral ischemia injury in rats, but the underlying mechanism is unclear. In this study, we investigated whether ARC ameliorated ischemic stroke by inhibiting NLRP3 inflammasome-derived neuroinflammation and whether SIRT1 signaling was involved in this process. ARC (20 mg/kg) or vehicle were intraperitoneally injected to Sprague-Dawley rats for 3 days before middle cerebral artery occlusion (MCAO) surgery performed. The infarct volume, neurological score, brain water content, neuroinflammation, NLRP3 inflammasome activation and SIRT1 protein expression were assessed. Furthermore, we also investigated whether ARC protected against cerebral ischemia via SIRT1-dependent inhibition of NLRP3 inflammasome by administrating EX527, a specific SIRT1 inhibitor, under oxygen-glucose deprivation (OGD) condition. We found that ARC pretreatment decreased infarct volume, neurological score and brain water content. Moreover, ARC treatment effectively inhibited cerebral ischemia induced NLRP3 inflammasome activation and IL-1β, IL-18 secretion both in vivo and in vitro. Futhermore, ARC treatment activated Silent information regulator 1 (SIRT1) singnaling in the brain. Importantly, suppress of SIRT1 reversed the inhibitory effect of ARC on NLRP3 inflammasome activation. Taken together our results demonstrated that ARC may confer protection against ischemic stroke by inhibiting NLRP3 inflammasome activation. The activation of SIRT1 signaling pathway may contribute to the neuroprotection of ARC in MCAO. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Hepatic SIRT1 attenuates hepatic steatosis and controls energy balance in mice by inducing fibroblast growth factor 21.

    Science.gov (United States)

    Li, Yu; Wong, Kimberly; Giles, Amber; Jiang, Jianwei; Lee, Jong Woo; Adams, Andrew C; Kharitonenkov, Alexei; Yang, Qin; Gao, Bin; Guarente, Leonard; Zang, Mengwei

    2014-02-01

    The hepatocyte-derived hormone fibroblast growth factor 21 (FGF21) is a hormone-like regulator of metabolism. The nicotinamide adenine dinucleotide-dependent deacetylase SIRT1 regulates fatty acid metabolism through multiple nutrient sensors. Hepatic overexpression of SIRT1 reduces steatosis and glucose intolerance in obese mice. We investigated mechanisms by which SIRT1 controls hepatic steatosis in mice. Liver-specific SIRT1 knockout (SIRT1 LKO) mice and their wild-type littermates (controls) were divided into groups that were placed on a normal chow diet, fasted for 24 hours, or fasted for 24 hours and then fed for 6 hours. Liver tissues were collected and analyzed by histologic examination, gene expression profiling, and real-time polymerase chain reaction assays. Human HepG2 cells were incubated with pharmacologic activators of SIRT1 (resveratrol or SRT1720) and mitochondrion oxidation consumption rate and immunoblot analyses were performed. FGF21 was overexpressed in SIRT1 LKO mice using an adenoviral vector. Energy expenditure was assessed by indirect calorimetry. Prolonged fasting induced lipid deposition in livers of control mice, but severe hepatic steatosis in SIRT1 LKO mice. Gene expression analysis showed that fasting up-regulated FGF21 in livers of control mice but not in SIRT1 LKO mice. Decreased hepatic and circulating levels of FGF21 in fasted SIRT1 LKO mice were associated with reduced hepatic expression of genes involved in fatty acid oxidation and ketogenesis, and increased expression of genes that control lipogenesis, compared with fasted control mice. Resveratrol or SRT1720 each increased the transcriptional activity of the FGF21 promoter (-2070/+117) and levels of FGF21 messenger RNA and protein in HepG2 cells. Surprisingly, SIRT1 LKO mice developed late-onset obesity with impaired whole-body energy expenditure. Hepatic overexpression of FGF21 in SIRT1 LKO mice increased the expression of genes that regulate fatty acid oxidation, decreased

  14. SIRT1 and metabolic syndrome

    Directory of Open Access Journals (Sweden)

    Katarzyna Mac-Marcjanek

    2011-04-01

    Full Text Available Both obesity and type 2 diabetes mellitus, two major components of metabolic syndrome, become healthepidemics in the world. Over the past decade, advances in understanding the role of some regulators participatingin lipid and carbohydrate homeostasis have been made.Of them, SIRT1, the mammalian orthologue of the yeast Sir2 protein has been identified. SIRT1 is a nuclearNAD+-dependent deacetylase that targets many transcriptional modulators, including PPAR-α and -γ (peroxisomeproliferator-activated receptors α and γ, PGC-1α (PPAR-γ coactivator-1α, FOXO (forkhead box O proteins,and nuclear factor κB (NF-κB, thereby this enzyme mediates a wide range of physiological processes like apoptosis,fat metabolism, glucose homeostasis, and neurodegeneration.In this article, we discuss how SIRT1 regulates lipid and carbohydrate metabolism, and insulin secretion indifferent metabolic organs/tissue, including liver, muscle, pancreas, and fat. Additionally, the role of this enzymein reduction of inflammatory signalling is highlighted.

  15. Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells.

    Science.gov (United States)

    Yen, Jui-Hung; Wu, Pei-Shan; Chen, Shu-Fen; Wu, Ming-Jiuan

    2017-04-17

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH₂-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor

  16. SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

    International Nuclear Information System (INIS)

    Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

    2012-01-01

    Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

  17. A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function

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    Mohamad A. Zaini

    2018-01-01

    Full Text Available Summary: Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα. Protein lysine acetylation is a key post-translational modification (PTM that integrates cellular metabolic cues with other physiological processes. Here, we show that C/EBPα is acetylated by the lysine acetyl transferase (KAT p300 and deacetylated by the lysine deacetylase (KDAC sirtuin1 (SIRT1. SIRT1 is activated in times of energy demand by high levels of nicotinamide adenine dinucleotide (NAD+ and controls mitochondrial biogenesis and function. A hypoacetylated mutant of C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. Our study identifies C/EBPα as a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply. : Zaini et al. show that the transcription factor C/EBPα is acetylated by p300 and deacetylated by the lysine deacetylase SIRT1. Hypoacetylated C/EBPα induces the transcription of mitochondrial genes and results in increased mitochondrial respiration. C/EBPα is a key mediator of SIRT1-controlled adaption of energy homeostasis to changes in nutrient supply. Keywords: C/EBPα, SIRT1, p300, lysine acetylation, mitochondrial function, cellular metabolism, NAD+, gene regulation

  18. Role of SIRT1/PGC-1α in mitochondrial oxidative stress in autistic spectrum disorder

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    Bu XS

    2017-06-01

    Full Text Available Xiaosong Bu, De Wu, Xiaomei Lu, Li Yang, Xiaoyan Xu, Juan Wang, Jiulai Tang Department of Pediatrics, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China Abstract: Autistic spectrum disorder (ASD is a neurodevelopmental disorder and has a high prevalence in children. Recently, mitochondrial oxidative stress has been proposed to be associated with ASD. Besides, SIRT1/PGC-1α signaling plays an important role in combating oxidative stress. In this study, we sought to determine the role of SIRT1/PGC-1α signaling in the ASD lymphoblastoid cell lines (LCLs. In this study, the mRNA and protein expressions of SIRT1/PGC-1α axis genes were assessed in 35 children with ASD and 35 healthy controls (matched for age, gender, and IQ. An immortalized LCL was established by transforming lymphocytes with Epstein–Barr virus. Next, we used ASD LCLs and control LCLs to detect SIRT1/PGC-1α axis genes expression and oxidative damage. Finally, the effect of overexpression of PGC-1α on oxidative injury in the ASD LCLs was determined. SIRT1/PGC-1α axis genes expression was downregulated at RNA and protein levels in ASD patients and LCLs. Besides, the translocation of cytochrome c and DIABLO from mitochondria to the cytosol was found in the ASD LCLs. Moreover, the intracellular reactive oxygen species (ROS and mitochondrial ROS and cell apoptosis were increased in the ASD LCLs. However, overexpression of PGC-1α upregulated the SIRT1/PGC-1α axis genes expression and reduced cytochrome c and DIABLO release in the ASD LCLs. Also, overexpression of PGC-1α reduced the ROS generation and cell apoptosis in the ASD LCLs. Overexpression of PGC-1α could reduce the oxidative injury in the ASD LCLs, and PGC-1α may act as a target for treatment. Keywords: autistic spectrum disorder, PGC-1α, SIRT1, mitochondria, oxidative stress

  19. sirt1-null mice develop an autoimmune-like condition

    International Nuclear Information System (INIS)

    Sequeira, Jedon; Boily, Gino; Bazinet, Stephanie; Saliba, Sarah; He Xiaohong; Jardine, Karen; Kennedy, Christopher; Staines, William; Rousseaux, Colin; Mueller, Rudi; McBurney, Michael W.

    2008-01-01

    The sirt1 gene encodes a protein deacetylase with a broad spectrum of reported substrates. Mice carrying null alleles for sirt1 are viable on outbred genetic backgrounds so we have examined them in detail to identify the biological processes that are dependent on SIRT1. Sera from adult sirt1-null mice contain antibodies that react with nuclear antigens and immune complexes become deposited in the livers and kidneys of these animals. Some of the sirt1-null animals develop a disease resembling diabetes insipidus when they approach 2 years of age although the relationship to the autoimmunity remains unclear. We interpret these observations as consistent with a role for SIRT1 in sustaining normal immune function and in this way delaying the onset of autoimmune disease

  20. Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

    Science.gov (United States)

    Schuster, Susanne; Penke, Melanie; Gorski, Theresa; Petzold-Quinque, Stefanie; Damm, Georg; Gebhardt, Rolf; Kiess, Wieland; Garten, Antje

    2014-01-01

    Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells. PMID:24603648

  1. Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Susanne Schuster

    Full Text Available Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382. Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

  2. Effect of chronic alcohol consumption on Hepatic SIRT1 and PGC-1α in rats

    International Nuclear Information System (INIS)

    Lieber, Charles S.; Leo, Maria A.; Wang Xiaolei; DeCarli, Leonore M.

    2008-01-01

    The nuclear genes, NAD-dependent deacetylase Sirtuis 1 (SIRT1) and the peroxisome proliferator-activated receptor-γ coactivator1α (PGC-1α) are regulators of energy metabolism. Here, we studied the role of alcohol consumption in expression of these sensing molecules. Alcohol significantly reduced hepatic SIRT1 mRNA by 50% and PGC-1α mRNA by 46% and it significantly inhibited the protein expression of SIRT1 and PGC-1α, while the transcription factor PPAR-γ remained unchanged. However, when the lipid composition of the alcohol diet was changed by replacing long-chain triglycerides (LCT) with medium chain triglycerides (MCT), SIRT1 and PGC-1α mRNA were restored to near control levels. This study demonstrates that alcohol reduces key energy sensing proteins and that replacement of LCT by MCT affects the transcription of these genes. Since there is a pathophysiological link between SIRT1 and PGC-1α and mitochondrial energy, the implication of the study is that mitochondrial dysfunction due to alcohol abuse can be treated by dietary modifications

  3. Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Kyung-Soo [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Park, Jun-Ik [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Dao, Trong Tuan; Oh, Won Keun [BK21 Project Team, College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Kang, Chi-Dug, E-mail: kcdshbw@pusan.ac.kr [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Kim, Sun-Hee, E-mail: ksh7738@pusan.ac.kr [Department of Biochemistry, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan (Korea, Republic of)

    2012-03-01

    SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

  4. Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

    International Nuclear Information System (INIS)

    Hong, Kyung-Soo; Park, Jun-Ik; Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won; Dao, Trong Tuan; Oh, Won Keun; Kang, Chi-Dug; Kim, Sun-Hee

    2012-01-01

    SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

  5. A CREB-Sirt1-Hes1 Circuitry Mediates Neural Stem Cell Response to Glucose Availability

    Directory of Open Access Journals (Sweden)

    Salvatore Fusco

    2016-02-01

    Full Text Available Summary: Adult neurogenesis plays increasingly recognized roles in brain homeostasis and repair and is profoundly affected by energy balance and nutrients. We found that the expression of Hes-1 (hairy and enhancer of split 1 is modulated in neural stem and progenitor cells (NSCs by extracellular glucose through the coordinated action of CREB (cyclic AMP responsive element binding protein and Sirt-1 (Sirtuin 1, two cellular nutrient sensors. Excess glucose reduced CREB-activated Hes-1 expression and results in impaired cell proliferation. CREB-deficient NSCs expanded poorly in vitro and did not respond to glucose availability. Elevated glucose also promoted Sirt-1-dependent repression of the Hes-1 promoter. Conversely, in low glucose, CREB replaced Sirt-1 on the chromatin associated with the Hes-1 promoter enhancing Hes-1 expression and cell proliferation. Thus, the glucose-regulated antagonism between CREB and Sirt-1 for Hes-1 transcription participates in the metabolic regulation of neurogenesis. : Using a combination of in vitro and in vivo studies, Fusco et al. find that excess glucose impairs the self-renewal capacity of neural stem cells through a molecular circuit that involves the transcription factor CREB and Sirtuin 1. The authors suggest that this circuitry may link nutrient excess with neurodegeneration and brain aging. Keywords: neural stem cells, adult neurogenesis, CREB, Sirt-1, nutrients, metabolism, diabetes

  6. An in vitro comparative study of the antioxidant activity and SIRT1 modulation of natural compounds.

    Science.gov (United States)

    Fusi, Jonathan; Bianchi, Sara; Daniele, Simona; Pellegrini, Silvia; Martini, Claudia; Galetta, Fabio; Giovannini, Luca; Franzoni, Ferdinando

    2018-05-01

    Oxidative stress arises from an imbalance between the production of free radicals and antioxidant defences. Several studies have suggested that dietary antioxidants (such as polyphenols and berberine) may counteract oxidative stress through the involvement of the Sirtuin 1/Adenosine Monophosphate-Activated Protein Kinase (SIRT1/AMPK) pathway. The aim of this study was to evaluate the direct and specific antioxidant activity of some natural compounds, as well as their ability to modulate the expression of SIRT1 and the activation of AMPK. Quercetin, tyrosol, ferulic acid, catechin, berberine and curcumin were evaluated for their specific and direct antioxidant activity with TOSC assay. Their ability to modulate SIRT1 and AMPK was assessed by immunoblotting assay, while their cytotoxicity by CellTiter-Blue Cell Viability Assay. No statistically significant decrease (p > 0.05) in the number of viable cells was found upon challenging with the natural compounds. Quercetin exhibited the highest antioxidant activity against peroxyl radical and peroxinitrate derivates, while curcumin showed the best anti-hydroxyl activity with respect to the other compounds and, most importantly, respect to the reference antioxidants. Finally, all the tested compounds significantly increased the SIRT1 expression and the activation of AMPK. Our results clearly disclose the specific antioxidant activity of these natural compounds and their ability to increase SIRT1 expression and AMPK activation. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  7. SIRT1 signalling protects mouse oocytes against oxidative stress and is deregulated during aging.

    Science.gov (United States)

    Di Emidio, Giovanna; Falone, Stefano; Vitti, Maurizio; D'Alessandro, Anna Maria; Vento, Marilena; Di Pietro, Cinzia; Amicarelli, Fernanda; Tatone, Carla

    2014-09-01

    mouse oocytes in response to OS. Moreover, increased intracellular ROS were observed when SIRT1 activity was inhibited by Ex527. In aged oocytes Sirt1 was expressed more than in young oocytes but SIRT1 protein was undetectable. Upon OS, significant changes in miR-132 micro-RNA, a validated Sirt1 modulator, were observed. A negative correlation between Sirt1 mRNA and miR-132 levels was observed when young oocytes exposed to OS were compared with young control oocytes, and when aged oocytes were compared with young control oocytes. FoxO3a and MnSod transcripts were increased upon OS with the same kinetics as Sirt1 transcripts, and up-regulation of MnSod gene was prevented by oocyte treatment with Ex527, indicating that SIRT1 acts upstream to the FoxO3a-MnSod axis. Finally, the results of the in vitro maturation assay suggested that SIRT1 might be involved in oocyte maturation by regulating the redox state and ensuring normal spindle assembly. The main limitation of this study was the absence of direct quantification of SIRT1 enzymatic activity due to the lack of an appropriately sensitive method. The present findings may provide a valuable background for studying the regulation of SIRT1 during oogenesis and its relevance as a sensor of oocyte redox state and energy status. The antioxidant response orchestrated by SIRT1 in oocytes seems to decrease with aging. This suggests that SIRT1 could be an excellent pharmacological target for improving oocyte quality and IVF outcome in aging or aging-like diseases. The work was supported by the Ministero dell'Università e della Ricerca Scientifica (MIUR) to C.T., F.A., C.D., A.M.D. The authors declare no conflict of interest. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

    International Nuclear Information System (INIS)

    Herbert, Katharine J.; Holloway, Adele; Cook, Anthony L.; Chin, Suyin P.; Snow, Elizabeth T.

    2014-01-01

    Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylation of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5 μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24 days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5 μM; > 5 weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24 h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity. - Highlights: • Submicromolar arsenic concentrations disrupt SIRT1 activity and expression in human keratinocytes. • Arsenic-induced chromatin remodelling at the miR-34a gene promoter is associated with hyperacetylation

  9. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J. [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia); Holloway, Adele [Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000 (Australia); Cook, Anthony L. [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia); Chin, Suyin P. [Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000 (Australia); Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia)

    2014-11-15

    Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylation of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5 μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24 days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5 μM; > 5 weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24 h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity. - Highlights: • Submicromolar arsenic concentrations disrupt SIRT1 activity and expression in human keratinocytes. • Arsenic-induced chromatin remodelling at the miR-34a gene promoter is associated with hyperacetylation

  10. The role of SIRT1 in diabetic kidney disease

    Directory of Open Access Journals (Sweden)

    Rabi eYacoub

    2014-10-01

    Full Text Available Sirtuins (SIRTs are members of the silent information regulator 2 (Sir2 family. In mammals, of the seven known SIRTs, SIRT1 function is most studied and has been shown to regulate wide range of cellular functions that affect metabolic homeostasis and aging. SIRT1 exerts anti-apoptotic, anti-oxidative, and anti-inflammatory effects against cellular injury, and protects the cells through the regulation of mitochondrial biogenesis, autophagy, and metabolism in response to the cellular energy and redox status. SIRT1 also promotes vasodilation and protects vascular tissues. In humans and animal models with diabetic kidney disease, its expression tends to be decreased in renal cells, and increased expression of SIRT1 was found to play a renal protective role in animal models with diabetic kidney disease. In this review we discuss the role and potential mechanisms by which SIRT1 protects against DKD.

  11. SIRT1 is a positive regulator of the master osteoblast transcription factor, RUNX2.

    Directory of Open Access Journals (Sweden)

    Kayvan Zainabadi

    Full Text Available Activation of SIRT1 has previously been shown to protect mice against osteoporosis through yet ill-defined mechanisms. In this study, we outline a role for SIRT1 as a positive regulator of the master osteoblast transcription factor, RUNX2. We find that ex vivo deletion of sirt1 leads to decreased expression of runx2 downstream targets, but not runx2 itself, along with reduced osteoblast differentiation. Reciprocally, treatment with a SIRT1 agonist promotes osteoblast differentiation, as well as the expression of runx2 downstream targets, in a SIRT1-dependent manner. Biochemical and luciferase reporter assays demonstrate that SIRT1 interacts with and promotes the transactivation potential of RUNX2. Intriguingly, mice treated with the SIRT1 agonist, resveratrol, show similar increases in the expression of RUNX2 targets in their calvaria (bone tissue, validating SIRT1 as a physiologically relevant regulator of RUNX2.

  12. Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

    Science.gov (United States)

    Pinho, Andreia V; Bensellam, Mohammed; Wauters, Elke; Rees, Maxine; Giry-Laterriere, Marc; Mawson, Amanda; Ly, Le Quan; Biankin, Andrew V; Wu, Jianmin; Laybutt, D Ross; Rooman, Ilse

    2015-01-01

    Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear. This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas. We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas. This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

  13. Pancreas-Specific Sirt1-Deficiency in Mice Compromises Beta-Cell Function without Development of Hyperglycemia.

    Directory of Open Access Journals (Sweden)

    Andreia V Pinho

    Full Text Available Sirtuin 1 (Sirt1 has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear.This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas.We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r as well as a marked down regulation of endoplasmic reticulum (ER chaperones that participate in the Unfolded Protein Response (UPR pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas.This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

  14. NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Wei-Xia; He, Min-Di; Mao, Lin [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China); Qian, Feng-Hua [Department of Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Li, Yu-Ming [Institute of Hepatobiliary Surgery, XinQiao Hospital, Third Military Medical University, Chongqing 400038 (China); Pi, Hui-Feng; Liu, Chuan; Chen, Chun-Hai; Lu, Yong-Hui; Cao, Zheng-Wang; Zhang, Lei; Yu, Zheng-Ping [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China); Zhou, Zhou, E-mail: lunazhou00@163.com [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China)

    2015-07-15

    With application of nano-sized nickel-containing particles (Nano-Ni) expanding, the health concerns about their adverse effects on the pulmonary system are increasing. However, the mechanisms for the pulmonary toxicity of these materials remain unclear. In the present study, we focused on the impacts of NiO nanoparticles (NiONPs) on sirtuin1 (SIRT1), a NAD-dependent deacetylase, and investigated whether SIRT1 was involved in NiONPs-induced apoptosis. Although the NiONPs tended to agglomerate in fluid medium, they still entered into the human bronchial epithelial cells (BEAS-2B) and released Ni{sup 2+} inside the cells. NiONPs at doses of 5, 10, and 20 μg/cm{sup 2} inhibited the cell viability. NiONPs' produced cytotoxicity was demonstrated through an apoptotic process, indicated by increased numbers of Annexin V positive cells and caspase-3 activation. The expression of SIRT1 was markedly down-regulated by the NiONPs, accompanied by the hyperacetylation of p53 (tumor protein 53) and overexpression of Bax (Bcl-2-associated X protein). However, overexpression of SIRT1 through resveratrol treatment or transfection clearly attenuated the NiONPs-induced apoptosis and activation of p53 and Bax. Our results suggest that the repression of SIRT1 may underlie the NiONPs-induced apoptosis via p53 hyperacetylation and subsequent Bax activation. Because SIRT1 participates in multiple biologic processes by deacetylation of dozens of substrates, this knowledge of the impact of NiONPs on SIRT1 may lead to an improved understanding of the toxic mechanisms of Nano-Ni and provide a molecular target to antagonize Nano-Ni toxicity. - Highlights: • NiONPs were taken up by BEAS-2B cells and released Ni{sup 2+}. • NiONPs produced cytotoxicity was demonstrated through an apoptotic process. • NiONPs repressed SIRT1 expression and activated p53 and Bax. • Overexpression of SIRT1 attenuated NiONPs-induced apoptosis via deacetylation p53.

  15. Down-regulated miR-448 relieves spinal cord ischemia/reperfusion injury by up-regulating SIRT1

    Directory of Open Access Journals (Sweden)

    Yun Wang

    2018-03-01

    Full Text Available MicroRNAs play a crucial role in the progression of spinal cord ischemia/reperfusion injury (SCII. The role of miR-448 and SIRT1 in SCII was investigated in this study, to provide further insights into prevention and improvement of this disorder. In this study, expressions of miR-448 and SIRT1 protein were determined by qRT-PCR and western blot, respectively. Flow cytometry was used to analyze cell apoptosis. The endogenous expression of genes was modulated by recombinant plasmids and cell transfection. Dual-luciferase reporter assay was performed to determine the interaction between miR-448 and SIRT1. The Basso, Beattie, and Bresnahan score was used to measure the hind-limb function of rat. The spinal cord ischemia reperfusion injury model of adult rats was developed by abdominal aorta clamping, and the nerve function evaluation was completed by motor deficit index score. In SCII tissues and cells treated with hypoxia, miR-448 was up-regulated while SIRT1 was down-regulated. Hypoxia treatment reduced the expression of SIRT1 through up-regulating miR-448 in nerve cells. Up-regulation of miR-448 induced by hypoxia promoted apoptosis of nerve cells through down-regulating SIRT1. Down-regulated miR-448 improved neurological function and hind-limb motor function of rats with SCII by up-regulating SIRT1. Down-regulated miR-448 inhibited apoptosis of nerve cells and improved neurological function by up-regulating SIRT1, which contributes to relieving SCII.

  16. Detection of Sirtuin-1 protein expression in peripheral blood leukocytes in dogs.

    Science.gov (United States)

    Yoshimura, Kuniko; Matsuu, Aya; Sasaki, Kai; Momoi, Yasuyuki

    2018-05-11

    Sirtuin-1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD + )-dependent histone deacetylase with a large number of protein substrates. It has attracted a lot of attention in association with extending lifespan. The objective of this study was to enable the evaluation of SIRT1 expression in peripheral blood mononuclear cells (PBMCs) from dogs by flow cytometry. Three transcript variants were amplified from PBMCs by reverse transcription PCR and the nucleotide sequences were analyzed. On the basis deduced amino acid sequence, a monoclonal antibody against human SIRT1, 1F3, was selected to detect canine SIRT1. Canine SIRT1 in peripheral blood mononuclear cells was successfully detected by western blotting using this antibody. Intracellular canine SIRT1 was also detected in permeabilized 293T cells transfected with a canine SIRT1 expression plasmid by flow cytometry using this antibody. SIRT1 was detected in all leukocyte subsets including lymphocytes, granulocytes and monocytes. The expression level was markedly different among individual dogs. These results indicated that the method applied in this study is useful for evaluating canine SIRT1 levels in PBMCs from dogs.

  17. SIRT1, 2, 3 protect mouse oocytes from postovulatory aging.

    Science.gov (United States)

    Zhang, Teng; Zhou, Yang; Li, Li; Wang, Hong-Hui; Ma, Xue-Shan; Qian, Wei-Ping; Shen, Wei; Schatten, Heide; Sun, Qing-Yuan

    2016-04-01

    The quality of metaphase II oocytes will undergo a time-dependent deterioration following ovulation as the result of the oocyte aging process. In this study, we determined that the expression of sirtuin family members (SIRT1, 2, 3) was dramatically reduced in mouse oocytes aged in vivo or in vitro. Increased intracellular ROS was observed when SIRT1, 2, 3 activity was inhibited. Increased frequency of spindle defects and disturbed distribution of mitochondria were also observed in MII oocytes aged in vitro after treatment with Nicotinamide (NAM), indicating that inhibition of SIRT1, 2, 3 may accelerate postovulatory oocyte aging. Interestingly, when MII oocytes were exposed to caffeine, the decline of SIRT1, 2, 3 mRNA levels was delayed and the aging-associated defective phenotypes could be improved. The results suggest that the SIRT1, 2, 3 pathway may play a potential protective role against postovulatory oocyte aging by controlling ROS generation.

  18. Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

    Science.gov (United States)

    Kim, Hak-Bong; Lee, Su-Hoon; Um, Jee-Hyun; Oh, Won Keun; Kim, Dong-Wan; Kang, Chi-Dug; Kim, Sun-Hee

    2015-01-01

    The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp. PMID:26416354

  19. Down-regulation of the miR-543 alleviates insulin resistance through targeting the SIRT1.

    Science.gov (United States)

    Hu, Xiaojing; Chi, Liyi; Zhang, Wentao; Bai, Tiao; Zhao, Wei; Feng, Zhanbin; Tian, Hongyan

    2015-12-25

    Insulin resistance plays an important role in the development of hypertension, which is seriously detrimental to human health. Recently, Sirtuin-1 (SIRT1) has been found to participate in regulation of insulin resistance. Therefore, further studies focused on the SIRT1 regulators might provide a potential approach for combating insulin resistance and hypertension. Interestingly, in this study, we found that SIRT1 was the target gene of the miR-543 by the Dual-Luciferase Reporter Assay. Moreover, the miR-543 expression notably increased in the insulin-resistant HepG2 cells induced by TNF-α. Further analysis showed that the overexpression of the miR-543 lowered the SIRT1 mRNA and protein levels, resulting in the insulin resistance in the HepG2 cells; the inhibition of miR-543, however, enhanced the mRNA and protein expression of the SIRT1, and alleviated the insulin resistance. Furthermore, the SIRT1 overexpression abrogated the effect of miR-543 on insulin resistance. In addition, the overexpression of the miR-543 by the lentivirus-mediated gene transfer markedly impaired the insulin signaling assessed by the Western blot analysis of the glycogen synthesis and the phosphorylation of Akt and GSK3β. In summary, our study suggested that the downregulation of the miR-543 could alleviate the insulin resistance via the modulation of the SIRT1 expression, which might be a potential new strategy for treating insulin resistance and a promising therapeutic method for hypertension. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK

    Directory of Open Access Journals (Sweden)

    Sung-Jun Park

    2017-04-01

    Full Text Available The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK, an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

  1. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-κB p65 subunit and cytotoxicity in renal proximal tubule cells

    International Nuclear Information System (INIS)

    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Lee, Sang Yong; Han, Myung Kwan; Kim, Duk Hoon; Kim, Won

    2012-01-01

    Highlights: ► Cisplatin increases acetylation of NF-κB p65 subunit in HK2 cells. ► SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. ► Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-κB (NF-κB) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD + )-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-κB and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-κB p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-κB during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-κB p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-κB through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  2. Mounting evidence validates Ursolic Acid directly activates SIRT1: A powerful STAC which mimic endogenous activator of SIRT1.

    Science.gov (United States)

    Bakhtiari, Nuredin; Mirzaie, Sako; Hemmati, Roohullah; Moslemee-Jalalvand, Elham; Noori, Ali Reza; Kazemi, Jahanfard

    2018-05-16

    Ursolic Acid (UA), a pentacyclic triterpenoid compound, plays a vital role in aging process. However, the role of UA in the regulation of aging and longevity is still controversial as we have previously demonstrated that UA increases SIRT1 protein level in aged-mice. Here, we reveal that UA directly activates SIRT1 in silico, in vitro and in vivo. We have identified that UA binds to outer surface of SIRT1 and leads to tight binding of substrates to enzyme in comparison with Resveratrol (RSV) and control. Furthermore, our results indicate that UA drives the structure of SIRT1 toward a closed state (an active form of enzyme). Interestingly, our experimental findings are in agreement with the molecular dynamic results. Based on our data, UA increases the affinity of enzyme for both substrates with decreasing Km value, while enhances the Vmax of enzyme. Additionally, we have determined that UA heightened SIRT1 catalytic efficiency by 2 folds compared with RSV. Thereby, to identify the endogenous activator of SIRT1, UA was administrated to aged-mice and then the tissues were isolated. According to our results, it can be concluded that UA increases SIRT1 activity and mimics Lamin A and AROS behavior in the living cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

    Science.gov (United States)

    Hubbard, Basil P; Loh, Christine; Gomes, Ana P; Li, Jun; Lu, Quinn; Doyle, Taylor LG; Disch, Jeremy S; Armour, Sean M; Ellis, James L; Vlasuk, George P; Sinclair, David A

    2013-01-01

    SIRT1 is an NAD+-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1. PMID:23892437

  4. SIRT1 interacts with and protects glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from nuclear translocation: Implications for cell survival after irradiation

    International Nuclear Information System (INIS)

    Joo, Hyun-Yoo; Woo, Seon Rang; Shen, Yan-Nan; Yun, Mi Yong; Shin, Hyun-Jin; Park, Eun-Ran; Kim, Su-Hyeon; Park, Jeong-Eun; Ju, Yeun-Jin; Hong, Sung Hee; Hwang, Sang-Gu; Cho, Myung-Haing; Kim, Joon; Lee, Kee-Ho

    2012-01-01

    Highlights: ► SIRT1 serves to retain GAPDH in the cytosol, preventing GAPDH nuclear translocation. ► When SIRT1 is depleted, GAPDH translocation occurs even in the absence of stress. ► Upon irradiation, SIRT1 interacts with GAPDH. ► SIRT1 prevents irradiation-induced nuclear translocation of GAPDH. ► SIRT1 presence rather than activity is essential for inhibiting GAPDH translocation. -- Abstract: Upon apoptotic stimulation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a cytosolic enzyme normally active in glycolysis, translocates into the nucleus and activates an apoptotic cascade therein. In the present work, we show that SIRT1 prevents nuclear translocation of GAPDH via interaction with GAPDH. SIRT1 depletion triggered nuclear translocation of cytosolic GAPDH even in the absence of apoptotic stress. Such translocation was not, however, observed when SIRT1 enzymatic activity was inhibited, indicating that SIRT1 protein per se, rather than the deacetylase activity of the protein, is required to inhibit GAPDH translocation. Upon irradiation, SIRT1 prevented irradiation-induced nuclear translocation of GAPDH, accompanied by interaction of SIRT1 and GAPDH. Thus, SIRT1 functions to retain GAPDH in the cytosol, protecting the enzyme from nuclear translocation via interaction with these two proteins. This serves as a mechanism whereby SIRT1 regulates cell survival upon induction of apoptotic stress by means that include irradiation.

  5. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yun; Wang, Jing [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Chen, Guian [Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Reproductive Medical Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Fan, Dongsheng, E-mail: dsfan@yahoo.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Deng, Min, E-mail: dengmin1706@yahoo.com.cn [Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China); Clinical Stem Cell Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191 (China)

    2011-01-14

    Research highlights: {yields} Nicotinamide inhibit Sirt1. {yields} MASH1 and Ngn2 activation. {yields} Increase the expression of HB9. {yields} Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and {beta}III-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 {mu}M) or inhibitor nicotinamide (100 {mu}M). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  6. Inhibition of Sirt1 promotes neural progenitors toward motoneuron differentiation from human embryonic stem cells

    International Nuclear Information System (INIS)

    Zhang, Yun; Wang, Jing; Chen, Guian; Fan, Dongsheng; Deng, Min

    2011-01-01

    Research highlights: → Nicotinamide inhibit Sirt1. → MASH1 and Ngn2 activation. → Increase the expression of HB9. → Motoneurons formation increases significantly. -- Abstract: Several protocols direct human embryonic stem cells (hESCs) toward differentiation into functional motoneurons, but the efficiency of motoneuron generation varies based on the human ESC line used. We aimed to develop a novel protocol to increase the formation of motoneurons from human ESCs. In this study, we tested a nuclear histone deacetylase protein, Sirt1, to promote neural precursor cell (NPC) development during differentiation of human ESCs into motoneurons. A specific inhibitor of Sirt1, nicotinamide, dramatically increased motoneuron formation. We found that about 60% of the cells from the total NPCs expressed HB9 and βIII-tubulin, commonly used motoneuronal markers found in neurons derived from ESCs following nicotinamide treatment. Motoneurons derived from ESC expressed choline acetyltransferase (ChAT), a positive marker of mature motoneuron. Moreover, we also examined the transcript levels of Mash1, Ngn2, and HB9 mRNA in the differentiated NPCs treated with the Sirt1 activator resveratrol (50 μM) or inhibitor nicotinamide (100 μM). The levels of Mash1, Ngn2, and HB9 mRNA were significantly increased after nicotinamide treatment compared with control groups, which used the traditional protocol. These results suggested that increasing Mash1 and Ngn2 levels by inhibiting Sirt1 could elevate HB9 expression, which promotes motoneuron differentiation. This study provides an alternative method for the production of transplantable motoneurons, a key requirement in the development of hESC-based cell therapy in motoneuron disease.

  7. miR-543 promotes gastric cancer cell proliferation by targeting SIRT1

    International Nuclear Information System (INIS)

    Li, Juan; Dong, Guoying; Wang, Bo; Gao, Wei; Yang, Qing

    2016-01-01

    SIRT1, a class III histone deacetylase, exerts inhibitory effects on tumorigenesis and is downregulated in gastric cancer. However, the role of microRNAs in the regulation of SIRT1 in gastric cancer is still largely unknown. Here, we identified miR-543 as a predicted upstream regulator of SIRT1 using 3 different bioinformatics databases. Mimics of miR-543 significantly inhibited the expression of SIRT1, whereas an inhibitor of miR-543 increased SIRT1 expression. MiR-543 directly targeted the 3′-UTR of SIRT1, and both of the two binding sites contributed to the inhibitory effects. In gastric epithelium-derived cell lines, miR-543 promoted cell proliferation and cell cycle progression, and overexpression of SIRT1 rescued the above effects of miR-543. The inhibitory effects of miR-543 on SIRT1 were also validated using clinical gastric cancer samples. Moreover, we found that miR-543 expression was positively associated with tumor size, clinical grade, TNM stage and lymph node metastasis in gastric cancer patients. Our results identify a new regulatory mechanism of miR-543 on SIRT1 expression in gastric cancer, and raise the possibility that the miR-543/SIRT1 pathway may serve as a potential target for the treatment of gastric cancer. - Highlights: • SIRT1 is a novel target of miR-543. • miR-543 promotes gastric cancer cell proliferation and cell cycle progression by targeting SIRT1. • miR-543 is upregulated in GC and positively associated with tumor size, clinical grade, TNM stage and lymph node metastasis. • miR-543 is negatively correlated with SIRT1 expression in gastric cancer tissues.

  8. Cardiac Sirt1 mediates the cardioprotective effect of caloric restriction by suppressing local complement system activation after ischemia-reperfusion.

    Science.gov (United States)

    Yamamoto, Tsunehisa; Tamaki, Kayoko; Shirakawa, Kohsuke; Ito, Kentaro; Yan, Xiaoxiang; Katsumata, Yoshinori; Anzai, Atsushi; Matsuhashi, Tomohiro; Endo, Jin; Inaba, Takaaki; Tsubota, Kazuo; Sano, Motoaki; Fukuda, Keiichi; Shinmura, Ken

    2016-04-15

    Caloric restriction (CR) confers cardioprotection against ischemia-reperfusion (I/R) injury. We previously found the essential roles of endothelial nitric oxide synthase in the development of CR-induced cardioprotection and Sirt1 activation during CR (Shinmura K, Tamaki K, Ito K, Yan X, Yamamoto T, Katsumata Y, Matsuhashi T, Sano M, Fukuda K, Suematsu M, Ishii I. Indispensable role of endothelial nitric oxide synthase in caloric restriction-induced cardioprotection against ischemia-reperfusion injury.Am J Physiol Heart Circ Physiol 308: H894-H903, 2015). However, the exact mechanism by which Sirt1 in cardiomyocytes mediates the cardioprotective effect of CR remains undetermined. We subjected cardiomyocyte-specific Sirt1 knockout (CM-Sirt1(-/-)) mice and the corresponding control mice to either 3-mo ad libitum feeding or CR (-40%). Isolated perfused hearts were subjected to 25-min global ischemia, followed by 60-min reperfusion. The recovery of left ventricle function after I/R was improved, and total lactate dehydrogenase release into the perfusate during reperfusion was attenuated in the control mice treated with CR, but a similar cardioprotective effect of CR was not observed in the CM-Sirt1(-/-)mice. The expression levels of cardiac complement component 3 (C3) at baseline and the accumulation of C3 and its fragments in the ischemia-reperfused myocardium were attenuated by CR in the control mice, but not in the CM-Sirt1(-/-)mice. Resveratrol treatment also attenuated the expression levels of C3 protein in cultured neonatal rat ventricular cardiomyocytes. Moreover, the degree of myocardial I/R injury in conventional C3 knockout (C3(-/-)) mice treated with CR was similar to that in the ad libitum-fed C3(-/-)mice, although the expression levels of Sirt1 were enhanced by CR. These results demonstrate that cardiac Sirt1 plays an essential role in CR-induced cardioprotection against I/R injury by suppressing cardiac C3 expression. This is the first report suggesting

  9. Fisetin up-regulates the expression of adiponectin in 3T3-L1 adipocytes via the activation of silent mating type information regulation 2 homologue 1 (SIRT1)-deacetylase and peroxisome proliferator-activated receptors (PPARs).

    Science.gov (United States)

    Jin, Taewon; Kim, Oh Yoen; Shin, Min-Jeong; Choi, Eun Young; Lee, Sung Sook; Han, Ye Sun; Chung, Ji Hyung

    2014-10-29

    Adiponectin, an adipokine, has been described as showing physiological benefits against obesity-related malfunctions and vascular dysfunction. Several natural compounds that promote the expression and secretion of adipokines in adipocytes could be useful for treating metabolic disorders. This study investigated the effect of fisetin, a dietary flavonoid, on the regulation of adiponectin in adipocytes using 3T3-L1 preadipocytes. The expression and secretion of adiponectin increased in 3T3-L1 cells upon treatment with fisetin in a dose-dependent manner. Fisetin-induced adiponectin secretion was inhibited by peroxisome proliferator-activated receptor (PPAR) antagonists. It was also revealed that fisetin increased the activities of PPARs and silent mating type information regulation 2 homologue 1 (SIRT1) in a dose-dependent manner. Furthermore, the up-regulation of adiponectin and the activation of PPARs induced by fisetin were prevented by a SIRT1 inhibitor. Fisetin also promoted deacetylation of PPAR γ coactivator 1 (PGC-1) and its interaction with PPARs. SIRT knockdown by siRNA significantly decreased both adiponectin production and PPARs-PGC-1 interaction. These results provide evidence that fisetin promotes the gene expression of adiponectin through the activation of SIRT1 and PPARs in adipocytes.

  10. Disruption of the Circadian Clock Alters Antioxidative Defense via the SIRT1-BMAL1 Pathway in 6-OHDA-Induced Models of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Yali Wang

    2018-01-01

    Full Text Available Parkinson’s disease (PD is the second most common neurodegenerative disease and is known to involve circadian dysfunction and oxidative stress. Although antioxidative defense is regulated by the molecular circadian clock, few studies have examined their function in PD and their regulation by silent information regulator 1 (SIRT1. We hypothesize that reduced antioxidative activity in models of PD results from dysfunction of the molecular circadian clock via the SIRT1 pathway. We treated rats and SH-SY5Y cells with 6-hydroxydopamine (6-OHDA and measured the expression of core circadian clock and associated nuclear receptor genes using real-time quantitative PCR as well as levels of SIRT1, brain and muscle Arnt-like protein 1 (BMAL1, and acetylated BMAL1 using Western blotting. We found that 6-OHDA treatment altered the expression patterns of clock and antioxidative molecules in vivo and in vitro. We also detected an increased ratio of acetylated BMAL1:BMAL1 and a decreased level of SIRT1. Furthermore, resveratrol, an activator of SIRT1, decreased the acetylation of BMAL1 and inhibited its binding with CRY1, thereby reversing the impaired antioxidative activity induced by 6-OHDA. These results suggest that a dysfunctional circadian clock contributes to an abnormal antioxidative response in PD via a SIRT1-dependent BMAL1 pathway.

  11. Resveratrol Induced Premature Senescence Is Associated with DNA Damage Mediated SIRT1 and SIRT2 Down-Regulation.

    Directory of Open Access Journals (Sweden)

    Mehtap Kilic Eren

    Full Text Available The natural polyphenolic compound resveratrol (3,4,5-trihydroxy-trans-stilbene has broad spectrum health beneficial activities including antioxidant, anti-inflammatory, anti-aging, anti-cancer, cardioprotective, and neuroprotective effects. Remarkably, resveratrol also induces apoptosis and cellular senescence in primary and cancer cells. Resveratrol's anti-aging effects both in vitro and in vivo attributed to activation of a (NAD-dependent histone deacetylase family member sirtuin-1 (SIRT1 protein. In mammals seven members (SIRT1-7 of sirtuin family have been identified. Among those, SIRT1 is the most extensively studied with perceptive effects on mammalian physiology and suppression of the diseases of aging. Yet no data has specified the role of sirtuins, under conditions where resveratrol treatment induces senescence. Current study was undertaken to investigate the effects of resveratrol in human primary dermal fibroblasts (BJ and to clarify the role of sirtuin family members in particular SIRT1 and SIRT2 that are known to be involved in cellular stress responses and cell cycle, respectively. Here, we show that resveratrol decreases proliferation of BJ cells in a time and dose dependent manner. In addition the increase in senescence associated β-galactosidase (SA-β-gal activity and methylated H3K9-me indicate the induction of premature senescence. A significant increase in phosphorylation of γ-H2AX, a surrogate of DNA double strand breaks, as well as in levels of p53, p21CIP1 and p16INK4A is also detected. Interestingly, at concentrations where resveratrol induced premature senescence we show a significant decrease in SIRT1 and SIRT2 levels by Western Blot and quantitative RT-PCR analysis. Conversely inhibition of SIRT1 and SIRT2 via siRNA or sirtinol treatment also induced senescence in BJ fibroblasts associated with increased SA-β-gal activity, γ-H2AX phosphorylation and p53, p21CIP1 and p16INK4A levels. Interestingly DNA damaging

  12. Spinal SIRT1 activation attenuates neuropathic pain in mice.

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    Haijun Shao

    Full Text Available Abnormal histone acetylation occurs during neuropathic pain through an epigenetic mechanism. Silent information regulator 1 (sir2 or SIRT1, a NAD-dependent deacetylase, plays complex systemic roles in a variety of processes through deacetylating acetylated histone and other specific substrates. But the role of SIRT1 in neuropathic pain is not well established yet. The present study was intended to detect SIRT1 content and activity, nicotinamide (NAM and nicotinamide adenine dinucleotide (NAD in the spinal cord using immunoblotting or mass spectroscopy over time in mice following chronic constriction injury (CCI or sham surgery. In addition, the effect of intrathecal injection of NAD or resveratrol on thermal hyperalgesia and mechanical allodynia was evaluated in CCI mice. Finally, we investigated whether SIRT1 inhibitor EX-527 could reverse the anti-nociceptive effect of NAD or resveratrol. It was found that spinal SIRT1 expression, deacetylase activity and NAD/NAM decreased significantly 1, 3, 7, 14 and 21 days after CCI surgery as compared with sham group. In addition, daily intrathecal injection of 5 µl 800 mM NAD 1 h before and 1 day after CCI surgery or single intrathecal injection of 5 µl 90 mM resveratrol 1 h before CCI surgery produced a transient inhibitory effect on thermal hyperalgesia and mechanical allodynia in CCI mice. Finally, an intrathecal injection of 5 µl 1.2 mM EX-527 1 h before NAD or resveratrol administration reversed the anti-nociceptive effect of NAD or resveratrol. These data indicate that the reduction in SIRT1 deacetylase activity may be a factor contributing to the development of neuropathic pain in CCI mice. Our findings suggest that the enhancement of spinal NAD/NAM and/or SIRT1 activity may be a potentially promising strategy for the prevention or treatment of neuropathic pain.

