Changes In Expression Of SIRT1 Research Articles

Silencing SIRT1 promotes the anti-HBV action of IFN-α by regulating Pol expression and activating the JAK-STAT signaling pathway

PurposeThe purpose this study is to investigate the impact of SIRT1 on the anti-HBV activity of IFN-α and further elucidate its underlying mechanism. MethodsHepG2.2.15 cells stably transfected with HBV virus were chosen as the primary study subject. IFN-α was used to stimulate the cells and regulate the expression of SIRT1, and the JAK-STAT pathway and HBV-related indices were measured by qRT-PCR, Western blotting and ELISA. Immunofluorescence (IF) was used to detect the nuclear translocation of STAT1 and STAT2. Coimmunoprecipitation (Co-IP) was used to detect the binding of SIRT1 to HBV Polymerase (Pol). ResultsIn HepG2.2.15 cells, we found changes in SIRT1 expression. We show that silencing SIRT1 promotes the IFN-α-triggered Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway and consequently enhances the antiviral effects of IFN-α against HBV replication. Importantly, SIRT1 can interact with Pol and increase JAK-STAT activity by regulating Pol expression. Additionally, the inhibition of SIRT1 activity by treatment with the SIRT1 inhibitor selisistat enhanced the anti-HBV effect of IFN-α and JAK-STAT pathway activity. ConclusionIn conclusion, our results demonstrate that silencing SIRT1 activates the JAK-STAT pathway and enhances the anti-HBV activity of IFN-α by inhibiting Pol expression. This would be a promising therapeutic target to improve the efficacy of IFN-α in the treatment of CHB.

SIRT1 Protects Against Particulate Matter-Induced Oxidative Stress in Human Corneal and Conjunctival Epithelial Cells.

PurposeSirtuin1 (SIRT1) as a hot therapeutic target for oxidative stress–associated diseases that has been extensively studied. This study aimed to determine the changes in SIRT1 expression in particulate matter (PM)–induced corneal and conjunctival epithelial cell damage and explore potential drugs to reduce PM-associated ocular surface injury.MethodsImmortalized human corneal epithelial cells (HCECs) and human conjunctival epithelial cells (HCjECs) were exposed to an ambient PM sample. Cytotoxicity was evaluated by water-soluble tetrazolium salt–8 assay. SIRT1 expression was measured by Western blot analysis. Reactive oxygen species (ROS) production, cell apoptosis, mitochondrial function, and cell senescence were assessed by using 2ʹ,7ʹ-dichlorofluorescein diacetate assay, annexin V apoptosis assay, tetramethylrhodamine ethyl ester assay, and senescence β-galactosidase staining, respectively.ResultsPM-induced cytotoxicity of HCECs and HCjECs occurred in a dose-dependent manner. Increased ROS production, as well as decreased SIRT1 expression, were observed in HCECs and HCjECs after 200 µg/mL PM exposure. In addition, PM induced oxidative stress-mediated cellular damage, including cell apoptosis, mitochondrial damage, and cell senescence. Interestingly, SRT1720, a SIRT1 activator, increased SIRT1 expression and decreased ROS production and attenuated PM-induced cell damage in HCECs and HCjECs.ConclusionsThis study determined that SIRT1 was involved in PM-induced oxidative stress in HCECs and HCjECs and found that ROS overproduction may a key factor in PM-induced SIRT1 downregulation. The SIRT1 activator, SRT1720, can effectively upregulate SIRT1 expression and inhibit ROS production, thereby reversing PM-induced cell damage. This study provides a new potential target for clinical treatment of PM-associated ocular surface diseases.

Chronic Exercise Mitigates the Effects of Sirtuin Inhibition by Salermide on Endothelium-Dependent Vasodilation.