  13. Depigmenting Effect of Resveratrol Is Dependent on FOXO3a Activation without SIRT1 Activation.

    Science.gov (United States)

    Kwon, Soon-Hyo; Choi, Hye-Ryung; Kang, Youn-A; Park, Kyoung-Chan

    2017-06-07

    Resveratrol exhibits not only anti-melanogenic property by inhibiting microphthalmia-associated transcription factor (MITF), but also anti-aging property by activating sirtuin-1 (SIRT1). In this study, the relationship between depigmenting effect of resveratrol and SIRT1/forkhead box O (FOXO) 3a activation and was investigated. Resveratrol suppressed melanogenesis by the downregulation of MITF and tyrosinase via ERK pathway. Results showed that the expression of both SIRT1 and FOXO3a were increased. It is reported that SIRT1 is critical regulator of FOXO-mediated transcription in response to oxidative stress. However in our study, FOXO3a activation appeared earlier than that of SIRT1. Furthermore, the effect of resveratrol on the levels of MITF and tyrosinase was suppressed when melanocytes were pre-treated with SP600125 (JNK inhibitor). However, pre-treatment with SIRT1 inhibitor (EX527, or sirtinol) did not affect the levels of MITF and tyrosinase. Therefore, resveratrol inhibits melanogenesis through the activation of FOXO3a but not by the activation of SIRT1. Although SIRT1 activation by resveratrol is a well-known mechanism of resveratrol-induced antiaging effects, our study showed that not SIRT1 but FOXO3a activation is involved in depigmenting effects of resveratrol.

  14. Opposing effects of sirtuins on neuronal survival: SIRT1-mediated neuroprotection is independent of its deacetylase activity.

    Directory of Open Access Journals (Sweden)

    Jason A Pfister

    Full Text Available BACKGROUND: Growing evidence suggests that sirtuins, a family of seven distinct NAD-dependent enzymes, are involved in the regulation of neuronal survival. Indeed, SIRT1 has been reported to protect against neuronal death, while SIRT2 promotes neurodegeneration. The effect of SIRTs 3-7 on the regulation of neuronal survival, if any, has yet to be reported. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the effect of expressing each of the seven SIRT proteins in healthy cerebellar granule neurons (CGNs or in neurons induced to die by low potassium (LK treatment. We report that SIRT1 protects neurons from LK-induced apoptosis, while SIRT2, SIRT3 and SIRT6 induce apoptosis in otherwise healthy neurons. SIRT5 is generally localized to both the nucleus and cytoplasm of CGNs and exerts a protective effect. In a subset of neurons, however, SIRT5 localizes to the mitochondria and in this case it promotes neuronal death. Interestingly, the protective effect of SIRT1 in neurons is not reduced by treatments with nicotinamide or sirtinol, two pharmacological inhibitors of SIRT1. Neuroprotection was also observed with two separate mutant forms of SIRT1, H363Y and H355A, both of which lack deacetylase activity. Furthermore, LK-induced neuronal death was not prevented by resveratrol, a pharmacological activator of SIRT1, at concentrations at which it activates SIRT1. We extended our analysis to HT-22 neuroblastoma cells which can be induced to die by homocysteic acid treatment. While the effects of most of the SIRT proteins were similar to that observed in CGNs, SIRT6 was modestly protective against homocysteic acid toxicity in HT-22 cells. SIRT5 was generally localized in the mitochondria of HT-22 cells and was apoptotic. CONCLUSIONS/SIGNIFICANCE: Overall, our study makes three contributions - (a it represents the first analysis of SIRT3-7 in the regulation of neuronal survival, (b it shows that neuroprotection by SIRT1 can be mediated by a novel, non

  15. Fisetin inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes through activating SIRT1 and attenuates the progression of osteoarthritis in mice.

    Science.gov (United States)

    Zheng, Wenhao; Feng, Zhenhua; You, Shengban; Zhang, Hui; Tao, Zhenyu; Wang, Quan; Chen, Hua; Wu, Yaosen

    2017-04-01

    Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Fisetin, a polyphenol extracted from fruits and vegetables, has been reported to have anti-inflammatory effects. Our study aimed to investigate the effect of fisetin on OA both in vitro and in vivo. In vitro, chondrocytes were pretreated with fisetin alone or fisetin combined with sirtinol (an inhibitor of SIRT1) for 2h before IL-1β stimulation. Production of NO, PGE2, TNF-α and IL-6 were evaluated by the Griess reaction and ELISAs. The mRNA (COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5, Sox-9, aggrecan and collagen-II) and protein expression (COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5 and SIRT1) were measured by qRT-PCR and Western blot respectively. Immunofluorescence was used to assess the expression of collagen-II and SIRT1. SIRT1 activity was quantified with SIRT1 fluorometric assay kit. The in vivo effect of fisetin was evaluated by gavage in mice OA models induced by destabilization of the medial meniscus (DMM). We found that fisetin inhibited IL-1β-induced expression of NO, PGE2, TNF-α, IL-6, COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5. Besides, fisetin remarkably decreased IL-1β-induced degradation of Sox-9, aggrecan and collagen-II. Furthermore, fisetin significantly inhibited IL-1β-induced SIRT1 decrease and inactivation. However, the inhibitory effect of fisetin was obvious abolished by sirtinol, suggesting that fisetin exerts anti-inflammatory effects through activating SIRT1. In vivo, fisetin-treated mice exhibited less cartilage destruction and lower OARSI scores. Moreover, fisetin reduced subchondral bone plate thickness and alleviated synovitis. Taken together, these findings indicate that fisetin may be a potential agent in the treatment of OA. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. SIRT1 inhibitory diterpenoids from the Vietnamese medicinal plant Croton tonkinensis

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Le, Thi Van; Nguyen, Phi Hung

    2010-01-01

    Silent information regulator two ortholog 1 (SIRT1) is a member of the sirtuin deacetylase family of enzymes that removes acetyl groups from the lysine residues in histones and other proteins. It has been suggested that SIRT1 inhibitors might be beneficial in the treatment of cancer and neurodege...

  17. Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats.

    Science.gov (United States)

    Ding, Shibin; Jiang, Jinjin; Zhang, Guofu; Bu, Yongjun; Zhang, Guanghui; Zhao, Xiangmei

    2017-01-01

    Studies have demonstrated that resveratrol (a natural polyphenol) and caloric restriction activate Sirtuin-1 (SIRT1) and induce autophagy. Furthermore, autophagy is induced by the SIRT1-FoxO signaling pathway and was recently shown to be a critical protective mechanism against non-alcoholic fatty liver disease (NAFLD) development. We aimed to compare the effects of resveratrol and caloric restriction on hepatic lipid metabolism and elucidate the mechanism by which resveratrol supplementation and caloric restriction alleviate hepatosteatosis by examining the molecular interplay between SIRT1 and autophagy. Eight-week-old male Wistar rats (40) were divided into four groups: the STD group, which was fed a standard chow diet; the HFD group, which was fed a high-fat diet; HFD-RES group, which was fed a high-fat diet plus resveratrol (200 mg/kg.bw); and the HFD-CR group, which was fed a high-fat diet in portions containing 70% of the mean intake of the HFD group rats. The groups were maintained for 18 weeks. Metabolic parameters, Oil Red O and hematoxylin-eosin staining of the liver, and the mRNA and protein expression of SIRT1, autophagy markers and endoplasmic reticulum(ER) stress-associated genes in the liver were assessed after the 18-week treatment. We found that resveratrol (200 mg/kg bw) and caloric restriction (30%) partially prevented hepatic steatosis and hepatocyte ballooning, increased the expression of SIRT1 and autophagy markers while decreasing ER stress markers in the liver and alleviated lipid metabolism disorder. Moreover, caloric restriction provided superior protection against HFD-induced hepatic fatty accumulation compared with resveratrol and the effects were associated with decreased total energy intake and body weight. We conclude that the SIRT1-autophagy pathway and decreased ER stress are universally required for the protective effects of moderate caloric restriction (30%) and resveratrol (a pharmacological SIRT1 activator) supplementation

  18. Resveratrol and caloric restriction prevent hepatic steatosis by regulating SIRT1-autophagy pathway and alleviating endoplasmic reticulum stress in high-fat diet-fed rats.

    Directory of Open Access Journals (Sweden)

    Shibin Ding

    Full Text Available Studies have demonstrated that resveratrol (a natural polyphenol and caloric restriction activate Sirtuin-1 (SIRT1 and induce autophagy. Furthermore, autophagy is induced by the SIRT1-FoxO signaling pathway and was recently shown to be a critical protective mechanism against non-alcoholic fatty liver disease (NAFLD development. We aimed to compare the effects of resveratrol and caloric restriction on hepatic lipid metabolism and elucidate the mechanism by which resveratrol supplementation and caloric restriction alleviate hepatosteatosis by examining the molecular interplay between SIRT1 and autophagy.Eight-week-old male Wistar rats (40 were divided into four groups: the STD group, which was fed a standard chow diet; the HFD group, which was fed a high-fat diet; HFD-RES group, which was fed a high-fat diet plus resveratrol (200 mg/kg.bw; and the HFD-CR group, which was fed a high-fat diet in portions containing 70% of the mean intake of the HFD group rats. The groups were maintained for 18 weeks. Metabolic parameters, Oil Red O and hematoxylin-eosin staining of the liver, and the mRNA and protein expression of SIRT1, autophagy markers and endoplasmic reticulum(ER stress-associated genes in the liver were assessed after the 18-week treatment. We found that resveratrol (200 mg/kg bw and caloric restriction (30% partially prevented hepatic steatosis and hepatocyte ballooning, increased the expression of SIRT1 and autophagy markers while decreasing ER stress markers in the liver and alleviated lipid metabolism disorder. Moreover, caloric restriction provided superior protection against HFD-induced hepatic fatty accumulation compared with resveratrol and the effects were associated with decreased total energy intake and body weight.We conclude that the SIRT1-autophagy pathway and decreased ER stress are universally required for the protective effects of moderate caloric restriction (30% and resveratrol (a pharmacological SIRT1 activator

  19. Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes

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    Chunzi Liang

    2014-01-01

    Full Text Available Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB, a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM, alanine (0.5 mM, valine (0.5 mM, EX527 (SIRT1 inhibitor, 25 μM, and Compound C (AMPK inhibitor, 25 μM alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.

  20. Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Xiaomei; Hu, Yan; Zhai, Xiaohan; Lin, Musen [Department of Pharmacology, Dalian Medical University, Dalian 116044 (China); Chen, Zhao; Tian, Xiaofeng; Zhang, Feng [Department of General Surgery, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Gao, Dongyan; Ma, Xiaochi [Department of Pharmacology, Dalian Medical University, Dalian 116044 (China); Lv, Li, E-mail: lv_li@126.com [Department of Pharmacology, Dalian Medical University, Dalian 116044 (China); Yao, Jihong, E-mail: Yaojihong65@hotmail.com [Department of Pharmacology, Dalian Medical University, Dalian 116044 (China)

    2013-11-15

    Salvianolic acid A (SalA) is a phenolic carboxylic acid derivative extracted from Salvia miltiorrhiza. It has many biological and pharmaceutical activities. The purpose of this study was to investigate the effect of SalA on concanavalin A (ConA)-induced acute hepatic injury in Kunming mice and to explore the role of SIRT1 in such an effect. The results showed that in vivo pretreatment with SalA significantly reduced ConA-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and decreased levels of the hepatotoxic cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Moreover, the SalA pretreatment ameliorated the increases in NF-κB and in cleaved caspase-3 caused by ConA exposure. Whereas, the pretreatment completely reversed expression of the B-cell lymphoma-extra large (Bcl-xL). More importantly, the SalA pretreatment significantly increased the expression of SIRT1, a NAD{sup +}-dependent deacetylase, which was known to attenuate acute hypoxia damage and metabolic liver diseases. In our study, the increase in SIRT1 was closely associated with down-regulation of the p66 isoform (p66shc) of growth factor adapter Shc at both protein and mRNA levels. In HepG2 cell culture, SalA pretreatment increased SIRT1 expression in a time and dose-dependent manner and such an increase was abrogated by siRNA knockdown of SIRT1. Additionally, inhibition of SIRT1 significantly reversed the decreased expression of p66shc, and attenuated SalA-induced p66shc down-regulation. Collectively, the present study indicated that SalA may be a potent activator of SIRT and that SalA can alleviate ConA-induced hepatitis through SIRT1-mediated repression of the p66shc pathway. - Highlights: • We report for the first time that SalA protects against ConA-induced hepatitis. • We find that SalA is a potential activator of SIRT1. • SalA's protection against hepatitis involves SIRT1-mediated repression of p

  1. Angiogenic dysfunction in bone marrow-derived early outgrowth cells from diabetic animals is attenuated by SIRT1 activation.

    Science.gov (United States)

    Yuen, Darren A; Zhang, Yanling; Thai, Kerri; Spring, Christopher; Chan, Lauren; Guo, Xiaoxin; Advani, Andrew; Sivak, Jeremy M; Gilbert, Richard E

    2012-12-01

    Impaired endothelial repair is a key contributor to microvascular rarefaction and consequent end-organ dysfunction in diabetes. Recent studies suggest an important role for bone marrow-derived early outgrowth cells (EOCs) in mediating endothelial repair, but the function of these cells is impaired in diabetes, as in advanced age. We sought to determine whether diabetes-associated EOC dysfunction might be attenuated by pharmacological activation of silent information regulator protein 1 (SIRT1), a lysine deacetylase implicated in nutrient-dependent life span extension in mammals. Despite being cultured in normal (5.5 mM) glucose for 7 days, EOCs from diabetic rats expressed less SIRT1 mRNA, induced less endothelial tube formation in vitro and neovascularization in vivo, and secreted less of the proangiogenic ELR(+) CXC chemokines CXCL1, CXCL3, and CXCL5. Ex vivo SIRT1 activation restored EOC chemokine secretion and increased the in vitro and in vivo angiogenic activity of EOC conditioned medium derived from diabetic animals to levels similar to that derived from control animals. These findings suggest a pivotal role for SIRT1 in diabetes-induced EOC dysfunction and that its pharmacologic activation may provide a new strategy for the restoration of EOC-mediated repair mechanisms.

  2. Reduction in podocyte SIRT1 accelerates kidney injury in aging mice.

    Science.gov (United States)

    Chuang, Peter Y; Cai, Weijing; Li, Xuezhu; Fang, Lu; Xu, Jin; Yacoub, Rabi; He, John Cijiang; Lee, Kyung

    2017-09-01

    Both the incidence and prevalence of chronic kidney disease are increasing in the elderly population. Although aging is known to induce kidney injury, the underlying molecular mechanisms remain unclear. Sirtuin 1 (Sirt1), a longevity gene, is known to protect kidney cell injury from various cellular stresses. In previous studies, we showed that the podocyte-specific loss of Sirt1 aggravates diabetic kidney injury. However, the role of Sirt1 in aging-induced podocyte injury is not known. Therefore, in this study we sought to determine the effects of podocyte-specific reduction of Sirt1 in age-induced kidney injury. We employed the inducible podocyte-specific Sirt1 knockdown mice that express shRNA against Sirt1 (Pod-Sirt1 RNAi ) and control mice that express shRNA for luciferase (Pod-Luci RNAi ). We found that reduction of podocyte Sirt1 led to aggravated aging-induced glomerulosclerosis and albuminuria. In addition, urinary level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, was markedly increased in aged Pod-Sirt1 RNAi mice compared with aged Pod-Luci RNAi mice. Although podocyte-specific markers decreased in aged mice compared with the young controls, the decrease was further exacerbated in aged Pod-Sirt1 RNAi compared with Pod-Luci RNAi mice. Interestingly, expression of cellular senescence markers was significantly higher in the glomeruli of Pod-Sirt1 RNAi mice than Pod-Luci RNAi mice, suggesting that cellular senescence may contribute to podocyte loss in aging kidneys. Finally, we confirmed that Pod-Sirt1 RNAi glomeruli were associated with reduced activation of the transcription factors peroxisome proliferator-activated receptor (PPAR)-α coactivador-1 (PGC1α)/PPARγ, forkhead box O (FOXO)3, FOXO4, and p65 NF-κB, through SIRT1-mediated deacetylation. Together, our data suggest that SIRT1 may be a potential therapeutic target to treat patients with aging-related kidney disease.

  3. SirT1 regulates energy metabolism and response to caloric restriction in mice.

    Directory of Open Access Journals (Sweden)

    Gino Boily

    Full Text Available The yeast sir2 gene and its orthologues in Drosophila and C. elegans have well-established roles in lifespan determination and response to caloric restriction. We have studied mice carrying two null alleles for SirT1, the mammalian orthologue of sir2, and found that these animals inefficiently utilize ingested food. These mice are hypermetabolic, contain inefficient liver mitochondria, and have elevated rates of lipid oxidation. When challenged with a 40% reduction in caloric intake, normal mice maintained their metabolic rate and increased their physical activity while the metabolic rate of SirT1-null mice dropped and their activity did not increase. Moreover, CR did not extend lifespan of SirT1-null mice. Thus, SirT1 is an important regulator of energy metabolism and, like its orthologues from simpler eukaryotes, the SirT1 protein appears to be required for a normal response to caloric restriction.

  4. The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth.

    Directory of Open Access Journals (Sweden)

    Ron Firestein

    2008-04-01

    Full Text Available Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD(+-dependent deacetylases is proposed to underlie the health benefits of calorie restriction (CR, a diet that broadly suppresses cancer in mammals. Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a beta-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates beta-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of beta-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of beta-catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in beta-catenin-driven malignancies.

  5. Distinct HIC1-SIRT1-p53 Loop Deregulation in Lung Squamous Carcinoma and Adenocarcinoma Patients

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    Ruo-Chia Tseng

    2009-08-01

    Full Text Available A HIC1-SIRT1-p53 circular loop in which hypermethylation in cancer 1 (HIC1 represses the transcription of SIRT1 that deacetylates and inactivates p53 thus leading to HIC1 inactivation has been identified in cell and animal models. However, the alteration and prognostic effects of HIC1-SIRT1-p53 circular loop have never been demonstrated in human cancer patients. We examine the HIC1-SIRT1-p53 alterations in 118 lung cancer patients to define their etiological roles in tumorigenesis. We found that patients with lung squamous cell carcinoma with low p53 acetylation and SIRT1 expression mostly showed low HIC1 expression, confirming deregulation of HIC1-SIRT1-p53 circular loop in the clinical model. Interestingly, the expression of deleted in breast cancer 1 (DBC1, which blocks the interaction between SIRT1 deacetylase and p53, led to acetylated p53 in patients with lung adenocarcinoma. However, epigenetic alteration of HIC1 promoter by posttranslational modifications of histones and promoter hypermethylation favoring the compacted chromatin production attenuated the transcriptional induction by acetylated p53. Importantly, lung cancer patients with altered HIC1-SIRT1-p53 circular regulation showed poor prognosis. Our data show the first valid clinical evidence of the deregulation of HIC1-SIRT1-p53 loop in lung tumorigenesis and prognosis. Distinct status of p53 acetylation/deacetylation and HIC1 alteration mechanism result from different SIRT1-DBC1 control and epigenetic alteration in lung squamous cell carcinoma and lung adenocarcinoma.

  6. Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver.

    Science.gov (United States)

    Choi, Sung E; Kwon, Sanghoon; Seok, Sunmi; Xiao, Zhen; Lee, Kwan-Woo; Kang, Yup; Li, Xiaoling; Shinoda, Kosaku; Kajimura, Shingo; Kemper, Byron; Kemper, Jongsook Kim

    2017-08-01

    Sirtuin1 (SIRT1) deacetylase delays and improves many obesity-related diseases, including nonalcoholic fatty liver disease (NAFLD) and diabetes, and has received great attention as a drug target. SIRT1 function is aberrantly low in obesity, so understanding the underlying mechanisms is important for drug development. Here, we show that obesity-linked phosphorylation of SIRT1 inhibits its function and promotes pathological symptoms of NAFLD. In proteomic analysis, Ser-164 was identified as a major serine phosphorylation site in SIRT1 in obese, but not lean, mice, and this phosphorylation was catalyzed by casein kinase 2 (CK2), the levels of which were dramatically elevated in obesity. Mechanistically, phosphorylation of SIRT1 at Ser-164 substantially inhibited its nuclear localization and modestly affected its deacetylase activity. Adenovirus-mediated liver-specific expression of SIRT1 or a phosphor-defective S164A-SIRT1 mutant promoted fatty acid oxidation and ameliorated liver steatosis and glucose intolerance in diet-induced obese mice, but these beneficial effects were not observed in mice expressing a phosphor-mimic S164D-SIRT1 mutant. Remarkably, phosphorylated S164-SIRT1 and CK2 levels were also highly elevated in liver samples of NAFLD patients and correlated with disease severity. Thus, inhibition of phosphorylation of SIRT1 by CK2 may serve as a new therapeutic approach for treatment of NAFLD and other obesity-related diseases. Copyright © 2017 American Society for Microbiology.

  7. Inhibition of SIRT1 Transcription inResveratrol-differentiated Medulloblastoma Cells

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    Jing-Xin Ma

    2013-05-01

    Full Text Available ABSTRACTBackgrounds: Medulloblastoma(MB is the commonestbrain malignancyin childhood with poor prognosis, because of itsrapid aggressive growth and frequent occurrence. The current chemotherapeutic regimens for medulloblastoma patients involve a combination of lomustine, cisplatin, carboplatin, vincristine or cyclophosphamide, which have distinct short-and long-term side-effects. It is therefore in urgent need to explore safer and more effective adjuvant approach(s.Resveratrol, a polyphenol rich in numerous plants, has multiple biological activities including anticancer effects. Our previous data confirmed that resveratrolinhibited proliferation and induced differentiation and apoptosis of medulloblastoma cells. SIRT1 is a deacetylase of class III HDACs and the supposed molecular effecter of resveratrol. SIRT1 involves in aging prevention and cancer formation in a cell-context specific manner.Nevertheless, the datum concerningthe role(s ofSIRT1 in formation and prognosis of medulloblastomais still missing.Objective:The present study aimed to address the expression patterna of SIRT1 in medulloblastoma tissuesand non-cancerous counterpartsand to explorewhether resveratrol exerts its anti-medulloblastoma effects via regulating SIRT1 expression and bioactivity.Methods:The expression of SIRT1 in medulloblastoma and non-cancerous counterparts was elucidatedby immunohistochemical ataining (IHC.To clarify the function of SIRT1 in medulloblastomas, SIRT1 expression in UW228-3 medulloblastoma cells were suppressed by RNA interference(RNAi. The influence of resveratrol in SIRT1 expressionsin UW228-3 cellswas analyzedby reverse transcription-polymerase chain reaction (RT-PCR,immunocytochemistry (ICCand Western blotting(WB. The catalytic activity of deacetylase SIRT1was examined by measuring the acetylation ofthe main substrate p53.Results: IHC staining revealedthat SIRT1 was expressed in 64.17% of MB tissues,which was higher than that in

  8. SIRT1 overexpression decreases cisplatin-induced acetylation of NF-{kappa}B p65 subunit and cytotoxicity in renal proximal tubule cells

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    Jung, Yu Jin; Lee, Jung Eun; Lee, Ae Sin [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Lee, Sang Yong [Department of Diagnostic Radiology, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Han, Myung Kwan [Department of Microbiology, Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Kim, Duk Hoon [Division of Forensic Medicine, National Forensic Service, Seoul (Korea, Republic of); Kim, Won, E-mail: kwon@jbnu.ac.kr [Department of Internal Medicine, Chonbuk National University Medical School, Jeonju (Korea, Republic of); Institute for Medical Sciences, Chonbuk National University Medical School, Jeonju (Korea, Republic of)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Cisplatin increases acetylation of NF-{kappa}B p65 subunit in HK2 cells. Black-Right-Pointing-Pointer SIRT1 overexpression decreases cisplatin-induced p65 acetylation and -cytotoxicity. Black-Right-Pointing-Pointer Resveratrol decreased cisplatin-induced cell viability through deacetylation of p65. -- Abstract: As the increased acetylation of p65 is linked to nuclear factor-{kappa}B (NF-{kappa}B) activation, the regulation of p65 acetylation can be a potential target for the treatment of inflammatory injury. Cisplatin-induced nephrotoxicity is an important issue in chemotherapy of cancer patients. SIRT1, nicotinamide adenine dinucleotide (NAD{sup +})-dependent protein deacetylase, has been implicated in a variety of cellular processes such as inflammatory injury and the control of multidrug resistance in cancer. However, there is no report on the effect of SIRT1 overexpression on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury. To investigate the effect of SIRT1 in on cisplatin-induced acetylation of p65 subunit of NF-{kappa}B and cell injury, HK2 cells were exposed with SIRT1 overexpression, LacZ adenovirus or dominant negative adenovirus after treatment with cisplatin. While protein expression of SIRT1 was decreased by cisplatin treatment compared with control buffer treatment, acetylation of NF-{kappa}B p65 subunit was significantly increased after treatment with cisplatin. Overexpression of SIRT1 ameliorated the increased acetylation of p65 of NF-{kappa}B during cisplatin treatment and cisplatin-induced cytotoxicity. Further, treatment of cisplatin-treated HK2 cells with resveratrol, a SIRT1 activator, also decreased acetylation of NF-{kappa}B p65 subunit and cisplatin-induced increase of the cell viability in HK2 cells. Our findings suggests that the regulation of acetylation of p65 of NF-{kappa}B through SIRT1 can be a possible target to attenuate cisplatin-induced renal cell damage.

  9. SIRT1 Suppresses Doxorubicin-Induced Cardiotoxicity by Regulating the Oxidative Stress and p38MAPK Pathways

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    Yang Ruan

    2015-02-01

    Full Text Available Background: SIRT1, which belongs to the Sirtuin family of NAD-dependent enzymes, plays diverse roles in aging, metabolism, and disease biology. It could regulate cell survival and has been shown to be a protective factor in heart function. Hence, we verified the mechanism by which SIRT1 regulates doxorubicin induced cardiomyocyte injury in vivo and in vitro. Methods: We analyzed SIRT1 expression in doxorubicin-induced neonatal rat cardiomyocyte injury model and adult mouse heart failure model. SIRT1 was over-expressed in cultured neonatal rat cardiomyocyte by adenovirus mediated gene transfer. SIRT1 agonist resveratrol was used to treat the doxorubicin-induced heart failure mouse model. Echocardiography, reactive oxygen species (ROS production, TUNEL, qRT-PCR, and Western blotting were performed to analyze cell survival, oxidative stress, and inflammatory signal pathways in cardiomyocytes. Results: SIRT1 expression was down-regulated in doxorubicin induced cardiomocyte injury, accompanied by elevated oxidative stress and cell apoptosis. SIRT1 over-expression reduced doxorubicin induced cardiomyocyte apoptosis with the attenuated ROS production. SIRT1 also reduced cell apoptosis by inhibition of p38MAPK phosphorylation and caspase-3 activation. The SIRT1 agonist resveratrol was able to prevent doxorubicin-induced heart function loss. Moreover, the SIRT1 inhibitor niacinamide could reverse SIRT1's protective effect in cultured neonatal rat cardiomyocytes. Conclusions: These results support the role of SIRT1 as an important regulator of cardiomyocyte apoptosis during doxorubicin-induced heart injury, which may represent a potential therapeutic target for doxorubicin-induced cardiomyopathy.

  10. Thyroid hormone regulation of Sirtuin 1 expression and implications to integrated responses in fasted mice.

    Science.gov (United States)

    Cordeiro, Aline; de Souza, Luana Lopes; Oliveira, Lorraine Soares; Faustino, Larissa Costa; Santiago, Letícia Aragão; Bloise, Flavia Fonseca; Ortiga-Carvalho, Tania Maria; Almeida, Norma Aparecida Dos Santos; Pazos-Moura, Carmen Cabanelas

    2013-02-01

    Sirtuin 1 (SIRT1), a NAD(+)-dependent deacetylase, has been connected to beneficial effects elicited by calorie restriction. Physiological adaptation to starvation requires higher activity of SIRT1 and also the suppression of thyroid hormone (TH) action to achieve energy conservation. Here, we tested the hypothesis that those two events are correlated and that TH may be a regulator of SIRT1 expression. Forty-eight-hour fasting mice exhibited reduced serum TH and increased SIRT1 protein content in liver and brown adipose tissue (BAT), and physiological thyroxine replacement prevented or attenuated the increment of SIRT1 in liver and BAT of fasted mice. Hypothyroid mice exhibited increased liver SIRT1 protein, while hyperthyroid ones showed decreased SIRT1 in liver and BAT. In the liver, decreased protein is accompanied by reduced SIRT1 activity and no alteration in its mRNA. Hyperthyroid and hypothyroid mice exhibited increases and decreases in food intake and body weight gain respectively. Food-restricted hyperthyroid animals (pair-fed to euthyroid group) exhibited liver and BAT SIRT1 protein levels intermediary between euthyroid and hyperthyroid mice fed ad libitum. Mice with TH resistance at the liver presented increased hepatic SIRT1 protein and activity, with no alteration in Sirt1 mRNA. These results suggest that TH decreases SIRT1 protein, directly and indirectly, via food ingestion control and, in the liver, this reduction involves TRβ. The SIRT1 reduction induced by TH has important implication to integrated metabolic responses to fasting, as the increase in SIRT1 protein requires the fasting-associated suppression of TH serum levels.

  11. mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway.

    Science.gov (United States)

    Zheng, Haixiang; Fu, Yucai; Huang, Yusheng; Zheng, Xinde; Yu, Wei; Wang, Wei

    2017-09-01

    Atherosclerosis (AS) is a chronic immuno‑inflammatory disease accompanied by dyslipidemia. The authors previously demonstrated that sirtuin 1 (SIRT1) may prevent atherogenesis through influencing the liver X receptor/C‑C chemokine receptor type 7/nuclear factor‑κB (LXR‑CCR7/NF‑κB) signaling pathway. Previous studies have suggested a role for mammalian target of rapamycin (mTOR) signaling in the pathogenesis of cardiovascular diseases. The present study investigated the potential association between mTOR signaling and SIRT1‑LXR‑CCR7/NF‑κB signaling (SIRT1 signaling) in AS pathogenesis. To induce foam cell formation, U937 cells were differentiated into macrophages by exposure to phorbol 12‑myristate 13‑acetate (PMA) for 24 h, followed by treatment with palmitate and oxidized low density lipoprotein for a further 24 h. Oil red O staining revealed a large accumulation of lipid droplets present in foam cells. Western blot analysis demonstrated increased protein levels of phosphorylated (p)‑mTOR and its downstream factor p‑ribosomal protein S6 kinase (p70S6K). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses additionally revealed decreased expression of SIRT1, LXRα and CCR7 and increased expression of NF‑κB and its downstream factor tumor necrosis factor‑α (TNF‑α) in an atherogenetic condition induced by lysophosphatidic acid (LPA). In addition, abundant lipid droplets accumulated in U937‑LPA‑treated foam cells. Rapamycin, an mTOR inhibitor, suppressed the expression and activity of mTOR and p70S6K, however enhanced expression of SIRT1, LXRα, and CCR7. Conversely, rapamycin deceased TNF‑α and NF‑κB activity, the latter of which was further confirmed by immunofluorescence analysis demonstrating increased levels of NF‑κB present in the cytoplasm compared with the nucleus. The findings of the present study suggest that mTOR signaling promotes foam cell formation and inhibits foam

  12. Molecular Insights into SIRT1 Protection Against UVB-Induced Skin Fibroblast Senescence by Suppression of Oxidative Stress and p53 Acetylation.

    Science.gov (United States)

    Chung, Ki Wung; Choi, Yeon Ja; Park, Min Hi; Jang, Eun Ji; Kim, Dae Hyun; Park, Byung Hyun; Yu, Byung Pal; Chung, Hae Young

    2015-08-01

    Stresses, such as exposure to ultraviolet radiation and those associated with aging, are known to cause premature cellular senescence that is characterized by growth arrest and morphological and gene expression changes. This study was designed to investigate the protective effect of Sirtuin1 (SIRT1) on the UVB-induced premature senescence. Under in vitro experimental conditions, exposure to a subcytotoxic dose of UVB enhanced human skin fibroblasts senescence, as characterized by increased β-galactosidase activity and increased levels of senescence-associated proteins. However, adenovirus-mediated SIRT1 overexpression significantly protected fibroblasts from UVB-induced cellular deterioration. Exposure to UVB-induced cell senescence was associated with oxidative stress and p38 mitogen-activated protein kinase activation. Molecular analysis demonstrated that deacetylation of Forkhead box O3α (FOXO3α) by SIRT1 changed the transcriptional activity of FOXO3α and increased resistance to the oxidative stress. In addition, SIRT1 suppressed UVB-induced p53 acetylation and its transcriptional activity, which directly affected the cell cycle arrest induced by UVB. Further study demonstrated that SIRT1 activation inhibited cell senescence in the skin of the HR1 hairless mouse exposed to UVB. The study identifies a new role for SIRT1 in the UVB-induced senescence of skin fibroblats and provides a potential target for skin protection through molecuar insights into the mechanisms responsible for UVB-induced photoaging. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. SIRT1 may play a crucial role in overload-induced hypertrophy of skeletal muscle.

    Science.gov (United States)

    Koltai, Erika; Bori, Zoltán; Chabert, Clovis; Dubouchaud, Hervé; Naito, Hisashi; Machida, Shuichi; Davies, Kelvin Ja; Murlasits, Zsolt; Fry, Andrew C; Boldogh, Istvan; Radak, Zsolt

    2017-06-01

    Silent mating type information regulation 2 homologue 1 (SIRT1) activity and content increased significantly in overload-induced hypertrophy. SIRT1-mediated signalling through Akt, the endothelial nitric oxide synthase mediated pathway, regulates anabolic process in the hypertrophy of skeletal muscle. The regulation of catabolic signalling via forkhead box O 1 and protein ubiquitination is SIRT1 dependent. Overload-induced changes in microRNA levels regulate SIRT1 and insulin-like growth factor 1 signalling. Significant skeletal muscle mass guarantees functional wellbeing and is important for high level performance in many sports. Although the molecular mechanism for skeletal muscle hypertrophy has been well studied, it still is not completely understood. In the present study, we used a functional overload model to induce plantaris muscle hypertrophy by surgically removing the soleus and gastrocnemius muscles in rats. Two weeks of muscle ablation resulted in a 40% increase in muscle mass, which was associated with a significant increase in silent mating type information regulation 2 homologue 1 (SIRT1) content and activity (P overload-induced hypertrophy. These findings, along with the well-known regulatory roles that SIRT1 plays in modulating both anabolic and catabolic pathways, allow us to propose the hypothesis that SIRT1 may actually play a crucial causal role in overload-induced hypertrophy of skeletal muscle. This hypothesis will now require rigorous direct and functional testing. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

  14. Overexpression of SIRT1 in mouse forebrain impairs lipid/glucose metabolism and motor function.

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    Dongmei Wu

    Full Text Available SIRT1 plays crucial roles in glucose and lipid metabolism, and has various functions in different tissues including brain. The brain-specific SIRT1 knockout mice display defects in somatotropic signaling, memory and synaptic plasticity. And the female mice without SIRT1 in POMC neuron are more sensitive to diet-induced obesity. Here we created transgenic mice overexpressing SIRT1 in striatum and hippocampus under the control of CaMKIIα promoter. These mice, especially females, exhibited increased fat accumulation accompanied by significant upregulation of adipogenic genes in white adipose tissue. Glucose tolerance of the mice was also impaired with decreased Glut4 mRNA levels in muscle. Moreover, the SIRT1 overexpressing mice showed decreased energy expenditure, and concomitantly mitochondria-related genes were decreased in muscle. In addition, these mice showed unusual spontaneous physical activity pattern, decreased activity in open field and rotarod performance. Further studies demonstrated that SIRT1 deacetylated IRS-2, and upregulated phosphorylation level of IRS-2 and ERK1/2 in striatum. Meanwhile, the neurotransmitter signaling in striatum and the expression of endocrine hormones in hypothalamus and serum T3, T4 levels were altered. Taken together, our findings demonstrate that SIRT1 in forebrain regulates lipid/glucose metabolism and motor function.

  15. Celastrol ameliorates liver metabolic damage caused by a high-fat diet through Sirt1

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    Yinliang Zhang

    2017-01-01

    Full Text Available Objective: Celastrol was recently identified as a potential novel treatment for obesity. However, the effect of Celastrol on nonalcoholic fatty liver disease (NAFLD remains elusive. The aim of this study is to evaluate the role of Celastrol in NAFLD. Methods: Functional studies were performed using wild-type C57BL/6J (WT mice and liver specific Sirt1-deficient (LKO mice. The molecular mechanism was explored in primary mouse liver and primary hepatocytes. Results: When WT mice receiving a high-fat diet (HFD were treated with Celastrol, reductions in body weight, subcutaneous and visceral fat content, and liver lipid droplet formation were observed, along with reduced hepatic intracellular triglyceride and serum triglyceride, free fatty acid, and ALT concentrations. Furthermore, Celastrol decreased hepatic sterol regulatory element binding protein 1c (Srebp-1c expression, enhanced the phosphorylation of hepatic AMP-activated protein kinase α (AMPKα, and increased the expression of hepatic serine–threonine liver kinase B1 (LKB1. Additionally, Celastrol treatment improved glucose tolerance and insulin sensitivity in WT mice fed the HFD. Celastrol administration also improved the anti-inflammatory and anti-oxidative status by inhibiting nuclear factor kappa B (NFκB activity and the mRNA levels of proinflammatory cytokines and increasing mitochondrial DNA copy number and anti-oxidative stress genes expression in WT mice liver, in vivo and in vitro. Moreover, Celastrol induced hepatic Sirt1 expression in WT mice, in vivo and in vitro. These Celastrol-mediated protective effects in WT mice fed a HFD were abolished in LKO mice fed a HFD. It was more interesting that Celastrol aggravated HFD-induced liver damage in LKO mice fed a HFD by inhibiting the phosphorylation of AMPKα and boosting the translocation of NFκB into the nucleus, thereby resulting in the increase of Srebp-1c expression and the mRNA levels of liver proinflammatory cytokines

  16. Impaired cardiac SIRT1 activity by carbonyl stress contributes to aging-related ischemic intolerance.

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    Chunhu Gu

    Full Text Available Reactive aldehydes can initiate protein oxidative damage which may contribute to heart senescence. Sirtuin 1 (SIRT1 is considered to be a potential interventional target for I/R injury management in the elderly. We hypothesized that aldehyde mediated carbonyl stress increases susceptibility of aged hearts to ischemia/reperfusion (I/R injury, and elucidate the underlying mechanisms with a focus on SIRT1. Male C57BL/6 young (4-6 mo and aged (22-24 mo mice were subjected to myocardial I/R. Cardiac aldehyde dehydrogenase (ALDH2, SIRT1 activity and protein carbonyls were assessed. Our data revealed that aged heart exhibited increased endogenous aldehyde/carbonyl stress due to impaired ALDH2 activity concomitant with blunted SIRT1 activity (P<0.05. Exogenous toxic aldehydes (4-HNE exposure in isolated cardiomyocyte verified that aldehyde-induced carbonyl modification on SIRT1 impaired SIRT1 activity leading to worse hypoxia/reoxygenation (H/R injury, which could all be rescued by Alda-1 (ALDH2 activator (all P<0.05. However, SIRT1 inhibitor blocked the protective effect of Alda-1 on H/R cardiomyocyte. Interestingly, myocardial I/R leads to higher carbonylation but lower activity of SIRT1 in aged hearts than that seen in young hearts (P<0.05. The application of Alda-1 significantly reduced the carbonylation on SIRT1 and markedly improved the tolerance to in vivo I/R injury in aged hearts, but failed to protect Sirt1(+/- knockout mice against myocardial I/R injury. This was verified by Alda-1 treatment improved postischemic contractile function recovery in ex vivo perfused aged but not in Sirt1(+/- hearts. Thus, aldehyde/carbonyl stress is accelerated in aging heart. These results provide a new insight that impaired cardiac SIRT1 activity by carbonyl stress plays a critical role in the increased susceptibility of aged heart to I/R injury. ALDH2 activation can restore this aging-related myocardial ischemic intolerance.

  17. Theobromine increases NAD⁺/Sirt-1 activity and protects the kidney under diabetic conditions.

    Science.gov (United States)

    Papadimitriou, Alexandros; Silva, Kamila C; Peixoto, Elisa B M I; Borges, Cynthia M; Lopes de Faria, Jacqueline M; Lopes de Faria, José B

    2015-02-01

    Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-β1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy. Copyright © 2015 the American Physiological Society.

  18. SirT1—A Sensor for Monitoring Self-Renewal and Aging Process in Retinal Stem Cells

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    Chi-Hsien Peng

    2010-06-01

    Full Text Available Retinal stem cells bear potency of proliferation, self-renewal, and differentiation into many retinal cells. Utilizing appropriate sensors one can effectively detect the self-renewal and aging process abilities. Silencing information regulator (SirT1, a member of the sirtuin family, is a NAD-dependent histone deacetylase and an essential mediator for longevity in normal cells by calorie restriction. We firstly investigate the SirT1 mRNA expression in retinal stem cells from rats and 19 human eyes of different ages. Results revealed that SirT1 expression was significantly decreased in in vivo aged eyes, associated with poor self-renewal abilities. Additionally, SirT1 mRNA levels were dose-dependently increased in resveratrol- treated retinal stem cells. The expression of SirT1 on oxidative stress-induced damage was significantly decreased, negatively correlated with the level of intracellular reactive oxygen species production. Treatment with resveratrol could effectively further reduce oxidative stress induced by H2O2 treatment in retinal stem cells. Importantly, the anti-oxidant effects of resveratrol in H2O2-treated retinal stem cells were significantly abolished by knockdown of SirT1 expression (sh-SirT1. SirT1 expression provides a feasible sensor in assessing self-renewal and aging process in retinal stem cells. Resveratrol can prevent reactive oxygen species-induced damages via increased retinal SirT1 expression.

  19. Sirtuin 1 stimulates the proliferation and the expression of glycolysis genes in pancreatic neoplastic lesions.

    Science.gov (United States)

    Pinho, Andreia V; Mawson, Amanda; Gill, Anthony; Arshi, Mehreen; Warmerdam, Max; Giry-Laterriere, Marc; Eling, Nils; Lie, Triyana; Kuster, Evelyne; Camargo, Simone; Biankin, Andrew V; Wu, Jianmin; Rooman, Ilse

    2016-11-15

    Metabolic reprogramming is a feature of neoplasia and tumor growth. Sirtuin 1 (SIRT1) is a lysine deacetylase of multiple targets including metabolic regulators such as p53. SIRT1 regulates metaplasia in the pancreas. Nevertheless, it is unclear if SIRT1 affects the development of neoplastic lesions and whether metabolic gene expression is altered.To assess neoplastic lesion development, mice with a pancreas-specific loss of Sirt1 (Pdx1-Cre;Sirt1-lox) were bred into a KrasG12D mutant background (KC) that predisposes to the development of pancreatic intra-epithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC). Similar grade PanIN lesions developed in KC and KC;Sirt1-lox mice but specifically early mucinous PanINs occupied 40% less area in the KC;Sirt1-lox line, attributed to reduced proliferation. This was accompanied by reduced expression of proteins in the glycolysis pathway, such as GLUT1 and GAPDH.The stimulatory effect of SIRT1 on proliferation and glycolysis gene expression was confirmed in a human PDAC cell line. In resected PDAC samples, higher proliferation and expression of glycolysis genes correlated with poor patient survival. SIRT1 expression per se was not prognostic but low expression of Cell Cycle and Apoptosis Regulator 2 (CCAR2), a reported SIRT1 inhibitor, corresponded to poor patient survival.These findings open perspectives for novel targeted therapies in pancreatic cancer.

  20. Sirtuin 1 stimulates the proliferation and the expression of glycolysis genes in pancreatic neoplastic lesions

    Science.gov (United States)

    Pinho, Andreia V.; Mawson, Amanda; Gill, Anthony; Arshi, Mehreen; Warmerdam, Max; Giry-Laterriere, Marc; Eling, Nils; Lie, Triyana; Kuster, Evelyne; Camargo, Simone; Biankin, Andrew V.; Wu, Jianmin; Rooman, Ilse

    2016-01-01

    Metabolic reprogramming is a feature of neoplasia and tumor growth. Sirtuin 1 (SIRT1) is a lysine deacetylase of multiple targets including metabolic regulators such as p53. SIRT1 regulates metaplasia in the pancreas. Nevertheless, it is unclear if SIRT1 affects the development of neoplastic lesions and whether metabolic gene expression is altered. To assess neoplastic lesion development, mice with a pancreas-specific loss of Sirt1 (Pdx1-Cre;Sirt1-lox) were bred into a KrasG12D mutant background (KC) that predisposes to the development of pancreatic intra-epithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC). Similar grade PanIN lesions developed in KC and KC;Sirt1-lox mice but specifically early mucinous PanINs occupied 40% less area in the KC;Sirt1-lox line, attributed to reduced proliferation. This was accompanied by reduced expression of proteins in the glycolysis pathway, such as GLUT1 and GAPDH. The stimulatory effect of SIRT1 on proliferation and glycolysis gene expression was confirmed in a human PDAC cell line. In resected PDAC samples, higher proliferation and expression of glycolysis genes correlated with poor patient survival. SIRT1 expression per se was not prognostic but low expression of Cell Cycle and Apoptosis Regulator 2 (CCAR2), a reported SIRT1 inhibitor, corresponded to poor patient survival. These findings open perspectives for novel targeted therapies in pancreatic cancer. PMID:27494892

  1. [Rosuvastatin improves insulin sensitivity in overweight rats induced by high fat diet. Role of SIRT1 in adipose tissue].

    Science.gov (United States)

    Valero-Muñoz, María; Martín-Fernández, Beatriz; Ballesteros, Sandra; Cachofeiro, Victoria; Lahera, Vicente; de Las Heras, Natalia

    2014-01-01

    To study the effects of rosuvastatin on insulin resistance in overweight rats induced by high fat diet, as well as potential mediators. We used male Wistar rats fed with a standard diet (CT) or high fat diet (33.5% fat) (HFD); half of the animals HFD were treated with rosuvastatin (15mg/kg/day) (HFD+Rosu) for 7 weeks. HFD rats showed increased body, epididymal and lumbar adipose tissue weights. Treatment with Rosu did not modify body weight or the weight of the adipose packages in HFD rat. Plasma glucose and insulin levels and HOMA index were higher in HFD rats, and rosuvastatin treatment reduced them. Leptin/adiponectin ratio in plasma and lumbar adipose tissue were higher in HDF rats, and were reduced by rosuvastatin. SIRT-1, PPAR-γ and GLUT-4 protein expression in lumbar adipose tissue were lower in HFD rats and Rosu normalized expression of the three mediators. Rosuvastatin ameliorates insulin sensitivity induced by HFD in rats. This effect is mediated by several mechanisms including reduction of leptin and enhancement of SIRT-1, PPAR-γ and GLUT-4 expression in white adipose tissue. SIRT1 could be considered a major mediator of the beneficial effects of rosuvastatin on insulin sensitivity in overweight rats induced by diet. Copyright © 2013 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

  2. The NAD+/PARP1/SIRT1 Axis in Aging.

    Science.gov (United States)

    Mendelsohn, Andrew R; Larrick, James W

    2017-06-01

    NAD+ levels decline with age in diverse animals from Caenorhabditis elegans to mice. Raising NAD+ levels by dietary supplementation with NAD+ precursors, nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN), improves mitochondrial function and muscle and neural and melanocyte stem cell function in mice, as well as increases murine life span. Decreased NAD+ levels with age reduce SIRT1 function and reduce the mitochondrial unfolded protein response, which can be overcome by NR supplementation. Decreased NAD+ levels cause NAD+-binding protein DBC1 to form a complex with PARP1, inhibiting poly(adenosine diphosphate-ribose) polymerase (PARP) catalytic activity. Old mice have increased amounts of DBC1-PARP1 complexes, lower PARP activity, increased DNA damage, and reduced nonhomologous end joining and homologous recombination repair. DBC1-PARP1 complexes in old mice can be broken by increasing NAD+ levels through treatment with NMN, reducing DNA damage and restoring PARP activity to youthful levels. The mechanism of declining NAD+ levels and its fundamental importance to aging are yet to be elucidated. There is a correlation of PARP activity with mammalian life span that suggests that NAD+/SIRT1/PARP1 may be more significant than the modest effects on life span observed for NR supplementation in old mice. The NAD+/PARP1/SIRT1 axis may link NAD+ levels and DNA damage with the apparent epigenomic DNA methylation clocks that have been described.

  3. PODOCYTE-SPECIFIC OVEREXPRESSION OF SIRT-1 INCREASES NEPHRIN IN OBESE MICE FED A HIGH-FAT DIET

    Directory of Open Access Journals (Sweden)

    Han Byoung-Geun

    2012-06-01

    Taken together the results, deterioration of the kidney disease caused by obesity and hyperglycemia could be prevented by increasing the level of the nephrin expression through SIRT-1 activation. SIRT-1 may have the ability to protect the podocyte from injuries caused by obesity and hyperglycemia.

  4. Rev1 contributes to proper mitochondrial function via the PARP-NAD(+)-SIRT1-PGC1 alpha axis

    DEFF Research Database (Denmark)

    Fakouri, Nima Borhan; Durhuus, Jon Ambaek; Regnell, Christine Elisabeth

    2017-01-01

    (ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via...... the PARP-NAD+-SIRT1-PGC1α axis....

  5. Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats.

    Directory of Open Access Journals (Sweden)

    Nady Braidy

    2011-04-01

    Full Text Available The cofactor nicotinamide adenine dinucleotide (NAD+ has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose polymerase (PARP, an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months compared to young (i.e. 3 month old rats. These changes in [NAD(H] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.

  6. A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

    Science.gov (United States)

    Song, Tuzz-Ying; Yeh, Shu-Lan; Hu, Miao-Lin; Chen, Mei-Yau; Yang, Nae-Cherng

    2015-12-01

    Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation.

  7. Identification of a SIRT1 mutation in a family with type 1 diabetes

    DEFF Research Database (Denmark)

    Biason-Lauber, Anna; Böni-Schnetzler, Marianne; Hubbard, Basil P

    2013-01-01

    Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members...... developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin....... Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations...

  8. Resveratrol Improved the Progression of Chronic Prostatitis via the Downregulation of c-kit/SCF by Activating Sirt1.

    Science.gov (United States)

    He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Duan, Xingping; Liu, Qi; Yang, Bo

    2017-07-19

    The regulation mechanism of inflammation inducing prostate carcinogenesis remains largely unknown. Therefore, we investigated the role of the c-kit/SCF pathway, which has been associated with the control of prostate carcinogenesis, in chronic prostatitis (CP) rats and evaluated the anti-prostatitis effect of resveratrol. We performed hemolysin and eosin staining to evaluate the histopathological changes in prostates. Multiple approaches evaluated the expression levels of c-kit, stem cell factor (SCF), Sirt1, and carcinogenesis-associated proteins. The CP group exhibited severe diffuse chronic inflammation. Meanwhile, the prostate cells appeared atypia; the activity of c-kit/SCF was upregulated, and carcinogenesis-associated proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. In summary, CP could further cause prostate carcinogenesis, which may be associated with activated c-kit/SCF signaling. Resveratrol treatment could improve the progression of CP via the downregulation of c-kit/SCF by activating Sirt1.

  9. MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Juan [Department of Gynecology, Changzhou Maternity and Children Health Hospital, Changzhou, Jiangsu 213003 (China); Li, Li; Yun, Hui-fang [Department of Anesthesiology, Changzhou No. 2 People' s Hospital, Changzhou, Jiangsu 213003 (China); Han, Ye-shan, E-mail: yeshanhan123@163.com [Department of Anesthesiology, Changzhou No. 2 People' s Hospital, Changzhou, Jiangsu 213003 (China)

    2015-08-07

    Background: Diabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. Methods: The expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. Results: We discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 3′-UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic. In addition, we examined that the up-regulation of NF-κB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Conclusion: In this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1. - Highlights: • Higher mRNA level of miR-138 was observed in SMCs from db/db mice. • The mRNA and protein level of SIRT1 in SMCs from db/db mice were greatly reduced. • miR-138 could regulate the expression of SIRT1 in SMCs. • SIRT1 overexpression reversed the up-regulation of acetylized p65 and NF-κB induced by high glucose. • MiR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose.

  10. Sirtuin 1 (SIRT1 activation mediates sildenafil induced delayed cardioprotection against ischemia-reperfusion injury in mice.

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    Mona Shalwala

    Full Text Available BACKGROUND: It has been well documented that phosphodiesterase-5 inhibitor, sildenafil (SIL protects against myocardial ischemia/reperfusion (I-R injury. SIRT1 is part of the class III Sirtuin family of histone deacetylases that deacetylates proteins involved in cellular stress response including those related to I-R injury. OBJECTIVE/HYPOTHESIS: We tested the hypothesis that SIL-induced cardioprotection may be mediated through activation of SIRT1. METHODS: Adult male ICR mice were treated with SIL (0.7 mg/kg, i.p., Resveratrol (RSV, 5 mg/kg, a putative activator of SIRT1 used as the positive control, or saline (0.2 mL. The hearts were harvested 24 hours later and homogenized for SIRT1 activity analysis. RESULTS: Both SIL- and RSV-treated mice had increased cardiac SIRT1 activity (P<0.001 as compared to the saline-treated controls 24 hours after drug treatment. In isolated ventricular cardiomyocytes, pretreatment with SIL (1 µM or RSV (1 µM for one hour in vitro also upregulated SIRT1 activity (P<0.05. We further examined the causative relationship between SIRT1 activation and SIL-induced late cardioprotection. Pretreatment with SIL (or RSV 24 hours prior to 30 min ischemia and 24 hours of reperfusion significantly reduced infarct size, which was associated with a significant increase in SIRT1 activity (P<0.05. Moreover, sirtinol (a SIRT1 inhibitor, 5 mg/kg, i.p. given 30 min before I-R blunted the infarct-limiting effect of SIL and RSV (P<0.001. CONCLUSION: Our study shows that activation of SIRT1 following SIL treatment plays an essential role in mediating the SIL-induced cardioprotection against I-R injury. This newly identified SIRT1-activating property of SIL may have enormous therapeutic implications.

  11. Accelerated recovery of renal mitochondrial and tubule homeostasis with SIRT1/PGC-1α activation following ischemia–reperfusion injury

    Energy Technology Data Exchange (ETDEWEB)

    Funk, Jason A., E-mail: funkj@musc.edu [Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Schnellmann, Rick G., E-mail: schnell@musc.edu [Center for Cell Death, Injury, and Regeneration, Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC 29425 (United States); Ralph H. Johnson VA Medical Center, Charleston, SC 29401 (United States)

    2013-12-01

    Kidney ischemia–reperfusion (I/R) injury elicits cellular injury in the proximal tubule, and mitochondrial dysfunction is a pathological consequence of I/R. Promoting mitochondrial biogenesis (MB) as a repair mechanism after injury may offer a unique strategy to restore both mitochondrial and organ function. Rats subjected to bilateral renal pedicle ligation for 22 min were treated once daily with the SIRT1 activator SRT1720 (5 mg/kg) starting 24 h after reperfusion until 72 h–144 h. SIRT1 expression was elevated in the renal cortex of rats after I/R + vehicle treatment (IRV), but was associated with less nuclear localization. SIRT1 expression was even further augmented and nuclear localization was restored in the kidneys of rats after I/R + SRT1720 treatment (IRS). PGC-1α was elevated at 72 h–144 h in IRV and IRS kidneys; however, SRT1720 treatment induced deacetylation of PGC-1α, a marker of activation. Mitochondrial proteins ATP synthase β, COX I, and NDUFB8, as well as mitochondrial respiration, were diminished 24 h–144 h in IRV rats, but were partially or fully restored in IRS rats. Urinary kidney injury molecule-1 (KIM-1) was persistently elevated in both IRV and IRS rats; however, KIM-1 tissue expression was attenuated in IRS rats. Additionally, sustained loss of Na{sup +},K{sup +}–ATPase expression and basolateral localization and elevated vimentin in IRV rats was normalized in IRS rats, suggesting restoration of a differentiated, polarized tubule epithelium. The results suggest that SRT1720 treatment expedited recovery of mitochondrial protein expression and function by enhancing MB, which was associated with faster proximal tubule repair. Targeting MB may offer unique therapeutic strategy following ischemic injury. - Highlights: • We examined recovery of mitochondrial and renal function after ischemia–reperfusion. • SRT1720 treatment after I/R induced mitochondrial biogenesis via SIRT1/PGC-1α. • Recovery of mitochondrial function was

  12. SIRT1 exhibits antioxidative effects in HT22 cells induced by tert-butyl alcohol.

    Science.gov (United States)

    Ma, Junxiang; Song, Dongmei; Zhang, Yuanyuan; Chen, Li; Zhang, Shixuan; Jia, Jiaxin; Chen, Tian; Guo, Caixia; Tian, Lin; Gao, Ai; Niu, Piye

    2018-02-01

    Tertiary butyl alcohol (TBA) is a principal metabolite of methyl tertiary-butyl ether (MTBE), a common pollutant worldwide in the ground or underground water, which is found to produce nervous system damage. Nevertheless, few data regarding the effects of TBA has been reported. Studies indicated that oxidative stress plays a pivotal role in MTBE neurotoxic mechanism. Sirtuin 1 (SIRT1) has been reported to exert a neuroprotective effect on various neurologic diseases via resistance to oxidative stress by deacetylating its substrates. In this study, we examined levels of oxidative stress after exposure to TBA for 6 h in HT22 cells and HT22 cells with SIRT1 silencing (transfected with SIRT1 siRNA) or high expression (preconditioned with agonists SRT1720). We found that TBA activated oxidative stress by increasing generation of intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and Oxidized glutathione (GSSG), and decreasing contents of superoxide dismutase (SOD) and glutathione reductase (GSH). In additional, levels of TBA-induced oxidative stress were aggravated when SIRT1 silenced but alleviated when SIRT1 enhanced. Our study indicated that SIRT1 mitigated oxidative stress induced by TBA. © 2017 Wiley Periodicals, Inc.

  13. Sirt1 overexpression suppresses fluoride-induced p53 acetylation to alleviate fluoride toxicity in ameloblasts responsible for enamel formation.

    Science.gov (United States)

    Suzuki, Maiko; Ikeda, Atsushi; Bartlett, John D

    2018-03-01

    Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8 Sirt/KO ) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8 Sirt1/over ) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8 Sirt/KO cells, whereas it decreased in LS8 Sirt1/over . Fluoride-induced Ac-p53 formation was also suppressed in LS8 Sirt1/over cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8 Sirt/KO and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8 Sirt1/over cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8 Sirt1/over cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8 Sirt1/KO cells, but was attenuated in LS8 Sirt1/over cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.

  14. SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kgamma Activity through Deacetylation of Specific Lysine Residues in Mammals.

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    Sayaka Akieda-Asai

    Full Text Available BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5Kgamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268 in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5P(2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.

  15. Kallistatin reduces vascular senescence and aging by regulating microRNA-34a-SIRT1 pathway.

    Science.gov (United States)

    Guo, Youming; Li, Pengfei; Gao, Lin; Zhang, Jingmei; Yang, Zhirong; Bledsoe, Grant; Chang, Eugene; Chao, Lee; Chao, Julie

    2017-08-01

    Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)-induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF-α-induced cellular senescence in EPCs, as indicated by reduced senescence-associated β-galactosidase activity and plasminogen activator inhibitor-1 expression, and elevated telomerase activity. Kallistatin blocked TNF-α-induced superoxide levels, NADPH oxidase activity, and microRNA-21 (miR-21) and p16 INK 4a synthesis. Kallistatin prevented TNF-α-mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR-34a synthesis, whereas miR-34a overexpression abolished kallistatin-induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR-34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ-induced aortic senescence, oxidative stress, and miR-34a and miR-21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild-type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR-34 or sir-2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR-34, but stimulated sir-2.1 and sod-3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR-34a-SIRT1

  16. SIRT1 mediates Sphk1/S1P-induced proliferation and migration of endothelial cells.

    Science.gov (United States)

    Gao, Zhan; Wang, Hua; Xiao, Feng-Jun; Shi, Xue-Feng; Zhang, Yi-Kun; Xu, Qin Qin; Zhang, Xiao-Yan; Ha, Xiao-Qin; Wang, Li-Sheng

    2016-05-01

    Angiogenesis is one of the most important components of embryonic organ formation and vessel growth after birth. Sphingosine kinase 1 (Sphk1) and S1P has been confirmed to participate in various cell signaling pathways and physiological processes including neovascularisation. However, the mechanisms that Sphk1/S1P regulates neovascularisation remain unclear. In this study, we elucidated that Sphk1/S1P upregulates sirtuin 1 (SIRT1), a NAD+ dependent deacetylases protease which exerts multiple cellular functions, to regulate the proliferation and migration of endothelial cells. By using CCK8 and Transwell assays, we demonstrated that Sphk1 and SIRT1 knockdown could significantly decrease proliferation and migration of HUVEC cells. Sphk1 inhibition results in SIRT1 downregulation which could be reversed by exogenous S1P in HUVEC cells. Treatment of HUVECs with S1P reverses the impaired proliferation and migration caused by SIRT1 knockdown. Furthermore, Sphk1 knockdown inhibits the phosphorylation of P38 MAPK, ERK and AKT. Treatment of HUVECs with PD98059, SB203580 and Wortmannin, which are the inhibitors of ERK, P38 MAPK and AKT respectively, resulted in decreased SIRT1 expression and reduced migration of HUVEC cells. Thus, we conclude that Sphk1/S1P induces SIRT1 upregulation through multiple pathways including P38 MAPK, ERK and AKT signals. This is the first report to disclose the existence and roles of Sphk1/S1P/SIRT1 axis in regulation of endothelial cell proliferation and migration, which may provide a theoretical basis for angiogenesis. Copyright © 2016. Published by Elsevier Ltd.

  17. Celecoxib sensitizes Staphylococcus aureus to antibiotics in macrophages by modulating SIRT1.

    Science.gov (United States)

    Annamanedi, Madhavi; Kalle, Arunasree M

    2014-01-01

    We have previously shown that celecoxib in combination with an antibiotic, increase the bacterial sensitivity to antibiotics. However, the underlying molecular mechanism remained elusive. Efficacy of the combinatorial treatment of celecoxib and ampicillin in vitro was evaluated on macrophage-phagocytosed S. aureus. To elucidate the mechanism, signaling pathway of infection and inflammation involving TLR2, JNK, SIRT1 and NF-κB was studied by FACS, Western blot, ELISA and activity assays. Combinatorial treatment of ampicillin and celecoxib reduced the bacterial load in the macrophages. Further studies clearly suggested the activation of the master regulator of oxidative stress and inflammation SIRT1, by celecoxib when used alone and/or in combination with ampicillin. Also, the results indicated that celecoxib inhibited JNK phosphorylation thereby stabilizing and activating SIRT1 protein that inhibited the COX-2 gene transcription with a significant decrease in the levels of protein inflammatory cytokines like IL-6, MIP-1α and IL-1β via inhibition of NF-κB. SIRT1 activation by celecoxib also resulted in increase of catalase and peroxidase activity with a decrease in Nitric oxide levels. In conclusion, we demonstrate a novel role of celecoxib in controlling inflammation as an enhancer of antibiotic activity against bacteria by modulating SIRT1.

  18. SIRT1 regulates MAPK pathways in vitiligo skin: insight into the molecular pathways of cell survival

    Science.gov (United States)

    Becatti, Matteo; Fiorillo, Claudia; Barygina, Victoria; Cecchi, Cristina; Lotti, Torello; Prignano, Francesca; Silvestro, Agrippino; Nassi, Paolo; Taddei, Niccolò

    2014-01-01

    Vitiligo is an acquired and progressive hypomelanotic disease that manifests as circumscribed depigmented patches on the skin. The aetiology of vitiligo remains unclear, but recent experimental data underline the interactions between melanocytes and other typical skin cells, particularly keratinocytes. Our previous results indicate that keratinocytes from perilesional skin show the features of damaged cells. Sirtuins (silent mating type information regulation 2 homolog) 1, well-known modulators of lifespan in many species, have a role in gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy ageing. In the literature there is no evidence for SIRT1 signalling in vitiligo and its possible involvement in disease progression. Here, biopsies were taken from the perilesional skin of 16 patients suffering from non-segmental vitiligo and SIRT1 signalling was investigated in these cells. For the first time, a new SIRT1/Akt, also known as Protein Kinase B (PKB)/mitogen-activated protein kinase (MAPK) signalling has been revealed in vitiligo. SIRT1 regulates MAPK pathway via Akt-apoptosis signal-regulating kinase-1 and down-regulates pro-apoptotic molecules, leading to decreased oxidative stress and apoptotic cell death in perilesional vitiligo keratinocytes. We therefore propose SIRT1 activation as a novel way of protecting perilesional vitiligo keratinocytes from damage. PMID:24410795

  19. SIRT1: A Novel Target for the Treatment of Muscular Dystrophies

    Directory of Open Access Journals (Sweden)

    Atsushi Kuno

    2016-01-01

    Full Text Available Muscular dystrophies are inherited myogenic disorders accompanied by progressive skeletal muscle weakness and degeneration. Duchenne muscular dystrophy (DMD is the most common and severe form of muscular dystrophy and is caused by mutations in the gene that encodes the cytoskeletal protein dystrophin. The treatment for DMD is limited to glucocorticoids, which are associated with multiple side effects. Thus, the identification of novel therapeutic targets is urgently needed. SIRT1 is an NAD+-dependent histone/protein deacetylase that plays roles in diverse cellular processes, including stress resistance and cell survival. Studies have shown that SIRT1 activation provides beneficial effects in the dystrophin-deficient mdx mouse, a model of DMD. SIRT1 activation leads to the attenuation of oxidative stress and inflammation, a shift from the fast to slow myofiber phenotype, and the suppression of tissue fibrosis. Although further research is needed to clarify the molecular mechanisms underlying the protective role of SIRT1 in mdx mice, we propose SIRT1 as a novel therapeutic target for patients with muscular dystrophies.

  20. The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription.

    Science.gov (United States)

    Curtil, Claire; Enache, Liviu S; Radreau, Pauline; Dron, Anne-Gaëlle; Scholtès, Caroline; Deloire, Alexandre; Roche, Didier; Lotteau, Vincent; André, Patrice; Ramière, Christophe

    2014-03-01

    Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.

  1. Aggravation of nonalcoholic steatohepatitis by moderate alcohol consumption is associated with decreased SIRT1 activity in rats

    Science.gov (United States)

    Chronic alcohol intake decreases adiponectin and sirtuin 1 (SIRT1) expressions, both of which have been implicated in various biological processes including inflammation, apoptosis and metabolism. We have previously shown that moderate consumption of alcohol aggravates liver inflammation and apoptos...

  2. Late-onset running biphasically improves redox balance, energy- and methylglyoxal-related status, as well as SIRT1 expression in mouse hippocampus.

    Directory of Open Access Journals (Sweden)

    Stefano Falone

    Full Text Available Despite the active research in this field, molecular mechanisms underlying exercise-induced beneficial effects on brain physiology and functions are still matter of debate, especially with regard to biological processes activated by regular exercise affecting the onset and progression of hippocampal aging in individuals unfamiliar with habitual physical activity. Since such responses seem to be mediated by changes in antioxidative, antiglycative and metabolic status, a possible exercise-induced coordinated response involving redox, methylglyoxal- and sirtuin-related molecular networks may be hypothesized. In this study, hippocampi of CD1 mice undergoing the transition from mature to middle age were analyzed for redox-related profile, oxidative and methylglyoxal-dependent damage patterns, energy metabolism, sirtuin1 and glyoxalase1 expression after a 2- or 4-mo treadmill running program. Our findings suggested that the 4-mo regular running lowered the chance of dicarbonyl and oxidative stress, activated mitochondrial catabolism and preserved sirtuin1-related neuroprotection. Surprisingly, the same cellular pathways were negatively affected by the first 2 months of exercise, thus showing an interesting biphasic response. In conclusion, the duration of exercise caused a profound shift in the response to regular running within the rodent hippocampus in a time-dependent fashion. This research revealed important details of the interaction between exercise and mammal hippocampus during the transition from mature to middle age, and this might help to develop non-pharmacological approaches aimed at retarding brain senescence, even in individuals unfamiliar with habitual exercise.

  3. Anti-Fibrotic Effect of Losartan, an Angiotensin II Receptor Blocker, Is Mediated through Inhibition of ER Stress via Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin

    Directory of Open Access Journals (Sweden)

    Hyosang Kim

    2017-01-01

    Full Text Available Endoplasmic reticulum (ER stress is increasingly identified as modulator of fibrosis. Losartan, an angiotensin II receptor blocker, has been widely used as the first choice of treatment in chronic renal diseases. We postulated that anti-fibrotic effect of losartan is mediated through inhibition of ER stress via SIRT1 (silent mating type information regulation 2 homolog 1 hemeoxygenase-1 (HO-1/thioredoxin pathway. Renal tubular cells, tunicamycin (TM-induced ER stress, and unilateral ureteral obstruction (UUO mouse model were used. Expression of ER stress was assessed by Western blot analysis and immunohistochemical stain. ER stress was induced by chemical ER stress inducer, tunicamycin, and non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin. Losartan suppressed the TM-induced ER stress, as shown by inhibition of TM-induced expression of GRP78 (glucose related protein 78 and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α, through up-regulation of SIRT1 via HO-1 and thioredoxin. Losartan also suppressed the ER stress by non-chemical inducers. In both animal models, losartan reduced the tubular expression of GRP78, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor. Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis. These findings provide new insights into renoprotective effects of losartan and suggest that SIRT1, HO-1, and thioredoxin may be potential pharmacological targets in kidney diseases under excessive ER stress condition.

  4. SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Jun [Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University (China); Department of Respiratory Medicine, Jiangsu Provincial Hospital of Chinese Traditional Medicine (China); Wu, Xiaoyan; Yang, Yuyu; Zhao, Yuhao [Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Pathophysiology, Nanjing Medical University (China); Fang, Mingming [Jiangsu Jiankang Vocational Institute (China); Xie, Weiping, E-mail: wpxienjmu@gmail.com [Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University (China); Wang, Hong, E-mail: hwangnjmu@gmail.com [Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University (China); Xu, Yong [Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Department of Pathophysiology, Nanjing Medical University (China)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanism underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.

  5. Role of SIRT1-mediated mitochondrial and Akt pathways in glioblastoma cell death induced by Cotinus coggygria flavonoid nanoliposomes

    Directory of Open Access Journals (Sweden)

    Wang G

    2015-08-01

    Full Text Available Gang Wang,1,2,* Jun Jie Wang,1,2,* Tony SS To,3 Hua Fu Zhao,3 Jing Wang3 1Department of Pharmaceutics, Shanghai Eighth People’s Hospital, Shanghai, People’s Republic of China; 2College of Pharmacy, Hubei University of Medicine, Shiyan, Hubei Province, People’s Republic of China; 3Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hong Kong SAR, People’s Republic of China *These authors contributed equally to this work Abstract: Flavonoids, the major polyphenol components in Cotinus coggygria (CC, have been found to show an anticancer effect in our previous study; however, the exact mechanisms of inducing human glioblastoma (GBM cell death remain to be resolved. In this study, a novel polyvinylpyrrolidone K-30/sodium dodecyl sulfate and polyethyleneglycol-coated liposome loaded with CC flavonoids (CCFs was developed to enhance solubility and the antibrain tumor effect, and the molecular mechanism regarding how CCF nanoliposomes (CCF-NLs induce apoptotic cell death in vitro was investigated. DBTRG-05MG GBM cell lines treated with CCF-NLs showed potential antiproliferative effects. Regarding the underlying mechanisms of inducing apoptosis in DBTRG-05MG GBM cells, CCF-NLs were shown to downregulate the expression of antiapoptotic B-cell lymphoma/leukemia 2 (Bcl-2, an apoptosis-related protein family member, but the expression of proapoptotic Bcl-2-associated X protein was enhanced compared with that in controls. CCF-NLs also inhibited the activity of caspase-3 and -9, which is the initiator caspase of the extrinsic and intrinsic apoptotic pathways. Blockade of caspase activation consistently induced apoptosis and inhibited growth in CCF-NL-treated DBTRG-05MG cells. This study further investigated the role of the Akt pathway in the apoptotic cell death by CCF-NLs, showing that CCF-NLs deactivated Akt. Specifically, CCF-NLs downregulated the expression of p-Akt and SIRT1 as well as the level of

  6. [Effect of microRNA-34a/SIRT1/p53 signal pathway on notoginsenoside R₁ delaying vascular endothelial cell senescence].

    Science.gov (United States)

    Lai, Xiao-Hua; Lei, Yan; Yang, Jing; Xiu, Cheng-Kui

    2018-02-01

    This study aimed to investigate the effect of notoginsenoside R₁ in delaying H₂O₂-induced vascular endothelial cell senescence through microRNA-34a/SIRT1/p53 signal pathway. In this study, human umbilical vein endothelial cells(HUVECs) were selected as the study object; the aging model induced by hydrogen peroxide(H₂O₂) was established, with resveratrol as the positive drug. HUVECs were randomly divided into four groups, youth group, senescence model group, notoginsenoside R₁ group and resveratrol group. Notoginsenoside R₁ group and resveratrol group were modeled with 100 μmoL·L⁻¹ H₂O₂ for 4 h after 24 h treatment with notoginsenoside R₁(30 μmoL·L⁻¹) and resveratrol(10 μmoL·L⁻¹) respectively. At the end, each group was cultured with complete medium for 24 h. The degree of cellular senescence was detected by senescence-associated β-galactosidase(SA-β-Gal) staining kit, the cell viability was detected by cell counting kit-8, the cell cycle distribution was analyzed by flow cytometry, and the cellular SOD activity was detected by WST-1 method in each group. The expressions of SIRT1, p53, p21 and p16 proteins in HUVECs were detected by Western blot. In addition, the mRNA expressions of miRNA-34a, SIRT1 and p53 in HUVECs were assayed by Real-time PCR. These results indicated that notoginsenoside R₁ significantly reduced the positive staining rate of senescent cells, enhanced the cell proliferation capacity and intracellular SOD activity, decreased the proportion of cells in G₀/G₁ phase, and increased the percentage of cells in S phase simultaneously compared with the senescence model group. Moreover, notoginsenoside R₁ decreased the mRNA expressions of miRNA-34a and p53 and the protein expression of p53, p21 and p16.At the same time, notoginsenoside R₁ increased the protein and mRNA expressions of SIRT1. The differences in these results between the senescence model group and the

  7. SIRT1 stimulation by polyphenols is affected by their stability and metabolism

    NARCIS (Netherlands)

    Boer, de V.C.J.; Goffau, de L.; Arts, I.C.W.; Hollman, P.C.H.; Keijer, J.

    2006-01-01

    Silent information regulator two ortholog 1 (SIRT1) is the human ortholog of the yeast sir2 protein; one of the most important regulators of lifespan extension by caloric restriction in several organisms. Dietary polyphenols, abundant in vegetables, fruits, cereals, wine and tea, were reported to

  8. Sirt1 Protects Stressed Adult Hematopoietic Stem Cells | Center for Cancer Research

    Science.gov (United States)

    The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic

  9. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    International Nuclear Information System (INIS)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo; Koya, Daisuke

    2012-01-01

    Highlights: ► SIRT1 inactivation decreases autophagy in THP-1 cell. ► Inhibition of autophagy induces inflammation. ► SIRT1 inactivation induces inflammation through NF-κB activation. ► The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-κB activation. ► SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD + -dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 through nuclear factor (NF)-κB signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-κB activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-κB activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and is implicated in decreased 5′-AMP activated kinase (AMPK) activation, leading to the impairment of autophagy. The mTOR inhibitor, rapamycin, abolishes

  10. SIRT1 inactivation induces inflammation through the dysregulation of autophagy in human THP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Takeda-Watanabe, Ai; Kitada, Munehiro; Kanasaki, Keizo [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan); Koya, Daisuke, E-mail: koya0516@kanazawa-med.ac.jp [Diabetology and Endocrinology, Kanazawa Medical University, Kahoku-Gun, Ishikawa (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer SIRT1 inactivation decreases autophagy in THP-1 cell. Black-Right-Pointing-Pointer Inhibition of autophagy induces inflammation. Black-Right-Pointing-Pointer SIRT1 inactivation induces inflammation through NF-{kappa}B activation. Black-Right-Pointing-Pointer The p62/Sqstm1 accumulation by impairment of autophagy is related to NF-{kappa}B activation. Black-Right-Pointing-Pointer SIRT1 inactivation is involved in the activation of mTOR and decreased AMPK activation. -- Abstract: Inflammation plays a crucial role in atherosclerosis. Monocytes/macrophages are some of the cells involved in the inflammatory process in atherogenesis. Autophagy exerts a protective effect against cellular stresses like inflammation, and it is regulated by nutrient-sensing pathways. The nutrient-sensing pathway includes SIRT1, a NAD{sup +}-dependent histone deacetylase, which is implicated in the regulation of a variety of cellular processes including inflammation and autophagy. The mechanism through which the dysfunction of SIRT1 contributes to the regulation of inflammation in relation to autophagy in monocytes/macrophages is unclear. In the present study, we demonstrate that treatment with 2-[(2-Hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benzamide (Sirtinol), a chemical inhibitor of SIRT1, induces the overexpression of inflammation-related genes such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-6 through nuclear factor (NF)-{kappa}B signaling activation, which is associated with autophagy dysfunction, as shown through p62/Sqstm1 accumulation and decreased expression of light chain (LC) 3 II in THP-1 cells. The autophagy inhibitor, 3-methyladenine, also induces inflammation-related NF-{kappa}B activation. In p62/Sqstm1 knockdown cells, Sirtinol-induced inflammation through NF-{kappa}B activation is blocked. In addition, inhibition of SIRT1 is involved in the activation of the mammalian target of rapamycin (mTOR) pathway and

  11. Fortifying the Link between SIRT1, Resveratrol, and Mitochondrial Function

    OpenAIRE

    Denu, John M.

    2012-01-01

    The molecular mechanisms behind the health benefits of resveratrol remain enigmatic and controversial. Here, Price et al. establish a clear chemical-genetic connection between SIRT1 and resveratrol, providing strong evidence that SIRT1 is critical for resveratrol to stimulate mitochondrial biogenesis and a switch toward oxidative muscle fibers (Price et al., 2012).

  12. Sirt1 Protects against Oxidative Stress-Induced Apoptosis in Fibroblasts from Psoriatic Patients: A New Insight into the Pathogenetic Mechanisms of Psoriasis

    Directory of Open Access Journals (Sweden)

    Matteo Becatti

    2018-05-01

    Full Text Available Psoriasis, a multisystem chronic disease characterized by abnormal keratinocyte proliferation, has an unclear pathogenesis where systemic inflammation and oxidative stress play mutual roles. Dermal fibroblasts, which are known to provide a crucial microenvironment for epidermal keratinocyte function, represented the selected experimental model in our study which aimed to clarify the potential role of SIRT1 in the pathogenetic mechanisms of the disease. We firstly detected the presence of oxidative stress (lipid peroxidation and total antioxidant capacity, significantly reduced SIRT1 expression level and activity, mitochondrial damage and apoptosis (caspase-3, -8 and -9 activities in psoriatic fibroblasts. Upon SIRT1 activation, redox balance was re-established, mitochondrial function was restored and apoptosis was no longer evident. Furthermore, we examined p38, ERK and JNK activation, which was strongly altered in psoriatic fibroblasts, in response to SIRT1 activation and we measured caspase-3 activity in the presence of specific MAPK inhibitors demonstrating the key role of the SIRT1 pathway against apoptotic cell death via MAPK modulation. Our results clearly demonstrate the involvement of SIRT1 in the protective mechanisms related to fibroblast injury in psoriasis. SIRT1 activation exerts an active role in restoring both mitochondrial function and redox balance via modulation of MAPK signaling. Hence, SIRT1 can be proposed as a specific tool for the treatment of psoriasis.

  13. Association of SIRT-1 Gene Polymorphism and Vitamin D Level in Egyptian Patients With Rheumatoid Arthritis.

    Science.gov (United States)

    Sabry, Dina; Kaddafy, Shereen Rashad; Abdelaziz, Ahmed Ali; Nassar, Abdelfattah Kasem; Rayan, Mohamed Moneer; Sadek, Sadek Mostafa; Abou-Elalla, Amany A

    2018-03-01

    We investigated SIRT-1 genetic variant and its association with vitamin D level in Egyptian patients with rheumatoid arthritis (RA). Seventy Egyptian subjects were enrolled in our study and divided into two groups: RA group (n = 50 patients) and healthy control group (n = 20 subjects). Five milliliter blood sample was withdrawn from each subject followed by laboratory investigation and DNA extraction for SIRT-1 gene polymorphism assessment (rs7895833 A>G, rs7069102 C>G and rs2273773 C>T) and vitamin D level expression. There was statistically significant difference between rheumatoid cases and controls with regard to vitamin D level with 88% of cases showing insufficient vitamin D versus all controls showing sufficient level. SIRT-1 different SNPs rs2273773, rs7895833and rs7069102 genotype frequencies were statistically significant in RA compared to control group (P = 0.001). There was no statistically significant difference between different genotypes of rs2273773, rs7895833 and rs7069102 with regard to vitamin D level. We concluded that there is a strong association between SIRT-1 polymorphism genotyping and RA. Vitamin D level was insufficient in Egyptian patients with RA.

  14. Hydrogen alleviates hyperoxic acute lung injury related endoplasmic reticulum stress in rats through upregulation of SIRT1.

    Science.gov (United States)

    Sun, Qiang; Han, Wenjie; Hu, Huijun; Fan, Danfeng; Li, Yanbo; Zhang, Yu; Lv, Yan; Li, Mingxin; Pan, Shuyi

    2017-06-01

    Hyperoxic acute lung injury (HALI) is a major clinical problem for patients undergoing supplemental oxygen therapy. Currently in clinical settings there exist no effective means of prevention or treatment methods. Our previous study found that: hydrogen could reduce HALI, as well as oxidative stress. This research will further explore the mechanism underlying the protective effect of hydrogen on oxygen toxicity. Rats were randomly assigned into three experimental groups and were exposed in a oxygen chamber for 60 continuous hours: 100% balanced air (control); 100% oxygen (HALI); 100% oxygen with hydrogen treatment (HALI + HRS). We examined lung function by wet to dry ratio of lung, lung pleural effusion and cell apoptosis. We also detected endoplasmic reticulum stress (ERS) by examining the expression of CHOP, GRP78 and XBP1. We further investigated the role of Sirtuin 1 (SIRT1) in HALI, which contributes to cellular regulation including ERS, by examining its expression after hydrogen treatment with SIRT1 inhibitor. Hydrogen could significantly reduce HALI by reducing lung edema and apoptosis, inhibiting the elevating of ERS and increased SIRT1 expression. By inhibition of SIRT1 expression, the effect of hydrogen on prevention of HALI is significantly weakened, the inhibition of the ERS was also reversed. Our findings indicate that hydrogen could reduce HALI related ERS and the mechanism of hydrogen may be associated with upregulation of SIRT1, this study reveals the molecular mechanisms underlying the protective effect of hydrogen, which provides a new theoretical basis for clinical application of hydrogen.

  15. A high-confidence interaction map identifies SIRT1 as a mediator of acetylation of USP22 and the SAGA coactivator complex.

    Science.gov (United States)

    Armour, Sean M; Bennett, Eric J; Braun, Craig R; Zhang, Xiao-Yong; McMahon, Steven B; Gygi, Steven P; Harper, J Wade; Sinclair, David A

    2013-04-01

    Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.

  16. Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

    Science.gov (United States)

    Li, Xian; Lee, Youn Ju; Jin, Fansi; Park, Young Na; Deng, Yifeng; Kang, Youra; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Chang, Young-Chae; Ko, Hyun-Jeong; Kim, Cheorl-Ho; Murakami, Makoto; Chang, Hyeun Wook

    2017-07-25

    Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

  17. Role and Possible Mechanisms of Sirt1 in Depression

    OpenAIRE

    Lu, Guofang; Li, Jianguo; Zhang, Hongmei; Zhao, Xin; Yan, Liang-Jun; Yang, Xiaorong

    2018-01-01

    Depression is a common, devastating illness. Due to complicated causes and limited treatments, depression is still a major problem that plagues the world. Silent information regulator 1 (Sirt1) is a deacetylase at the consumption of NAD+ and is involved in gene silencing, cell cycle, fat and glucose metabolism, cellular oxidative stress, and senescence. Sirt1 has now become a critical therapeutic target for a number of diseases. Recently, a genetic study has received considerable attention fo...

  18. Curcumin as therapeutics for the treatment of head and neck squamous cell carcinoma by activating SIRT1

    Science.gov (United States)

    Hu, An; Huang, Jing-Juan; Li, Rui-Lin; Lu, Zhao-Yang; Duan, Jun-Li; Xu, Wei-Hua; Chen, Xiao-Ping; Fan, Jing-Ping

    2015-01-01

    SIRT1 is one of seven mammalian homologs of Sir2 that catalyzes NAD+-dependent protein deacetylation. The aim of the present study is to explore the effect of SIRT1 small molecule activator on the anticancer activity and the underlying mechanism. We examined the anticancer activity of a novel oral agent, curcumin, which is the principal active ingredient of the traditional Chinese herb Curcuma Longa. Treatment of FaDu and Cal27 cells with curcumin inhibited growth and induced apoptosis. Mechanistic studies showed that anticancer activity of curcumin is associated with decrease in migration of HNSCC and associated angiogenesis through activating of intrinsic apoptotic pathway (caspase-9) and extrinsic apoptotic pathway (caspase-8). Our data demonstrating that anticancer activity of curcumin is linked to the activation of the ATM/CHK2 pathway and the inhibition of nuclear factor-κB. Finally, increasing SIRT1 through small molecule activator curcumin has shown beneficial effects in xenograft mouse model, indicating that SIRT1 may represent an attractive therapeutic target. Our studies provide the preclinical rationale for novel therapeutics targeting SIRT1 in HNSCC. PMID:26299580

  19. The combination of blueberry juice and probiotics reduces apoptosis of alcoholic fatty liver of mice by affecting SIRT1 pathway

    Science.gov (United States)

    Zhu, Juanjuan; Ren, Tingting; Zhou, Mingyu; Cheng, Mingliang

    2016-01-01

    Purpose To explore the effects of the combination of blueberry juice and probiotics on the apoptosis of alcoholic fatty liver disease (AFLD). Methods Healthy C57BL/6J mice were used in the control group (CG). AFLD mice models were established with Lieber–DeCarli ethanol diet and evenly assigned to six groups with different treatments: MG (model), SI (SIRT1 [sirtuin type 1] small interfering RNA [siRNA]), BJ (blueberry juice), BJSI (blueberry juice and SIRT1 siRNA), BJP (blueberry juice and probiotics), and BJPSI (blueberry juice, probiotics, and SIRT1 siRNA). Hepatic tissue was observed using hematoxylin and eosin (HE) and Oil Red O (ORO) staining. Biochemical indexes of the blood serum were analyzed. The levels of SIRT1, caspase-3, forkhead box protein O1 (FOXO1), FasL (tumor necrosis factor ligand superfamily member 6), BAX, and Bcl-2 were measured by reverse transcription-polymerase chain reaction and Western blotting. Results HE and ORO staining showed that the hepatocytes were heavily destroyed with large lipid droplets in MG and SI groups, while the severity was reduced in the CG, BJ, and BJP groups (Pjuice and probiotics reduces apoptosis in AFLD by suppressing FOXO1, phosphorylated FOXO1, acetylated FOXO1, FasL, caspase-3, BAX, and Bcl-2 via the upregulation of SIRT1. PMID:27274198

  20. The combination of blueberry juice and probiotics reduces apoptosis of alcoholic fatty liver of mice by affecting SIRT1 pathway.

    Science.gov (United States)

    Zhu, Juanjuan; Ren, Tingting; Zhou, Mingyu; Cheng, Mingliang

    2016-01-01

    To explore the effects of the combination of blueberry juice and probiotics on the apoptosis of alcoholic fatty liver disease (AFLD). Healthy C57BL/6J mice were used in the control group (CG). AFLD mice models were established with Lieber-DeCarli ethanol diet and evenly assigned to six groups with different treatments: MG (model), SI (SIRT1 [sirtuin type 1] small interfering RNA [siRNA]), BJ (blueberry juice), BJSI (blueberry juice and SIRT1 siRNA), BJP (blueberry juice and probiotics), and BJPSI (blueberry juice, probiotics, and SIRT1 siRNA). Hepatic tissue was observed using hematoxylin and eosin (HE) and Oil Red O (ORO) staining. Biochemical indexes of the blood serum were analyzed. The levels of SIRT1, caspase-3, forkhead box protein O1 (FOXO1), FasL (tumor necrosis factor ligand superfamily member 6), BAX, and Bcl-2 were measured by reverse transcription-polymerase chain reaction and Western blotting. HE and ORO staining showed that the hepatocytes were heavily destroyed with large lipid droplets in MG and SI groups, while the severity was reduced in the CG, BJ, and BJP groups (Pblueberry juice and probiotics reduces apoptosis in AFLD by suppressing FOXO1, phosphorylated FOXO1, acetylated FOXO1, FasL, caspase-3, BAX, and Bcl-2 via the upregulation of SIRT1.

  1. Suppression of Sirtuin-1 Increases IL-6 Expression by Activation of the Akt Pathway During Allergic Asthma

    Directory of Open Access Journals (Sweden)

    Lingling Tang

    2017-10-01

    Full Text Available Background/Aims: A growing number of studies have demonstrated that the activity and expression level of sirtuin-1 (SIRT1 are decreased in asthma patients; however, the mechanisms underlying decreased SIRT1 expression and function are still not completely understood. Interleukin (IL-6 plays important roles in inflammation during allergic asthma. In this study, we examined whether loss of SIRT1 activity regulated the expression of IL-6 and further verified the underlying mechanisms. Methods: The human airway epithelial cell line 16HBE was used to test the effects of the SIRT1 inhibitor (salermide on expression of IL-6. IL-6 mRNA and protein expression were assessed with real-time polymerase chain reaction (PCR, immunochemistry, and ELISA. OVA-challenged mice were used as an asthma model to investigate the effect of SIRT1 activation on IL-6 and relative Akt phosphorylation level. Results: We found that inhibition of SIRT1 increased IL-6 mRNA and protein levels in a time-dependent manner, which was accompanied by increased Akt pathway activation in 16HBE cells. Furthermore activation of Akt showed upregulated expression of the IL-6 protein whereas Akt inhibitor, LY294002 or Akt siRNA significantly inhibited SIRT1-regulated IL-6 expression. Conversely, activation of SIRT1 inhibited Akt activation and IL-6 expression in an asthmatic mice model and 16HBE cells. Conclusion: Our results indicate the potential role of SIRT1 in regulating inflammation by modulation of IL-6 expression in an Akt-dependent manner during allergic asthma.

  2. Curcumin protects against myocardial infarction-induced cardiac fibrosis via SIRT1 activation in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Xiao J

    2016-03-01

    Full Text Available Jie Xiao, Xi Sheng, Xinyu Zhang, Mengqi Guo, Xiaoping JiKey Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of ChinaAbstract: Curcumin, a polyphenolic compound derived from turmeric, protects against myocardial injury by alleviating oxidative stress, inflammation, apoptosis, and fibrosis. However, the role of curcumin and its mechanism of action on interstitial fibrosis after myocardial infarction (MI are poorly understood. To clarify, MI was induced by a permanent ligation of the left anterior descending coronary artery in adult mice, and the effects of curcumin were evaluated 4 weeks after the MI event. In vitro, we treated cardiac fibroblasts (CFs with Ang II, and investigated the anti-fibrotic mechanism of curcumin. Our results showed that curcumin significantly attenuated collagen deposition in vivo and inhibited CF proliferation and migration, and MMP expression. In addition, we found that the down-regulation of SIRT1 after MI was attenuated by curcumin pretreatment, which indicated that the activation of SIRT1 might be involved in the protective action of curcumin. This hypothesis was confirmed by genetic inhibition of SIRT1 (siRNA-SIRT1 in Ang II-treated CFs. Our results provide new insights into the mechanism underlying the anti-fibrotic effects of curcumin in the heart.Keywords: curcumin, myocardial infarction, angiotensin II, cardiac fibroblasts, fibrosis, SIRT1

  3. Role of Sirt1 during the ageing process: relevance to protection of synapses in the brain.

    Science.gov (United States)

    Godoy, Juan A; Zolezzi, Juan M; Braidy, Nady; Inestrosa, Nibaldo C

    2014-12-01

    Ageing is a stochastic process associated with a progressive decline in physiological functions which predispose to the pathogenesis of several neurodegenerative diseases. The intrinsic complexity of ageing remains a significant challenge to understand the cause of this natural phenomenon. At the molecular level, ageing is thought to be characterized by the accumulation of chronic oxidative damage to lipids, proteins and nucleic acids caused by free radicals. Increased oxidative stress and misfolded protein formations, combined with impaired compensatory mechanisms, may promote neurodegenerative disorders with age. Nutritional modulation through calorie restriction has been shown to be effective as an anti-ageing factor, promoting longevity and protecting against neurodegenerative pathology in yeast, nematodes and murine models. Calorie restriction increases the intracellular levels of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD(+)), a co-substrate for the sirtuin 1 (Sirt1, silent mating-type information regulator 2 homolog 1) activity and a cofactor for oxidative phosphorylation and ATP synthesis. Promotion of intracellular NAD(+) anabolism is speculated to induce neuroprotective effects against amyloid-β-peptide (Aβ) toxicity in some models for Alzheimer's disease (AD). The NAD(+)-dependent histone deacetylase, Sirt1, has been implicated in the ageing process. Sirt1 serves as a deacetylase for numerous proteins involved in several cellular pathways, including stress response and apoptosis, and plays a protective role in neurodegenerative disorders, such as AD.

  4. Mangiferin Improves Hepatic Lipid Metabolism Mainly Through Its Metabolite-Norathyriol by Modulating SIRT-1/AMPK/SREBP-1c Signaling

    Directory of Open Access Journals (Sweden)

    Jian Li

    2018-03-01

    Full Text Available Objective: Mangiferin (MGF is a natural xanthone, with regulation effect on lipid metabolism. However, the molecular mechanism remains unclear. We purposed after oral administration, MGF is converted to its active metabolite(s, which contributes to the effects on lipid metabolism.Methods: KK-Ay mice were used to validate the effects of MGF on lipid metabolic disorders. Liver biochemical indices and gene expressions were determined. MGF metabolites were isolated from MGF administrated rat urine. Mechanism studies were carried out using HepG2 cells treated by MGF and its metabolite with or without inhibitors or small interfering RNA (siRNA. Western blot and immunoprecipitation methods were used to determine the lipid metabolism related gene expression. AMP/ATP ratios were measured by HPLC. AMP-activated protein kinase (AMPK activation were identified by homogeneous time resolved fluorescence (HTRF assays.Results: MGF significantly decreased liver triglyceride and free fatty acid levels, increased sirtuin-1 (SIRT-1 and AMPK phosphorylation in KK-Ay mice. HTRF studies indicated that MGF and its metabolites were not direct AMPK activators. Norathyriol, one of MGF’s metabolite, possess stronger regulating effect on hepatic lipid metabolism than MGF. The mechanism was mediated by activation of SIRT-1, liver kinase B1, and increasing the intracellular AMP level and AMP/ATP ratio, followed by AMPK phosphorylation, lead to increased phosphorylation level of sterol regulatory element-binding protein-1c.Conclusion: These results provided new insight into the molecular mechanisms of MGF in protecting against hepatic lipid metabolic disorders via regulating SIRT-1/AMPK pathway. Norathyriol showed potential therapeutic in treatment of non-alcoholic fatty liver disease.

  5. SIRT1-mediated deacetylation of PGC1α attributes to the protection of curcumin against glutamate excitotoxicity in cortical neurons

    International Nuclear Information System (INIS)

    Jia, Ning; Sun, Qinru; Su, Qian; Chen, Guomin

    2016-01-01

    It is widely accepted that accumulation of extracellular glutamate mediates neuronal injuries in a number of neurological disorders via binding glutamate receptors. However, usage of the glutamate receptor antagonists aimed to prevent glutamate excitotoxicity is still controversial. As a polyphenol natural product, curcumin, has been implied multiple bioactivities. In this study, we explored whether the silent information regulator 1 (SIRT1)-peroxisome proliferator-activated receptor-coactivator 1α (PGC1α) pathway participated in the protection of curcumin against glutamate excitotoxicity. The cultured primary cortical neurons were treated with glutamate to set up a neuronal excitotoxicity model. The MTT and TUNEL methods were employed to measure cell viability and apoptosis, respectively. The mitochondrial function, the expression levels of SIRT1, PGC1α and acetylated PGC1α (ac-PGC1α) were measured to explore the mechanism of curcumin against glutamate excitotoxicity. The results showed that glutamate significantly induced cell death and apoptosis, which was blocked by pretreatment with curcumin. Meanwhile, curcumin preserved mitochondrial function, increased the expression level of SIRT1 and reduced the level of ac-PGC1α in the presence of glutamate. These results suggest that SIRT1-mediated deacetylation of PGC1α attributes to the neuroprotection of curcumin against glutamate excitotoxicity. - Highlights: • Curcumin attenuates glutamate induced cell death and apoptosis in cultured neurons. • Curcumin preserves mitochondrial function in the presence of glutamate. • Curcumin enhanced the expression of SIRT1 in the glutamate rich environment. • SIRT1-mediated deacetylation of PGC1α attributes to the neuroprotection of curcumin.

  6. Sildenafil protects against bile duct ligation induced hepatic fibrosis in rats: Potential role for silent information regulator 1 (SIRT1).

    Science.gov (United States)

    Abd El Motteleb, Dalia M; Ibrahim, Islam A A E-H; Elshazly, Shimaa M

    2017-11-15

    Hepatic fibrosis is a potential health problem that may end with life-threatening cirrhosis and primary liver cancer. Recent studies point out to the protective effects of silent information regulator1 (SIRT1), against different models of organs fibrosis. This work aimed to investigate the possible protective effect of sildenafil (SIRT1 activator) against hepatic fibrosis induced by bile duct ligation (BDL). Firstly, three different doses of sildenafil (5, 10, 20mg/kg/day) were investigated; to detect the most protective one against BDL induced liver dysfunction and hepatic fibrosis. The most protective dose is then used; to study its effect on BDL induced SIRT1 downregulation, imbalance of oxidant/antioxidant status, increased inflammatory cytokines and fibrosis. Sildenafil (20mg/kg/day) was the most protective one, it caused upregulation of SIRT1, reduction of hepatic malondialdehyde (MDA) content, increase in expression of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenease (HO)-1, reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Hepatic content of tumor necrosis factor-α (TNF-α) and nuclear factor κB (NFκB) expression & content displayed significant reductions with sildenafil treatment, Furthermore, sildenafil caused marked reductions of transforming growth factor (TGF)-β content, expression of plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), α-smooth muscle actin (α-SMA), fibronectin, collagen I (α1) and hydroxyproline content. However, sildenafil protective effects were significantly reduced by co-administration of EX527 (SIRT1 inhibitor). Our work showed, for the first time that, sildenafil has promising protective effects against BDL induced liver dysfunction and hepatic fibrosis. These effects may be, in part, mediated by up regulation of SIRT1. Copyright © 2017. Published by Elsevier Inc.

  7. Activation of type 2 cannabinoid receptors (CB2R) promotes fatty acid oxidation through the SIRT1/PGC-1α pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Xuqin [Department of Endocrinology, First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu Province 210029 (China); Sun, Tao [Department of Neurology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, Jiangsu Province 210002 (China); Wang, Xiaodong, E-mail: xdwang666@hotmail.com [Department of Endocrinology, First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu Province 210029 (China)

    2013-07-05

    Highlights: •TC, a CB2R specific agonist, stimulates SIRT1 activity by PKA/CREB pathway. •TC promotes PGC-1α transcriptional activity by increasing its deacetylation. •TC increases the expression of genes linked to FAO and promotes the rate of FAO. •The effects of TC in FAO are dependent on CB2R. •Suggesting CB2R as a target to treat diseases with lipid dysregulation. -- Abstract: Abnormal fatty acid oxidation has been associated with obesity and type 2 diabetes. At the transcriptional level, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) has been reported to strongly increase the ability of hormone nuclear receptors PPARα and ERRα to drive transcription of fatty acid oxidation enzymes. In this study, we report that a specific agonist of the type 2 cannabinoid receptor (CB2R) can lead to fatty acid oxidation through the PGC-1α pathway. We have found that CB2R is expressed in differentiated C2C12 myotubes, and that use of the specific agonist trans-caryophyllene (TC) stimulates sirtuin 1 (SIRT1) deacetylase activity by increasing the phosphorylation of cAMP response element-binding protein (CREB), thus leading to increased levels of PGC-1α deacetylation. This use of TC treatment increases the expression of genes linked to the fatty acid oxidation pathway in a SIRT1/PGC-1α-dependent mechanism and also drastically accelerates the rate of complete fatty acid oxidation in C2C12 myotubes, neither of which occur when CB2R mRNA is knocked down using siRNA. These results reveal that activation of CB2R by a selective agonist promotes lipid oxidation through a signaling/transcriptional pathway. Our findings imply that pharmacological manipulation of CB2R may provide therapeutic possibilities to treat metabolic diseases associated with lipid dysregulation.

  8. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death.

    Science.gov (United States)

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2015-11-01

    Resveratrol (3,5,4'-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. The epigenetic regulation of HIF-1α by SIRT1 in MPP{sup +} treated SH-SY5Y cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Su-Yan; Guo, Yan-Jie; Feng, Ya; Cui, Xin-Xin [Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080 (China); Kuo, Sheng-Han [Department of Neurology, College of Physicians and Surgeons, Columbia University, New York (United States); Liu, Te, E-mail: liute1979@126.com [Shanghai Geriatric Institute of Chinese Medicine, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200031 (China); Wu, Yun-Cheng, E-mail: yunchw@medmail.com.cn [Department of Neurology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200080 (China)

    2016-02-05

    Both silent information regulator 1 (SIRT1) and hypoxia inducible factor 1 (HIF-1) have been found to play important roles in the pathophysiology of Parkinson's disease (PD). However, their mechanisms and their relationship still require further study. In the present study, we focused on the change and relationship of SIRT1 and HIF-1α in PD. PD cell models were established by using methyl-4-phenylpyridinium (MPP{sup +}), which induced inhibition of cell proliferation, cell cycle arrest and apoptosis. We found that the expression of HIF-1α and its target genes VEGFA and LDHA increased and that SIRT1 expression was inhibited in MPP{sup +} treated cells. With further analysis, we found that the acetylation of H3K14 combined with the HIF-1α promoter was dramatically increased in cells treated with MPP{sup +}, which resulted in the transcriptional activation of HIF-1α. Moreover, the acetylation of H3K14 and the expression of HIF-1α increased when SIRT1 was knocked down, suggesting that SIRT1 was involved in the epigenetic regulation of HIF-1α. At last, phenformin, another mitochondrial complex1 inhibitor, was used to testify that the increased HIF-1a was not due to off target effects of MPP{sup +}. Therefore, our results support a link between PD and SIRT1/HIF-1α signaling, which may serve as a clue for understanding PD.

  10. The Sirt1 activator SRT1720 attenuates angiotensin II-induced atherosclerosis in apoE{sup −/−} mice through inhibiting vascular inflammatory response

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yi xi; Zhang, Man; Cai, Yuehua; Zhao, Qihui; Dai, Wenjian, E-mail: wjdai@126.com

    2015-10-02

    Activation of the silent mating type information regulation 2 homolog 1 (SIRT1) has been shown consistent antiinflammatory function. However, little information is available on the function of SIRT1 during Angiotensin II (AngII)-induced atherosclerosis. Here we report atheroprotective effects of sirt1 activation in a model of AngII-accelerated atherosclerosis, characterized by suppression pro-inflammatory transcription factors Nuclear transcription factor (NF)-κB and Signal Transducers and Activators of Transcription. (STAT) signaling pathway, and atherosclerotic lesion macrophage content. In this model, administration of the SIRT1 agonist SRT1720 substantially attenuated AngII-accelerated atherosclerosis with decreasing blood pressure and inhibited NF-κB and STAT3 activation, which was associated with suppression of inflammatory factor and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in AngII-treated VSMCs and macrophages: SIRT1 activation inhibited the expression levels of proinflammatory factor. These studies uncover crucial proinflammatory mechanisms of AngII and highlight actions of SIRT1 activation to inhibit AngII signaling, which is atheroprotective. - Highlights: • SRT1720 reduced atherosclerotic lesion size in aortic arches and atherosclerotic lesion macrophage content. • SRT1720 could inhibit the phosphorylation of STAT3 and p65 phosphorylation and translocation. • SRT1720 could inhibit the expression of proinflammatory factor.

  11. Mechanism of Anti-glioblastoma Effect of Temzolomide Involved in ROS-Mediated SIRT 1 Pathway

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    Yuan Jiang

    2014-03-01

    Full Text Available Objective: To explore the new molecular mechanism of anti-tumor effect of temzolomide (TMZon glioblastoma cell strain. Methods: MTT methods and Hoechst 33342 staining method were applied to determine the effect of TMZ on the proliferation and apoptosis of glioblastoma cell strains U251 and SHG44, while flow cytometry was used to detect the impact of TMZ on cellular cycles. Additionally, DCFH-DA probe was adopted to test intracellular reactive oxygen species (ROS level while Real-time PCR and Western blot tests were applied to determine the influence of TMZ on SIRT1 expression. Results: TMZ in different concentrations added into glioblastoma cell strain for 72 h could concentration-dependently inhibit the proliferation of glioblastoma cells, 100 μmol/L of which could also block cells in phase G2/M and improve cellular apoptosis. In addition, TMZ could evidently increase intracellular ROS level so as to activate SIRT1. Conclusion: The mechanism of anti-tumor effect of TMZ on glioblastoma may be associated with ROS-induced SIRT1 pathway, providing theoretical basis for the clinical efficacy of TMZ.

  12. SIRT1 genetic variants associate with the metabolic response of Caucasians to a controlled lifestyle intervention – the TULIP Study

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    Stefan Norbert

    2008-11-01

    Full Text Available Abstract Background Sirtuin1 (SIRT1 regulates gene expression in distinct metabolic pathways and mediates beneficial effects of caloric restriction in animal models. In humans, SIRT1 genetic variants associate with fasting energy expenditure. To investigate the relevance of SIRT1 for human metabolism and caloric restriction, we analyzed SIRT1 genetic variants in respect to the outcome of a controlled lifestyle intervention in Caucasians at risk for type 2 diabetes. Methods A total of 1013 non-diabetic Caucasians from the Tuebingen Family Study (TUEF were genotyped for four tagging SIRT1 SNPs (rs730821, rs12413112, rs7069102, rs2273773 for cross-sectional association analyses with prediabetic traits. SNPs that associated with basal energy expenditure in the TUEF cohort were additionally analyzed in 196 individuals who underwent a controlled lifestyle intervention (Tuebingen Lifestyle Intervention Program; TULIP. Multivariate regressions analyses with adjustment for relevant covariates were performed to detect associations of SIRT1 variants with the changes in anthropometrics, weight, body fat or metabolic characteristics (blood glucose, insulin sensitivity, insulin secretion and liver fat, measured by magnetic resonance techniques after the 9-month follow-up test in the TULIP study. Results Minor allele (X/A carriers of rs12413112 (G/A had a significantly lower basal energy expenditure (p = 0.04 and an increased respiratory quotient (p = 0.02. This group (rs12413112: X/A was resistant against lifestyle-induced improvement of fasting plasma glucose (GG: -2.01%, X/A: 0.53%; p = 0.04, had less increase in insulin sensitivity (GG: 17.3%, X/A: 9.6%; p = 0.05 and an attenuated decline in liver fat (GG: -38.4%, X/A: -7.5%; p = 0.01. Conclusion SIRT1 plays a role for the individual lifestyle intervention response, possibly owing to decreased basal energy expenditure and a lower lipid-oxidation rate in rs12413112 X/A allele carriers. SIRT1 genetic

  13. Aerobic endurance capacity affects spatial memory and SIRT1 is a potent modulator of 8-oxoguanine repair.

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    Sarga, L; Hart, N; Koch, L G; Britton, S L; Hajas, G; Boldogh, I; Ba, X; Radak, Z

    2013-11-12

    Regular exercise promotes brain function via a wide range of adaptive responses, including the increased expression of antioxidant and oxidative DNA damage-repairing systems. Accumulation of oxidized DNA base lesions and strand breaks is etiologically linked to for example aging processes and age-associated diseases. Here we tested whether exercise training has an impact on brain function, extent of neurogenesis, and expression of 8-oxoguanine DNA glycosylase-1 (Ogg1) and SIRT1 (silent mating-type information regulation 2 homolog). To do so, we utilized strains of rats with low- and high-running capacity (LCR and HCR) and examined learning and memory, DNA synthesis, expression, and post-translational modification of Ogg1 hippocampal cells. Our results showed that rats with higher aerobic/running capacity had better spatial memory, and expressed less Ogg1, when compared to LCR rats. Furthermore, exercise increased SIRT1 expression and decreased acetylated Ogg1 (AcOgg1) levels, a post-translational modification important for efficient repair of 8-oxo-7,8-dihydroguanine (8-oxoG). Our data on cell cultures revealed that nicotinamide, a SIRT1-specific inhibitor, caused the greatest increase in the acetylation of Ogg1, a finding further supported by our other observations that silencing SIRT1 also markedly increased the levels of AcOgg1. These findings imply that high-running capacity is associated with increased hippocampal function, and SIRT1 level/activity and inversely correlates with AcOgg1 levels and thereby the repair of genomic 8-oxoG. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Terpenylated coumarins as SIRT1 activators isolated from Ailanthus altissima

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    Dao, Trong-Tuan; Tran, Tien-Lam; Kim, Jayeon

    2012-01-01

    Four new terpenylated coumarins (1-4) were isolated from the stem bark of Ailanthus altissima by bioactivity-guided fractionation using an in vitro SIRT1 deacetylation assay. Their structures were identified as (2'R,3'R)-7-(2',3'-dihydroxy-3',7'-dimethylocta-6'-enyloxy)-6,8-dimethoxycoumarin (1),...

  15. TNF-α and LPS activate angiogenesis via VEGF and SIRT1 signalling in human dental pulp cells.

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    Shin, M R; Kang, S K; Kim, Y S; Lee, S Y; Hong, S C; Kim, E-C

    2015-07-01

    To assess whether SIRT1 and VEGF are responsible for tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS)-induced angiogenesis and to examine the molecular mechanism(s) of action in human dental pulp cells (HDPCs). Immortalized HDPCs obtained from Prof. Takashi Takata (Hiroshima University, Japan) were treated with LPS (1 μg mL(-1) ) and TNF-α (10 ng mL(-1) ) for 24 h. mRNA and protein levels were examined by RT-PCR and Western blotting, respectively. Migration and tube formation were examined in human umbilical vein endothelial cells (HUVECs). The data were analysed by one-way anova. Statistical analysis was performed at α = 0.05. LPS and TNF-α upregulated VEGF and SIRT1 mRNA and protein levels. Inhibition of SIRT1 activity by sirtinol and SIRT1 siRNA or inhibition of the VEGF receptor by CBO-P11 significantly attenuated LPS + TNF-α-stimulated MMPs production in HDPCs, as well as migration and tube formation in HUVECs (P disease. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  16. Low-Frequency Electroacupuncture Improves Insulin Sensitivity in Obese Diabetic Mice through Activation of SIRT1/PGC-1α in Skeletal Muscle

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    Fengxia Liang

    2011-01-01

    Full Text Available Electroacupuncture (EA has been observed to reduce insulin resistance in obesity and diabetes. However, the biochemical mechanism underlying this effect remains unclear. This study investigated the effects of low-frequency EA on metabolic action in genetically obese and type 2 diabetic db/db mice. Nine-week-old db/m and db/db mice were randomly divided into four groups, namely, db/m, db/m + EA, db/db, and db/db + EA. db/m + EA and db/db + EA mice received 3-Hz electroacupuncture five times weekly for eight consecutive weeks. In db/db mice, EA tempered the increase in fasting blood glucose, food intake, and body mass and maintained insulin levels. In EA-treated db/db mice, improved insulin sensitivity was established through intraperitoneal insulin tolerance test. EA was likewise observed to decrease free fatty acid levels in db/db mice; it increased protein expression in skeletal muscle Sirtuin 1 (SIRT1 and induced gene expression of peroxisome proliferator-activated receptor coactivator (PGC-, nuclear respiratory factor 1 (NRF1, and acyl-CoA oxidase (ACOX. These results indicated that EA offers a beneficial effect on insulin resistance in obese and diabetic db/db mice, at least partly, via stimulation of SIRT1/PGC-, thus resulting in improved insulin signal.

  17. Resveratrol Targeting of Carcinogen-Induced Brain Endothelial Cell Inflammation Biomarkers MMP-9 and COX-2 is Sirt1-Independent

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    Borhane Annabi

    2012-01-01

    Full Text Available The occurrence of a functional relationship between the release of metalloproteinases (MMPs and the expression of cyclooxygenase (COX-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB, increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Tissue scan microarray analysis of grade I–IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I–III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA, a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IκB phosphorylation in siSirt1-transfected HBMEC was however observed. Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1.

  18. Sirtuin1 Over-Expression Does Not Impact Retinal Vascular and Neuronal Degeneration in a Mouse Model of Oxygen-Induced Retinopathy

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    Michan, Shaday; Juan, Aimee M.; Hurst, Christian G.; Cui, Zhenghao; Evans, Lucy P.; Hatton, Colman J.; Pei, Dorothy T.; Ju, Meihua; Sinclair, David A.; Smith, Lois E. H.; Chen, Jing

    2014-01-01

    Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy. PMID:24416337

  19. Sirtuin1 over-expression does not impact retinal vascular and neuronal degeneration in a mouse model of oxygen-induced retinopathy.

    Science.gov (United States)

    Michan, Shaday; Juan, Aimee M; Hurst, Christian G; Cui, Zhenghao; Evans, Lucy P; Hatton, Colman J; Pei, Dorothy T; Ju, Meihua; Sinclair, David A; Smith, Lois E H; Chen, Jing

    2014-01-01

    Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy.

  20. Acute and long-term nutrient-led modifications of gene expression: potential role of SIRT1 as a central co-ordinator of short and longer-term programming of tissue function.

    Science.gov (United States)

    Holness, Mark J; Caton, Paul W; Sugden, Mary C

    2010-05-01

    Environmental factors can influence the acute and longer-term risks of developing diseases, including type 2 diabetes mellitus and cardiovascular disease; however, the underlying mechanism remains elusive. Increasing evidence suggests that these effects can be achieved by modification of metabolic gene expression. These include acute changes in histone methylation, acetylation, phosphorylation, and ubiquitination and longer-term DNA silencing elicited by DNA methylation. Thus, an increased risk of disease may reflect acute or chronic stable modification of genes that regulate nutrient handling, leading to altered nutrient utilization (increased lipid oxidation at the expense of glucose utilization) and/or changes in the balance between nutrient storage and energy production, thereby favoring the development of obesity. The review addresses the hypothesis that early-life epigenetic programming of gene expression could be mirrored by changes in acute function of nuclear receptors, in particular the peroxisome proliferator-activated receptors, achieved by enzymes that are more conventionally involved in regulating DNA methylation and post-transcriptional modification of histones. Emphasis is placed on the potential importance of the protein deacetylase sirtuin-1 as a central co-ordinator. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle

    DEFF Research Database (Denmark)

    Cantó, Carles; Jiang, Lake Q; Deshmukh, Atul S

    2010-01-01

    During fasting and after exercise, skeletal muscle efficiently switches from carbohydrate to lipid as the main energy source to preserve glycogen stores and blood glucose levels for glucose-dependent tissues. Skeletal muscle cells sense this limitation in glucose availability and transform...... and lipid utilization genes. Deficient AMPK activity compromises SIRT1-dependent responses to exercise and fasting, resulting in impaired PGC-1alpha deacetylation and blunted induction of mitochondrial gene expression. Thus, we conclude that AMPK acts as the primordial trigger for fasting- and exercise...

  2. Resveratrol Ameliorates Palmitate-Induced Inflammation in Skeletal Muscle Cells by Attenuating Oxidative Stress and JNK/NF-κB Pathway in a SIRT1-Independent Mechanism.

    Science.gov (United States)

    Sadeghi, Asie; Seyyed Ebrahimi, Shadi Sadat; Golestani, Abolfazl; Meshkani, Reza

    2017-09-01

    Resveratrol has been shown to exert anti-inflammatory and anti-oxidant effects in a variety of cell types, however, its role in prevention of inflammatory responses mediated by palmitate in skeletal muscle cells remains unexplored. In the present study, we investigated the effects of resveratrol on palmitate-induced inflammation and elucidated the underlying mechanisms in skeletal muscle cells. The results showed that palmitate significantly enhanced TNF-α and IL-6 mRNA expression and protein secretion from C2C12 cells at 12, 24, and 36 h treatments. Increased expression of cytokines was accompanied by an enhanced phosphorylation of JNK, P38, ERK1/2, and IKKα/IKKβ. In addition, JNK and P38 inhibitors could significantly attenuate palmitate-induced mRNA expression of TNF-α and IL-6, respectively, whereas NF-κB inhibitor reduced the expression of both cytokines in palmitate-treated cells. Resveratrol pretreatment significantly prevented palmitate-induced TNF-α and IL-6 mRNA expression and protein secretion in C2C12 cells. Importantly, pre-treatment of the cells with resveratrol completely abrogated the phosphorylation of ERK1/2, JNK, and IKKα/IKKβ in palmitate treated cells. The protection from palmitate-induced inflammation by resveratrol was accompanied by a decrease in the generation of reactive oxygen species (ROS). N-acetyl cysteine (NAC), a known scavenger of ROS, could protect palmitate-induced expression of TNF-α and IL-6. Furthermore, inhibition of SIRT1 by shRNA or sirtinol demonstrated that the anti-inflammatory effect of resveratrol in muscle cells is mediated through a SIRT1-independent mechanism. Taken together, these findings suggest that resveratrol may represent a promising therapy for prevention of inflammation in skeletal muscle cells. J. Cell. Biochem. 118: 2654-2663, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Metformin and Resveratrol Inhibited High Glucose-Induced Metabolic Memory of Endothelial Senescence through SIRT1/p300/p53/p21 Pathway.

    Science.gov (United States)

    Zhang, Erli; Guo, Qianyun; Gao, Haiyang; Xu, Ruixia; Teng, Siyong; Wu, Yongjian

    2015-01-01

    Endothelial senescence plays crucial roles in diabetic vascular complication. Recent evidence indicated that transient hyperglycaemia could potentiate persistent diabetic vascular complications, a phenomenon known as "metabolic memory." Although SIRT1 has been demonstrated to mediate high glucose-induced endothelial senescence, whether and how "metabolic memory" would affect endothelial senescence through SIRT1 signaling remains largely unknown. In this study, we investigated the involvement of SIRT1 axis as well as the protective effects of resveratrol (RSV) and metformin (MET), two potent SIRT1 activators, during the occurrence of "metabolic memory" of cellular senescence (senescent "memory"). Human umbilical vascular endothelial cells (HUVECs) were cultured in either normal glucose (NG)/high glucose (HG) media for 6 days, or 3 days of HG followed by 3 days of NG (HN), with or without RSV or MET treatment. It was shown that HN incubation triggered persistent downregulation of deacetylase SIRT1 and upregulation of acetyltransferase p300, leading to sustained hyperacetylation (at K382) and activation of p53, and subsequent p53/p21-mediated senescent "memory." In contrast, senescent "memory" was abrogated by overexpression of SIRT1 or knockdown of p300. Interestingly, we found that SIRT1 and p300 could regulate each other in response to HN stimulation, suggesting that a delicate balance between acetyltransferases and deacetylases may be particularly important for sustained acetylation and activation of non-histone proteins (such as p53), and eventually the occurrence of "metabolic memory." Furthermore, we found that RSV or MET treatment prevented senescent "memory" by modulating SIRT1/p300/p53/p21 pathway. Notably, early and continuous treatment of MET, but not RSV, was particularly important for preventing senescent "memory." In conclusion, short-term high glucose stimulation could induce sustained endothelial senescence via SIRT1/p300/p53/p21 pathway. RVS or MET

  4. SIRT1 activation with neuroheal is neuroprotective but SIRT2 inhibition with AK7 is detrimental for disconnected motoneurons.

    Science.gov (United States)

    Romeo-Guitart, David; Leiva-Rodríguez, Tatiana; Espinosa-Alcantud, María; Sima, Núria; Vaquero, Alejandro; Domínguez-Martín, Helena; Ruano, Diego; Casas, Caty

    2018-05-10

    Sirtuin 1 (SIRT1) activity is neuroprotective, and we have recently demonstrated its role in the retrograde degenerative process in motoneurons (MNs) in the spinal cord of rats after peripheral nerve root avulsion (RA) injury. SIRT2 has been suggested to exert effects opposite those of SIRT1; however, its roles in neurodegeneration and neuron response after nerve injury remain unclear. Here we compared the neuroprotective potentials of SIRT1 activation and SIRT2 inhibition in a mouse model of hypoglossal nerve axotomy. This injury induced a reduction of around half MN population within the hypoglossal nucleus by a non-apoptotic neurodegenerative process triggered by endoplasmic reticulum (ER) stress that resulted in activation of the unfolded protein response mediated by IRE1α and XBP1 by 21 days post injury. Both SIRT1 activation with NeuroHeal and SIRT2 inhibition with AK7 protected NSC-34 motor neuron-like cells against ER stress in vitro. In agreement with the in vitro results, NeuroHeal treatment or SIRT1 overexpression was neuroprotective of axotomized hypoglossal MNs in a transgenic mouse model. In contrast, AK7 treatment or SIRT2 genetic depletion in mice inhibited damaged MN survival. To resolve the in vitro/in vivo discrepancies, we used an organotypic spinal cord culture system that preserves glial cells. In this system, AK7 treatment of ER-stressed organotypic cultures was detrimental for MNs and increased microglial nuclear factor-κB and the consequent transcription of cytotoxic pro-inflammatory factors similarly. The results highlight the importance of glial cells in determining the neuroprotective impact of any treatment.

  5. HGF and BFGF Secretion by Human Adipose-Derived Stem Cells Improves Ovarian Function During Natural Aging via Activation of the SIRT1/FOXO1 Signaling Pathway.

    Science.gov (United States)

    Ding, Chenyue; Zou, Qinyan; Wang, Fuxin; Wu, Huihua; Wang, Wei; Li, Hong; Huang, Boxian

    2018-01-01

    Human adipose-derived stem cells (hADSCs) are a potential therapeutic option for clinical applications because of their ability to produce cytokines and their capacity for trilineage differentiation. To date, few researchers have investigated the effects of hADSCs on natural ovarian aging (NOA). An NOA mouse model and human ovarian granule cells (hGCs) collected from individuals with NOA were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing NOA. Enzyme-linked immunosorbent assay was used to detect the serum levels of sex hormones and antioxidative enzymes. The proliferation rate and marker expression level of hGCs were measured by flow cytometry (FACS). Cytokines were measured by a protein antibody array methodology. Western blot assays were used to determine the protein expression levels of SIRT1 and FOXO1. Our results showed that hADSCs displayed therapeutic activity against ovarian function in an NOA mouse model, increasing the proliferation rate and marker expression level of hGCs. Furthermore, the yields of hADSC-secreted HGF and bFGF were higher than those of other growth factors. FACS showed that combination treatment with the growth factors HGF and bFGF more strongly promoted proliferation and inhibited apoptosis in hGCs than HGF or bFGF treatment alone. FACS and ELISA revealed that the combination treatment with both growth factors inhibited oxidative stress more forcefully than treatments with only one of these growth factors. In addition, protein assays demonstrated that combination treatment with both growth factors suppressed oxidative stress by up-regulating the expression of SIRT1 and FOXO1. These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which HGF and bFGF derived from hADSCs improved ovarian function during natural aging via reduction of oxidative stress by activating the SIRT1/FOXO1 signaling pathway. © 2018 The Author

  6. Supplementation with an insoluble fiber obtained from carob pod (Ceratonia siliqua L.) rich in polyphenols prevents dyslipidemia in rabbits through SIRT1/PGC-1α pathway.

    Science.gov (United States)

    Valero-Muñoz, María; Ballesteros, Sandra; Ruiz-Roso, Baltasar; Pérez-Olleros, Lourdes; Martín-Fernández, Beatriz; Lahera, Vicente; de Las Heras, Natalia

    2017-12-22

    To investigate the mechanism implicated in the effect of an insoluble fiber (obtained from carob pod) rich in polyphenols (IFCP) in lipid metabolism in the liver. Male New Zealand rabbits were fed with the following diets for 8 weeks: control diet (CT group), dyslipidemic diet supplemented with 0.5% cholesterol + 14% coconut oil (DL group) and dyslipidemic diet containing 0.5% cholesterol + 14% coconut oil plus 3% IFCP (DL + IFCP group). Dyslipidemic diet with IFCP was able to reduce development of mixed dyslipidemia, liver relative weight and collagen I protein expression compared to DL rabbits. Analyses of the main enzymes implicated in cholesterol and triglycerides metabolism revealed that IFCP increased hepatic concentration of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cytochrome P450, family 7, subfamily a, polypeptide 1C (CYP7A1) (82.34, 114.42%, respectively) as well as protein expression of LDL receptor (42.48%) in DL rabbits. Importantly, IFCP also increased hepatic lipase (HL) levels (91.43%) and decreased glycerol phosphate acyltransferase (GPAT) and sterol regulatory element-binding protein 1C (SREBP1c) liver expression levels (20.38 and 41.20%, respectively). Finally, sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α) hepatic expression increased in DL + IFCP group compared with DL (159.81 and 48.00%, respectively). These findings show that IFCP is able to abrogate the deleterious effects of hepatic dyslipidemia by modulating SIRT1 and PGC-1α pathways.

  7. Tanshinone IIA suppresses FcεRI-mediated mast cell signaling and anaphylaxis by activation of the Sirt1/LKB1/AMPK pathway.

    Science.gov (United States)

    Li, Xian; Park, Soon Jin; Jin, Fansi; Deng, Yifeng; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Lee, Youn Ju; Murakami, Makoto; Son, Kun Ho; Chang, Hyeun Wook

    2018-06-01

    AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2 -/- or Sirt1 -/- mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2 -/- and Sirt1 -/- mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FcεRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2 -/- or Sirt1 -/- mice. In conclusion, Tan IIA suppresses FcεRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Endogenous Nampt upregulation is associated with diabetic nephropathy inflammatory-fibrosis through the NF-κB p65 and Sirt1 pathway; NMN alleviates diabetic nephropathy inflammatory-fibrosis by inhibiting endogenous Nampt.

    Science.gov (United States)

    Chen, Ye; Liang, Yuzhen; Hu, Tingting; Wei, Riming; Cai, Congjie; Wang, Ping; Wang, Lingyu; Qiao, Wei; Feng, Leping

    2017-11-01

    Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme in the nicotinamide adenine dinucleotide (NAD + ) biosynthetic pathway. Exogenous extra cellular Nampt has been reported to increase the synthesis of pro-fibrotic molecules in various types of renal cells. However, the role of endogenous Namptenzymatic activity in diabetic renal cells, particularly those associated with inflammation and fibrosis through the nuclear factor (NF)-κB p65 and sirtuin 1 (Sirt1) pathway is still unknown. In the present study, a possible mechanism by which endogenous Nampt upregulation affects the expression of pro-inflammatory and pro-fibrotic cytokines in vivo and in vitro , is reported. The present results demonstrate that the expression of vimentin and fibronectin was directly implicated in endogenous Nampt upregulation. The expression levels of Poly(ADP-ribose) polymerase-1, NF-κB p65, forkhead box protein O1 and B-cell lymphoma 2-like protein 4 were also significantly increased at 96 h compared with control group (Pendogenous Nampt upregulation. Furthermore, the expression level of Sirt1 was significantly reduced (Pendogenous Nampt upregulation may be critical in the treatment of DN pro-inflammatory fibrosis and NMN is likely to be a potential pharmacological agent for the treatment of resistant DN nephritic fibrosis.

  9. Capsaicin Inhibits Multiple Bladder Cancer Cell Phenotypes by Inhibiting Tumor-Associated NADH Oxidase (tNOX and Sirtuin1 (SIRT1

    Directory of Open Access Journals (Sweden)

    Ming-Hung Lin

    2016-06-01

    Full Text Available Bladder cancer is one of the most frequent cancers among males, and its poor survival rate reflects problems with aggressiveness and chemo-resistance. Recent interest has focused on the use of chemopreventatives (nontoxic natural agents that may suppress cancer progression to induce targeted apoptosis for cancer therapy. Capsaicin, which has anti-cancer properties, is one such agent. It is known to preferentially inhibit a tumor-associated NADH oxidase (tNOX that is preferentially expressed in cancer/transformed cells. Here, we set out to elucidate the correlation between tNOX expression and the inhibitory effects of capsaicin in human bladder cancer cells. We showed that capsaicin downregulates tNOX expression and decreases bladder cancer cell growth by enhancing apoptosis. Moreover, capsaicin was found to reduce the expression levels of several proteins involved in cell cycle progression, in association with increases in the cell doubling time and enhanced cell cycle arrest. Capsaicin was also shown to inhibit the activation of ERK, thereby reducing the phosphorylation of paxillin and FAK, which leads to decreased cell migration. Finally, our results indicate that RNA interference-mediated tNOX depletion enhances spontaneous apoptosis, prolongs cell cycle progression, and reduces cell migration and the epithelial-mesenchymal transition. We also observed a downregulation of sirtuin 1 (SIRT1 in these tNOX-knockdown cells, a deacetylase that is important in multiple cellular functions. Taken together, our results indicate that capsaicin inhibits the growth of bladder cancer cells by inhibiting tNOX and SIRT1 and thereby reducing proliferation, attenuating migration, and prolonging cell cycle progression.

  10. Evidence for a Common Mechanism of SIRT1 Regulation by Allosteric Activators

    Science.gov (United States)

    Hubbard, Basil P.; Gomes, Ana P.; Dai, Han; Li, Jun; Case, April W.; Considine, Thomas; Riera, Thomas V.; Lee, Jessica E.; Sook Yen, E; Lamming, Dudley W.; Pentelute, Bradley L.; Schuman, Eli R.; Stevens, Linda A.; Ling, Alvin J. Y.; Armour, Sean M.; Michan, Shaday; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M.; Blum, Charles A.; Disch, Jeremy S.; Ng, Pui Yee; Howitz, Konrad T.; Rolo, Anabela P.; Hamuro, Yoshitomo; Moss, Joel; Perni, Robert B.; Ellis, James L.; Vlasuk, George P.; Sinclair, David A.

    2013-01-01

    A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu230, located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs. PMID:23471411

  11. Evidence for a common mechanism of SIRT1 regulation by allosteric activators.

    Science.gov (United States)

    Hubbard, Basil P; Gomes, Ana P; Dai, Han; Li, Jun; Case, April W; Considine, Thomas; Riera, Thomas V; Lee, Jessica E; E, Sook Yen; Lamming, Dudley W; Pentelute, Bradley L; Schuman, Eli R; Stevens, Linda A; Ling, Alvin J Y; Armour, Sean M; Michan, Shaday; Zhao, Huizhen; Jiang, Yong; Sweitzer, Sharon M; Blum, Charles A; Disch, Jeremy S; Ng, Pui Yee; Howitz, Konrad T; Rolo, Anabela P; Hamuro, Yoshitomo; Moss, Joel; Perni, Robert B; Ellis, James L; Vlasuk, George P; Sinclair, David A

    2013-03-08

    A molecule that treats multiple age-related diseases would have a major impact on global health and economics. The SIRT1 deacetylase has drawn attention in this regard as a target for drug design. Yet controversy exists around the mechanism of sirtuin-activating compounds (STACs). We found that specific hydrophobic motifs found in SIRT1 substrates such as PGC-1α and FOXO3a facilitate SIRT1 activation by STACs. A single amino acid in SIRT1, Glu(230), located in a structured N-terminal domain, was critical for activation by all previously reported STAC scaffolds and a new class of chemically distinct activators. In primary cells reconstituted with activation-defective SIRT1, the metabolic effects of STACs were blocked. Thus, SIRT1 can be directly activated through an allosteric mechanism common to chemically diverse STACs.

  12. A high-fat diet and NAD+ activate sirt1 to rescue premature aging in cockayne syndrome

    DEFF Research Database (Denmark)

    Scheibye-Knudsen, Morten; Mitchell, Sarah J.; Fang, Evandro F.

    2014-01-01

    -fat, caloric-restricted, or resveratrol-supplemented diet. High-fat feeding rescued the metabolic, transcriptomic, and behavioral phenotypes of Csbm/m mice. Furthermore, premature aging in CS mice, nematodes, and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased......Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in genes encoding the DNA repair proteins CS group A or B (CSA or CSB). Since dietary interventions can alter neurodegenerative processes, Csbm/m mice were given a high...... SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high-fat diet, and β-hydroxybutyrate, PARP inhibition, or NAD+ supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB can displace activated PARP1 from damaged DNA...

  13. Multi-regulatory network of ROS: the interconnection of ROS, PGC-1 alpha, and AMPK-SIRT1 during exercise.

    Science.gov (United States)

    Thirupathi, Anand; de Souza, Claudio Teodoro

    2017-11-01

    Transcriptional factors are easily susceptible to any stimuli, including exercise. Exercise can significantly influence PGC-1 α and AMPK-SIRT1 pathway, as it is involved in the regulation of energy metabolism and mitochondrial biogenesis. Exercise is a major energy deprivation process by which many of transcription factors get tuned positively. However, how transcription factors help to boost the antioxidant defense system at cellular level is elusive. It is well known that physical exercise can induce reactive oxygen species, but how these reactive oxygen species can help to regulate multiple transcription factors during exercise is an important area to be discussed yet. This review mainly focuses on interconnecting role of PGC-1 α and AMPK-SIRT1 pathway during exercise and how these proteins are getting tuned by reactive oxygen species in exercise condition.

  14. Ionizing Radiation Induces Cellular Senescence of Articular Chondrocytes via Negative Regulation of SIRT1 by p38 Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Eun Hee; Hwang, Sang Gu [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2009-05-15

    Senescent cells exhibit irreversible growth arrest, large flat morphology, and up-regulated senescence-associated {beta}-galactosidase activity at pH 6.0. Several conditions, including oncogenic stress, oxidative stress, and DNA damage are associated with cellular senescence. Massive acute DNA double-strand breaks occurring as a result of mechanical and chemical stress can be repaired, but some DNA damage persists, eventually triggering premature senescence. Since ionizing radiation directly induces DBS, it is possible that cellular senescence is activated under these conditions. The biological events in chondrocytes following irradiation are poorly understood, and limited information is available on the molecular signal transduction mechanisms of cellular senescence at present. In this study, we identify SIRT1 as a target molecule of p38 kinase and demonstrate that the interactions between p38 kinase and SIRT1 protein play an important role in the regulation of cellular senescence in response to IR.

  15. Silibinin Restores NAD⁺ Levels and Induces the SIRT1/AMPK Pathway in Non-Alcoholic Fatty Liver.

    Science.gov (United States)

    Salomone, Federico; Barbagallo, Ignazio; Godos, Justyna; Lembo, Vincenzo; Currenti, Walter; Cinà, Diana; Avola, Roberto; D'Orazio, Nicolantonio; Morisco, Filomena; Galvano, Fabio; Li Volti, Giovanni

    2017-09-30

    Nicotinamide adenine dinucleotide (NAD⁺) homeostasis is emerging as a key player in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) and is tightly linked to the SIRT1/5'-AMP-activated protein kinase (AMPK) pathway. Silibinin, the main component of silymarin, has been proposed as a nutraceutical for the treatment of NAFLD. In this study, we aimed to identify whether silibinin may influence the NAD⁺/SIRT1 axis. To this end, C57BL/6 mice were fed a high fat diet (HFD) for 16 weeks, and were treated with silibinin or vehicle during the last 8 weeks. HepG2 cells were treated with 0.25 mM palmitate for 24 h with silibinin 25 µM or vehicle. HFD and palmitate administration led to oxidative stress, poly-(ADP-ribose)-polymerase (PARP) activation, NAD⁺ consumption, and lower SIRT1 activity. In mice fed the HFD, and in HepG2 treated with palmitate, we consistently observed lower levels of phospho-AMPK Thr172 and phospho-acetyl-CoA carboxylase Ser79 and higher levels of nuclear sterol regulatory element-binding protein 1 activity, indicating de novo lipogenesis. Treatment of mice and HepG2 with silibinin abolished oxidative stress, and inhibited PARP activation thus restoring the NAD⁺ pool. In agreement with preserved NAD⁺ levels, SIRT1 activity and AMPK phosphorylation returned to control levels in mice and HepG2. Our results further indicate silibinin as a promising molecule for the treatment of NAFLD.

  16. Differential roles of Sirt1 in HIF-1α and HIF-2α mediated hypoxic responses

    International Nuclear Information System (INIS)

    Yoon, Haejin; Shin, Seung-Hyun; Shin, Dong Hoon; Chun, Yang-Sook; Park, Jong-Wan

    2014-01-01

    Highlights: • Roles of SIRT1 in HIF-1α and HIF-2α regulations are reevaluated using specific antibodies and Gal4 reporters. • SIRT1 represses the HIF-1α-driven transcription constantly in ten cancer cell-lines. • SIRT1 regulates the HIF-2α-driven transcription cell context-dependently. • SIRT1 determines cell growth under hypoxia by regulating HIF-1α and HIF-2α activities. - Abstract: Hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) determine cancer cell fate under hypoxia. Despite the similarities of their structures, HIF-1α and HIF-2α have distinct roles in cancer growth under hypoxia, that is, HIF-1α induces growth arrest whereas HIF-2α promotes cell growth. Recently, sirtuin 1 (Sirt1) was reported to fine-tune cellular responses to hypoxia by deacetylating HIF-1α and HIF-2α. Yet, the roles of Sirt1 in HIF-1α and HIF-2α functions have been controversial. We here investigated the precise roles of Sirt1 in HIF-1α and HIF-2α regulations. Immunological analyses revealed that HIF-1α K674 and HIF-2α K741 are acetylated by PCAF and CBP, respectively, but are deacetylated commonly by Sirt1. In the Gal4 reporter systems, Sirt1 was found to repress HIF-1α activity constantly in ten cancer cell-lines but to regulate HIF-2α activity cell type-dependently. Moreover, Sirt1 determined cell growth under hypoxia depending on HIF-1α and HIF-2α. Under hypoxia, Sirt1 promoted cell proliferation of HepG2, in which Sirt1 differentially regulates HIF-1α and HIF-2α. In contrast, such an effect of Sirt1 was not shown in HCT116, in which Sirt1 inactivates both HIF-1α and HIF-2α because conflicting actions of HIF-1α and HIF-2α on cell growth may be offset. Our results provide a better understanding of the roles of Sirt1 in HIF-mediated hypoxic responses and also a basic concept for developing anticancer strategy targeting Sirt1

  17. Ablation of systemic SIRT1 activity promotes nonalcoholic fatty liver disease by affecting liver-mesenteric adipose tissue fatty acid mobilization

    Science.gov (United States)

    The incidence of nonalcoholic fatty liver disease (NAFLD) is escalating paralleled with obesity rates in both adults and children. Mammalian sirtuin 1 (SIRT1), a highly conserved NAD+-dependent protein deacetylase, has been identified as a metabolic regulator of lipid homeostasis and a potential tar...

  18. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

    Energy Technology Data Exchange (ETDEWEB)

    Guida, Natascia [IRCSS SDN, Naples 80131 (Italy); Laudati, Giusy [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Anzilotti, Serenella [IRCSS SDN, Naples 80131 (Italy); Secondo, Agnese [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Montuori, Paolo [Department of Public Health, ‘Federico II’ University of Naples, Naples (Italy); Di Renzo, Gianfranco [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Canzoniero, Lorella M.T. [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy); Formisano, Luigi, E-mail: cformisa@unisannio.it [Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, “Federico II” University of Naples, Via Pansini, 5, 80131 Naples (Italy); Division of Pharmacology, Department of Science and Technology, University of Sannio, Via Port' Arsa 11, 82100 Benevento (Italy)

    2015-11-01

    Resveratrol (3,5,4′-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. - Highlights: • Resveratrol via SIRT1/c-Jun downregulates REST mRNA and protein in SH-SY5Y cells. • Non-dioxin-like (NDL) PCB-95 is cytotoxic to

  19. Resveratrol via sirtuin-1 downregulates RE1-silencing transcription factor (REST) expression preventing PCB-95-induced neuronal cell death

    International Nuclear Information System (INIS)

    Guida, Natascia; Laudati, Giusy; Anzilotti, Serenella; Secondo, Agnese; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2015-01-01

    Resveratrol (3,5,4′-trihydroxystilbene) (RSV), a polyphenol widely present in plants, exerts a neuroprotective function in several neurological conditions; it is an activator of class III histone deacetylase sirtuin1 (SIRT1), a crucial regulator in the pathophysiology of neurodegenerative diseases. By contrast, the RE1-silencing transcription factor (REST) is involved in the neurotoxic effects following exposure to polychlorinated biphenyl (PCB) mixture A1254. The present study investigated the effects of RSV-induced activation of SIRT1 on REST expression in SH-SY5Y cells. Further, we investigated the possible relationship between the non-dioxin-like (NDL) PCB-95 and REST through SIRT1 to regulate neuronal death in rat cortical neurons. Our results revealed that RSV significantly decreased REST gene and protein levels in a dose- and time-dependent manner. Interestingly, overexpression of SIRT1 reduced REST expression, whereas EX-527, an inhibitor of SIRT1, increased REST expression and blocked RSV-induced REST downregulation. These results suggest that RSV downregulates REST through SIRT1. In addition, RSV enhanced activator protein 1 (AP-1) transcription factor c-Jun expression and its binding to the REST promoter gene. Indeed, c-Jun knockdown reverted RSV-induced REST downregulation. Intriguingly, in SH-SY5Y cells and rat cortical neurons the NDL PCB-95 induced necrotic cell death in a concentration-dependent manner by increasing REST mRNA and protein expression. In addition, SIRT1 knockdown blocked RSV-induced neuroprotection in rat cortical neurons treated with PCB-95. Collectively, these results indicate that RSV via SIRT1 activates c-Jun, thereby reducing REST expression in SH-SY5Y cells under physiological conditions and blocks PCB-95-induced neuronal cell death by activating the same SIRT1/c-Jun/REST pathway. - Highlights: • Resveratrol via SIRT1/c-Jun downregulates REST mRNA and protein in SH-SY5Y cells. • Non-dioxin-like (NDL) PCB-95 is cytotoxic to

  20. Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells.

    Science.gov (United States)

    Dey, Swatee; Bakthavatchalu, Vasudevan; Tseng, Michael T; Wu, Peng; Florence, Rebecca L; Grulke, Eric A; Yokel, Robert A; Dhar, Sanjit Kumar; Yang, Hsin-Sheng; Chen, Yumin; St Clair, Daret K

    2008-10-01

    The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

  1. Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet.

    Directory of Open Access Journals (Sweden)

    Komal Sodhi

    NAFLD-associated vascular dysfunction; effects that are mediated by activation of SIRT1 gene expression.

  2. Resveratrol reduces senescence-associated secretory phenotype by SIRT1/NF-κB pathway in gut of the annual fish Nothobranchius guentheri.

    Science.gov (United States)

    Liu, Shan; Zheng, Zhaodi; Ji, Shuhua; Liu, Tingting; Hou, Yanhan; Li, Shasha; Li, Guorong

    2018-06-13

    Senescent cells display a senescence-associated secretory phenotype (SASP), which contributes to aging. Resveratrol, an activator of SIRT1, has anti-aging, anti-inflammatory, anti-oxidant, anti-free radical and other pharmacological effects. The genus of the annual fish Nothobranchius has become an emerging animal model for studying aging. However, the underlying mechanism for resveratrol to delay aging by SASP regulation has not been elucidated in vertebrates. In this study, the annual fish N. guentheri were fed with resveratrol for long-term treatment. The results showed that resveratrol reversed intensive senescence-associated β-galactosidase activity with aging process, down-regulated levels of SASP-associated proinflammatory cytokines IL-8 and TNFα, and up-regulated expression of anti-inflammatory cytokine IL-10 in gut of the fish. Resveratrol increased SIRT1 expression, and inhibited NF-κB by decreasing RelA/p65, Ac-RelA/p65 and p-IκBα levels and by increasing the interaction between SIRT1 and RelA/p65. Moreover, resveratrol reversed the decline of intestinal epithelial cells (IECs) and intestinal stem cells (ISCs) caused by aging in gut of the fish. Together, our results implied that resveratrol inhibited SASP through SIRT1/NF-κB signaling pathway and delayed aging of the annual fish N. guentheri. Copyright © 2018. Published by Elsevier Ltd.

  3. The combination of blueberry juice and probiotics reduces apoptosis of alcoholic fatty liver of mice by affecting SIRT1 pathway

    Directory of Open Access Journals (Sweden)

    Zhu J

    2016-05-01

    Full Text Available Juanjuan Zhu,1,2,* Tingting Ren,3,* Mingyu Zhou,2 Mingliang Cheng2 1First Hospital Affiliated to Suzhou University, Suzhou, 2Department of Infectious Diseases, 3Biochemistry Department, Affiliated Hospital of Guiyang Medical College, Guiyang, People’s Republic of China *These authors contributed equally to this work Purpose: To explore the effects of the combination of blueberry juice and probiotics on the apoptosis of alcoholic fatty liver disease (AFLD.Methods: Healthy C57BL/6J mice were used in the control group (CG. AFLD mice models were established with Lieber–DeCarli ethanol diet and evenly assigned to six groups with ­different treatments: MG (model, SI (SIRT1 [sirtuin type 1] small interfering RNA [siRNA], BJ (blueberry juice, BJSI (blueberry juice and SIRT1 siRNA, BJP (blueberry juice and probiotics, and BJPSI (blueberry juice, probiotics, and SIRT1 siRNA. Hepatic tissue was observed using hematoxylin and eosin (HE and Oil Red O (ORO staining. Biochemical indexes of the blood serum were analyzed. The levels of SIRT1, caspase-3, forkhead box protein O1 (FOXO1, FasL (tumor necrosis factor ligand superfamily member 6, BAX, and Bcl-2 were measured by reverse transcription-polymerase chain reaction and Western blotting.Results: HE and ORO staining showed that the hepatocytes were heavily destroyed with large lipid droplets in MG and SI groups, while the severity was reduced in the CG, BJ, and BJP groups (P<0.05. The levels of superoxide dismutase (SOD, reduced glutathione (GSH, and high-density lipoprotein-cholesterol (HDL-C were increased in BJ and BJP groups when compared with the model group (P<0.05. In contrast, the levels of aspartate aminotransferase (AST and alanine aminotransferase (ALT, total triglycerides (TGs, total cholesterol, low-density lipoprotein-cholesterol (LDL-C, and malondialdehyde (MDA were lower in BJ and BJP groups than in the model group (P<0.05. The level of SIRT1 was increased, while the levels of FOXO1

  4. Beraprost sodium, a prostacyclin analogue, reduces fructose-induced hepatocellular steatosis in mice and in vitro via the microRNA-200a and SIRT1 signaling pathway.

    Science.gov (United States)

    Zhang, Pengyuan; Xu, Lijuan; Guan, Hongyu; Liu, Liehua; Liu, Juan; Huang, Zhimin; Cao, Xiaopei; Liao, Zhihong; Xiao, Haipeng; Li, Yanbing

    2017-08-01

    To determine whether beraprost sodium, a prostacyclin analogue, could reduce hepatic lipid accumulation induced by fructose in mice and cultured human hepatocytes, and to investigate the expression of microRNAs and the sirtuin 1 (SIRT1) pathway. Male C57BL/6JNju mice were divided into three groups and fed one of the following diets: a normal diet, a high fructose diet, or a high fructose diet with beraprost sodium treatment. In addition, human-derived HepG2 cells were cultured and treated with fructose (25mmol/L) with or without beraprost sodium (10μmol/L) for 24h, and transfected with small interfering RNA (siRNA) against SIRT1, miR-200a mimic, or miR-200a inhibitor for 48h. The miRNA microarray analysis was performed on the HepG2 cells, and the expression profiles of miRNAs were analyzed using Gene Cluster 3.0 and verified using qPCR. Beraprost sodium treatment attenuated hepatic steatosis, induced the transcription of genes involved in lipid metabolism in C57BL/6 mice (Pfructose. These effects were blocked in HepG2 cells after transfection with siRNA against SIRT1. MiR-200a was highly expressed during fructose treatment and was down regulated by beraprost sodium (Pfructose and revealed the primary role of miR-200a in the regulation of hepatic SIRT1 by beraprost sodium. Our findings suggested that SIRT1 might be a therapeutic target of fructose-related metabolism disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. The loss of Sirt1 in mouse pancreatic beta cells impairs insulin secretion by disrupting glucose sensing

    DEFF Research Database (Denmark)

    Luu, L; Dai, F F; Prentice, K J

    2013-01-01

    Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin sec...

  6. SIRT1 Gain of Function Does Not Mimic or Enhance the Adaptations to Intermittent Fasting

    Directory of Open Access Journals (Sweden)

    Marie Boutant

    2016-03-01

    Full Text Available Caloric restriction (CR has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD+-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF. Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can be even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different.

  7. SIRT1 Gain of Function Does Not Mimic or Enhance the Adaptations to Intermittent Fasting.

    Science.gov (United States)

    Boutant, Marie; Kulkarni, Sameer S; Joffraud, Magali; Raymond, Frédéric; Métairon, Sylviane; Descombes, Patrick; Cantó, Carles

    2016-03-08

    Caloric restriction (CR) has been shown to prevent the onset of insulin resistance and to delay age-related physiological decline in mammalian organisms. SIRT1, a NAD(+)-dependent deacetylase enzyme, has been suggested to mediate the adaptive responses to CR, leading to the speculation that SIRT1 activation could be therapeutically used as a CR-mimetic strategy. Here, we used a mouse model of moderate SIRT1 overexpression to test whether SIRT1 gain of function could mimic or boost the metabolic benefits induced by every-other-day feeding (EODF). Our results indicate that SIRT1 transgenesis does not affect the ability of EODF to decrease adiposity and improve insulin sensitivity. Transcriptomic analyses revealed that SIRT1 transgenesis and EODF promote very distinct adaptations in individual tissues, some of which can be even be metabolically opposite, as in brown adipose tissue. Therefore, whereas SIRT1 overexpression and CR both improve glucose metabolism and insulin sensitivity, the etiologies of these benefits are largely different. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Screening SIRT1 Activators from Medicinal Plants as Bioactive Compounds against Oxidative Damage in Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2016-01-01

    Full Text Available Sirtuin type 1 (SIRT1 belongs to the family of NAD+ dependent histone deacetylases and plays a critical role in cellular metabolism and response to oxidative stress. Traditional Chinese medicines (TCMs, as an important part of natural products, have been reported to exert protective effect against oxidative stress in mitochondria. In this study, we screened SIRT1 activators from TCMs and investigated their activities against mitochondrial damage. 19 activators were found in total by in vitro SIRT1 activity assay. Among those active compounds, four compounds, ginsenoside Rb2, ginsenoside F1, ginsenoside Rc, and schisandrin A, were further studied to validate the SIRT1-activation effects by liquid chromatography-mass spectrometry and confirm their activities against oxidative damage in H9c2 cardiomyocytes exposed to tert-butyl hydroperoxide (t-BHP. The results showed that those compounds enhanced the deacetylated activity of SIRT1, increased ATP content, and inhibited intracellular ROS formation as well as regulating the activity of Mn-SOD. These SIRT1 activators also showed moderate protective effects on mitochondrial function in t-BHP cells by recovering oxygen consumption and increasing mitochondrial DNA content. Our results suggested that those compounds from TCMs attenuated oxidative stress-induced mitochondrial damage in cardiomyocytes through activation of SIRT1.

  9. Inhibition of SIRT1 combined with gemcitabine therapy for pancreatic carcinoma

    Directory of Open Access Journals (Sweden)

    Gong DJ

    2013-07-01

    Full Text Available Dao-Jun Gong,1 Jia-Min Zhang,1 Min Yu,1 Bo Zhuang,1 Qing-Qu Guo21Department of Hepatobiliary-Pancreatic Surgery, Jinhua Hospital of Zhejiang University, Jinhua, People's Republic of China; 2Department of Surgery, Second Affiliated Hospital of Zhejiang University College of Medicine, Hangzhou, People's Republic of ChinaBackground: Pancreatic carcinoma possesses one of the highest lethality rates, highest drug-resistance, and highest incidence rates. The objective of this research was to enhance the efficacy and drug-resistance for pancreatic carcinoma by using inhibition of SIRT1 combined with gemcitabine therapy methods.Methods: Three pancreatic carcinoma cells (PANC-1 cells, BxPC-3 cells, and SW1990 cells received treatment with physiological saline, inhibition of SIRT1, gemcitabine, and combination therapy with inhibition of SIRT1 and gemcitabine in vitro; then BxPC-3 pancreatic cancer xenogeneic mice also received treatment with physiological saline, inhibition of SIRT1, gemcitabine, and combination therapy with inhibition of SIRT1 and gemcitabine in vivo.Results: The cleaved poly ADP ribose polymerase (PARP-1 effect of drug in pancreatic carcinoma cells was significantly different (P < 0.05 and the efficacy in descending order was the combination therapy with inhibition of SIRT1 and gemcitabine, inhibition of SIRT1, and gemcitabine. The BxPC-3 pancreatic cancer xenogeneic mice model received treatment with physiological saline, inhibition of SIRT1, gemcitabine, and combination therapy with inhibition of SIRT1 and gemcitabine in vivo and the results showed that the tumor volumes decreased and the survival rate within 45 days increased according to the order of the given drugs and the difference was significant (P < 0.05.Conclusion: Combination therapy with inhibition of SIRT1 and gemcitabine could improve efficacy and survival time in a BxPC-3 pancreatic cancer xenogeneic mice model, compared with single inhibition of SIRT1, or single

  10. Protective Effect of Unacylated Ghrelin on Compression-Induced Skeletal Muscle Injury Mediated by SIRT1-Signaling

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    Felix N. Ugwu

    2017-11-01

    Full Text Available Unacylated ghrelin, the predominant form of circulating ghrelin, protects myotubes from cell death, which is a known attribute of pressure ulcers. In this study, we investigated whether unacylated ghrelin protects skeletal muscle from pressure-induced deep tissue injury by abolishing necroptosis and apoptosis signaling and whether these effects were mediated by SIRT1 pathway. Fifteen adult Sprague Dawley rats were assigned to receive saline or unacylated ghrelin with or without EX527 (a SIRT1 inhibitor. Animals underwent two 6-h compression cycles with 100 mmHg static pressure applied over the mid-tibialis region of the right limb whereas the left uncompressed limb served as the intra-animal control. Muscle tissues underneath the compression region, and at the similar region of the opposite uncompressed limb, were collected for analysis. Unacylated ghrelin attenuated the compression-induced muscle pathohistological alterations including rounding contour of myofibers, extensive nucleus accumulation in the interstitial space, and increased interstitial space. Unacylated ghrelin abolished the increase in necroptosis proteins including RIP1 and RIP3 and attenuated the elevation of apoptotic proteins including p53, Bax, and AIF in the compressed muscle. Furthermore, unacylated ghrelin opposed the compression-induced phosphorylation and acetylation of p65 subunit of NF-kB. The anti-apoptotic effect of unacylated ghrelin was shown by a decrease in apoptotic DNA fragmentation and terminal dUTP nick-end labeling index in the compressed muscle. The protective effects of unacylated ghrelin vanished when co-treated with EX527. Our findings demonstrated that unacylated ghrelin protected skeletal muscle from compression-induced injury. The myoprotective effects of unacylated ghrelin on pressure-induced tissue injury were associated with SIRT1 signaling.

  11. Telmisartan ameliorates insulin sensitivity by activating the AMPK/SIRT1 pathway in skeletal muscle of obese db/db mice

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    Shiota Asuka

    2012-11-01

    Full Text Available Abstract Background Telmisartan is a well-established angiotensin II type 1 receptor blocker that improves insulin sensitivity in animal models of obesity and insulin resistance, as well as in humans. Telmisartan has been reported to function as a partial agonist of the peroxisome proliferator-activated receptor (PPAR γ, which is also targeted by the nicotinamide adenine dinucleotide (NAD-dependent deacetylase (SIRT1. Here, we investigated the pathways through which telmisartan acts on skeletal muscle, in vitro as well as in vivo. Methods Nine-week-old male db/db mice were fed a 60% high-fat diet, with orally administrated either vehicle (carboxymethyl-cellulose, CMC, 5 mg/kg telmisartan, or 5 mg/kg telmisartan and 1 mg/kg GW9662, a selective irreversible antagonist of PPARγ, for 5 weeks. Effects of telmisartan on Sirt1 mRNA, AMPK phosphorylation, and NAD+/NADH ratio were determined in C2C12 cultured myocytes. Results and discussion Telmisartan treatment improved insulin sensitivity in obese db/db mice fed a high-fat diet and led to reduction in the size of hypertrophic pancreatic islets in these mice. Moreover, in vitro treatment with telmisartan led to increased expression of Sirt1 mRNA in C2C12 skeletal muscle cells; the increase in Sirt1 mRNA in telmisartan-treated C2C12 myoblasts occurred concomitantly with an increase in AMPK phosphorylation, an increase in NAD+/NADH ratio, and increases in the mRNA levels of PGC1α, FATP1, ACO, and GLUT4. Conclusions Our results indicate that telmisartan acts through a PPARγ-independent pathway, but at least partially exerts its effects by acting directly on skeletal muscle AMPK/SIRT1 pathways.

  12. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kim, Dohoon; Nguyen, Minh Dang; Dobbin, Matthew M; Fischer, Andre; Sananbenesi, Farahnaz; Rodgers, Joseph T; Delalle, Ivana; Baur, Joseph A; Sui, Guangchao; Armour, Sean M; Puigserver, Pere; Sinclair, David A; Tsai, Li-Huei

    2007-07-11

    A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.

  13. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

    Science.gov (United States)

    Kim, Dohoon; Nguyen, Minh Dang; Dobbin, Matthew M; Fischer, Andre; Sananbenesi, Farahnaz; Rodgers, Joseph T; Delalle, Ivana; Baur, Joseph A; Sui, Guangchao; Armour, Sean M; Puigserver, Pere; Sinclair, David A; Tsai, Li-Huei

    2007-01-01

    A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention. PMID:17581637

  14. Hepatitis C virus core protein induces dysfunction of liver sinusoidal endothelial cell by down-regulation of silent information regulator 1.

    Science.gov (United States)

    Sun, Li-Jie; Yu, Jian-Wu; Shi, Yu-Guang; Zhang, Xiao-Yu; Shu, Meng-Ni; Chen, Mo-Yang

    2018-05-01

    Hepatic fibrosis is a frequent feature of chronic hepatitis C virus (HCV) infection. Some evidence has suggested the potential role of silent information regulator 1 (SIRT1) in organ fibrosis. The aim of this study was to investigate the effect of HCV core protein on expression of SIRT1 of liver sinusoidal endothelial cell (LSEC) and function of LSEC. LSECs were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein and LSECs cultured alone were used as controls. After co-culture, the activity and expression levels of mRNA and protein of SIRT1 in LSEC were detected by a SIRT1 fluorometric assay kit, real time-PCR (RT-PCR), Western blot, respectively. The levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by Western blot. Cluster of differentiation 31 (CD31), CD14, and von Willebrand factor (vWf) of LSECs was performed by flow cytometry. The level of reactive oxygen species (ROS) was assayed. Malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO), and endothelin-1 (ET-1) levels in the co-culture supernatant were measured. The co-culture supernatant was then used to cultivate LX-2 cells. The levels of α-smooth muscle actin (ASMA) and transforming growth factor-β1 (TGF-β1) protein in LX-2 cells were measured by Western blot. Compared with LSEC co-cultured with HepG2 cells group, in LSEC co-cultured with HepG2-core cells group, the activity and expression level of mRNA and protein of SIRT1 reduced; the level of adiponectin reduced and the expression level of AdipoR2 protein decreased; ROS levels increased; the expression level of eNOS, VEGF protein decreased; and the expression level of CD14 decreased; the expression level of vWf and CD31 increased; NO and SOD levels decreased; whereas ET-1 and MDA levels increased; the levels of ASMA and TGF-β1 protein in LX-2 cells increased. SIRT1 activator improved the above-mentioned changes

  15. Neuronal SIRT1 (Silent Information Regulator 2 Homologue 1) Regulates Glycolysis and Mediates Resveratrol-Induced Ischemic Tolerance.

    Science.gov (United States)

    Koronowski, Kevin B; Khoury, Nathalie; Saul, Isabel; Loris, Zachary B; Cohan, Charles H; Stradecki-Cohan, Holly M; Dave, Kunjan R; Young, Juan I; Perez-Pinzon, Miguel A

    2017-11-01

    Resveratrol, at least in part via SIRT1 (silent information regulator 2 homologue 1) activation, protects against cerebral ischemia when administered 2 days before injury. However, it remains unclear if SIRT1 activation must occur, and in which brain cell types, for the induction of neuroprotection. We hypothesized that neuronal SIRT1 is essential for resveratrol-induced ischemic tolerance and sought to characterize the metabolic pathways regulated by neuronal Sirt1 at the cellular level in the brain. We assessed infarct size and functional outcome after transient 60 minute middle cerebral artery occlusion in control and inducible, neuronal-specific SIRT1 knockout mice. Nontargeted primary metabolomics analysis identified putative SIRT1-regulated pathways in brain. Glycolytic function was evaluated in acute brain slices from adult mice and primary neuronal-enriched cultures under ischemic penumbra-like conditions. Resveratrol-induced neuroprotection from stroke was lost in neuronal Sirt1 knockout mice. Metabolomics analysis revealed alterations in glucose metabolism on deletion of neuronal Sirt1 , accompanied by transcriptional changes in glucose metabolism machinery. Furthermore, glycolytic ATP production was impaired in acute brain slices from neuronal Sirt1 knockout mice. Conversely, resveratrol increased glycolytic rate in a SIRT1-dependent manner and under ischemic penumbra-like conditions in vitro. Our data demonstrate that resveratrol requires neuronal SIRT1 to elicit ischemic tolerance and identify a novel role for SIRT1 in the regulation of glycolytic function in brain. Identification of robust neuroprotective mechanisms that underlie ischemia tolerance and the metabolic adaptations mediated by SIRT1 in brain are crucial for the translation of therapies in cerebral ischemia and other neurological disorders. © 2017 American Heart Association, Inc.

  16. p16(INK4a suppression by glucose restriction contributes to human cellular lifespan extension through SIRT1-mediated epigenetic and genetic mechanisms.

    Directory of Open Access Journals (Sweden)

    Yuanyuan Li

    2011-02-01

    Full Text Available Although caloric restriction (CR has been shown to increase lifespan in various animal models, the mechanisms underlying this phenomenon have not yet been revealed. We developed an in vitro system to mimic CR by reducing glucose concentration in cell growth medium which excludes metabolic factors and allows assessment of the effects of CR at the cellular and molecular level. We monitored cellular proliferation of normal WI-38, IMR-90 and MRC-5 human lung fibroblasts and found that glucose restriction (GR can inhibit cellular senescence and significantly extend cellular lifespan compared with cells receiving normal glucose (NG in the culture medium. Moreover, GR decreased expression of p16(INK4a (p16, a well-known senescence-related gene, in all of the tested cell lines. Over-expressed p16 resulted in early replicative senescence in glucose-restricted cells suggesting a crucial role of p16 regulation in GR-induced cellular lifespan extension. The decreased expression of p16 was partly due to GR-induced chromatin remodeling through effects on histone acetylation and methylation of the p16 promoter. GR resulted in an increased expression of SIRT1, a NAD-dependent histone deacetylase, which has positive correlation with CR-induced longevity. The elevated SIRT1 was accompanied by enhanced activation of the Akt/p70S6K1 signaling pathway in response to GR. Furthermore, knockdown of SIRT1 abolished GR-induced p16 repression as well as Akt/p70S6K1 activation implying that SIRT1 may affect p16 repression through direct deacetylation effects and indirect regulation of Akt/p70S6K1 signaling. Collectively, these results provide new insights into interactions between epigenetic and genetic mechanisms on CR-induced longevity that may contribute to anti-aging approaches and also provide a general molecular model for studying CR in vitro in mammalian systems.

  17. Diallyl trisulfide ameliorates myocardial ischemia-reperfusion injury by reducing oxidative stress and endoplasmic reticulum stress-mediated apoptosis in type 1 diabetic rats: role of SIRT1 activation.

    Science.gov (United States)

    Yu, Liming; Li, Shu; Tang, Xinlong; Li, Zhi; Zhang, Jian; Xue, Xiaodong; Han, Jinsong; Liu, Yu; Zhang, Yuji; Zhang, Yong; Xu, Yinli; Yang, Yang; Wang, Huishan

    2017-07-01

    Diallyl trisulfide (DATS) protects against apoptosis during myocardial ischemia-reperfusion (MI/R) injury in diabetic state, although the underlying mechanisms remain poorly defined. Previously, we and others demonstrated that silent information regulator 1 (SIRT1) activation inhibited oxidative stress and endoplasmic reticulum (ER) stress during MI/R injury. We hypothesize that DATS reduces diabetic MI/R injury by activating SIRT1 signaling. Streptozotocin (STZ)-induced type 1 diabetic rats were subjected to MI/R surgery with or without perioperative administration of DATS (40 mg/kg). We found that DATS treatment markedly improved left ventricular systolic pressure and the first derivative of left ventricular pressure, reduced myocardial infarct size as well as serum creatine kinase and lactate dehydrogenase activities. Furthermore, the myocardial apoptosis was also suppressed by DATS as evidenced by reduced apoptotic index and cleaved caspase-3 expression. However, these effects were abolished by EX527 (the inhibitor of SIRT1 signaling, 5 mg/kg). We further found that DATS effectively upregulated SIRT1 expression and its nuclear distribution. Additionally, PERK/eIF2α/ATF4/CHOP-mediated ER stress-induced apoptosis was suppressed by DATS treatment. Moreover, DATS significantly activated Nrf-2/HO-1 antioxidant signaling pathway, thus reducing Nox-2/4 expressions. However, the ameliorative effects of DATS on oxidative stress and ER stress-mediated myocardial apoptosis were inhibited by EX527 administration. Taken together, these data suggest that perioperative DATS treatment effectively ameliorates MI/R injury in type 1 diabetic setting by enhancing cardiac SIRT1 signaling. SIRT1 activation not only upregulated Nrf-2/HO-1-mediated antioxidant signaling pathway but also suppressed PERK/eIF2α/ATF4/CHOP-mediated ER stress level, thus reducing myocardial apoptosis and eventually preserving cardiac function.

  18. Regulatory effects of the AMPKα-SIRT1 molecular pathway on insulin resistance in PCOS mice: An in vitro and in vivo study.

    Science.gov (United States)

    Tao, Xin; Chen, Lei; Cai, Lisi; Ge, Shuqi; Deng, Xuanying

    2017-12-16

    In order to preliminarily explore the correlation between the AMPKα-SIRT1 pathway and insulin resistance and reproductive function in PCOS mice and find the pathogenesis molecular mechanism and potential therapeutic target of PCOS, we carried out in vitro study of human granulosa KGN cells and in vivo study of PCOS mouse model which was constructed with DHEA, and AICAR and Compound C were applied. We have found that SIRT1 and AMPKα expression in KGN cells gradually decreased as DHEA concentration increased; Mice of the PCOS model were in an obvious status of IR (P PCOS and may serve as a therapeutic target for the development of potential treatments for improving metabolic and reproductive function in PCOS. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Dietary Blueberry and Bifidobacteria Attenuate Nonalcoholic Fatty Liver Disease in Rats by Affecting SIRT1-Mediated Signaling Pathway

    Science.gov (United States)

    Ren, Tingting; Huang, Chao; Cheng, Mingliang

    2014-01-01

    NAFLD model rats were established and divided into NAFLD model (MG group), SIRT1 RNAi (SI group), blueberry juice (BJ group), blueberry juice + bifidobacteria (BJB group), blueberry juice + SIRT1 RNAi (BJSI group), and blueberry juice + bifidobacteria + SIRT1 RNAi groups (BJBSI group). A group with normal rats was a control group (CG). BJB group ameliorated NAFLD, which was better than BJ group (P Blueberry juice and bifidobacteria improve NAFLD by activating SIRTI-mediating signaling pathway. PMID:25544867

  20. miR-23b-3p induces the cellular metabolic memory of high glucose in diabetic retinopathy through a SIRT1-dependent signalling pathway.

    Science.gov (United States)

    Zhao, Shuzhi; Li, Tao; Li, Jun; Lu, Qianyi; Han, Changjing; Wang, Na; Qiu, Qinghua; Cao, Hui; Xu, Xun; Chen, Haibing; Zheng, Zhi

    2016-03-01

    The mechanisms underlying the cellular metabolic memory induced by high glucose remain unclear. Here, we sought to determine the effects of microRNAs (miRNAs) on metabolic memory in diabetic retinopathy. The miRNA microarray was used to examine human retinal endothelial cells (HRECs) following exposure to normal glucose (N) or high glucose (H) for 1 week or transient H for 2 days followed by N for another 5 days (H→N). Levels of sirtuin 1 (SIRT1) and acetylated-nuclear factor κB (Ac-NF-κB) were examined following transfection with miR-23b-3p inhibitor or with SIRT1 small interfering (si)RNA in the H→N group, and the apoptotic HRECs were determined by flow cytometry. Retinal tissues from diabetic rats were similarly studied following intravitreal injection of miR-23b-3p inhibitor. Chromatin immunoprecipitation (ChIP) analysis was performed to detect binding of NF-κB p65 to the potential binding site of the miR-23b-27b-24-1 gene promoter in HRECs. High glucose increased miR-23b-3p expression, even after the return to normal glucose. Luciferase assays identified SIRT1 as a target mRNA of miR-23b-3p. Reduced miR-23b-3p expression inhibited Ac-NF-κB expression by rescuing SIRT1 expression and also relieved the effect of metabolic memory induced by high glucose in HRECs. The results were confirmed in the retina using a diabetic rat model of metabolic memory. High glucose facilitated the recruitment of NF-κB p65 and promoted transcription of the miR-23b-27b-24-1 gene, which can be suppressed by decreasing miR-23b-3p expression. These studies identify a novel mechanism whereby miR-23b-3p regulates high-glucose-induced cellular metabolic memory in diabetic retinopathy through a SIRT1-dependent signalling pathway.

  1. SIRT1 Regulates Cognitive Performance and Ability of Learning and Memory in Diabetic and Nondiabetic Models

    Directory of Open Access Journals (Sweden)

    Yue Cao

    2017-01-01

    Full Text Available Type 2 diabetes mellitus is a complex age-related metabolic disease. Cognitive dysfunction and learning and memory deficits are main characteristics of age-related metabolic diseases in the central nervous system. The underlying mechanisms contributing to cognitive decline are complex, especially cognitive dysfunction associated with type 2 diabetes mellitus. SIRT1, as one of the modulators in insulin resistance, is indispensable for learning and memory. In the present study, deacetylation, oxidative stress, mitochondrial dysfunction, inflammation, microRNA, and tau phosphorylation are considered in the context of mechanism and significance of SIRT1 in learning and memory in diabetic and nondiabetic murine models. In addition, future research directions in this field are discussed, including therapeutic potential of its activator, resveratrol, and application of other compounds in cognitive improvement. Our findings suggest that SIRT1 might be a potential therapeutic target for the treatment of cognitive impairment induced by type 2 diabetes mellitus.

  2. Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kalle, Arunasree M., E-mail: arunasreemk@ilsresearch.org [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Mallika, A. [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Badiger, Jayasree [HKE' s Smt. V.G. College for Women, Aiwan-E-Shahi Area, Gulbarga, KA 585 102 (India); Alinakhi [Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, AP 500 046 (India); Talukdar, Pinaki [Department of Chemistry, Indian Institute of Science Education and Research, First Floor, Central Tower, Sai Trinity Building Garware Circle, Sutarwadi, PashanPune, Maharashtra 411 021 (India); Sachchidanand [Lupin Research Park, 46/47, A, Village Nande, Taluka Mulshi, Dist. Pune 411 042 (India)

    2010-10-08

    Research highlights: {yields} Novel small molecule SIRT1 inhibitor better than sirtinol. {yields} IC{sub 50} 500 nM. {yields} Specific tumor cytotoxicity towards breast cancer cells. {yields} Restoration of H3K9 acetylation levels to baseline when co-treated with SIRT1 activator (Activator X) and inhibitor (ILS-JGB-1741). -- Abstract: Overexpression of SIRT1, a NAD{sup +}-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC{sub 50} of 1, 10 and 0.5 {mu}M, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.

  3. Dioscin alleviates BDL- and DMN-induced hepatic fibrosis via Sirt1/Nrf2-mediated inhibition of p38 MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Lina; Tao, Xufeng; Xu, Youwei; Han, Xu; Qi, Yan; Xu, Lina; Yin, Lianhong; Peng, Jinyong, E-mail: jinyongpeng2014@163.com

    2016-02-01

    Oxidative stress is involved in hepatic stellate cells (HSCs) activation and extracellular matrix overproduction. We previously reported the promising effects of dioscin against CCl{sub 4}-induced liver fibrosis, but its effects and mechanisms on BDL- and DMN-induced liver fibrosis remain unknown. The results in the present study indicated that dioscin significantly inhibited HSCs activation and attenuated hepatic fibrosis in rats. Furthermore, dioscin markedly up-regulated the levels of sirtuin 1 (Sirt1), HO-1, GST, GCLC and GCLM via increasing the nuclear translocation of nuclear erythroid factor 2-related factor 2 (Nrf2), which in turn inhibited mitogen-activated protein kinase 14 (p38 MAPK) phosphorylation and reduced the levels of COL1A1, COL3A1, α-SMA and fibronectin. These results were further validated by knockdown of Sirt1 and Nrf2 using siRNAs silencing, and abrogation of p38 MAPK using SB-203580 (a p38 MAPK inhibitor) in HSC-T6 and LX-2 cells. Collectively, our findings confirmed the potent effects of dioscin against liver fibrosis and also provided novel insights into the mechanisms of this compound as a candidate for the prevention of liver fibrosis in the future. - Highlights: • Dioscin showed potent effects against BDL- and DMN-induced liver fibrosis in rats. • Dioscin significantly suppressed oxidative stress. • Dioscin triggered Sirt1/Nrf2-mediated inhibition of p38 MAPK pathway. • Dioscin should be developed as a novel candidate to treat liver fibrosis.

  4. Resveratrol (trans-3,5,4'-trihydroxystilbene) suppresses EL4 tumor growth by induction of apoptosis involving reciprocal regulation of SIRT1 and NF-κB.

    Science.gov (United States)

    Singh, Narendra P; Singh, Udai P; Hegde, Venkatesh L; Guan, Hongbing; Hofseth, Lorne; Nagarkatti, Mitzi; Nagarkatti, Prakash S

    2011-08-01

    Understanding the molecular mechanisms through which natural products and dietary supplements exhibit anticancer properties is crucial and can lead to drug discovery and chemoprevention. The current study sheds new light on the mode of action of resveratrol (RES), a plant-derived polyphenolic compound, against EL-4 lymphoma growth. Immuno-compromised NOD/SCID mice injected with EL-4 tumor cells and treated with RES (100 mg/kg body weight) showed delayed development and progression of tumor growth and increased mean survival time. RES caused apoptosis in EL4 cells through activation of aryl hydrocarbon receptor (AhR) and upregulation of Fas and FasL expression in vitro. Blocking of RES-induced apoptosis in EL4 cells by FasL mAb, cleavage of caspases and PARP, and release of cytochorme c, demonstrated the participation of both extrinsic and intrinsic pathways of apoptosis. RES also induced upregulation of silent mating type information regulation 2 homolog, 1 (SIRT1) and downregulation of nuclear factor kappa B (NF-κB) in EL4 cells. siRNA-mediated downregulation of SIRT1 in EL4 cells increased the activation of NF-κB but decreased RES-mediated apoptosis, indicating the critical role of SIRT1 in apoptosis via blocking activation of NF-κB. These data suggest that RES-induced SIRT1 upregulation promotes tumor cell apoptosis through negative regulation of NF-κB, leading to suppression of tumor growth. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Effect of regional muscle location but not adiposity on mitochondrial biogenesis-regulating proteins

    DEFF Research Database (Denmark)

    Ponce-González, Jesús Gustavo; Ara, Ignacio; Larsen, Steen

    2016-01-01

    PURPOSE: The aim of this study was to determine if the expression of the mitochondrial biogenesis-regulating proteins SIRT1, SIRT3 and PGC-1alpha in human skeletal muscle is influenced by adiposity. METHOD: Twenty-nine male subjects were recruited into three groups: control (n = 10), obese (n = 10...

  6. Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation

    NARCIS (Netherlands)

    Feige, Jérôme N.; Lagouge, Marie; Canto, Carles; Strehle, Axelle; Houten, Sander M.; Milne, Jill C.; Lambert, Philip D.; Mataki, Chikage; Elliott, Peter J.; Auwerx, Johan

    2008-01-01

    The NAD(+)-dependent deacetylase SIRT1 controls metabolic processes in response to low nutrient availability. We report the metabolic phenotype of mice treated with SRT1720, a specific and potent synthetic activator of SIRT1 that is devoid of direct action on AMPK. SRT1720 administration robustly

  7. Adipocyte SIRT1 knockout promotes PPARγ activity, adipogenesis and insulin sensitivity in chronic-HFD and obesity

    Directory of Open Access Journals (Sweden)

    Rafael Mayoral

    2015-05-01

    Conclusion: Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.

  8. SIRT1/FoxO3 axis alteration leads to aberrant immune responses in bronchial epithelial cells

    NARCIS (Netherlands)

    Di Vincenzo, Serena; Heijink, Irene H; Noordhoek, Jacobien A; Cipollina, Chiara; Siena, Liboria; Bruno, Andreina; Ferraro, Maria; Postma, Dirkje S; Gjomarkaj, Mark; Pace, Elisabetta

    Inflammation and ageing are intertwined in chronic obstructive pulmonary disease (COPD). The histone deacetylase SIRT1 and the related activation of FoxO3 protect from ageing and regulate inflammation. The role of SIRT1/FoxO3 in COPD is largely unknown. This study evaluated whether cigarette smoke,

  9. Upregulated miR-132 in Lgr5+ gastric cancer stem cell-like cells contributes to cisplatin-resistance via SIRT1/CREB/ABCG2 signaling pathway.

    Science.gov (United States)

    Zhang, Lanfang; Guo, Xiaohe; Zhang, Dezhong; Fan, Yingying; Qin, Lei; Dong, Shuping; Zhang, Lanfang

    2017-09-01

    Cisplatin resistance has long been a major problem that restricts its use. A novel paradigm in tumor biology suggests that gastric tumor chemo-resistance is driven by gastric cancer stem cell-like (GCSCs). Growing evidence has indicated that microRNAs (miRNAs) contributes to chemo-resistance in gastric cancer (GC). Here, Lgr5 + cells derived from gastric cancer cell lines displayed stem cell-like features. Flow cytometry demonstrated the presence of a variable fraction of Lgr5 in 19 out of 20 GC specimens. By comparing the miRNA expression profiles of Lgr5 + GCSCs and Lrg5 - cells, we established the upregulation of miR-132 in Lgr5 + GCSCs. The enhanced miR-132 expression correlated chemo-resistance in GC patients. Kaplan-Meier survival curve showed that patients with low miR-132 expression survived obviously longer. Functional assays results indicated that miR-132 promoted cisplatin resistance in Lgr5 + GCSCs in vitro and in vivo. Further dual-luciferase reporter gene assays revealed that SIRT1 was the direct target of miR-132. The expression of miR-132 was inversely correlated with SIRT1 in gastric cancer specimens. Furthermore, through PCR array we discovered ABCG2 was one of the downstream targets of SIRT1. Overexpression of SIRT1 down-regulated ABCG2 expression by promoting the de-acetylation of the transcription factor CREB. CREB was further activated ABCG2 via binding to the promoter of ABCG2 to induce transcription. Thus, we concluded that miR-132 regulated SIRT1/CREB/ABCG2 signaling pathway contributing to the cisplatin resistance and might serve as a novel therapeutic target against gastric cancer. © 2017 Wiley Periodicals, Inc.

  10. SIRT1 gene is associated with cardiovascular disease in the Iranian ...

    African Journals Online (AJOL)

    Background: Sirtuins (SIRT) have recently been identified as the pivotal regulators of lifespan and health. SIRT1 has protective effects against cardiovascular disease (CVD) and through its deacetylase activity it regulates numerous essential pathways including regulating blood pressure, reducing atherosclerosis, heart ...

  11. Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells.

    Science.gov (United States)

    Kalle, Arunasree M; Mallika, A; Badiger, Jayasree; Alinakhi; Talukdar, Pinaki; Sachchidanand

    2010-10-08

    Overexpression of SIRT1, a NAD+-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38

    International Nuclear Information System (INIS)

    Aksoy, Pinar; Escande, Carlos; White, Thomas A.; Thompson, Michael; Soares, Sandra; Benech, Juan Claudio; Chini, Eduardo N.

    2006-01-01

    The SIRT 1 enzyme is a NAD dependent deacetylase implicated in ageing, cell protection, and energy metabolism in mammalian cells. How the endogenous activity of SIRT 1 is modulated is not known. The enzyme CD38 is a multifunctional enzyme capable of synthesis of the second messenger, cADPR, NAADP, and ADPR. However, the major enzymatic activity of CD38 is the hydrolysis of NAD. Of particular interest is the fact that CD38 is present on the inner nuclear membrane. Here, we investigate the modulation of the SIRT 1 activity by CD38. We propose that by modulating availability of NAD to the SIRT1 enzyme, CD38 may regulate SIRT1 enzymatic activity. We observed that in CD38 knockout mice, tissue levels of NAD are significantly increased. We also observed that incubation of purified recombinant SIRT1 enzyme with CD38 or nuclear extracts of wild-type mice led to a significant inhibition of its activity. In contrast, incubation of SIRT1 with cellular extract from CD38 knockout mice was without effect. Furthermore, the endogenous activity of SIRT1 was several time higher in nuclear extracts from CD38 knockout mice when compared to wild-type nuclear extracts. Finally, the in vivo deacetylation of the SIRT1 substrate P53 is increased in CD38 knockout mice tissue. Our data support the novel concept that nuclear CD38 is a major regulator of cellular/nuclear NAD level, and SIRT1 activity. These findings have strong implications for understanding the basic mechanisms that modulate intracellular NAD levels, energy homeostasis, as well as ageing and cellular protection modulated by the SIRT enzymes

  13. Cafestol Inhibits Cyclic-Strain-Induced Interleukin-8, Intercellular Adhesion Molecule-1, and Monocyte Chemoattractant Protein-1 Production in Vascular Endothelial Cells

    Science.gov (United States)

    Hao, Wen-Rui; Sung, Li-Chin; Chen, Chun-Chao; Chen, Jin-Jer

    2018-01-01

    Moderate coffee consumption is inversely associated with cardiovascular disease mortality; however, mechanisms underlying this causal effect remain unclear. Cafestol, a diterpene found in coffee, has various properties, including an anti-inflammatory property. This study investigated the effect of cafestol on cyclic-strain-induced inflammatory molecule secretion in vascular endothelial cells. Cells were cultured under static or cyclic strain conditions, and the secretion of inflammatory molecules was determined using enzyme-linked immunosorbent assay. The effects of cafestol on mitogen-activated protein kinases (MAPK), heme oxygenase-1 (HO-1), and sirtuin 1 (Sirt1) signaling pathways were examined using Western blotting and specific inhibitors. Cafestol attenuated cyclic-strain-stimulated intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein- (MCP-) 1, and interleukin- (IL-) 8 secretion. Cafestol inhibited the cyclic-strain-induced phosphorylation of extracellular signal-regulated kinase and p38 MAPK. By contrast, cafestol upregulated cyclic-strain-induced HO-1 and Sirt1 expression. The addition of zinc protoporphyrin IX, sirtinol, or Sirt1 silencing (transfected with Sirt1 siRNA) significantly attenuated cafestol-mediated modulatory effects on cyclic-strain-stimulated ICAM-1, MCP-1, and IL-8 secretion. This is the first study to report that cafestol inhibited cyclic-strain-induced inflammatory molecule secretion, possibly through the activation of HO-1 and Sirt1 in endothelial cells. The results provide valuable insights into molecular pathways that may contribute to the effects of cafestol. PMID:29854096

  14. Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice.

    NARCIS (Netherlands)

    Escande, C.; Chini, C.C.; Nin, V.; Dykhouse, K.M.; Novak, C.M.; Levine, J.; Deursen, J.M.A. van; Gores, G.J.; Chen, J.; Lou, Z.; Chini, E.N.

    2010-01-01

    The enzyme sirtuin 1 (SIRT1) is a critical regulator of many cellular functions, including energy metabolism. However, the precise mechanisms that modulate SIRT1 activity remain unknown. As SIRT1 activity in vitro was recently found to be negatively regulated by interaction with the deleted in

  15. ERG protein expression over time

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Brasso, Klaus; Thomsen, Frederik Birkebæk

    2015-01-01

    AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed by immunohistochem......AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed...

  16. Ethylenediamine diacetate (EDDA) mediated synthesis of aurones under ultrasound: their evaluation as inhibitors of SIRT1.

    Science.gov (United States)

    Manjulatha, Khanapur; Srinivas, S; Mulakayala, Naveen; Rambabu, D; Prabhakar, M; Arunasree, Kalle M; Alvala, Mallika; Basaveswara Rao, M V; Pal, Manojit

    2012-10-01

    An improved synthesis of functionalized aurones has been accomplished via the reaction of benzofuran-3(2H)-one with a range of benzaldehydes in the presence of a mild base EDDA under ultrasound. A number of aurones were synthesized (within 5-30min) and the molecular structure of a representative compound determined by single crystal X-ray diffraction study confirmed Z-geometry of the C-C double bond present within the molecule. Some of the compounds synthesized have shown SIRT1 inhibiting as well as anti proliferative properties against two cancer cell lines in vitro. Compound 3a [(Z)-2-(5-bromo-2-hydroxybenzylidene) benzofuran-3(2H)-one] was identified as a potent inhibitor of SIRT1 (IC(50)=1μM) which showed a dose dependent increase in the acetylation of p53 resulting in induction of apoptosis. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Does SIRT-1 Mediate Calorie Restriction and Prolong Life? – A Mini Review

    Directory of Open Access Journals (Sweden)

    Kordala Anna

    2014-09-01

    Full Text Available Calorie restriction is the only intervention proved to prolong both average and maximum lifespan in yeast, worms, fish, rodents and possibly primates. Not only does the regimen prolong life, but it also reduces the incident of numerous age-related diseases like diabetes, atherosclerosis or cancer and slows down ageing. Mechanisms by which that is thought to occur have not yet been elucidated, but they probably involve reactive oxygen species signaling, insulin growth factor and transcriptional factors. Here, special emphasis is given to SIRT1 - silent information regulator. There is sound evidence showing that SIRT1 is a key player in mediating physiological response to calorie restriction and that its overexpression is correlated with extended lifespan. The possible mechanism leading to its elevated levels is high NAD/NADH ratio, observed in Sir2 in yeast. SIRT1 increases glucose production, enhances fat mobilization, stimulates angiogenesis, prevents neuronal degeneration and rises insulin sensitivity. Therefore, it seems to be a very beneficial factor activated by such a simple intervention that is calorie restriction.

  18. Ligand-based virtual screening and inductive learning for identification of SIRT1 inhibitors in natural products.

    Science.gov (United States)

    Sun, Yunan; Zhou, Hui; Zhu, Hongmei; Leung, Siu-wai

    2016-01-25

    Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase, and its dysregulation can lead to ageing, diabetes, and cancer. From 346 experimentally confirmed SIRT1 inhibitors, an inhibitor structure pattern was generated by inductive logic programming (ILP) with DMax Chemistry Assistant software. The pattern contained amide, amine, and hetero-aromatic five-membered rings, each of which had a hetero-atom and an unsubstituted atom at a distance of 2. According to this pattern, a ligand-based virtual screening of 1 444 880 active compounds from Chinese herbs identified 12 compounds as inhibitors of SIRT1. Three compounds (ZINC08790006, ZINC08792229, and ZINC08792355) had high affinity (-7.3, -7.8, and -8.6 kcal/mol, respectively) for SIRT1 as estimated by molecular docking software AutoDock Vina. This study demonstrated a use of ILP and background knowledge in machine learning to facilitate virtual screening.

  19. High-Fat Diet Triggers Inflammation-Induced Cleavage of SIRT1 in Adipose Tissue To Promote Metabolic Dysfunction

    OpenAIRE

    Chalkiadaki, Angeliki; Guarente, Leonard

    2012-01-01

    Adipose tissue plays an important role in storing excess nutrients and preventing ectopic lipid accumulation in other organs. Obesity leads to excess lipid storage in adipocytes, resulting in the generation of stress signals and the derangement of metabolic functions. SIRT1 is an important regulatory sensor of nutrient availability in many metabolic tissues. Here we report that SIRT1 functions in adipose tissue to protect from inflammation and obesity under normal feeding conditions, and to f...

  20. Benzylglucosinolate Derived Isothiocyanate from Tropaeolum majus Reduces Gluconeogenic Gene and Protein Expression in Human Cells.

    Directory of Open Access Journals (Sweden)

    Valentina Guzmán-Pérez

    Full Text Available Nasturtium (Tropaeolum majus L. contains high concentrations of benzylglcosinolate. We found that a hydrolysis product of benzyl glucosinolate-the benzyl isothiocyanate (BITC-modulates the intracellular localization of the transcription factor Forkhead box O 1 (FOXO1. FoxO transcription factors can antagonize insulin effects and trigger a variety of cellular processes involved in tumor suppression, longevity, development and metabolism. The current study evaluated the ability of BITC-extracted as intact glucosinolate from nasturtium and hydrolyzed with myrosinase-to modulate i the insulin-signaling pathway, ii the intracellular localization of FOXO1 and, iii the expression of proteins involved in gluconeogenesis, antioxidant response and detoxification. Stably transfected human osteosarcoma cells (U-2 OS with constitutive expression of FOXO1 protein labeled with GFP (green fluorescent protein were used to evaluate the effect of BITC on FOXO1. Human hepatoma HepG2 cell cultures were selected to evaluate the effect on gluconeogenic, antioxidant and detoxification genes and protein expression. BITC reduced the phosphorylation of protein kinase B (AKT/PKB and FOXO1; promoted FOXO1 translocation from cytoplasm into the nucleus antagonizing the insulin effect; was able to down-regulate the gene and protein expression of gluconeogenic enzymes; and induced the gene expression of antioxidant and detoxification enzymes. Knockdown analyses with specific siRNAs showed that the expression of gluconeogenic genes was dependent on nuclear factor (erythroid derived-like2 (NRF2 and independent of FOXO1, AKT and NAD-dependent deacetylase sirtuin-1 (SIRT1. The current study provides evidence that BITC might have a role in type 2 diabetes T2D by reducing hepatic glucose production and increasing antioxidant resistance.

  1. Intracerebroventricular tempol administration in older rats reduces oxidative stress in the hypothalamus but does not change STAT3 signalling or SIRT1/AMPK pathway.

    Science.gov (United States)

    Toklu, Hale Z; Scarpace, Philip J; Sakarya, Yasemin; Kirichenko, Nataliya; Matheny, Michael; Bruce, Erin B; Carter, Christy S; Morgan, Drake; Tümer, Nihal

    2017-01-01

    Hypothalamic inflammation and increased oxidative stress are believed to be mechanisms that contribute to obesity. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol), a free radical scavenger, has been shown to reduce inflammation and oxidative stress. We hypothesized that brain infusion of tempol would reduce oxidative stress, and thus would reduce food intake and body weight and improve body composition in rats with age-related obesity and known elevated oxidative stress. Furthermore, we predicted an associated increase in markers of leptin signalling, including the silent mating type information regulator 2 homolog 1 (SIRT1)/5'AMP-activated protein kinase (AMPK) pathway and the signal transducer and activator of transcription 3 (STAT3) pathway. For this purpose, osmotic minipumps were placed in the intracerebroventricular region of young (3 months) and aged (23 months) male Fischer 344 x Brown Norway rats for the continuous infusion of tempol or vehicle for 2 weeks. Tempol significantly decreased (p < 0.01) nicotinamide adenine dinucleotide phosphate oxidase activity in the hypothalamus but failed to reduce food intake or weight gain and did not alter body composition. SIRT1 activity and Acetyl p53 were decreased and phosphorylation of AMPK was increased with age, but they were unchanged with tempol. Basal phosphorylation of STAT3 was unchanged with age or tempol. These results indicate that tempol decreases oxidative stress but fails to alter feeding behaviour, body weight, or body composition. Moreover, tempol does not modulate the SIRT1/AMPK/p53 pathway and does not change leptin signalling. Thus, a reduction in hypothalamic oxidative stress is not sufficient to reverse age-related obesity.

  2. Long non-coding RNA TUG1 promotes cervical cancer progression by regulating the miR-138-5p-SIRT1 axis.

    Science.gov (United States)

    Zhu, Jie; Shi, Huirong; Liu, Huina; Wang, Xiaojuan; Li, Fengmei

    2017-09-12

    Increasing evidences showed that long non-coding RNAs (lncRNAs) play vital roles in tumor progression. Recent studies indicated that lncRNA TUG1 was upregulated and promoted tumor processes in several cancers. However, the expression and underlying mechanism of TUG1 in cervical cancer remain unclear. In the present study, we found that TUG1 expression was upregulated in cervical cancer tissues and correlated with advanced clinical features and poor overall survival. TUG1 knockdown suppressed cervical cancer cell growth and metastasis in vitro and tumor growth in vivo . In addition, our results indicated that TUG1 could act as an endogenous sponge by directly binding to miR-138-5p and suppressed miR-138-5p expression. Furthermore, we found that TUG1 could reverse the inhibitory effect of miR-138-5p on cervical cancer cells processes, which might be involved in the activation of SIRT1, a target gene of miR-138-5p, and activation of Wnt/β-catenin signaling pathway. Taken together, we elucidated that TUG1 might promote cervical cancer malignant progression via miR-138-5p-SIRT1-Wnt/β-catenin signaling pathway axis.

  3. Regulation of Sirtuin-Mediated Protein Deacetylation by Cardioprotective Phytochemicals

    OpenAIRE

    Treviño-Saldaña, Niria; García-Rivas, Gerardo

    2017-01-01

    Modulation of posttranslational modifications (PTMs), such as protein acetylation, is considered a novel therapeutic strategy to combat the development and progression of cardiovascular diseases. Protein hyperacetylation is associated with the development of numerous cardiovascular diseases, including atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. In addition, decreased expression and activity of the deacetylases Sirt1, Sirt3, and Sirt6 have been linked to the developm...

  4. Cardioprotection of CAPE-oNO2 against myocardial ischemia/reperfusion induced ROS generation via regulating the SIRT1/eNOS/NF-κB pathway in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Dejuan Li

    2018-05-01

    Full Text Available Caffeic acid phenethyl ester (CAPE could ameliorate myocardial ischemia/reperfusion injury (MIRI by various mechanisms, but there hadn’t been any reports on that CAPE could regulate silent information regulator 1 (SIRT1 and endothelial nitric oxide synthase (eNOS to exert cardioprotective effect. The present study aimed to investigate the cardioprotective potential of caffeic acid o-nitro phenethyl ester (CAPE-oNO2 on MIRI and the possible mechanism based on the positive control of CAPE. The SD rats were subjected to left coronary artery ischemia /reperfusion (IR and the H9c2 cell cultured in hypoxia/reoxygenation (HR to induce the MIRI model. Prior to the procedure, vehicle, CAPE or CAPE-oNO2 were treated in the absence or presence of a SIRT1 inhibitor nicotinamide (NAM and an eNOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME. In vivo, CAPE and CAPE-oNO2 conferred a cardioprotective effect as shown by reduced myocardial infarct size, cardiac marker enzymes and structural abnormalities. From immunohistochemical and sirius red staining, above two compounds ameliorated the TNF-α release and collagen deposition of IR rat hearts. They could agitate SIRT1 and eNOS expression, and consequently enhance NO release and suppress NF-κB signaling, to reduce the malondialdehyde content and cell necrosis. In vitro, they could inhibit HR-induced H9c2 cell apoptosis and ROS generation by activating SIRT1/eNOS pathway and inhabiting NF-κB expression. Emphatically, CAPE-oNO2 presented the stronger cardioprotection than CAPE both in vivo and in vitro. However, NAM and L-NAME eliminated the CAPE-oNO2-mediated cardioprotection by restraining SIRT1 and eNOS expression, respectively. It suggested that CAPE-oNO2 ameliorated MIRI by suppressing the oxidative stress, inflammatory response, fibrosis and necrocytosis via the SIRT1/eNOS/NF-κB pathway.

  5. The effect of exercise, resveratrol or their combination on Sarcopenia in aged rats via regulation of AMPK/Sirt1 pathway.

    Science.gov (United States)

    Liao, Zhi-Yin; Chen, Jin-Liang; Xiao, Ming-Han; Sun, Yue; Zhao, Yu-Xing; Pu, Die; Lv, An-Kang; Wang, Mei-Li; Zhou, Jing; Zhu, Shi-Yu; Zhao, Ke-Xiang; Xiao, Qian

    2017-11-01

    Sarcopenia is an age-related syndrome characterized by progressive loss of muscle mass and function. Exercise is an important strategy to prolong life and increase muscle mass, and resveratrol has been shown a variety beneficial effects on skeletal muscle. In the present study, we investigated the potential efficacy of using short-term exercise (six weeks), resveratrol (150mg/kg/day), or combined exercise+resveratrol (150mg/kg/day) on gastrocnemius muscle mass, grip strength, cross-sectional area and microscopic morphology in aged rats, and explored the potential mechanism at the apoptosis level. Six months old SD rats were used as young control group and 24months old SD rats were adopted as aged group. After six weeks intervention, the data provide evidence that exercise, resveratrol or their combination significantly increase the relative grip strength and muscle mass in aged rats (Presveratrol or their combination significantly reduced the increasement (Presveratrol did not show significant effects on them (P>0.05). Furthermore, exercise, resveratrol or their combination significantly increased the expression of p-AMPK and SIRT1, decreased the expression of acetyl P53 and Bax/Bcl-2 ratio in aged rats (Presveratrol and their combination are probably associated with anti-apoptotic signaling pathways through activation of AMPK/Sirt1. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

    Science.gov (United States)

    Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

    2014-01-01

    Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

  7. Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction

    DEFF Research Database (Denmark)

    Fang, Evandro Fei; Scheibye-Knudsen, Morten; Brace, Lear E

    2014-01-01

    with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition...... or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration...

  8. RhNRG-1β Protects the Myocardium against Irradiation-Induced Damage via the ErbB2-ERK-SIRT1 Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Anxin Gu

    Full Text Available Radiation-induced heart disease (RIHD, which is a serious side effect of the radiotherapy applied for various tumors due to the inevitable irradiation of the heart, cannot be treated effectively using current clinical therapies. Here, we demonstrated that rhNRG-1β, an epidermal growth factor (EGF-like protein, protects myocardium tissue against irradiation-induced damage and preserves cardiac function. rhNRG-1β effectively ameliorated irradiation-induced myocardial nuclear damage in both cultured adult rat-derived cardiomyocytes and rat myocardium tissue via NRG/ErbB2 signaling. By activating ErbB2, rhNRG-1β maintained mitochondrial integrity, ATP production, respiratory chain function and the Krebs cycle status in irradiated cardiomyocytes. Moreover, the protection of irradiated cardiomyocytes and myocardium tissue by rhNRG-1β was at least partly mediated by the activation of the ErbB2-ERK-SIRT1 signaling pathway. Long-term observations further showed that rhNRG-1β administered in the peri-irradiation period exerts continuous protective effects on cardiac pump function, the myocardial energy metabolism, cardiomyocyte volume and interstitial fibrosis in the rats receiving radiation via NRG/ErbB2 signaling. Our findings indicate that rhNRG-1β can protect the myocardium against irradiation-induced damage and preserve cardiac function via the ErbB2-ERK-SIRT1 signaling pathway.

  9. Melatonin protects chondrocytes from impairment induced by glucocorticoids via NAD+-dependent SIRT1.

    Science.gov (United States)

    Yang, Wei; Kang, Xiaomin; Qin, Na; Li, Feng; Jin, Xinxin; Ma, Zhengmin; Qian, Zhuang; Wu, Shufang

    2017-10-01

    Intra-articular injection of glucocorticoids is used to relieve pain and inflammation in osteoarthritis patients, which is occasionally accompanied with the serious side effects of glucocorticoids in collagen-producing tissue. Melatonin is the major hormone released from the pineal gland and its beneficial effects on cartilage has been suggested. In the present study, we investigated the protective role of melatonin on matrix degeneration in chondrocytes induced by dexamethasone (Dex). The chondrocytes isolated from mice knee joint were treated with Dex, melatonin, EX527 and siRNA targeted for SIRT6, respectively. Dex treatment induced the loss of the extracellular matrix, NAD + /NADH ratio and NADPH concentration in chondrocytes. Melatonin alone have no effect on the quantity of proteoglycans and collagen type IIa1, however, the pretreatment of melatonin reversed the negative effects induced by Dex. Meanwhile, the significant decrease in NAD + /NADH ratio and NADPH concentration in Dex group were up-regulated by pretreatment of melatonin. Furthermore, it was revealed that inhibition of SIRT1 blocked the protective effects of melatonin. The enhancement of NAD + -dependent SIRT1 activity contributes to the chondroprotecfive effects of melatonin, which has a great benefit to prevent dexamethasone-induced chondrocytes impairment. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Omega-3 polyunsaturated fatty acid attenuates the inflammatory response by modulating microglia polarization through SIRT1-mediated deacetylation of the HMGB1/NF-κB pathway following experimental traumatic brain injury.

    Science.gov (United States)

    Chen, Xiangrong; Chen, Chunnuan; Fan, Sining; Wu, Shukai; Yang, Fuxing; Fang, Zhongning; Fu, Huangde; Li, Yasong

    2018-04-20

    Microglial polarization and the subsequent neuroinflammatory response are contributing factors for traumatic brain injury (TBI)-induced secondary injury. High mobile group box 1 (HMGB1) mediates the activation of the NF-κB pathway, and it is considered to be pivotal in the late neuroinflammatory response. Activation of the HMGB1/NF-κB pathway is closely related to HMGB1 acetylation, which is regulated by the sirtuin (SIRT) family of proteins. Omega-3 polyunsaturated fatty acids (ω-3 PUFA) are known to have antioxidative and anti-inflammatory effects. We previously demonstrated that ω-3 PUFA inhibited TBI-induced microglial activation and the subsequent neuroinflammatory response by regulating the HMGB1/NF-κB signaling pathway. However, no studies have elucidated if ω-3 PUFA affects the HMGB1/NF-κB pathway in a HMGB1 deacetylation of dependent SIRT1 manner, thus regulating microglial polarization and the subsequent neuroinflammatory response. The Feeney DM TBI model was adopted to induce brain injury in rats. Modified neurological severity scores, rotarod test, brain water content, and Nissl staining were employed to determine the neuroprotective effects of ω-3 PUFA supplementation. Assessment of microglia polarization and pro-inflammatory markers, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and HMGB1, were used to evaluate the neuroinflammatory responses and the anti-inflammatory effects of ω-3 PUFA supplementation. Immunofluorescent staining and western blot analysis were used to detect HMGB1 nuclear translocation, secretion, and HMGB1/NF-κB signaling pathway activation to evaluate the effects of ω-3 PUFA supplementation. The impact of SIRT1 deacetylase activity on HMGB1 acetylation and the interaction between HMGB1 and SIRT1 were assessed to evaluate anti-inflammation effects of ω-3 PUFAs, and also, whether these effects were dependent on a SIRT1-HMGB1/NF-κB axis to gain further insight into the mechanisms underlying the

  11. microRNA-494 is a potential prognostic marker and inhibits cellular proliferation, migration and invasion by targeting SIRT1 in epithelial ovarian cancer.

    Science.gov (United States)

    Yang, Aijun; Wang, Xuenan; Yu, Chunna; Jin, Zhenzhen; Wei, Lingxia; Cao, Jinghe; Wang, Qin; Zhang, Min; Zhang, Lin; Zhang, Lei; Hao, Cuifang

    2017-09-01

    Ovarian cancer is one of the most common types of gynecological malignancy worldwide, and is the fourth leading cause of cancer-associated mortality among women. Despite improvements in therapeutic treatments, the prognosis for epithelial ovarian cancer (EOC) remains poor, mainly due to the rapid growth and metastasis of ovarian cancer tumors. An increasing number of studies have indicated that microRNAs (miRNAs) are involved in the carcinogenesis and progression of human cancer, suggesting that miRNAs may be used in clinical prognosis and as a therapeutic target in EOC. The aim of the present study was to investigate the expression levels of miRNA-494 in EOC tissues and cell lines. The clinical significance of miRNA-494 in patients with EOC was also evaluated. The results demonstrated that miRNA-494 was significantly downregulated in EOC tissues and cell lines. Low expression levels of miRNA-494 were associated with poor prognostic features, including International Federation of Gynecology and Obstetrics stage, tumor size and lymph node metastasis. In vitro functional studies demonstrated that overexpression of miRNA-494 inhibited proliferation, migration and invasion in EOC cells. By contrast, knockdown of miRNA-494 enhanced cell growth, migration and invasion in EOC cells. Notably, sirtuin 1 (SIRT1) was identified as a direct target of miRNA-494 in EOC. Furthermore, MTT, cell migration and invasion assays verified that EOC cell proliferation, migration and invasion were completely restored with forced miRNA-494 expression and SIRT1 restoration. Together, these findings suggest that miRNA-494 is a potential prognostic marker, and may provide novel therapeutic regimens of targeted therapy for EOC.

  12. Identification of a novel polyprenylated acylphloroglucinol‑derived SIRT1 inhibitor with cancer‑specific anti-proliferative and invasion-suppressing activities.

    Science.gov (United States)

    Zhu, Lijia; Qi, Ji; Chiao, Christine Ya-Chi; Zhang, Qiang; Porco, John A; Faller, Douglas V; Dai, Yan

    2014-11-01

    SIRT1, a class III histone deacetylase, plays a critical role in regulating cancer cell growth, migration and invasion, which makes it a potential target for cancer therapeutics. In this study, we screened derivatives of several groups of natural products and identified a novel SIRT1 inhibitor JQ-101, a synthetic derivative of the polyprenylated acylphloroglucinol (PPAP) natural products, with an IC(50) for SIRT1 of 30 µM in vitro, with 5-fold higher activity for SIRT1 vs. SIRT2. Exposure of tumor cells to JQ-101 significantly enhanced acetylation of p53 and histone H4K16 at known sites of SIRT1 deacetylation, validating SIRT1 as its cellular target. JQ-101 suppressed cancer cell growth and survival by targeting SIRT1, and also exhibited selective cytotoxicity towards a panel of human tumor cell lines, while producing no toxicity in two normal human cell types at comparable concentrations. JQ-101 induced both apoptosis and cell senescence, and suppressed cancer cell invasion in vitro. In summary, we have identified JQ-101 as a new SIRT1 inhibitor which may have potential application in cancer treatment through its ability to induce tumor cell apoptosis and senescence and suppress cancer cell invasion.

  13. Identification of a novel polyprenylated acylphloroglucinol-derived SIRT1 inhibitor with cancer-specific anti-proliferative and invasion-suppressing activities

    Science.gov (United States)

    ZHU, LIJIA; QI, JI; CHIAO, CHRISTINE YA-CHI; ZHANG, QIANG; PORCO, JOHN A.; FALLER, DOUGLAS V.; DAI, YAN

    2014-01-01

    SIRT1, a class III histone deacetylase, plays a critical role in regulating cancer cell growth, migration and invasion, which makes it a potential target for cancer therapeutics. In this study, we screened derivatives of several groups of natural products and identified a novel SIRT1 inhibitor JQ-101, a synthetic derivative of the polyprenylated acylphloroglucinol (PPAP) natural products, with an IC50 for SIRT1 of 30 μM in vitro, with 5-fold higher activity for SIRT1 vs. SIRT2. Exposure of tumor cells to JQ-101 significantly enhanced acetylation of p53 and histone H4K16 at known sites of SIRT1 deacetylation, validating SIRT1 as its cellular target. JQ-101 suppressed cancer cell growth and survival by targeting SIRT1, and also exhibited selective cytotoxicity towards a panel of human tumor cell lines, while producing no toxicity in two normal human cell types at comparable concentrations. JQ-101 induced both apoptosis and cell senescence, and suppressed cancer cell invasion in vitro. In summary, we have identified JQ-101 as a new SIRT1 inhibitor which may have potential application in cancer treatment through its ability to induce tumor cell apoptosis and senescence and suppress cancer cell invasion. PMID:25189993

  14. The histone deacetylase SIRT1 controls male fertility in mice through regulation of hypothalamic-pituitary gonadotropin signaling

    NARCIS (Netherlands)

    Kolthur-Seetharam, Ullas; Teerds, Katja; de Rooij, Dirk G.; Wendling, Olivia; McBurney, Michael; Sassone-Corsi, Paolo; Davidson, Irwin

    2009-01-01

    Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis

  15. The Histone Deacetylase SIRT1 Controls Male Fertility in Mice Through Regulation of Hypothalamic-Pituitary Gonadotropin Signaling

    NARCIS (Netherlands)

    Teerds, K.J.; Kolthur-Seerharam, U.; Rooij, de D.G.

    2009-01-01

    Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis

  16. A TagSNP in SIRT1 gene confers susceptibility to myocardial infarction in a Chinese Han population.

    Directory of Open Access Journals (Sweden)

    Jie Cheng

    Full Text Available SIRT1 exerts protective effects against endothelial cells dysfunction, inflammation and atherosclerosis, indicating an important role on myocardial infarction (MI pathogenesis. Nonetheless, the effects of SIRT1 variants on MI risk remain poorly understood. Here we aimed to investigate the influence of SIRT1 polymorphisms on individual susceptibility to MI. Genotyping of three tagSNPs (rs7069102, rs3818292 and rs4746720 in SIRT1 gene was performed in a Chinese Han population, consisting of 287 MI cases and 654 control subjects. In a logistic regression analysis, we found that G allele of rs7069102 had increased MI risk with odds ratio (OR of 1.57 [95% confidence interval (CI = 1.15-2.16, Bonferroni corrected P (Pc = 0.015] after adjustment for conventional risk factors compared to C allele. Similarly, the combined CG/GG genotypes was associated with the increased MI risk (OR = 1.64, 95% CI = 1.14-2.35, Pc = 0.021 compared to the CC genotype. Further stratified analysis revealed a more significant association with MI risk among younger subjects (≤ 55 years old. Consistent with these results, the haplotype rs7069102G-rs3818292A-rs4746720T containing the rs7069102 G allele was also associated with the increased MI risk (OR = 1.41, 95% CI = 1.09-1.84, Pc = 0.040. However, we did not detect any association of rs3818292 and rs4746720 with MI risk. Our study provides the first evidence that the tagSNP rs7069102 and haplotype rs7069102G-rs3818292A-rs4746720T in SIRT1 gene confer susceptibility to MI in the Chinese Han population.

  17. Sirt1 S-nitrosylation induces acetylation of HMGB1 in LPS-activated RAW264.7 cells and endotoxemic mice.

    Science.gov (United States)

    Kim, Young Min; Park, Eun Jung; Kim, Hye Jung; Chang, Ki Churl

    2018-06-18

    Excessive inflammation plays a detrimental role in endotoxemia. A recent study indicated that alarmins such as high mobility group box 1 (HMGB1) have drawn attention as therapeutic targets of sepsis. Post-translational modification (i.e., acetylation of lysine residues) of HMGB1 leads to the release of HMGB1 into the cellular space, operating as a warning signal that induces inflammation. Sirtuin 1 (SIRT1) has been shown to negatively regulate HMGB1 hyperacetylation and its extracellular release in sepsis. Therefore, we hypothesized that the S-nitrosylation (SNO) of SIRT1 may disrupt the ability of SIRT1 to negatively regulate the hyperacetylation of HMGB1. As long as the S-nitrosylation of SIRT1 occurs during septic conditions, it may worsen the situation. We found that the activity of SIRT1 decreased as the SNO-SIRT1 levels increased, resulting in HMGB1 release by LPS in RAW264.7 cells. Both the iNOS inhibitor (1400 W) and silencing iNOS significantly inhibited SNO-SIRT1, allowing increases in SIRT1 activity that decreased the HMGB1 release by LPS. SNAP, a NO donor, significantly increased both SNO-SIRT1 levels and the HMGB1 release that was accompanied by decreased sirt1 activity. However, sirtinol, a Sirt1 inhibitor, by itself decreased Sirt1 activity compared to that of the control, so that it did not affect already increased SNO-SIRT levels by SNAP. Most importantly, in lung tissues of LPS-endotoxic mice, significantly increased levels of SNO-SIRT were found, which was inhibited by 1400 W treatment. Plasma nitrite and HMGB1 levels were significantly higher than those in the sham controls, and the elevated levels were significantly lowered in the presence of 1400 W. We concluded that the S-nitrosylation of Sirt1 under endotoxic conditions may uninhibit the acetylation of HMGB1 and its extracellular release. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Wld(S reduces paraquat-induced cytotoxicity via SIRT1 in non-neuronal cells by attenuating the depletion of NAD.

    Directory of Open Access Journals (Sweden)

    Qiujing Yu

    Full Text Available Wld(S is a fusion protein with NAD synthesis activity, and has been reported to protect axonal and synaptic compartments of neurons from various mechanical, genetic and chemical insults. However, whether Wld(S can protect non-neuronal cells against toxic chemicals is largely unknown. Here we found that Wld(S significantly reduced the cytotoxicity of bipyridylium herbicides paraquat and diquat in mouse embryonic fibroblasts, but had no effect on the cytotoxicity induced by chromium (VI, hydrogen peroxide, etoposide, tunicamycin or brefeldin A. Wld(S also slowed down the death of mice induced by intraperitoneal injection of paraquat. Further studies demonstrated that Wld(S markedly attenuated mitochondrial injury including disruption of mitochondrial membrane potential, structural damage and decline of ATP induced by paraquat. Disruption of the NAD synthesis activity of Wld(S by an H112A or F116S point mutation resulted in loss of its protective function against paraquat-induced cell death. Furthermore, Wld(S delayed the decrease of intracellular NAD levels induced by paraquat. Similarly, treatment with NAD or its precursor nicotinamide mononucleotide attenuated paraquat-induced cytotoxicity and decline of ATP and NAD levels. In addition, we showed that SIRT1 was required for both exogenous NAD and Wld(S-mediated cellular protection against paraquat. These findings suggest that NAD and SIRT1 mediate the protective function of Wld(S against the cytotoxicity induced by paraquat, which provides new clues for the mechanisms underlying the protective function of Wld(S in both neuronal and non-neuronal cells, and implies that attenuation of NAD depletion may be effective to alleviate paraquat poisoning.

  19. Pharmacologic investigations on the role of Sirt-1 in neuroprotective mechanism of postconditioning in mice.

    Science.gov (United States)

    Kaur, Harpreet; Kumar, Amit; Jaggi, Amteshwar S; Singh, Nirmal

    2015-07-01

    Cerebral ischemia-reperfusion (I-R) injury is one of the primary causes of ischemic stroke. Ischemic postconditioning (iPoCo) is evolving as an important adaptive technique to contain I-R injury. Some recent studies have shown neuroprotective effects of iPoCo. However, the neuroprotective mechanism of iPoCo is not clear. So, the present study has been undertaken to investigate the possible role of Sirtinol, a selective class III histone deacetylase (HDAC) inhibitor in the neuroprotective mechanism of iPoCo in mice. Bilateral carotid artery occlusion (BCAO) for 12 min followed by reperfusion for 24 h was used to produce I-R-induced cerebral injury in Swiss albino mice. iPoCo involving three episodes of 10-s carotid artery occlusion and reperfusion instituted immediately after BCAO just before prolonged reperfusion of 24 h. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was evaluated using a Morris water maze test. Rotarod test, inclined beam-walking test, and neurologic severity score (NSS) were used to assess motor incoordination. Acetylcholine esterase levels, brain thiobarbituric acid reactive species (TBARS), and glutathione level were also estimated. BCAO for 12 min followed by reperfusion for 24 h produced a significant rise in cerebral infarct size and NSS along with impairment of memory and motor coordination and biochemical alteration (↑acetylcholine esterase, ↓glutathione, and ↑TBARS). iPoCo, involving three episodes of 10-s carotid artery occlusion with intermittent reperfusion of 10 s applied just after ischemic insult of 12 min produced a significant decrease in cerebral infarct size and NSS along with the reversal of I-R-induced impairment of memory and motor coordination. iPoCo-induced neuroprotective effects were significantly abolished by pretreatment with selective SIRT 1 (class III HDAC) blocker Sirtinol (10 mg/kg intraperitoneal). It may be concluded that the neuroprotective effect of iPoCo probably

  20. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    International Nuclear Information System (INIS)

    Xu, Ying; Nie, Ling; Yin, Yang-Guang; Tang, Jian-Lin; Zhou, Ji-Yin; Li, Dan-Dan; Zhou, Shi-Wen

    2012-01-01

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  1. Resveratrol protects against hyperglycemia-induced oxidative damage to mitochondria by activating SIRT1 in rat mesangial cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ying [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Nie, Ling [Department of Nephrology, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Yin, Yang-Guang [Emergency Department, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Tang, Jian-Lin; Zhou, Ji-Yin; Li, Dan-Dan [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China); Zhou, Shi-Wen, E-mail: Zhoushiwen1956@yahoo.cn [Base for Drug Clinical Trial, Xinqiao Hospital, Third Military Medical University, Chongqing, 400037 (China)

    2012-03-15

    Oxidative stress and mitochondrial dysfunction are involved in the pathogenesis of diabetic nephropathy (DN). Resveratrol has potent protective effects on diabetes and diabetic complications including diabetic nephropathy. We aimed to investigate the protective effects of resveratrol on mitochondria and the underlying mechanisms by using an in vitro model of hyperglycemia. We exposed primary cultured rat mesangial cells to high glucose (30 mM) for 48 h. We found that pretreatment with resveratrol (10 μM) 6 h prior to high glucose treatment significantly reduced hyperglycemia-induced increase in reactive oxygen species (ROS) production and mitochondrial superoxide generation, as well as stimulated MnSOD activity. In addition, resveratrol pretreatment significantly reversed the decrease of mitochondrial complex III activity in glucose-treated mesangial cells, which is considered to be the major source of mitochondrial oxidative stress in glucose-treated cells. Furthermore, resveratrol pretreatment efficiently restored the hyperpolarization of ∆Ψm, increased ATP production and preserved the mtDNA content. All of these protective effects of resveratrol were successfully blocked by siRNA targeting SIRT1 and EX-527, a specific inhibitor of SIRT1 activity. Our results indicated that resveratrol efficiently reduced oxidative stress and maintained mitochondrial function related with activating SIRT1 in glucose-treated mesangial cells. It suggested that resveratrol is pharmacologically promising for treating diabetic nephropathy. -- Highlights: ► We treat mesangial cells with glucose as an in vitro model of diabetic nephropathy. ► We find that the nephroprotective effects of resveratrol relate with mitochondria. ► The beneficial effect of resveratrol was prevented by siRNA SIRT1 or its inhibitor.

  2. SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuexia [Institute of Radiation Medicine, Fudan University, Shanghai (China); Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai (China); Tu, Wenzhi; Zhang, Jianghong; He, Mingyuan; Ye, Shuang; Dong, Chen [Institute of Radiation Medicine, Fudan University, Shanghai (China); Shao, Chunlin, E-mail: clshao@shmu.edu.cn [Institute of Radiation Medicine, Fudan University, Shanghai (China)

    2015-02-15

    Highlights: • γ-Irradiation induced bystander effects between hepatoma cells and hepatocyte cells. • SirT1 played a protective role in regulating this bystander effect. • SirT1 contributed to the protective effects via elimination the accumulation of ROS. • The activity of c-Myc is critical for maintaining the protective role of SirT1. - Abstract: Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved.

  3. SirT1 knockdown potentiates radiation-induced bystander effect through promoting c-Myc activity and thus facilitating ROS accumulation

    International Nuclear Information System (INIS)

    Xie, Yuexia; Tu, Wenzhi; Zhang, Jianghong; He, Mingyuan; Ye, Shuang; Dong, Chen; Shao, Chunlin

    2015-01-01

    Highlights: • γ-Irradiation induced bystander effects between hepatoma cells and hepatocyte cells. • SirT1 played a protective role in regulating this bystander effect. • SirT1 contributed to the protective effects via elimination the accumulation of ROS. • The activity of c-Myc is critical for maintaining the protective role of SirT1. - Abstract: Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the bystander signaling processes, especially under hypoxic condition, are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 and SK-Hep-1 cells under either normoxia or hypoxia. This bystander response was dramatically diminished or enhanced when the SirT1 gene of irradiated hepatoma cells was overexpressed or knocked down, respectively, especially under hypoxia. Meanwhile, SirT1 knockdown promoted transcriptional activity for c-Myc and facilitated ROS accumulation. But both of the increased bystander responses and ROS generation due to SirT1-knockdown were almost completely suppressed by c-Myc interference. Moreover, ROS scavenger effectively abolished the RIBE triggered by irradiated hepatoma cells even with SirT1 depletion. These findings provide new insights that SirT1 has a profound role in regulating RIBE where a c-Myc-dependent release of ROS may be involved

  4. Reverse chemomodulatory effects of the SIRT1 activators resveratrol and SRT1720 in Ewing's sarcoma cells: resveratrol suppresses and SRT1720 enhances etoposide- and vincristine-induced anticancer activity.

    Science.gov (United States)

    Sonnemann, Jürgen; Kahl, Melanie; Siranjeevi, Priyanka M; Blumrich, Annelie; Blümel, Lisa; Becker, Sabine; Wittig, Susan; Winkler, René; Krämer, Oliver H; Beck, James F

    2016-01-01

    SIRT1-activating compounds (STACs) may have potential in the management of cancer. However, the best-studied STAC, the naturally occurring compound resveratrol, is reported to have contradictory effects in combination chemotherapy regimens: It has been shown both to increase and to decrease the action of anticancer agents. To shed more light on this issue, we comparatively investigated the impact of resveratrol and the synthetic STAC SRT1720 on the responsiveness of Ewing's sarcoma (ES) cells to the chemotherapeutic drugs etoposide and vincristine. Because the effects of STACs can depend on the functionality of the tumor suppressor protein p53, we used three ES cell lines differing in their p53 status, i.e., wild-type p53 WE-68 cells, mutant p53 SK-ES-1 cells and p53 null SK-N-MC cells. Single agent and combination therapy effects were assessed by flow cytometric analyses of propidium iodide uptake and mitochondrial depolarization, by measuring caspase 3/7 activity and by gene expression profiling. When applied as single agents, both STACs were effective in ES cells irrespective of their p53 status. Strikingly, however, when applied in conjunction with cytostatic agents, the STACs displayed reverse effects: SRT1720 largely enhanced etoposide- and vincristine-induced cell death, while resveratrol inhibited it. Combination index analyses validated the antipodal impact of the STACs on the effectiveness of the chemotherapeutics. These findings suggest that the synthetic STAC SRT1720 may be useful to enhance the efficacy of anticancer therapy in ES. But they also suggest that the dietary intake of the natural STAC resveratrol may be detrimental during chemotherapy of ES.

  5. Maternal Diabetes Alters Expression of MicroRNAs that Regulate Genes Critical for Neural Tube Development

    Directory of Open Access Journals (Sweden)

    Seshadri Ramya

    2017-07-01

    Full Text Available Maternal diabetes is known to cause neural tube defects (NTDs in embryos and neuropsychological deficits in infants. Several metabolic pathways and a plethora of genes have been identified to be deregulated in developing brain of embryos by maternal diabetes, although the exact mechanism remains unknown. Recently, miRNAs have been shown to regulate genes involved in brain development and maturation. Therefore, we hypothesized that maternal diabetes alters the expression of miRNAs that regulate genes involved in biological pathways critical for neural tube development and closure during embryogenesis. To address this, high throughput miRNA expression profiling in neural stem cells (NSCs isolated from the forebrain of embryos from normal or streptozotocin-induced diabetic pregnancy was carried out. It is known that maternal diabetes results in fetal hypoglycemia/hyperglycemia or hypoxia. Hence, NSCs from embryos of control pregnant mice were exposed to low or high glucose or hypoxia in vitro. miRNA pathway analysis revealed distinct deregulation of several biological pathways, including axon guidance pathway, which are critical for brain development in NSCs exposed to different treatments. Among the differentially expressed miRNAs, the miRNA-30 family members which are predicted to target genes involved in brain development was upregulated in NSCs from embryos of diabetic pregnancy when compared to control. miRNA-30b was found to be upregulated while its target gene Sirtuin 1 (Sirt1, as revealed by luciferase assay, was down regulated in NSCs from embryos of diabetic pregnancy. Further, overexpression of miRNA-30b in NSCs, resulted in decreased expression of Sirt1 protein, and altered the neuron/glia ratio. On the other hand, siRNA mediated knockdown of Sirt1 in NSCs promoted astrogenesis, indicating that miRNA-30b alters lineage specification via Sirt1. Overall, these results suggest that maternal diabetes alters the genes involved in neural tube

  6. Expression of multiple proteins in transgenic plants

    Science.gov (United States)

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  7. Repressed SIRT1/PGC-1α pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration.

    Science.gov (United States)

    Golestaneh, Nady; Chu, Yi; Cheng, Shuk Kei; Cao, Hong; Poliakov, Eugenia; Berinstein, Daniel M

    2016-12-20

    Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms. Generation of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping. The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin

  8. Predictable tuning of protein expression in bacteria

    DEFF Research Database (Denmark)

    Bonde, Mads; Pedersen, Margit; Klausen, Michael Schantz

    2016-01-01

    We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expressi...

  9. Sugary Kefir Strain Lactobacillus mali APS1 Ameliorated Hepatic Steatosis by Regulation of SIRT-1/Nrf-2 and Gut Microbiota in Rats.

    Science.gov (United States)

    Chen, Yung-Tsung; Lin, Yu-Chun; Lin, Jin-Seng; Yang, Ning-Sun; Chen, Ming-Ju

    2018-04-01

    Non-alcoholic fatty liver disease (NAFLD) is a common disease that is concomitant with obesity, resulting in increased mortality. To date, the efficiency of NAFLD treatment still needs to be improved. Therefore, we aimed to evaluate the effect of Lactobacillus mali APS1, which was isolated from sugary kefir, on hepatic steatosis in rats fed a high-fat diet (HFD). Sprague Dawley rats were fed a control diet, a HFD with saline, and a HFD with APS1 intervention by gavage daily for 12 weeks. The results showed that APS1 significantly reduced body weight and body weight gain in HFD-fed rats. APS1 reduced hepatic lipid accumulation by regulating SIRT-1/PGC-1α/SREBP-1 expression. Moreover, APS1 increased hepatic antioxidant activity by modulating Nrf-2/HO-1 expression. Notably, APS1 manipulated the gut microbiota, resulting in increasing proportions of the phylum Bacteroidetes/Firmicutes and reducing the abundance of specific NAFLD-associated bacteria. These results suggested that APS1 ameliorated hepatic steatosis by modulating lipid metabolism and antioxidant activity via manipulating specific NAFLD-associated gut microbiota in vivo. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. The NAD-Dependent Deacetylase Sirtuin-1 Regulates the Expression of Osteogenic Transcriptional Activator Runt-Related Transcription Factor 2 (Runx2 and Production of Matrix Metalloproteinase (MMP-13 in Chondrocytes in Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Koh Terauchi

    2016-06-01

    Full Text Available Aging is one of the major pathologic factors associated with osteoarthritis (OA. Recently, numerous reports have demonstrated the impact of sirtuin-1 (Sirt1, which is the NAD-dependent deacetylase, on human aging. It has been demonstrated that Sirt1 induces osteogenic and chondrogenic differentiation of mesenchymal stem cells. However, the role of Sirt1 in the OA chondrocytes still remains unknown. We postulated that Sirt1 regulates a hypertrophic chondrocyte lineage and degeneration of articular cartilage through the activation of osteogenic transcriptional activator Runx2 and matrix metalloproteinase (MMP-13 in OA chondrocytes. To verify whether sirtuin-1 (Sirt1 regulates chondrocyte activity in OA, we studied expressions of Sirt1, Runx2 and production of MMP-13, and their associations in human OA chondrocytes. The expression of Sirt1 was ubiquitously observed in osteoarthritic chondrocytes; in contrast, Runx2 expressed in the osteophyte region in patients with OA and OA model mice. OA relating catabolic factor IL-1βincreased the expression of Runx2 in OA chondrocytes. OA chondrocytes, which were pretreated with Sirt1 inhibitor, inhibited the IL-1β-induced expression of Runx2 compared to the control. Since the Runx2 is a promotor of MMP-13 expression, Sirt1 inactivation may inhibit the Runx2 expression and the resultant down-regulation of MMP-13 production in chondrocytes. Our findings suggest thatSirt1 may regulate the expression of Runx2, which is the osteogenic transcription factor, and the production of MMP-13 from chondrocytes in OA. Since Sirt1 activity is known to be affected by several stresses, including inflammation and oxidative stress, as well as aging, SIRT may be involved in the development of OA.

  11. The long non-coding RNA MALAT1 promotes the migration and invasion of hepatocellular carcinoma by sponging miR-204 and releasing SIRT1.

    Science.gov (United States)

    Hou, Zhouhua; Xu, Xuwen; Zhou, Ledu; Fu, Xiaoyu; Tao, Shuhui; Zhou, Jiebin; Tan, Deming; Liu, Shuiping

    2017-07-01

    Increasing evidence supports the significance of long non-coding RNA in cancer development. Several recent studies suggest the oncogenic activity of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in hepatocellular carcinoma. In this study, we explored the molecular mechanisms by which MALAT1 modulates hepatocellular carcinoma biological behaviors. We found that microRNA-204 was significantly downregulated in sh-MALAT1 HepG2 cell and 15 hepatocellular carcinoma tissues by quantitative real-time polymerase chain reaction analysis. Through bioinformatic screening, luciferase reporter assay, RNA-binding protein immunoprecipitation, and RNA pull-down assay, we identified microRNA-204 as a potential interacting partner for MALAT1. Functionally, wound-healing and transwell assays revealed that microRNA-204 significantly inhibited the migration and invasion of hepatocellular carcinoma cells. Notably, sirtuin 1 was recognized as a direct downstream target of microRNA-204 in HepG2 cells. Moreover, si-SIRT1 significantly inhibited cell invasion and migration process. These data elucidated, by sponging and competitive binding to microRNA-204, MALAT1 releases the suppression on sirtuin 1, which in turn promotes hepatocellular carcinoma migration and invasion. This study reveals a novel mechanism by which MALAT1 stimulates hepatocellular carcinoma progression and justifies targeting metastasis-associated lung adenocarcinoma transcript 1 as a potential therapy for hepatocellular carcinoma.

  12. Treatment with NAD(+) inhibited experimental autoimmune encephalomyelitis by activating AMPK/SIRT1 signaling pathway and modulating Th1/Th17 immune responses in mice.

    Science.gov (United States)

    Wang, Jueqiong; Zhao, Congying; Kong, Peng; Sun, Huanhuan; Sun, Zhe; Bian, Guanyun; Sun, Yafei; Guo, Li

    2016-10-01

    Nicotinamide adenine dinucleotide (NAD(+)) plays vital roles in mitochondrial functions, cellular energy metabolism and calcium homeostasis. In this study, we investigated the effect of NAD(+) administration for the treatment of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. EAE, a classical animal model of multiple sclerosis (MS), was induced by subcutaneous injection of myelin oligodendrocyteglycoprotein (MOG). The mice were treated with 250mg/kg (body weight) NAD(+) in PBS administered intraperitoneally once daily. We observed that NAD(+) treatment could lessen the severity of EAE. Additionally, NAD(+) treatment attenuated pathological injuries of EAE mice. We also found that the AMP-activated protein kinase (AMPK)/silent mating-type information regulation 2 homolog 1(SIRT1) pathway was activated in the NAD(+)-treated mice and NAD(+) treatment suppressed pro-inflammatory T cell responses. Our findings demonstrated that NAD(+) could be an effective and promising agent to treat multiple sclerosis and its effects on other autoimmune diseases should be explored. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. TRPM4 protein expression in prostate cancer

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Soldini, Davide; Jung, Maria

    2016-01-01

    BACKGROUND: Transient receptor potential cation channel, subfamily M, member 4 (TRPM4) messenger RNA (mRNA) has been shown to be upregulated in prostate cancer (PCa) and might be a new promising tissue biomarker. We evaluated TRPM4 protein expression and correlated the expression level.......79-2.62; p = 0.01-0.03 for the two observers) when compared to patients with a lower staining intensity. CONCLUSIONS: TRPM4 protein expression is widely expressed in benign and cancerous prostate tissue, with highest staining intensities found in PCa. Overexpression of TRPM4 in PCa (combination of high...

  14. Temporal protein expression pattern in intracellular signalling ...

    Indian Academy of Sciences (India)

    Supplementary figure 1. Protein expression dynamics observed in Experiment, Synchronous and. Asynchronous simulation. .... molecular basis for T cell suppression by IL-10: CD28-asso- ciated IL-10 receptor inhibits CD28 tyrosine ...

  15. Role of resveratrol in FOXO1-mediated gluconeogenic gene expression in the liver

    Energy Technology Data Exchange (ETDEWEB)

    Park, Joo-Man; Kim, Tae-Hyun [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Bae, Jin-Sik; Kim, Mi-Young [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Kim, Kyung-Sup [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); The Institute of Genetic Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemoon-gu, Seoul 120-752 (Korea, Republic of); Ahn, Yong-Ho, E-mail: yha111@yuhs.ac [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2010-12-17

    Research highlights: {yields} Insulin-suppression of PEPCK and G6Pase gene expression is counteracted by resveratrol. {yields} Resveratrol upregulates hepatic gluconeogenic genes by attenuating insulin signaling and deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively. {yields} Resveratrol increases the binding activity of Foxo1 to the IRE of PEPCK and G6Pase. -- Abstract: During a state of fasting, the blood glucose level is maintained by hepatic gluconeogenesis. SIRT1 is an important metabolic regulator during nutrient deprivation and the liver-specific knockdown of SIRT1 resulted in decreased glucose production. We hypothesize that SIRT1 is responsible for the upregulation of insulin-suppressed gluconeogenic genes through the deacetylation of FOXO1. Treatment of primary cultured hepatocytes with resveratrol increased insulin-repressed PEPCK and G6Pase mRNA levels, which depend on SIRT1 activity. We found that the resveratrol treatment resulted in a decrease in the phosphorylation of Akt and FOXO1, which are independent of SIRT1 action. Fluorescence microscopy revealed that resveratrol caused the nuclear localization of FOXO1. In the nucleus, FOXO1 is deacetylated by SIRT1, which might make it more accessible to the IRE of the PEPCK and G6Pase promoter, causing an increase in their gene expression. Our results indicate that resveratrol upregulates the expression of gluconeogenic genes by attenuating insulin signaling and by deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively.

  16. Role of resveratrol in FOXO1-mediated gluconeogenic gene expression in the liver

    International Nuclear Information System (INIS)

    Park, Joo-Man; Kim, Tae-Hyun; Bae, Jin-Sik; Kim, Mi-Young; Kim, Kyung-Sup; Ahn, Yong-Ho

    2010-01-01

    Research highlights: → Insulin-suppression of PEPCK and G6Pase gene expression is counteracted by resveratrol. → Resveratrol upregulates hepatic gluconeogenic genes by attenuating insulin signaling and deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively. → Resveratrol increases the binding activity of Foxo1 to the IRE of PEPCK and G6Pase. -- Abstract: During a state of fasting, the blood glucose level is maintained by hepatic gluconeogenesis. SIRT1 is an important metabolic regulator during nutrient deprivation and the liver-specific knockdown of SIRT1 resulted in decreased glucose production. We hypothesize that SIRT1 is responsible for the upregulation of insulin-suppressed gluconeogenic genes through the deacetylation of FOXO1. Treatment of primary cultured hepatocytes with resveratrol increased insulin-repressed PEPCK and G6Pase mRNA levels, which depend on SIRT1 activity. We found that the resveratrol treatment resulted in a decrease in the phosphorylation of Akt and FOXO1, which are independent of SIRT1 action. Fluorescence microscopy revealed that resveratrol caused the nuclear localization of FOXO1. In the nucleus, FOXO1 is deacetylated by SIRT1, which might make it more accessible to the IRE of the PEPCK and G6Pase promoter, causing an increase in their gene expression. Our results indicate that resveratrol upregulates the expression of gluconeogenic genes by attenuating insulin signaling and by deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively.

  17. Finding Potent Sirt Inhibitor in Coffee: Isolation, Confirmation and Synthesis of Javamide-II (N-Caffeoyltryptophan as Sirt1/2 Inhibitor.

    Directory of Open Access Journals (Sweden)

    Jae B Park

    Full Text Available Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC50; 8.7 μM better than Sirt1(IC50; 34μM. Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD+ (Ki = 9.8 μM and showed competitive inhibition against the peptide substrate (Ki = 5.3 μM. Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382 in p53, but not (K305. This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.

  18. Expression of multidrug resistance proteins in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Swati Shukla

    2017-11-01

    Full Text Available AIM: To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS: Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS: Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1 expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION: Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  19. Expression of multidrug resistance proteins in retinoblastoma.

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy.

  20. Expression of multidrug resistance proteins in retinoblastoma

    Science.gov (United States)

    Shukla, Swati; Srivastava, Arpna; Kumar, Sunil; Singh, Usha; Goswami, Sandeep; Chawla, Bhavna; Bajaj, Mandeep Singh; Kashyap, Seema; Kaur, Jasbir

    2017-01-01

    AIM To elucidate the mechanism of multidrug resistance in retinoblastoma, and to acquire more insights into in vivo drug resistance. METHODS Three anticancer drug resistant Y79 human RB cells were generated against vincristine, etoposide or carboplatin, which are used for conventional chemotherapy in RB. Primary cultures from enucleated eyes after chemotherapy (PCNC) were also prepared. Their chemosensitivity to chemotherapeutic agents (vincristine, etoposide and carboplatin) were measured using MTT assay. Western blot analysis was performed to evaluate the expression of p53, Bcl-2 and various multidrug resistant proteins in retinoblastoma cells. RESULTS Following exposure to chemotherapeutic drugs, PCNC showed less sensitivity to drugs. No significant changes observed in the p53 expression, whereas Bcl-2 expression was found to be increased in the drug resistant cells as well as in PCNC. Increased expression of P-glycoprotein (P-gp) was observed in drug resistant Y79 cells; however there was no significant change in the expression of P-gp found between primary cultures of primarily enucleated eyes and PCNC. Multidrug resistance protein 1 (Mrp-1) expression was found to be elevated in the drug resistant Y79 cells as well as in PCNC. No significant change in the expression of lung resistance associated protein (Lrp) was observed in the drug resistant Y79 cells as well as in PCNC. CONCLUSION Our results suggest that multidrug resistant proteins are intrinsically present in retinoblastoma which causes treatment failure in managing retinoblastoma with chemotherapy. PMID:29181307

  1. A Novel Role of SIRT1/ FGF-21 in Taurine Protection Against Cafeteria Diet-Induced Steatohepatitis in Rats.

    Science.gov (United States)

    Abd Elwahab, Azza H; Ramadan, Basma K; Schaalan, Mona F; Tolba, Amina M

    2017-01-01

    Non-alcoholic fatty liver disease (NAFLD) is one of the alarmingly rising clinical problems in the 21st century with no effective drug treatment until now. Taurine is an essential amino acid in humans that proved efficacy as a non-pharmacological therapy in a plethora of diseases; however, its impact on NAFLD remains elusive. The aim of the current study is to evaluate the protective mechanism of taurine in experimental steatohepatitis induced by junk food given as cafeteria-diet (CAF-D) in male albino rats. Forty adult male albino rats of local strain between 8-10 weeks old, weighing 150 ± 20 g, were divided into four equal groups: Group I (control group), Group II (Taurine group), Group III (CAF-D for 12 weeks) and Group IV (CAF-D +Taurine). CAF-D was given in addition to the standard chow for 12 weeks, where each rat was given one piece of beef burger fried in 15 g of sunflower oil, one teaspoonful of mayonnaise, and one piece of petit pan bread, weighing 60g/ piece. In the serum, liver function tests; ALT, AST, ALP, GGT and the lipid profile; TG, TC, HDL-C added to reduced glutathione (GSH) were assessed colorimetrically, while fibroblast growth factor (FGF)-21, adiponectin & interleukin (IL)-6 via ELISA. The same technique was used for the assays of the hepatic levels of FGF-21, silent information regulator (SIRT1), malondialdehyde (MDA),IL-10, tumor necrosis factor-α (TNF-α) as well as the apoptotic markers; caspase-3 and B-cell lymphoma (Bcl-2). The cafeteria-diet induced steatohepatitis was reflected by significantly increased body and liver weight gain, elevation of liver enzymes; ALT, AST, ALP and GGT added to the dyslipidemic panel, presented as increased TC, TG, LDL-C and decreased HDL-C levels. The steatosis-induced inflammatory milieu, marked by elevated serum levels of FGF-21, IL-6, hepatic TNF-α, as well as reduced IL-10 and adiponectin, was associated with steatosis- induced hepatic oxidative stress, reflected by increased hepatic MDA and

  2. A Novel Role of SIRT1/ FGF-21 in Taurine Protection Against Cafeteria Diet-Induced Steatohepatitis in Rats

    Directory of Open Access Journals (Sweden)

    Azza H. Abd Elwahab

    2017-09-01

    Full Text Available Background: Non-alcoholic fatty liver disease (NAFLD is one of the alarmingly rising clinical problems in the 21st century with no effective drug treatment until now. Taurine is an essential amino acid in humans that proved efficacy as a non-pharmacological therapy in a plethora of diseases; however, its impact on NAFLD remains elusive. The aim of the current study is to evaluate the protective mechanism of taurine in experimental steatohepatitis induced by junk food given as cafeteria-diet (CAF-D in male albino rats. Methods: Forty adult male albino rats of local strain between 8-10 weeks old, weighing 150 ± 20 g, were divided into four equal groups: Group I (control group, Group II (Taurine group, Group III (CAF-D for 12 weeks and Group IV (CAF-D +Taurine. CAF-D was given in addition to the standard chow for 12 weeks, where each rat was given one piece of beef burger fried in 15 g of sunflower oil, one teaspoonful of mayonnaise, and one piece of petit pan bread, weighing 60g/ piece. In the serum, liver function tests; ALT, AST, ALP, GGT and the lipid profile; TG, TC, HDL-C added to reduced glutathione (GSH were assessed colorimetrically, while fibroblast growth factor (FGF-21, adiponectin & interleukin (IL-6 via ELISA. The same technique was used for the assays of the hepatic levels of FGF-21, silent information regulator (SIRT1, malondialdehyde (MDA,IL-10, tumor necrosis factor-α (TNF-α as well as the apoptotic markers; caspase-3 and B-cell lymphoma (Bcl-2. Results: The cafeteria-diet induced steatohepatitis was reflected by significantly increased body and liver weight gain, elevation of liver enzymes; ALT, AST, ALP and GGT added to the dyslipidemic panel, presented as increased TC, TG, LDL-C and decreased HDL-C levels. The steatosis-induced inflammatory milieu, marked by elevated serum levels of FGF-21, IL-6, hepatic TNF-α, as well as reduced IL-10 and adiponectin, was associated with steatosis- induced hepatic oxidative stress

  3. Parts Characterization for Tunable Protein Expression

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Sommer, Morten Otto Alexander

    2018-01-01

    Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Construc......Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression...

  4. Resveratrol alleviates cerebral ischemia/reperfusion injury in rats by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy induction.

    Science.gov (United States)

    He, Qi; Li, Zhenyu; Wang, Yueting; Hou, Yanghao; Li, Lingyu; Zhao, Jing

    2017-09-01

    Resveratrol has been reported to protect against cerebral ischemia/reperfusion (I/R) injury in rats, but the underlying mechanism is unclear. In the current study, we examined whether resveratrol ameliorates cerebral I/R injury by inhibiting NLRP3 inflammasome-derived inflammation and whether autophagy is involved in this process. In addition, we explored the role of Sirt1 in resveratrol-mediated protective effects. To answer these questions, healthy male Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 1h followed by 24h reperfusion. We found that cerebral I/R increased levels of activated NLRP3 inflammasome, caspase-1, IL-1β, and IL-18 and enhanced autophagy activity (ratio of LC3B-II/LC3B-I and p62/SQSTM1). Treatment with resveratrol, a specific Sirt1 agonist, attenuated I/R-induced NLRP3 inflammasome-derived inflammation but upregulated autophagy. Furthermore, resveratrol treatment clearly reduced cerebral infarct volume, decreased brain water content, and improved neurological scores. In addition, inhibition of autophagy using 3-MA intracerebroventricular injection blocked the inhibitory effect of resveratrol on NLRP3 inflammasome activation. Finally, Sirt1 knockdown with siRNA significantly blocked resveratrol-induced enhancement of autophagy activity and suppression of NLRP3 inflammasome activation. In conclusion, our results demonstrate that resveratrol protects against cerebral I/R injury by inhibiting NLRP3 inflammasome activation through Sirt1-dependent autophagy activity. Copyright © 2017. Published by Elsevier B.V.

  5. The Natural Carotenoid Crocetin and the Synthetic Tellurium Compound AS101 Protect the Ovary against Cyclophosphamide by Modulating SIRT1 and Mitochondrial Markers

    Directory of Open Access Journals (Sweden)

    Giovanna Di Emidio

    2017-01-01

    Full Text Available Cancer therapies are associated with increased infertility risk due to accelerated reproductive aging. Oxidative stress (OS is a potential mechanism behind ovarian toxicity by cyclophosphamide (CPM, the most ovotoxic anticancer drug. An important sensor of OS is SIRT1, a NAD+-dependent deacetylase which regulates cellular defence and cell fate. This study investigated whether the natural carotenoid crocetin and the synthetic compound AS101 protect the ovary against CPM by modulating SIRT1 and mitochondrial markers. We found that the number of primordial follicles of female CD1 mice receiving crocetin plus CPM increased when compared with CPM alone and similar to AS101, whose protective effects are known. SIRT1 increased in CPM mouse ovaries revealing the occurrence of OS. Similarly, mitochondrial SIRT3 rose, whilst SOD2 and the mitochondrial biogenesis activator PGC1-α decreased, suggesting the occurrence of mitochondrial damage. Crocetin and AS101 administration prevented SIRT1 burst suggesting that preservation of redox balance can help the ovary to counteract ovarian damage by CPM. Decreased SIRT3 and increased SOD2 and PGC1-α in mice receiving crocetin or AS101 prior to CPM provide evidence for mitochondrial protection. Present results improve the knowledge of ovarian damage by CPM and may help to develop interventions for preserving fertility in cancer patients.

  6. Differentially expressed proteins on postoperative 3

    Directory of Open Access Journals (Sweden)

    Jialili Ainuer

    2011-04-01

    Full Text Available 【Abstract】Objectives: Surgical repair of Achilles tendon (AT rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far. Methods: Forty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n=16 received postoperative cast immobilization; Group B (early motion group, n=16 received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C. The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing twodimensional polyacrylamide gel electrophoresis (2D-PAGE. PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI protein database retrieval and then for bioinformatics analysis. Results: A mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS. We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1

  7. Immunohistochemical expression of latent membrane protein 1 ...

    African Journals Online (AJOL)

    Methods: Archival formalin-fixed, paraffin-embedded NPC biopsies were evaluated in 23 Moroccan patients for the presence of LMP1 and p53 using immunohistochemistry (IHC). Results: No LMP1 expression was observed whereas 8 of 23 cases (34. 7%) had detectable p53 protein in the nuclei of tumor cells.

  8. SIRT1 protects cardiac cells against apoptosis induced by zearalenone or its metabolites α- and β-zearalenol through an autophagy-dependent pathway

    International Nuclear Information System (INIS)

    Ben Salem, Intidhar; Boussabbeh, Manel; Da Silva, Julie Pires; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2017-01-01

    Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). In the present study, we investigated the underlying mechanism of the toxicity induced by ZEN, α-ZOL and β-ZOL in cardiac cells (H9c2). We show that treatment with ZEN or its metabolites induces the activation of the mitochondrial pathway of apoptosis as characterized by an increase in ROS generation, a loss of mitochondrial transmembrane potential (ΔΨm) and an activation of caspases. Besides, we demonstrate that these mycotoxins promote the activation of autophagy before the onset of apoptosis. Indeed, we observed that a short-time (6 h) treatment with ZEN, α-ZOL or β-ZOL, increased the level of Beclin-1 and LC3-II and induced the accumulation of the CytoID® autophagy detection probe. Moreover, the inhibition of autophagy by Chloroquine significantly increased cell death induced by ZEN, α-ZOL or β-ZOL, suggesting that the activation of autophagy serves as a cardioprotective mechanism against these mycotoxins. In addition, we found that the inhibition (EX527) or the knockdown of SIRT1 (siRNA) significantly increased apoptosis induced by ZEN or its derivatives, whereas SIRT1 activation with RSV greatly prevents the cytotoxic effects of these mycotoxins. By contrast, when autophagy was inhibited by CQ, the activation of SIRT1 by RSV had no protection against the cardiotoxicity of ZEN or its metabolites, suggesting that SIRT1 protects cardiac cells by an autophagy-dependent pathway. - Highlights: • ZEN, α- and β-ZOL induce the mitochondrial pathway of apoptosis in cardiac cells. • Inhibition of autophagy enhanced ZEN-, α-ZOL- and β-ZOL-induced apoptosis. • SIRT1 activates autophagy to protect cells from ZEN, α- and β-ZOL-induced toxicity.

  9. Deleted in breast cancer 1 (DBC1) protein regulates hepatic gluconeogenesis.

    Science.gov (United States)

    Nin, Veronica; Chini, Claudia C S; Escande, Carlos; Capellini, Verena; Chini, Eduardo N

    2014-02-28

    Liver gluconeogenesis is essential to provide energy to glycolytic tissues during fasting periods. However, aberrant up-regulation of this metabolic pathway contributes to the progression of glucose intolerance in individuals with diabetes. Phosphoenolpyruvate carboxykinase (PEPCK) expression plays a critical role in the modulation of gluconeogenesis. Several pathways contribute to the regulation of PEPCK, including the nuclear receptor Rev-erbα and the histone deacetylase SIRT1. Deleted in breast cancer 1 (DBC1) is a nuclear protein that binds to and regulates both Rev-erbα and SIRT1 and, therefore, is a candidate to participate in the regulation of PEPCK. In this work, we provide evidence that DBC1 regulates glucose metabolism and the expression of PEPCK. We show that DBC1 levels decrease early in the fasting state. Also, DBC1 KO mice display higher gluconeogenesis in a normal and a high-fat diet. DBC1 absence leads to an increase in PEPCK mRNA and protein expression. Conversely, overexpression of DBC1 results in a decrease in PEPCK mRNA and protein levels. DBC1 regulates the levels of Rev-erbα, and manipulation of Rev-erbα activity or levels prevents the effect of DBC1 on PEPCK. In addition, Rev-erbα levels decrease in the first hours of fasting. Finally, knockdown of the deacetylase SIRT1 eliminates the effect of DBC1 knockdown on Rev-erbα levels and PEPCK expression, suggesting that the mechanism of PEPCK regulation is, at least in part, dependent on the activity of this enzyme. Our results point to DBC1 as a novel regulator of gluconeogenesis.

  10. Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor–κB in osteoblasts

    Science.gov (United States)

    Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

    2017-01-01

    ABSTRACT Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity. PMID:28473882

  11. Sirtuin 1 regulates matrix metalloproteinase-13 expression induced by Porphyromonas endodontalis lipopolysaccharide via targeting nuclear factor-κB in osteoblasts.

    Science.gov (United States)

    Qu, Liu; Yu, Yaqiong; Qiu, Lihong; Yang, Di; Yan, Lu; Guo, Jiajie; Jahan, Rabita

    2017-01-01

    Porphyromonas endodontalis lipopolysaccharide (P.e LPS) is an important initiating factor for periapical inflammation and bone destruction. Matrix metalloproteinase-13 (MMP-13) has been shown to participate in the formation and diffusion of periapical bone lesion in chronic apical periodontitis. Sirtuin 1 (SIRT1) is a key regulator of inflammation in mammalian cells which suppresses the release of inflammatory mediators. This study aimed to explore the role of SIRT1 in regulating MMP-13 expression induced by P.e LPS in osteoblasts. P.e LPS stimulated MMP-13 expression in MC3T3-E1 cells. Knockdown of SIRT1 reinforced the increase of MMP-13mRNA expression induced by P.e LPS. SIRT1 activator resveratrol significantly reduced the expression of MMP-13 and SIRT1 inhibitor EX-527 enhanced the expression of MMP-13. Moreover, SIRT1 activation with resveratrol inhibited acetylation of NF-κB p65 and NF-κB transcriptional activity, which were enhanced by P.e LPS. In addition, NF-κB p65 was involved in P.e LPS-induced MMP-13 expression via directly binding to the MMP-13 promoter. However, SIRT1 activation significantly interfered with this binding. These findings strongly suggest that P.e LPS induces MMP-13 expression in osteoblasts, and SIRT1 suppresses this expression of MMP-13 through targeting NF-κB p65. This provides new insights into understanding the actions of SIRT1 on anti-inflammatory and anti-bone resorption activity.

  12. A p300 and SIRT1 Regulated Acetylation Switch of C/EBPα Controls Mitochondrial Function

    NARCIS (Netherlands)

    Zaini, Mohamad A; Müller, Christine; de Jong, Tristan V; Ackermann, Tobias; Hartleben, Götz; Kortman, Gertrud; Gührs, Karl-Heinz; Fusetti, Fabrizia; Krämer, Oliver H; Guryev, Victor; Calkhoven, Cornelis F

    2018-01-01

    Cellular metabolism is a tightly controlled process in which the cell adapts fluxes through metabolic pathways in response to changes in nutrient supply. Among the transcription factors that regulate gene expression and thereby cause changes in cellular metabolism is the basic leucine-zipper (bZIP)

  13. Resveratrol attenuates bone cancer pain through regulating the expression levels of ASIC3 and activating cell autophagy.

    Science.gov (United States)

    Zhu, Haili; Ding, Jieqiong; Wu, Ji; Liu, Tingting; Liang, Jing; Tang, Qiong; Jiao, Ming

    2017-11-01

    Bone cancer pain (BCP) is one of the most common pains in patients with malignant cancers. The mechanism underlying BCP is largely unknown. Our previous studies and the increasing evidence both have shown that acid-sensing ion channels 3 (ASIC3) is an important protein in the pathological pain state in some pain models. We hypothesized that the expression change of ASIC3 might be one of the factors related to BCP. In this study, we established the BCP model through intrathecally injecting rat mammary gland carcinoma cells (MRMT-1) into the left tibia of Sprague-Dawley female rats, and found that the BCP rats showed bone destruction, increased mechanical pain sensitivities and up-regulated ASIC3 protein expression levels in L4-L6 dorsal root ganglion. Then, resveratrol, which was intraperitoneally injected into the BCP rats on post-operative Day 21, dose-dependently increased the paw withdrawal threshold of BCP rats, reversed the pain behavior, and had an antinociceptive effect on BCP rats. In ASIC3-transfected SH-SY5Y cells, the ASIC3 protein expression levels were regulated by resveratrol in a dose- and time-dependent manner. Meanwhile, resveratrol also had an antinociceptive effect in ASIC3-mediated pain rat model. Furthermore, resveratrol also enhanced the phosphorylation of AMPK, SIRT1, and LC3-II levels in ASIC3-transfected SH-SY5Y cells, indicating that resveratrol could activate the AMPK-SIRT1-autophagy signal pathway in ASIC3-transfected SH-SY5Y cells. In BCP rats, SIRT1 and LC3-II were also down-regulated. These findings provide new evidence for the use of resveratrol as a therapeutic treatment during BCP states. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Regulation of Sirtuin-Mediated Protein Deacetylation by Cardioprotective Phytochemicals

    Directory of Open Access Journals (Sweden)

    Niria Treviño-Saldaña

    2017-01-01

    Full Text Available Modulation of posttranslational modifications (PTMs, such as protein acetylation, is considered a novel therapeutic strategy to combat the development and progression of cardiovascular diseases. Protein hyperacetylation is associated with the development of numerous cardiovascular diseases, including atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. In addition, decreased expression and activity of the deacetylases Sirt1, Sirt3, and Sirt6 have been linked to the development and progression of cardiac dysfunction. Several phytochemicals exert cardioprotective effects by regulating protein acetylation levels. These effects are mainly exerted via activation of Sirt1 and Sirt3 and inhibition of acetyltransferases. Numerous studies support a cardioprotective role for sirtuin activators (e.g., resveratrol, as well as other emerging modulators of protein acetylation, including curcumin, honokiol, oroxilyn A, quercetin, epigallocatechin-3-gallate, bakuchiol, tyrosol, and berberine. Studies also point to a cardioprotective role for various nonaromatic molecules, such as docosahexaenoic acid, alpha-lipoic acid, sulforaphane, and caffeic acid ethanolamide. Here, we review the vast evidence from the bench to the clinical setting for the potential cardioprotective roles of various phytochemicals in the modulation of sirtuin-mediated deacetylation.

  15. Regulation of Sirtuin-Mediated Protein Deacetylation by Cardioprotective Phytochemicals

    Science.gov (United States)

    2017-01-01

    Modulation of posttranslational modifications (PTMs), such as protein acetylation, is considered a novel therapeutic strategy to combat the development and progression of cardiovascular diseases. Protein hyperacetylation is associated with the development of numerous cardiovascular diseases, including atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. In addition, decreased expression and activity of the deacetylases Sirt1, Sirt3, and Sirt6 have been linked to the development and progression of cardiac dysfunction. Several phytochemicals exert cardioprotective effects by regulating protein acetylation levels. These effects are mainly exerted via activation of Sirt1 and Sirt3 and inhibition of acetyltransferases. Numerous studies support a cardioprotective role for sirtuin activators (e.g., resveratrol), as well as other emerging modulators of protein acetylation, including curcumin, honokiol, oroxilyn A, quercetin, epigallocatechin-3-gallate, bakuchiol, tyrosol, and berberine. Studies also point to a cardioprotective role for various nonaromatic molecules, such as docosahexaenoic acid, alpha-lipoic acid, sulforaphane, and caffeic acid ethanolamide. Here, we review the vast evidence from the bench to the clinical setting for the potential cardioprotective roles of various phytochemicals in the modulation of sirtuin-mediated deacetylation. PMID:29234485

  16. De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells.

    Science.gov (United States)

    Liu, Huiying; Xing, Rong; Cheng, Xuefang; Li, Qingran; Liu, Fang; Ye, Hui; Zhao, Min; Wang, Hong; Wang, Guangji; Hao, Haiping

    2016-09-20

    Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress.

  17. A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

    Science.gov (United States)

    Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

    2011-01-01

    While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

  18. Activation of the AMPK/Sirt1 pathway by a leucine-metformin combination increases insulin sensitivity in skeletal muscle, and stimulates glucose and lipid metabolism and increases life span in Caenorhabditis elegans.

    Science.gov (United States)

    Banerjee, Jheelam; Bruckbauer, Antje; Zemel, Michael B

    2016-11-01

    We have previously shown leucine (Leu) to activate Sirt1 by lowering its K M for NAD + , thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving ~80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction. Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met-Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. Leu (0.5mmol/L)+Met (50-100μmol/L) synergistically activated Sirt1 (pmetformin exerted no independent effect at any concentration (0.1-0.5mmol/L). Thus, Leu and Met synergize to enable Sirt1 activation at low NAD + concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met-Leu's full action, which result in improvements in energy metabolism and insulin sensitivity. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Recombinant Expression Screening of P. aeruginosa Bacterial Inner Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Jeffery Constance J

    2010-11-01

    Full Text Available Abstract Background Transmembrane proteins (TM proteins make up 25% of all proteins and play key roles in many diseases and normal physiological processes. However, much less is known about their structures and molecular mechanisms than for soluble proteins. Problems in expression, solubilization, purification, and crystallization cause bottlenecks in the characterization of TM proteins. This project addressed the need for improved methods for obtaining sufficient amounts of TM proteins for determining their structures and molecular mechanisms. Results Plasmid clones were obtained that encode eighty-seven transmembrane proteins with varying physical characteristics, for example, the number of predicted transmembrane helices, molecular weight, and grand average hydrophobicity (GRAVY. All the target proteins were from P. aeruginosa, a gram negative bacterial opportunistic pathogen that causes serious lung infections in people with cystic fibrosis. The relative expression levels of the transmembrane proteins were measured under several culture growth conditions. The use of E. coli strains, a T7 promoter, and a 6-histidine C-terminal affinity tag resulted in the expression of 61 out of 87 test proteins (70%. In this study, proteins with a higher grand average hydrophobicity and more transmembrane helices were expressed less well than less hydrophobic proteins with fewer transmembrane helices. Conclusions In this study, factors related to overall hydrophobicity and the number of predicted transmembrane helices correlated with the relative expression levels of the target proteins. Identifying physical characteristics that correlate with protein expression might aid in selecting the "low hanging fruit", or proteins that can be expressed to sufficient levels using an E. coli expression system. The use of other expression strategies or host species might be needed for sufficient levels of expression of transmembrane proteins with other physical

  20. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    Science.gov (United States)

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  1. Sirt1 suppresses RNA synthesis after UV irradiation in combined xeroderma pigmentosum group D/Cockayne syndrome (XP-D/CS) cells.

    Science.gov (United States)

    Vélez-Cruz, Renier; Zadorin, Anton S; Coin, Frédéric; Egly, Jean-Marc

    2013-01-15

    Specific mutations in the XPD subunit of transcription factor IIH result in combined xeroderma pigmentosum (XP)/Cockayne syndrome (CS), a severe DNA repair disorder characterized at the cellular level by a transcriptional arrest following UV irradiation. This transcriptional arrest has always been thought to be the result of faulty transcription-coupled repair. In the present study, we showed that, following UV irradiation, XP-D/CS cells displayed a gross transcriptional dysregulation compared with "pure" XP-D cells or WT cells. Furthermore, global RNA-sequencing analysis showed that XP-D/CS cells repressed the majority of genes after UV, whereas pure XP-D cells did not. By using housekeeping genes as a model, we demonstrated that XP-D/CS cells were unable to reassemble these gene promoters and thus to restart transcription after UV irradiation. Furthermore, we found that the repression of these promoters in XP-D/CS cells was not a simple consequence of deficient repair but rather an active heterochromatinization process mediated by the histone deacetylase Sirt1. Indeed, RNA-sequencing analysis showed that inhibition of and/or silencing of Sirt1 changed the chromatin environment at these promoters and restored the transcription of a large portion of the repressed genes in XP-D/CS cells after UV irradiation. Our work demonstrates that a significant part of the transcriptional arrest displayed by XP-D/CS cells arises as a result of an active repression process and not simply as a result of a DNA repair deficiency. This dysregulation of Sirt1 function that results in transcriptional repression may be the cause of various severe clinical features in patients with XP-D/CS that cannot be explained by a DNA repair defect.

  2. A pilot randomized, placebo controlled, double blind phase I trial of the novel SIRT1 activator SRT2104 in elderly volunteers.

    Directory of Open Access Journals (Sweden)

    Vincenzo Libri

    Full Text Available SRT2104 has been developed as a selective small molecule activator of SIRT1, a NAD(+-dependent deacetylase involved in the regulation of energy homeostasis and the modulation of various metabolic pathways, including glucose metabolism, oxidative stress and lipid metabolism. SIRT1 has been suggested as putative therapeutic target in multiple age-related diseases including type 2 diabetes and dyslipidemias. We report the first clinical trial of SRT2104 in elderly volunteers.Oral doses of 0.5 or 2.0 g SRT2104 or matching placebo were administered once daily for 28 days. Pharmacokinetic samples were collected through 24 hours post-dose on days 1 and 28. Multiple pharmacodynamic endpoints were explored with oral glucose tolerance tests (OGTT, serum lipid profiles, magnetic resonance imaging (MRI for assessment of whole body visceral and subcutaneous fat, maximal aerobic capacity test and muscle 31P magnetic resonance spectroscopy (MRS for estimation of mitochondrial oxidative capacity.SRT2104 was generally safe and well tolerated. Pharmacokinetic exposure increased less than dose-proportionally. Mean Tmax was 2-4 hours with elimination half-life of 15-20 hours. Serum cholesterol, LDL levels and triglycerides decreased with treatment. No significant changes in OGTT responses were observed. 31P MRS showed trends for more rapid calculated adenosine diphosphate (ADP and phosphocreatine (PCr recoveries after exercise, consistent with increased mitochondrial oxidative phosphorylation.SRT2104 can be safely administered in elderly individuals and has biological effects in humans that are consistent with SIRT1 activation. The results of this study support further development of SRT2104 and may be useful in dose selection for future clinical trials in patients.ClinicalTrials.gov NCT00964340.

  3. Perturbations in the p53/miR-34a/SIRT1 pathway in the R6/2 Huntington's disease model

    DEFF Research Database (Denmark)

    Reynolds, Regina Hertfelder; Petersen, Maria Hvidberg; Willert, Cecilie Wennemoes

    2018-01-01

    The three factors, p53, the microRNA-34 family and Sirtuin 1 (SIRT1), interact in a positive feedback loop involved in cell cycle progression, cellular senescence and apoptosis. Each factor in this triad has roles in metabolic regulation, maintenance of mitochondrial function, and regulation...... of brain-derived neurotrophic factor (BDNF). Thus, this regulatory network holds potential importance for the pathophysiology of Huntington's disease (HD), an inherited neurodegenerative disorder in which both mitochondrial dysfunction and impaired neurotrophic signalling are observed. We investigated...

  4. The predictive nature of transcript expression levels on protein expression in adult human brain.

    Science.gov (United States)

    Bauernfeind, Amy L; Babbitt, Courtney C

    2017-04-24

    Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R 2 values of < 10%), transcripts coding for protein kinases and membrane-associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples.

  5. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    International Nuclear Information System (INIS)

    Hayashi, Kokoro; Kojima, Chojiro

    2010-01-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in 1 H- 15 N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  6. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Kokoro [Fujifilm Corporation, Analysis Technology Center (Japan); Kojima, Chojiro, E-mail: kojima@protein.osaka-u.ac.j [Nara Institute of Science and Technology (NAIST), Graduate School of Biological Sciences (Japan)

    2010-11-15

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in {sup 1}H-{sup 15}N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  7. Ligand Binding Domain Protein in Tetracycline-Inducible Expression

    African Journals Online (AJOL)

    Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

  8. Identification of differentially expressed proteins in response to Pb ...

    African Journals Online (AJOL)

    In response to Pb, a total of 76 proteins, out of the 95 differentially expressed proteins, were subjected to MALDI-TOF-MS Of these, 46 identities were identified by PMF and 19 identities were identified by microsequencing. Basic metabolisms such as photosynthesis, photorespiration and protein biosynthesis in C. roseus ...

  9. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    Science.gov (United States)

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Evolution, diversification and expression of KNOX proteins in plants

    Directory of Open Access Journals (Sweden)

    Jie eGao

    2015-10-01

    Full Text Available The KNOX (KNOTTED1-like homeobox transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification.

  11. Major cancer protein amplifies global gene expression

    Science.gov (United States)

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  12. Effects of immunosuppressive treatment on protein expression in rat kidney

    Directory of Open Access Journals (Sweden)

    Kędzierska K

    2014-09-01

    Full Text Available Karolina Kędzierska,1 Katarzyna Sporniak-Tutak,2 Krzysztof Sindrewicz,2 Joanna Bober,3 Leszek Domański,1 Mirosław Parafiniuk,4 Elżbieta Urasińska,5 Andrzej Ciechanowicz,6 Maciej Domański,1 Tomasz Smektała,2 Marek Masiuk,5 Wiesław Skrzypczak,6 Małgorzata Ożgo,6 Joanna Kabat-Koperska,1 Kazimierz Ciechanowski1 1Department of Nephrology, Transplantology, and Internal Medicine, 2Department of Dental Surgery, 3Department of Medical Chemistry, 4Department of Forensic Medicine, 5Department of Pathomorphology, Pomeranian Medical University, 6Department of Physiology, Cytobiology, and Proteomics, West Pomeranian University of Technology, Szczecin, Poland Abstract: The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents' toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins' synthesis. Very slight differences

  13. Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21(WAF1) (/) (CIP1) ) and SIRT1 genes.

    Science.gov (United States)

    Wu, Dinglan; Yu, Shan; Jia, Lin; Zou, Chang; Xu, Zhenyu; Xiao, Lijia; Wong, Kam-Bo; Ng, Chi-Fai; Chan, Franky L

    2015-05-01

    Oncogene-induced senescence is an important tumour-suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene-induced senescence. We determined that TLX exhibited an increased expression in high-grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over-expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin-induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene-induced senescence induced either by activated oncogene H-Ras(G12V) or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence-associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene-induced senescence, and also suggests that targeting the senescence-regulatory TLX is of potential therapeutic significance in prostate cancer. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  14. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  15. Heterologous Protein Expression by Lactococcus lactis

    NARCIS (Netherlands)

    Villatoro-Hernández, J.; Kuipers, O.P.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.

    2012-01-01

    This chapter describes the use of Lactococcus lactis as a safe and efficient cell factory to produce heterologous proteins of medical interest. The relevance of the use of this lactic acid bacterium (LAB) is that it is a noncolonizing, nonpathogenic microorganism that can be delivered in vivo at a

  16. Genome-wide screens for expressed hypothetical proteins

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Durhuus, Jon Ambæk; Rasmussen, Lene Juel

    2012-01-01

    A hypothetical protein (HP) is defined as a protein that is predicted to be expressed from an open reading frame, but for which there is no experimental evidence of translation. HPs constitute a substantial fraction of proteomes of human as well as of other organisms. With the general belief that...... that the majority of HPs are the product of pseudogenes, it is essential to have a tool with the ability of pinpointing the minority of HPs with a high probability of being expressed....

  17. CURCUMIN DECREASES SPECIFICITY PROTEIN (Sp) EXPRESSION IN BLADDER CANCER CELLS

    OpenAIRE

    Chadalapaka, Gayathri; Jutooru, Indira; Chintharlapalli, Sudhakar; Papineni, Sabitha; Smith, Roger; Li, Xiangrong; Safe, Stephen

    2008-01-01

    Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. In this study, 10 – 25 µM curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of...

  18. Isocaloric pair-fed high-carbohydrate diet induced more hepatic steatosis and inflammation than high-fat diet mediated by miR- 34a/SIRT1 axis in mice

    Science.gov (United States)

    To investigate the different effects of isocaloric high-fat diet (HFD) and high-carbohydrate diet (HCD) on hepatic steatosis and the underlying mechanisms, especially the role of microRNA- 34a/silent information regulator T1 (SIRT1) axis, C57BL/6J mice (n = 12/group) were isocaloric pair-fed with Li...

  19. Resveratrol-Induced AMP-Activated Protein Kinase Activation Is Cell-Type Dependent: Lessons from Basic Research for Clinical Application.

    Science.gov (United States)

    Lan, Fan; Weikel, Karen A; Cacicedo, Jose M; Ido, Yasuo

    2017-07-14

    Despite the promising effects of resveratrol, its efficacy in the clinic remains controversial. We were the first group to report that the SIRT1 activator resveratrol activates AMP-activated protein kinase (AMPK) (Diabetes 2005; 54: A383), and we think that the variability of this cascade may be responsible for the inconsistency of resveratrol's effects. Our current studies suggest that the effect of SIRT1 activators such as resveratrol may not be solely through activation of SIRT1, but also through an integrated effect of SIRT1-liver kinase B1 (LKB1)-AMPK. In this context, resveratrol activates SIRT1 (1) by directly binding to SIRT1; and (2) by increasing NAD⁺ levels by upregulating the salvage pathway through Nampt activation, an effect mediated by AMPK. The first mechanism promotes deacetylation of a limited number of SIRT1 substrate proteins (e.g., PGC-1). The second mechanism (which may be more important than the first) activates other sirtuins in addition to SIRT1, which affects a broad spectrum of substrates. Despite these findings, detailed mechanisms of how resveratrol activates AMPK have not been reported. Here, we show that (1) resveratrol-induced activation of AMPK requires the presence of functional LKB1; (2) Resveratrol increases LKB1 activity, which involves translocation and phosphorylation at T336 and S428; (3) Activation of LKB1 causes proteasomal degradation of LKB1; (4) At high concentrations (50-100 µM), resveratrol also activates AMPK through increasing AMP levels; and (5) The above-mentioned activation mechanisms vary among cell types, and in some cell types, resveratrol fails to activate AMPK. These results suggest that resveratrol-induced activation of AMPK is not a ubiquitous phenomenon. In addition, AMPK-mediated increases in NAD⁺ in the second mechanism require several ATPs, which may not be available in many pathological conditions. These phenomena may explain why resveratrol is not always consistently beneficial in a clinical

  20. Recombinant Brucella abortus gene expressing immunogenic protein

    Energy Technology Data Exchange (ETDEWEB)

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  1. Improved means and methods for expressing recombinant proteins

    NARCIS (Netherlands)

    Poolman, Berend; Martinez Linares, Daniel; Gul, Nadia

    2014-01-01

    The invention relates to the field of genetic engineering and the production of recombinant proteins in microbial host cells. Provided is a method for enhanced expression of a recombinant protein of interest in a microbial host cell, comprising providing a microbial host cell wherein the function of

  2. Expression of green fluorescent protein (GFPuv) in Escherichia coli ...

    African Journals Online (AJOL)

    Administrator

    The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-α grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by ...

  3. The proteome response to amyloid protein expression in vivo.

    Directory of Open Access Journals (Sweden)

    Ricardo A Gomes

    Full Text Available Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE. We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

  4. Induction of cell cycle arrest and apoptosis with downregulation of Hsp90 client proteins and histone modification by 4β-hydroxywithanolide E isolated from Physalis peruviana.

    Science.gov (United States)

    Park, Eun-Jung; Sang-Ngern, Mayuramas; Chang, Leng Chee; Pezzuto, John M

    2016-06-01

    Physalis peruviana (Solanaceae) is used for culinary and medicinal purposes. We currently report withanolides, isolated from P. peruviana, inhibit the growth of colon cancer monolayer and spheroid cultures. A detailed mechanistic evaluation was performed with 4β-hydroxywithanolide E (4HWE). Treatment of HT-29 cells with low concentrations of 4HWE inhibited growth while enhancing levels of p21(Waf1/Cip1) and reducing levels of several cell cycle-related proteins. Apoptosis was induced at higher concentrations. In addition, 4HWE treatment downregulated the levels of Hsp90 client proteins. Nuclear sirtuin 1 (SIRT1) was increased and histone H3 acetylated at lysine 9 was decreased. An additional consequence of SIRT1 elevation in the nucleus may be inhibition of c-Jun activity. The expression of 21 genes was altered, including downregulation of PTGS2, and this correlated with reduced protein levels of cyclooxygenase-2 (COX-2). Overall, efficacious induction of G0/G1 cell cycle arrest at low concentrations, and induction of apoptosis at higher concentrations are interesting 4HWE-mediated phenomena that are accompanied by a complex array of molecular events. Considering the worldwide prevalence of colon cancer, and the unique mode of action mediated by 4HWE, it is reasonable to investigate additional mechanistic details and the potential utility of this compound. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. The clinical expression of hereditary protein C and protein S deficiency: : a relation to clinical thrombotic risk-factors and to levels of protein C and protein S

    NARCIS (Netherlands)

    Henkens, C. M. A.; van der Meer, J.; Hillege, J. L.; Bom, V. J. J.; Halie, M. R.; van der Schaaf, W.

    We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were

  6. Protein expression analysis of inflammation-related colon carcinogenesis

    Directory of Open Access Journals (Sweden)

    Yasui Yumiko

    2009-01-01

    Full Text Available Background: Chronic inflammation is a risk factor for colorectal cancer (CRC development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM and dextran sodium sulfate (DSS using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight, followed by 2% (w/v DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins. Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.

  7. Hypoxic-induced stress protein expression in rat cardiac myocytes

    International Nuclear Information System (INIS)

    Howard, G.; Geoghegan, T.E.

    1986-01-01

    Mammalian stress proteins can be induced in cells and tissues exposed to a variety of conditions including hyperthermia and diminished O 2 supply. The authors have previously shown that the expression of three stress proteins (71, 85, and 95 kDa) was induced in cardiac tissue from mice exposed to hypoxic conditions. The expression of mRNAs coding for the 85 and 95 kDa proteins increase with time of exposure to hypoxia, while the mRNA coding for the 71 kDa protein is transiently induced. The authors extended these studies to investigate the expression of stress proteins in isolated rat cardiac myocytes. Freshly prepared myocytes were exposed to control, hypoxic, anoxic, or heat-shock environments for up to 16 h. The proteins were then labeled for 6 hours with [ 35 S]methionine. Analysis of the solubilized proteins by SDS-PAGE and autoradiography showed that there was a 6-fold increase in synthesis of the 85 kDa protein upon exposure to hypoxia but not heat-shock conditions. The 71 kDa protein was present at high levels in both control and treated myocyte protein preparations, and presumably had been induced during the isolation procedure. Total RNA isolated from intact rat heart and isolated myocytes was compared by cell-free translation analysis and showed induction of RNAs coding for several stress proteins in the myocyte preparation. The induced proteins at 85 and 95 kDa have molecular weights similar to reported cell stress and/or glucose-regulated proteins

  8. Variation in Protein Intake Induces Variation in Spider Silk Expression

    Science.gov (United States)

    Blamires, Sean J.; Wu, Chun-Lin; Tso, I-Min

    2012-01-01

    Background It is energetically expensive to synthesize certain amino acids. The proteins (spidroins) of spider major ampullate (MA) silk, MaSp1 and MaSp2, differ in amino acid composition. Glutamine and proline are prevalent in MaSp2 and are expensive to synthesize. Since most orb web spiders express high proline silk they might preferentially attain the amino acids needed for silk from food and shift toward expressing more MaSp1 in their MA silk when starved. Methodology/Principal Findings We fed three spiders; Argiope aetherea, Cyrtophora moluccensis and Leucauge blanda, high protein, low protein or no protein solutions. A. aetherea and L. blanda MA silks are high in proline, while C. moluccesnsis MA silks are low in proline. After 10 days of feeding we determined the amino acid compositions and mechanical properties of each species' MA silk and compared them between species and treatments with pre-treatment samples, accounting for ancestry. We found that the proline and glutamine of A. aetherea and L. blanda silks were affected by protein intake; significantly decreasing under the low and no protein intake treatments. Glutmaine composition in C. moluccensis silk was likewise affected by protein intake. However, the composition of proline in their MA silk was not significantly affected by protein intake. Conclusions Our results suggest that protein limitation induces a shift toward different silk proteins with lower glutamine and/or proline content. Contradictions to the MaSp model lie in the findings that C. moluccensis MA silks did not experience a significant reduction in proline and A. aetherea did not experience a significant reduction in serine on low/no protein. The mechanical properties of the silks could not be explained by a MaSp1 expressional shift. Factors other than MaSp expression, such as the expression of spidroin-like orthologues, may impact on silk amino acid composition and spinning and glandular processes may impact mechanics. PMID:22363691

  9. Differential Protein Expression in Congenital and Acquired Cholesteatomas.

    Directory of Open Access Journals (Sweden)

    Seung-Ho Shin

    Full Text Available Congenital cholesteatomas are epithelial lesions that present as an epithelial pearl behind an intact eardrum. Congenital and acquired cholesteatomas progress quite differently from each other and progress patterns can provide clues about the unique origin and pathogenesis of the abnormality. However, the exact pathogenic mechanisms by which cholesteatomas develop remain unknown. In this study, key proteins that directly affect cholesteatoma pathogenesis are investigated with proteomics and immunohistochemistry. Congenital cholesteatoma matrices and retroauricular skin were harvested during surgery in 4 patients diagnosed with a congenital cholesteatoma. Tissue was also harvested from the retraction pocket in an additional 2 patients during middle ear surgery. We performed 2-dimensional (2D electrophoresis to detect and analyze spots that are expressed only in congenital cholesteatoma and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS to separate proteins by molecular weight. Protein expression was confirmed by immunohistochemical staining. The image analysis of 2D electrophoresis showed that 4 congenital cholesteatoma samples had very similar protein expression patterns and that 127 spots were exclusively expressed in congenital cholesteatomas. Of these 127 spots, 10 major spots revealed the presence of titin, forkhead transcription activator homolog (FKH 5-3, plectin 1, keratin 10, and leucine zipper protein 5 by MALDI-TOF/MS analysis. Immunohistochemical staining showed that FKH 5-3 and titin were expressed in congenital cholesteatoma matrices, but not in acquired cholesteatomas. Our study shows that protein expression patterns are completely different in congenital cholesteatomas, acquired cholesteatomas, and skin. Moreover, non-epithelial proteins, including FKH 5-3 and titin, were unexpectedly expressed in congenital cholesteatoma tissue. Our data indicates that congenital cholesteatoma origins

  10. Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed

    Directory of Open Access Journals (Sweden)

    Schininà Maria

    2010-09-01

    Full Text Available Abstract Background Several mutations have been described as responsible for rifampicin resistance in Neisseria meningitidis. However, the intriguing question on why these strains are so rare remains open. The aim of this study was to investigate the protein content and to identify differential expression in specific proteins in two rifampicin resistant and one susceptible meningococci using two-dimensional electrophoresis (2-DE combined with mass spectrometry. Results In our experimental conditions, able to resolve soluble proteins with an isoelectric point between 4 and 7, twenty-three proteins have been found differentially expressed in the two resistant strains compared to the susceptible. Some of them, involved in the main metabolic pathways, showed an increased expression, mainly in the catabolism of pyruvate and in the tricarboxylic acid cycle. A decreased expression of proteins belonging to gene regulation and to those involved in the folding of polypeptides has also been observed. 2-DE analysis showed the presence of four proteins displaying a shift in their isoelectric point in both resistant strains, confirmed by the presence of amino acid changes in the sequence analysis, absent in the susceptible. Conclusions The analysis of differentially expressed proteins suggests that an intricate series of events occurs in N. meningitidis rifampicin resistant strains and the results here reported may be considered a starting point in understanding their decreased invasion capacity. In fact, they support the hypothesis that the presence of more than one protein differentially expressed, having a role in the metabolism of the meningococcus, influences its ability to infect and to spread in the population. Different reports have described and discussed how a drug resistant pathogen shows a high biological cost for survival and that may also explain why, for some pathogens, the rate of resistant organisms is relatively low considering the

  11. Myocardin-related transcription factor regulates Nox4 protein expression

    DEFF Research Database (Denmark)

    Rozycki, Matthew; Bialik, Janne Folke; Speight, Pam

    2016-01-01

    translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application...... TGFβ/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFβ to facilitate...... MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFβ-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFβ-activated Smad3 and TAZ/YAP, two contact...

  12. EXPRESSION AND SIGNIFICANCE OF ERK PROTEIN IN HUMAN BREAST CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    张秀梅; 李柏林; 宋敏; 宋继谒

    2004-01-01

    Objective: To investigate the expression of ERK and p-ERK protein in human breast cancer and their corresponding tissue, to assess the significance of ERK signal pathway in tumorigenesis and progression of breast carcinoma. Methods: 40 breast cancer cases were used in S-P immunohistochemistry technique and Western Blot study. Results: The expression of ERK1, ERK2, and p- ERK protein levels increased remarkably in breast cancer tissues in comparison to normal tissues (P<0.01). The expression was upregulated by 1.32-, 1.53-and 4.27-fold, respectively. The overexpressions of ERK1, ERK2, and p- ERK proteins were obviously correlated with clinical stage of breast cancer. Protein levels of ERK and p-ERK were higher in stage III patients than in stage I and stage II patients (P<0.05). These proteins were strongly related with axillary lymph node metastasis of breast cancer, but not correlated with histopathological type and status of ER and PR of breast cancer. Expression of ERK1, and ERK2, protein showed a positive linear correlation. Conclusion: ERK signal transduction pathway is a key factor during human breast tumorigenesis and breast cancer progression.

  13. Genome engineering for improved recombinant protein expression in Escherichia coli.

    Science.gov (United States)

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-12-19

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

  14. BAX protein expression and clinical outcome in epithelial ovarian cancer.

    Science.gov (United States)

    Tai, Y T; Lee, S; Niloff, E; Weisman, C; Strobel, T; Cannistra, S A

    1998-08-01

    Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.

  15. Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Athanasakis, Emmanouil; Aloisio, Michelangelo; Monasta, Lorenzo; Ricci, Giuseppe

    2016-05-01

    Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two‑dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches.

  16. SIRT1/PGC1α-Dependent Increase in Oxidative Phosphorylation Supports Chemotherapy Resistance of Colon Cancer

    NARCIS (Netherlands)

    Vellinga, Thomas T.; Borovski, Tijana; de Boer, Vincent C. J.; Fatrai, Szabolcs; van Schelven, Susanne; Trumpi, Kari; Verheem, Andre; Snoeren, Nikol; Emmink, Benjamin L.; Koster, Jan; Rinkes, Inne H. M. Borel; Kranenburg, Onno

    2015-01-01

    Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms. We studied gene expression differences between groups of liver metastases

  17. High-level transient expression of recombinant protein in lettuce.

    Science.gov (United States)

    Joh, Lawrence D; Wroblewski, Tadeusz; Ewing, Nicholas N; VanderGheynst, Jean S

    2005-09-30

    Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. Copyright 2005 Wiley Periodicals, Inc

  18. Regenerating human muscle fibres express GLUT3 protein

    DEFF Research Database (Denmark)

    Gaster, M; Beck-Nielsen, H; Schrøder, H D

    2002-01-01

    The presence of the GLUT3 glucose transporter protein in human muscle cells is a matter of debate. The present study was designed to establish whether GLUT3 is expressed in mature human skeletal muscle fibres and, if so, whether its expression changes under different conditions, such as metabolic...... muscle fibres, nor did metabolic stress, training or de- and re-innervation induce GLUT3 expression, while a few GLUT3 expressing fibres were seen in some cases of polymyositis. In contrast, GLUT4 was expressed in all investigated muscle fibres. GLUT3 immunoreactivity was found in perineural...... and endoneural cells, indicating that GLUT3 is important for glucose transport into nerves through the perineurium. Taken together, these data suggest that GLUT3 expression is restricted to regenerating muscle fibres and nerves in adult human muscle. Although the significance of GLUT3 in adult human muscle...

  19. Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

    Science.gov (United States)

    Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

    2017-10-01

    Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene

  20. Kiss-1/GPR54 protein expression in breast cancer.

    Science.gov (United States)

    Papaoiconomou, Eleni; Lymperi, Maria; Petraki, Constantina; Philippou, Anastassios; Msaouel, Pavlos; Michalopoulou, Fani; Kafiri, Georgia; Vassilakos, George; Zografos, Georgios; Koutsilieris, Michael

    2014-03-01

    Numerous studies have shown that the Kiss-1 gene countervails the metastatic aptitude of several cancer cell lines and solid-tumor neoplasias. However, there still remains ambiguity regarding its role in breast cancer and literature has arisen asserting that Kiss-1 expression may be linked to an aggressive phenotype and malignant progression. Herein, we investigated the protein expression of Kiss-1 and its receptor GPR54 in breast cancer tissues compared to non-cancerous mammary tissues. Paraffin-fixed cancer tissues from 43 women with resected breast adenocarcinomas and 11 specimens derived from women suffering from fibrocystic disease, serving as controls, were immunostained with Kiss-1 and GPR54 antibodies. Kiss-1 and GPR54 protein expression levels were significantly higher in breast cancer compared to fibrocystic tissues (pbreast cancer and fibrocystic disease specimens. Kiss-1/GPR54 expression was found to be significantly higher in breast cancer compared to non-malignant mammary tissues.

  1. Bcl-2 Protein Expression in Egyptian Acute Myeloid Leukemia

    International Nuclear Information System (INIS)

    El-Shakankiry, N.; El-Sayed, Gh.M.M.; El-Maghraby, Sh.; Moneer, M.M.

    2009-01-01

    Objective: The primary cause of treatment failure in acute myeloid leukemia (AML) is the emergence of both resistant disease and early relapse. The bcl-2 gene encodes a 26-kDa protein that promotes cell survival by blocking programmed cell death (apoptosis). In the present study, bcl-2 protein expression was evaluated in newly diagnosed AML patients and correlated with the induction of remission and overall survival (OS), in an attempt to define patients who might benefit from modified therapeutic strategies. Patients and methods: Pretreatment cellular bcl-2 protein expression was measured in bone marrow samples obtained from 68 patients of newly diagnosed acute myeloid leukemia and 10 healthy controls by western blotting. Results: The mean bcl-2 protein expression was significantly higher in patients (0.68610.592) compared to controls (0.313±0.016) (p=0.002). The overall survival for patients with mean bcl-2 expression of less, and more than or equal to 0.315, was 67% and 56%, respectively, with no significant difference between the two groups 0»=0.86). Conclusion: Even though we did not observe a significant difference in overall survival between patients with high and low levels of bcl-2, modulation of this protein might still be considered as an option for enhancing the effectiveness of conventional chemotherapy.

  2. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    International Nuclear Information System (INIS)

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio

    2007-01-01

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation

  3. Amurensin G, a potent natural SIRT1 inhibitor, rescues doxorubicin responsiveness via down-regulation of multidrug resistance 1

    DEFF Research Database (Denmark)

    Oh, Won Keun; Cho, Kyoung Bin; Hien, Tran Thi

    2010-01-01

    The transition from a chemotherapy-responsive cancer to a chemotherapy-resistant one is accompanied by increased expression of multidrug resistance 1 (MDR1, p-glycoprotein), which plays an important role in the efflux from the target cell of many anticancer agents. We recently showed that a Forkh...

  4. SIRT1/PGC1a-dependent increase in oxidative phosphorylation supports chemotherapy resistance of colon cancer

    NARCIS (Netherlands)

    Vellinga, T.T.; Borovski, T.; Boer, de V.C.J.; Kranenburg, O.

    2015-01-01

    PURPOSE:
    Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms.
    EXPERIMENTAL DESIGN:
    We studied gene expression

  5. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    Science.gov (United States)

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  6. Different Cells Make Different Proteins: A Laboratory Exercise Illustrating Tissue-Specific Protein Expression in Animals

    Science.gov (United States)

    Ibarguren, Izaskun; Villamarín, Antonio

    2017-01-01

    All the cells of higher organisms have the same DNA but not the same proteins. Each type of specialised cell that forms a tissue has its own pattern of gene expression and, consequently, it contains a particular set of proteins that determine its function. Here, we describe a laboratory exercise addressed to undergraduate students that aims to…

  7. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    International Nuclear Information System (INIS)

    Tsutsui, Shinichi; Yasuda, Kazuhiro; Suzuki, Kosuke; Takeuchi, Hideya; Nishizaki, Takashi; Higashi, Hidefumi; Era, Shoichi

    2006-01-01

    Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

  8. Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

    Directory of Open Access Journals (Sweden)

    Nishizaki Takashi

    2006-07-01

    Full Text Available Abstract Background Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. Methods The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. Results The Bcl-2 protein expression was found to be decreased in 105 (42% cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089 associated with a worse disease free survival (DFS, while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. Conclusion The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer.

  9. Cloning and expression analysis of a blue copperbinding protein ...

    African Journals Online (AJOL)

    Adifferentially expressed fragment EST145 was isolated by suppression subtractive hybridization (SSH) method. Using EST145 as the probe, a blue copper-binding protein gene designated as DvBCB was screened from Dasypyrum villosum cDNA Library. The DvBCB gene was 845 bp in length with an open reading frame ...

  10. Lipid transfer proteins from fruit: cloning, expression and quantification

    NARCIS (Netherlands)

    Zuidmeer, Laurian; van Leeuwen, W. Astrid; Budde, Ilona Kleine; Cornelissen, Jessica; Bulder, Ingrid; Rafalska, Ilona; Besolí, Noèlia Telléz; Akkerdaas, Jaap H.; Asero, Riccardo; Fernandez Rivas, Montserrat; Rivas, Montserrat Fernandez; Gonzalez Mancebo, Eloina; Mancebo, Eloina Gonzalez; van Ree, Ronald

    2005-01-01

    BACKGROUND: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for

  11. Regulation of autophagy by AMP-activated protein kinase/ sirtuin 1 pathway reduces spinal cord neurons damage

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    Peng Yan

    2017-09-01

    Full Text Available Objective(s: AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1 signaling pathway has been proved to be involved in the regulation of autophagy in various models. The aim of this study was to evaluate the effect of AMPK/SIRT1 pathway on autophagy after spinal cord injury (SCI. Materials and Methods:The SCI model was established in rats in vivo and the primary spinal cord neurons were subjected to mechanical injury (MI in vitro. The apoptosis in spinal cord tissue and neurons was assessed by TUNEL staining and Hoechst 33342 staining, respectively. The autophagy-related proteins levels were detected by Western blot. The activation of AMPK/SIRT1 pathway was determined by Western blot and immunohistochemical staining. Results: We found that the apoptosis of spinal cord tissue and cell damage of spinal cord neurons was obvious after the trauma. The ratio of LC3II/LC3I and level of p62 were first increased significantly and then decreased after the trauma in vivo and in vitro, indicating the defect in autophagy. The levels of p-AMPK and SIRT1 were increased obviously after the trauma in vivo and in vitro. Further activation of the AMPK/SIRT1 pathway by pretreatment with resveratrol, a confirmed activator of the AMPK/SIRT1 pathway, alleviated the cell damage and promoted the autophagy flux via downregulation of p62 in spinal cord neurons at 24 hr after MI. Conclusion: Our results demonstrate that regulation of autophagy by AMPK/SIRT1 pathway can restrain spinal cord neurons damage, which may be a potential intervention of SCI.

  12. Regulation of autophagy by AMP-activated protein kinase/sirtuin 1 pathway reduces spinal cord neurons damage.

    Science.gov (United States)

    Yan, Peng; Bai, Liangjie; Lu, Wei; Gao, Yuzhong; Bi, Yunlong; Lv, Gang

    2017-09-01

    AMP-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway has been proved to be involved in the regulation of autophagy in various models. The aim of this study was to evaluate the effect of AMPK/SIRT1 pathway on autophagy after spinal cord injury (SCI). The SCI model was established in rats in vivo and the primary spinal cord neurons were subjected to mechanical injury (MI) in vitro . The apoptosis in spinal cord tissue and neurons was assessed by TUNEL staining and Hoechst 33342 staining, respectively. The autophagy-related proteins levels were detected by Western blot. The activation of AMPK/SIRT1 pathway was determined by Western blot and immunohistochemical staining. We found that the apoptosis of spinal cord tissue and cell damage of spinal cord neurons was obvious after the trauma. The ratio of LC3II/LC3I and level of p62 were first increased significantly and then decreased after the trauma in vivo and in vitro , indicating the defect in autophagy. The levels of p-AMPK and SIRT1 were increased obviously after the trauma in vivo and in vitro . Further activation of the AMPK/SIRT1 pathway by pretreatment with resveratrol, a confirmed activator of the AMPK/SIRT1 pathway, alleviated the cell damage and promoted the autophagy flux via downregulation of p62 in spinal cord neurons at 24 hr after MI. Our results demonstrate that regulation of autophagy by AMPK/SIRT1 pathway can restrain spinal cord neurons damage, which may be a potential intervention of SCI.

  13. Adiponectin Suppresses T Helper 17 Cell Differentiation and Limits Autoimmune CNS Inflammation via the SIRT1/PPARγ/RORγt Pathway.

    Science.gov (United States)

    Zhang, Kai; Guo, Yawei; Ge, Zhenzhen; Zhang, Zhihui; Da, Yurong; Li, Wen; Zhang, Zimu; Xue, Zhenyi; Li, Yan; Ren, Yinghui; Jia, Long; Chan, Koon-Ho; Yang, Fengrui; Yan, Jun; Yao, Zhi; Xu, Aimin; Zhang, Rongxin

    2017-09-01

    T helper 17 (Th17) cells are vital components of the adaptive immune system involved in the pathogenesis of most autoimmune and inflammatory syndromes, and adiponectin(ADN) is correlated with inflammatory diseases such as multiple sclerosis (MS) and type II diabetes. However, the regulatory effects of adiponectin on pathogenic Th17 cell and Th17-mediated autoimmune central nervous system (CNS) inflammation are not fully understood. In this study, we demonstrated that ADN could inhibit Th1 and Th17 but not Th2 cells differentiation in vitro. In the in vivo study, we demonstrated that ADN deficiency promoted CNS inflammation and demyelination and exacerbated experimental autoimmune encephalomyelitis (EAE), an animal model of human MS. Furthermore, ADN deficiency increased the Th1 and Th17 cell cytokines of both the peripheral immune system and CNS in mice suffering from EAE. It is worth mentioning that ADN deficiency predominantly promoted the antigen-specific Th17 cells response in autoimmune encephalomyelitis. In addition, in vitro and in vivo, ADN upregulated sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ (PPARγ) and inhibited retinoid-related orphan receptor-γt (RORγt); the key transcription factor during Th17 cell differentiation. These results systematically uncovered the role and mechanism of adiponectin on pathogenic Th17 cells and suggested that adiponectin could inhibit Th17 cell-mediated autoimmune CNS inflammation.

  14. Heterogeneity of proteins expressed by Brazilian Sporothrix schenckii isolates.

    Science.gov (United States)

    Fernandes, Geisa Ferreira; Do Amaral, Cristiane Candida; Sasaki, Alexandre; Godoy, Patrício Martinez; De Camargo, Zoilo Pires

    2009-12-01

    The profiles of proteins present in the exoantigens of Brazilian Sporothrix schenckii isolates were studied and compared by electrophoresis (SDS-PAGE). Thirteen isolates from five different regions of Brazil (1,000 to 2,000 km apart) and ten from a more limited region (200 to 400 km apart within the state of São Paulo) were cultured in Sabouraud, M199 and minimum (MM) media. Qualitative and quantitative differences in the expression of proteins, which varied according to the medium and the isolate, were observed. Fractions with the same MW but varying in intensity were detected, as well as fractions present in 1 isolate but absent in others. Dendrograms were constructed and isolates grouped based on the fractions obtained, irrespective of the intensity. The results showed that Brazilian S. schenckii isolates express different protein profiles, a feature also present in isolates from a more restricted region. The exoantigens were found to have a maximum of 15 protein fractions, ranging in MW from 19-220 KDaltons depending on the medium used for the cultures. These data show the great heterogeneity of Brazilian S. schenckii protein expression.

  15. Optimization of translation profiles enhances protein expression and solubility.

    Directory of Open Access Journals (Sweden)

    Anne-Katrin Hess

    Full Text Available mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  16. The E4 protein; structure, function and patterns of expression

    Energy Technology Data Exchange (ETDEWEB)

    Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  17. Developmental expression of Drosophila Wiskott-Aldrich Syndrome family proteins

    Science.gov (United States)

    Rodriguez-Mesa, Evelyn; Abreu-Blanco, Maria Teresa; Rosales-Nieves, Alicia E.; Parkhurst, Susan M.

    2012-01-01

    Background Wiskott-Aldrich Syndrome (WASP) family proteins participate in many cellular processes involving rearrangements of the actin cytoskeleton. To the date, four WASP subfamily members have been described in Drosophila: Wash, WASp, SCAR, and Whamy. Wash, WASp, and SCAR are essential during early Drosophila development where they function in orchestrating cytoplasmic events including membrane-cytoskeleton interactions. A mutant for Whamy has not yet been reported. Results We generated monoclonal antibodies that are specific to Drosophila Wash, WASp, SCAR, and Whamy, and use these to describe their spatial and temporal localization patterns. Consistent with the importance of WASP family proteins in flies, we find that Wash, WASp, SCAR, and Whamy are dynamically expressed throughout oogenesis and embryogenesis. For example, we find that Wash accumulates at the oocyte cortex. WASp is highly expressed in the PNS, while SCAR is the most abundantly expressed in the CNS. Whamy exhibits an asymmetric subcellular localization that overlaps with mitochondria and is highly expressed in muscle. Conclusion All four WASP family members show specific expression patterns, some of which reflect their previously known roles and others revealing new potential functions. The monoclonal antibodies developed offer valuable new tools to investigate how WASP family proteins regulate actin cytoskeleton dynamics. PMID:22275148

  18. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  19. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  20. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    Science.gov (United States)

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  1. Roles of HMGA proteins in cancer: Expression, pathways, and redundancies

    Directory of Open Access Journals (Sweden)

    Giancotti V

    2016-10-01

    Full Text Available The expression of the High Mobility Group A (HMGA proteins, their participation in cancer signalling pathways, and their redundant functions have been reviewed in seven types of cancer: breast, colorectal, prostate, lung, ovarian, thyroid, and brain. The analysis of cell lines and tumours revealed an elevated level of their expression in all fully transformed cancer systems, which represents a step of the main cancer signalling pathways. In breast, colorectal, prostate, and lung cancers Wnt/β-catenin pathway is a master inducer of cell transformation in which are deeply involved HMG A1 and A2 proteins. On the other hand, IL-6/Stat3 pathway is responsible for cancer transformation in breast, lung, and prostate. The expression of HMGA1 in lung and ovarian cancers is due to an active PI3K/Akt pathway. The let-7 family of microRNA represses the expression of HMGA showing specificity by its different forms: the let-7b form is able to inhibit both proteins A1 and A2, the last also inhibited by a, c, d, and g forms. Moreover, both proteins are down-regulated by the repressor couple p53/microRNA-34a. The protein A1 and A2 participate to the Epithelial-Mesenchymal Transition cooperating with the three couples of factors Twist1/2, Snai1/2, and Zeb1/2. Through a combination of pathways, there is the simultaneous presence of high levels of both A1 and A2 together with the expression of other factors: a high co-operating efficiency is reached that supplies the tumour cells with properties of self-renewal, resistance, and invasiveness.

  2. Expression of Sirtuins in the Retinal Neurons of Mice, Rats, and Humans

    Directory of Open Access Journals (Sweden)

    Hongdou Luo

    2017-11-01

    Full Text Available Sirtuins are a class of histone deacetylases (HDACs that have been shown to regulate a range of pathophysiological processes such as cellular aging, inflammation, metabolism, and cell proliferation. There are seven mammalian Sirtuins (SIRT1-7 that play important roles in stress response, aging, and neurodegenerative diseases. However, the location and function of Sirtuins in neurons are not well defined. This study assessed the retinal expression of Sirtuins in mice, rats, and humans and measured the expression of Sirtuins in aged and injured retinas. Expression of all 7 Sirtuins was confirmed by Western blot and Real-Time PCR analysis in all three species. SIRT1 is highly expressed in mouse, rat, and human retinas, whereas SIRT2-7 expression was relatively lower in human retinas. Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons. Importantly, we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion. On the other hand, none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5, which was significantly higher in the retinas of adults compared to both young and aged rats. Our work presents the first composite analysis of Sirtuins in the retinal neurons of mice, rats, and humans, and suggests that increasing the expression and activity of SIRT1 may be beneficial for the treatment of glaucoma and other age-related eye dysfunction.

  3. Expression of uncoupling protein 1 in bovine muscle cells.

    Science.gov (United States)

    Abd Eldaim, M A; Hashimoto, O; Ohtsuki, H; Yamada, T; Murakami, M; Onda, K; Sato, R; Kanamori, Y; Qiao, Y; Tomonaga, S; Matsui, T; Funaba, M

    2016-12-01

    Uncoupling protein 1 (Ucp1) is predominantly expressed in brown/beige adipocytes in mammals. Although myogenic cells have been suggested to commit to a brown adipocyte lineage through the induction of Prdm16 expression, Prdm16 is also expressed in skeletal muscle. Thus, we examined expression of Ucp1 in bovine myogenic cells. Considering that Ucp1 is a principle molecule that induces energy expenditure in brown/beige adipocytes, expression of Ucp1 is not preferable in beef cattle because of potential decrease in energy (fattening) efficiency. The RT-PCR analyses revealed the expression of Ucp1 in the skeletal muscle of cattle; expression levels were markedly lower than those in the brown fat of calves. Immunohistochemical analyses showed that Ucp1 surrounded muscle fibers, but not adipocytes residing in skeletal muscle. Myosatellite cells cultured in myogenic medium showed an increase in the expression levels of myogenic regulatory factors ( levels were greater in cells after myogenic culture for 12 d than in those after myogenic culture for 6 d ( bovine skeletal muscle, which suggests the necessity for further studies on Ucp1-mediated energy expenditure in bovine skeletal muscle.

  4. Novel leukocyte protein, Trojan, differentially expressed during thymocyte development.

    Science.gov (United States)

    Petrov, Petar; Motobu, Maki; Salmi, Jussi; Uchida, Tatsuya; Vainio, Olli

    2010-04-01

    "Trojan" is a novel cell surface protein, discovered from chicken embryonic thymocytes on the purpose to identify molecules involved in T cell differentiation. The molecule is predicted as a type I transmembrane protein having a Sushi and two fibronectin type III domains and a pair of intracellular phosphorylation sites. Its transcript expression is specific for lymphoid tissues and the presence of the protein on the surface of recirculating lymphocytes and macrophages was confirmed by immunofluorescence analysis. In thymus, about half of the double negative (CD4(-) CD8(-)) and CD8 single positive and the majority of CD4 single positive cells express Trojan with a relatively high intensity. However, only a minority of the double positive (CD4(+) CD8(+)) cells are positive for Trojan. This expression pattern, similar to that of some proteins with anti-apoptotic and function, like IL-7Ralpha, makes Trojan an attractive candidate of having an anti-apoptotic role. Copyright 2010 Elsevier Ltd. All rights reserved.

  5. Nociceptive DRG neurons express muscle lim protein upon axonal injury.

    Science.gov (United States)

    Levin, Evgeny; Andreadaki, Anastasia; Gobrecht, Philipp; Bosse, Frank; Fischer, Dietmar

    2017-04-04

    Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.

  6. Comparative temporospatial expression profiling of murine amelotin protein during amelogenesis.

    Science.gov (United States)

    Somogyi-Ganss, Eszter; Nakayama, Yohei; Iwasaki, Kengo; Nakano, Yukiko; Stolf, Daiana; McKee, Marc D; Ganss, Bernhard

    2012-01-01

    Tooth enamel is formed in a typical biomineralization process under the guidance of specific organic components. Amelotin (AMTN) is a recently identified, secreted protein that is transcribed predominantly during the maturation stage of enamel formation, but its protein expression profile throughout amelogenesis has not been described in detail. The main objective of this study was to define the spatiotemporal expression profile of AMTN during tooth development in comparison with other known enamel proteins. A peptide antibody against AMTN was raised in rabbits, affinity purified and used for immunohistochemical analyses on sagittal and transverse paraffin sections of decalcified mouse hemimandibles. The localization of AMTN was compared to that of known enamel proteins amelogenin, ameloblastin, enamelin, odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4. Three-dimensional images of AMTN localization in molars at selected ages were reconstructed from serial stained sections, and transmission electron microscopy was used for ultrastructural localization of AMTN. AMTN was detected in ameloblasts of molars in a transient fashion, declining at the time of tooth eruption. Prominent expression in maturation stage ameloblasts of the continuously erupting incisor persisted into adulthood. In contrast, amelogenin, ameloblastin and enamelin were predominantly found during the early secretory stage, while odontogenic ameloblast-associated/amyloid in Pindborg tumors and kallikrein 4 expression in maturation stage ameloblasts paralleled that of AMTN. Secreted AMTN was detected at the interface between ameloblasts and the mineralized enamel. Recombinant AMTN protein did not mediate cell attachment in vitro. These results suggest a primary role for AMTN in the late stages of enamel mineralization. Copyright © 2011 S. Karger AG, Basel.

  7. Differentially expressed genes in iron-induced prion protein conversion

    International Nuclear Information System (INIS)

    Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

    2016-01-01

    The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

  8. Monitoring the effects of toxic chemicals on protein expression

    International Nuclear Information System (INIS)

    Giometti, C.S.; Taylor, J.

    1987-01-01

    Two-dimensional gel electrophoresis coupled with computer-assisted image and data analysis was used to monitor protein populations for both qualitative and quantitative changes induced by exposure to chemicals. For mutagenesis studies designed to screen for heritable mutations, a computer-assisted search of the optical density data from 2DE patterns was used to look for (a) new protein spots, (b) missing protein spots and/or (c) altered expression of normal protein spots. Using this approach, 320 mice were screened for mutations induced by treatment of sires with 150 mg/kg body weight of ethylnitrosourea (ENU) and four different mutations were identified. Protein patterns from 105 offspring from untreated male mice (controls) and 369 offspring from irradiated male mice (3 Gy gamma) were also screened. No heritable mutations were found in those data sets, however. In addition, protein changes were observed in livers of animals exposed to the hepatocellular peroxisomal proliferation agents (and carcinogens) Wy-14,643 and DEHP. The de novo synthesis of a new protein by these agents was demonstrated and quantitated

  9. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  10. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    International Nuclear Information System (INIS)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

  11. Heat Shock Protein 90 (Hsp90 Expression and Breast Cancer

    Directory of Open Access Journals (Sweden)

    Christos A. Papadimitriou

    2012-09-01

    Full Text Available Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc. are currently under evaluation.

  12. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    Science.gov (United States)

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi

  13. Manipulating heat shock protein expression in laboratory animals.

    Science.gov (United States)

    Tolson, J Keith; Roberts, Stephen M

    2005-02-01

    Upregulation of heat shock proteins (Hsps) has been observed to impart resistance to a wide variety of physical and chemical insults. Elucidation of the role of Hsps in cellular defense processes depends, in part, on the ability to manipulate Hsp expression in laboratory animals. Simple methods of inducing whole body hyperthermia, such as warm water immersion or heating pad application, are effective in producing generalized expression of Hsps. Hsps can be upregulated locally with focused direct or indirect heating, such as with ultrasound or with laser or microwave radiation. Increased Hsp expression in response to toxic doses of xenobiotics has been commonly observed. Some pharmacologic agents are capable of altering Hsps more specifically by affecting processes involved in Hsp regulation. Gene manipulation offers the ability to selectively increase or decrease individual Hsps. Knockout mouse strains and Hsp-overexpressing transgenics have been used successfully to examine the role of specific Hsps in protection against hyperthermia, chemical insults, and ischemia-reperfusion injury. Gene therapy approaches also offer the possibility of selective alteration of Hsp expression. Some methods of increasing Hsp expression have application in specialized areas of research, such cold response, myocardial protection from exercise, and responses to stressful or traumatic stimuli. Each method of manipulating Hsp expression in laboratory animals has advantages and disadvantages, and selection of the best method depends upon the experimental objectives (e.g., the alteration in Hsp expression needed, its timing, and its location) and resources available.

  14. A new essential protein discovery method based on the integration of protein-protein interaction and gene expression data

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    Li Min

    2012-03-01

    Full Text Available Abstract Background Identification of essential proteins is always a challenging task since it requires experimental approaches that are time-consuming and laborious. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which have produced unprecedented opportunities for detecting proteins' essentialities from the network level. There have been a series of computational approaches proposed for predicting essential proteins based on network topologies. However, the network topology-based centrality measures are very sensitive to the robustness of network. Therefore, a new robust essential protein discovery method would be of great value. Results In this paper, we propose a new centrality measure, named PeC, based on the integration of protein-protein interaction and gene expression data. The performance of PeC is validated based on the protein-protein interaction network of Saccharomyces cerevisiae. The experimental results show that the predicted precision of PeC clearly exceeds that of the other fifteen previously proposed centrality measures: Degree Centrality (DC, Betweenness Centrality (BC, Closeness Centrality (CC, Subgraph Centrality (SC, Eigenvector Centrality (EC, Information Centrality (IC, Bottle Neck (BN, Density of Maximum Neighborhood Component (DMNC, Local Average Connectivity-based method (LAC, Sum of ECC (SoECC, Range-Limited Centrality (RL, L-index (LI, Leader Rank (LR, Normalized α-Centrality (NC, and Moduland-Centrality (MC. Especially, the improvement of PeC over the classic centrality measures (BC, CC, SC, EC, and BN is more than 50% when predicting no more than 500 proteins. Conclusions We demonstrate that the integration of protein-protein interaction network and gene expression data can help improve the precision of predicting essential proteins. The new centrality measure, PeC, is an effective essential protein discovery method.

  15. Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec

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    Yu-Sheng Chen

    2018-01-01

    Full Text Available Proinsulin-transferrin fusion protein (ProINS-Tf has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf. It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L., was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity.

  16. Ku70 acetylation and modulation of c-Myc/ATF4/CHOP signaling axis by SIRT1 inhibition lead to sensitization of HepG2 cells to TRAIL through induction of DR5 and down-regulation of c-FLIP

    DEFF Research Database (Denmark)

    Kim, Mi-Ju; Hong, Kyung-Soo; Kim, Hak-Bong

    2013-01-01

    In this study, we investigated the role of c-Myc/ATF4/CHOP signaling pathway in sensitization of human hepatoma HepG2 cells to TRAIL. Knockdown of SIRT1 or treatment with SIRT1 inhibitor caused the up-regulation of DR5 and down-regulation of c-FLIP through modulation of c-Myc/ATF4/CHOP pathway, a...

  17. Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system

    Science.gov (United States)

    Fujiwara, Kei; Katayama, Tsutomu; Nomura, Shin-ichiro M.

    2013-01-01

    Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds. PMID:23737447

  18. Expression of a fatty acid-binding protein in yeast

    International Nuclear Information System (INIS)

    Scholz, H.

    1991-06-01

    The unicellular eukaryotic microorganism, Saccharomyces cerevisiae, transformed with a plasmid containing a cDNA fragment encoding bovine heart fatty acid-binding protein (H-FABP C ) under the control of the inducible yeast GAL10 promoter, expressed FABP during growth on galactose. The maximum level of immunoreactive FABP, identical in size and isoelectric point to native protein, was reached after approximately 16 hours of induction. In contrast, transcription of the gene was induced within half an hour. Both, protein and mRNA were unstable and degraded within 1 h after repression of transcription. Analysis of subcellular fractions showed that FABP was exclusively associated with the cytosol. FABP expressed in yeast cells was functional as was demonstrated by its capacity to bind long chain fatty acids in an in vitro assay. Growth of all transformants on galactose as the carbon source showed no phenotype at temperatures up to 37 deg C, but the growth of FABP-expressing cells at 37 deg C was significantly retarded. Among the biochemical effects of FABP expression on lipid metabolism is a marked reduction of chain elongation and desaturation of exogenously added 14 C-palmitic acid. This effect is most pronounced in triacylglycerols and phospholipids when cells grow at 30 deg C and 37 deg C, respectively. In an in vitro assay determining the desaturation of palmitoyl CoA by microsomal membranes cytosol with or without exo- or endogenous FABP showed the same stimulation of the reaction. The desaturation of exogenously added 14 C-stearic acid, the pattern of unlabelled fatty acids (saturated vs. unsaturated) and the distribution of exogenously added radioactive fatty acids (palmitic, stearic or oleic acid) among lipid classes was not significantly affected. Using high concentrations (1 mM) the uptake of fatty acids was first stimulated and then inhibited when FABP was expressed. (author)

  19. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    Science.gov (United States)

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears. Copyright 2010 Elsevier Inc. All rights reserved.

  20. Identification of differentially expressed proteins in vitamin B 12

    Directory of Open Access Journals (Sweden)

    Swati Varshney

    2015-01-01

    Full Text Available Background: Vitamin B 12 (cobalamin is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC (Holo-TC [HoloTC] enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.

  1. Sperm protein 17 is expressed in the sperm fibrous sheath

    Directory of Open Access Journals (Sweden)

    Albani Elena

    2009-07-01

    Full Text Available Abstract Background Sperm protein 17 (Sp17 is a highly conserved mammalian protein characterized in rabbit, mouse, monkey, baboon, macaque, human testis and spermatozoa. mRNA encoding Sp17 has been detected in a range of murine and human somatic tissues. It was also recognized in two myeloma cell lines and in neoplastic cells from patients with multiple myeloma and ovarian carcinoma. These data all indicate that Sp17 is widely distributed in humans, expressed not only in germinal cells and in a variety of somatic tissues, but also in neoplastic cells of unrelated origin. Methods Sp17 expression was analyzed by immunocytochemistry and transmission electron microscopy on spermatozoa. Results Here, we demonstrate the ultrastructural localization of human Sp17 throughout the spermatozoa flagellar fibrous sheath, and its presence in spermatozoa during in vitro states from their ejaculation to the oocyte fertilization. Conclusion These findings suggest a possible role of Sp17 in regulating sperm maturation, capacitation, acrosomal reaction and interactions with the oocyte zona pellucida during the fertilization process. Further, the high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP-binding motif within this region, suggest that Sp17 might play a regulatory role in a protein kinase A-independent AKAP complex in both germinal and somatic cells.

  2. Complement inhibitory proteins expression in placentas of thrombophilic women Complement inhibitory proteins expression in placentas of thrombophilic women

    Directory of Open Access Journals (Sweden)

    Przemysław Krzysztof Wirstlein

    2012-10-01

    Full Text Available Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining
    of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.
    Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.
    The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western
    blot. We observed a higher transcript (p < 0.05 and protein (p < 0.001 levels of DAF and MCP in the placentas
    of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast
    membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of
    proteins that control activation of complement control proteins is only seemingly contradictory to the changes
    observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes
    associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is
    an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.Factors controlling complement activation appear to exert a protective effect on pregnancy. This is
    particularly important in women with thrombophilia. The aim of this study was to determine the transcript and
    protein levels of complement decay-accelerating factor (DAF and membrane cofactor protein (MCP in the
    placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry

  3. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    Science.gov (United States)

    Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  4. Unique expression of cytoskeletal proteins in human soft palate muscles.

    Science.gov (United States)

    Shah, Farhan; Berggren, Diana; Holmlund, Thorbjörn; Levring Jäghagen, Eva; Stål, Per

    2016-03-01

    The human oropharyngeal muscles have a unique anatomy with diverse and intricate functions. To investigate if this specialization is also reflected in the cytoarchitecture of muscle fibers, intermediate filament proteins and the dystrophin-associated protein complex have been analyzed in two human palate muscles, musculus uvula (UV) and musculus palatopharyngeus (PP), with immunohistochenmical and morphological techniques. Human limb muscles were used as reference. The findings show that the soft palate muscle fibers have a cytoskeletal architecture that differs from the limb muscles. While all limb muscles showed immunoreaction for a panel of antibodies directed against different domains of cytoskeletal proteins desmin and dystrophin, a subpopulation of palate muscle fibers lacked or had a faint immunoreaction for desmin (UV 11.7% and PP 9.8%) and the C-terminal of the dystrophin molecule (UV 4.2% and PP 6.4%). The vast majority of these fibers expressed slow contractile protein myosin heavy chain I. Furthermore, an unusual staining pattern was also observed in these fibers for β-dystroglycan, caveolin-3 and neuronal nitric oxide synthase nNOS, which are all membrane-linking proteins associated with the dystrophin C-terminus. While the immunoreaction for nNOS was generally weak or absent, β-dystroglycan and caveolin-3 showed a stronger immunostaining. The absence or a low expression of cytoskeletal proteins otherwise considered ubiquitous and important for integration and contraction of muscle cells indicate a unique cytoarchitecture designed to meet the intricate demands of the upper airway muscles. It can be concluded that a subgroup of muscle fibers in the human soft palate appears to have special biomechanical properties, and their unique cytoarchitecture must be taken into account while assessing function and pathology in oropharyngeal muscles. © 2015 Anatomical Society.

  5. Transcript and protein expression profile of PF11_0394, a Plasmodium falciparum protein expressed in salivary gland sporozoites

    Directory of Open Access Journals (Sweden)

    Schlarman Maggie S

    2012-03-01

    Full Text Available Abstract Background Plasmodium falciparum malaria is a significant problem around the world today, thus there is still a need for new control methods to be developed. Because the sporozoite displays dual infectivity for both the mosquito salivary glands and vertebrate host tissue, it is a good target for vaccine development. Methods The P. falciparum gene, PF11_0394, was chosen as a candidate for study due to its potential role in the invasion of host tissues. This gene, which was selected using a data mining approach from PlasmoDB, is expressed both at the transcriptional and protein levels in sporozoites and likely encodes a putative surface protein. Using reverse transcription-polymerase chain reaction (RT-PCR and green fluorescent protein (GFP-trafficking studies, a transcript and protein expression profile of PF11_0394 was determined. Results The PF11_0394 protein has orthologs in other Plasmodium species and Apicomplexans, but none outside of the group Apicomplexa. PF11_0394 transcript was found to be present during both the sporozoite and erythrocytic stages of the parasite life cycle, but no transcript was detected during axenic exoerythrocytic stages. Despite the presence of transcript throughout several life cycle stages, the PF11_0394 protein was only detected in salivary gland sporozoites. Conclusions PF11_0394 appears to be a protein uniquely detected in salivary gland sporozoites. Even though a specific function of PF11_0394 has not been determined in P. falciparum biology, it could be another candidate for a new vaccine.

  6. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    Science.gov (United States)

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Expression of livin protein in lung cancer and its relation with the expression of pro-caspase3 protein

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    Hongru LI

    2008-10-01

    Full Text Available Background and objective Livin is a novel inhibitor of apoptosis protein (IAP, recent studies showed it overexpresses in a variety of carcinomas including lung cancer and contributes much to the cancerous development. The objective of this study is to explore the expression of livin in tissues of lung cancer and its relationshipwith histological types, chemotherapy, Lymph node metastasis and to study its correlation with the expression of pro-caspase3 as well. Methods Expressions of Livin and caspase3 were detected by Western blot assay in lung cancer tissues as well as in controls. Results Livin was expressed in 15 of 27 lung cancer, significantly more than those in lung para-cancerous (1/5 or benign disease lung tissues (2/12 (P 0.05. Conclusion Livin are differently expressed in different histological types of lung cancer; High levels of livin expression do not relate to chemotherapy, lymph node metastasis (P >0.05. The levels of livin tends to be positively associated with those of accordingly pro-caspase3, it is presumed that livin could bind pro-caspase3 and suppress its activation.

  8. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    Science.gov (United States)

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  9. Intermediate filament protein nestin is expressed in developing meninges.

    Science.gov (United States)

    Yay, A; Ozdamar, S; Canoz, O; Baran, M; Tucer, B; Sonmez, M F

    2014-01-01

    Nestin is a type VI intermediate filament protein known as a marker for progenitor cells that can be mostly found in tissues during the embryonic and fetal periods. In our study, we aimed to determine the expression of nestin in meninges covering the brain tissue at different developmental stages and in the new born. In this study 10 human fetuses in different development stages between developmental weeks 9-34 and a newborn brain tissue were used. Fetuses in paraffin section were stained with H+E and nestin immunohistochemical staining protocol was performed. In this study, in the human meninges intense nestin expression was detected as early as in the 9th week of development. Intensity of this expression gradually decreased in later stages of development and nestin expression still persisted in a small population of newborn meningeal cells. In the present study, nestin positive cells gradually diminished in the developing and maturing meninges during the fetal period. This probably depends on initiation of a decrease in nestin expression and replacement with other tissue-specific intermediate filaments while the differentiation process continues. These differences can make significant contributions to the investigation and diagnosis of various pathological disorders (Tab. 1, Fig. 3, Ref. 36).

  10. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    2011-04-01

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  11. Functional modules by relating protein interaction networks and gene expression.

    Science.gov (United States)

    Tornow, Sabine; Mewes, H W

    2003-11-01

    Genes and proteins are organized on the basis of their particular mutual relations or according to their interactions in cellular and genetic networks. These include metabolic or signaling pathways and protein interaction, regulatory or co-expression networks. Integrating the information from the different types of networks may lead to the notion of a functional network and functional modules. To find these modules, we propose a new technique which is based on collective, multi-body correlations in a genetic network. We calculated the correlation strength of a group of genes (e.g. in the co-expression network) which were identified as members of a module in a different network (e.g. in the protein interaction network) and estimated the probability that this correlation strength was found by chance. Groups of genes with a significant correlation strength in different networks have a high probability that they perform the same function. Here, we propose evaluating the multi-body correlations by applying the superparamagnetic approach. We compare our method to the presently applied mean Pearson correlations and show that our method is more sensitive in revealing functional relationships.

  12. Simvastatin enhances bone morphogenetic protein receptor type II expression

    International Nuclear Information System (INIS)

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N.

    2006-01-01

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

  13. Expression and Production of SH2 Domain Proteins.

    Science.gov (United States)

    Liu, Bernard A; Ogiue-Ikeda, Mari; Machida, Kazuya

    2017-01-01

    The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

  14. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Science.gov (United States)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  15. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

  16. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  17. HER 2/neu protein expression in colorectal cancer

    International Nuclear Information System (INIS)

    Schuell, B; Gruenberger, T; Scheithauer, W; Zielinski, Ch; Wrba, F

    2006-01-01

    Conflicting data exist about the prevalence of HER-2/neu overexpression in colorectal cancer ranging from 0 to 83 %. In our study we tried to clarify the extent of expression and its relationship to clinicopathological parameters. This study involved 77 specimens of malignant colorectal cancer lesions of surgically resected patients. HER-2/neu immunohistochemistry was performed using the Hercep-Test Kit. Out of 77 specimens, 56 were Her-2/neu negative (70%), 20 (26%) showed a barely immunostaining (1+), only 1 (1%) was moderately (2+) and 2 (3%) were strongly positive (3+). Her-2/neu staining (moderately and strongly positive) was only detected in primary tumours of patients with confirmed metastases. No relationship was found between membranous HER-2 expression and patients' gender or differentiation. The median survival time of patients with positive HER-2/neu immunostaining was 21 versus 39 months in patients without HER-2/neu expression (p = 0.088). The c-erbB protein expression was observed in colorectal cancer but rarely in the therapeutic range (2+ and 3+). There was no significant association with tumour grade, gender, localization of the primary tumour or survival. These data indicate that c-erbB-2 is unlikely to play a major role in the therapeutic management of colorectal cancer

  18. Sperm protein 17 is expressed in human nervous system tumours

    International Nuclear Information System (INIS)

    Grizzi, Fabio; Baena, Riccardo Rodriguez y; Dioguardi, Nicola; Chiriva-Internati, Maurizio; Gaetani, Paolo; Franceschini, Barbara; Di Ieva, Antonio; Colombo, Piergiuseppe; Ceva-Grimaldi, Giorgia; Bollati, Angelo; Frezza, Eldo E; Cobos, E

    2006-01-01

    Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma),. A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells

  19. Sirtuin-1 regulates acinar-to-ductal metaplasia and supports cancer cell viability in pancreatic cancer.

    Science.gov (United States)

    Wauters, Elke; Sanchez-Arévalo Lobo, Victor J; Pinho, Andreia V; Mawson, Amanda; Herranz, Daniel; Wu, Jianmin; Cowley, Mark J; Colvin, Emily K; Njicop, Erna Ngwayi; Sutherland, Rob L; Liu, Tao; Serrano, Manuel; Bouwens, Luc; Real, Francisco X; Biankin, Andrew V; Rooman, Ilse

    2013-04-01

    The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD(+)-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. ©2012 AACR.

  20. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    Science.gov (United States)

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes. Copyright © 2016 Elsevier B.V. All rights reserved.