Sirtuins are regulators of eNOS and endothelial function; however, no studies have examined the influence of exercise on sirtuin regulation of endothelial function. Effects of the novel sirtuin inhibitor, salermide, on vascular reactivity in rat aortas were investigated following exercise training of different durations. Male Wistar rats (8-9months old) were divided into four groups (n = 10-12/group): sedentary (SED), 1 day (1D), 2 weeks (2WK), or 6weeks (6WK) of exercise. Exercise consisted of running on a motor-driven treadmill at 15m/min, 15% grade, for 40min (1D) increased up to 1h at the end of 2weeks (2WK) and sustained for an additional 4weeks (6WK). Dose responses to phenylephrine, sodium nitroprusside, and acetylcholine in the presence or absence of salermide (30µM) were analyzed. SIRT1 and eNOS protein expression as well as nitrotyrosine levels were determined by immunoblotting. Superoxide dismutase activity was determined by colorimetric assay. Sirtuin inhibition significantly impaired acetylcholine-induced vasorelaxtion in aortas in SED, 1D, and 2WK endurance trained rats but not in 6WK. eNOS expression significantly increased ~ 2.0-fold in 1D, 2WK, and 6WK groups. SIRT1 expression and 3-nitrotyrosine levels were significantly increased in 1D and 2WK but were not significantly elevated in 6WK. SOD levels were significantly elevated in 6WK. These data suggest that chronic endurance training diminishes the role of sirtuins in regulating endothelium-dependent relaxation and appears to be related to changes in SIRT1 expression as well as redox status.

Functional Link Between Heat Shock Protein HSP90alpha and Sirtuin 1 (SIRT1) in the Pathogenesis of Diffuse Large B Cell Lymphoma

Diffuse large B-cell lymphomas (DLBCL) are heterogenous tumors. A large subset of DLBCL is reliant on B-cell receptor activity ('BCR'-DLBCLs). Additional subset of tumors that does not exhibit addiction to BCR signaling features transcriptional program is associated with increased oxidative phosphorylation ('OxPhos'-DLBCLs, Blood 2005; 105: 1851-61). These OxPhos tumros are resistant to BCR inhibition and remain functionally poorly defined. We and others found that OxPhos-DLBCLs, compared to BCR subtype, exhibit overexpression of heat shock protein HSP90alpha (Br J Haematol 2009; 144: 358-66). Expression of multiple heat shock proteins is regulated by the activity of a stress-responsive, acetylation-dependent transcription factor HSF1. Acetylation blocks, whereas deacetylation by sirtuins increases HSF1 activity. However, molecular mechanisms responsible for increased HSP90alpha expression in OxPhos-DLBCLs remain unknown.We investigated the abundance of NAD-dependent deacetylase SIRT1 in a panel of DLBCL cell lines and found that SIRT1 protein level was higher in OxPhos- than BCR-DLBCLs and correlated positively with HSP90alpha gene expression (p=0.02, r=0.7857). To assess the role of SIRT1 in transcriptional regulation of HSP90alpha, we used a small molecule SIRT1 inhibitor tenovin-6 or interfering RNA, which dramatically reduced HSP90alpha induction after heat shock. We next characterized the mechanisms of SIRT1 overexpression in OxPhos cells. SIRT1 transcript abundance was similar in OxPhos and BCR cells, suggesting that changes in SIRT1 expression result from different protein half-life/stability. To test this assumption, OxPhos and BCR cells were incubated with a protein synthesis inhibitor, cycloheximide, and assessed for SIRT1 protein decays over time. Our experiments revealed increased stability of SIRT1 protein in OxPhos-DLBCLs, compared to BCR-DLBCLs. Addition of an HSP90 inhibitor 17-AAG accelerated SIRT1 protein degradation, suggesting that HSP90 might chaperone and thus protect SIRT1 against degradation. We further found that SIRT1 coimmunoprecipitated with HSP90alpha, confirming interaction between these proteins and highlighting a self-reinforcing feedback loop linking HSP90alpha and SIRT1 in OxPhos-DLBCLs.We next assessed the therapeutic potential of SIRT1 inhibition in DLBCLs. SIRT1 knockdown with small interfering RNA or chemical SIRT1 inhibition (tenovin-6) decreased proliferation of OxPhos-DLBCL cells, but also of BCR-DLBCLs, indicating that SIRT1 inhibition exhibits broader, subtype-independent activity in DLBCL cell lines. Surprisingly, BCR-type DLBCLs were significantly more sensitive to SIRT1 inhibitor, tenovin-6. We thus sought to identify a mechanism responsible for SIRT1 inhibition in BCR-DLBCLs. Since SIRT1 deacetylase modulates the transcriptional activity of BCL6 oncogene, critical for survival of BCR-type DLBCLs (PNAS 2007; 104: 3207-12), we assessed the expression of BCL6-regulated genes in these cells. SIRT1 inhibition upregulated the abundance of multiple BCL6-dependent transcripts (including FCER2, CCL3, BLIMP1 and CD74), suggesting that toxicity of SIRT1 inhibition in BCL6-dependent DLBCLs is, at least partially, mediated by decreased BCL6 repressor activity.Although OxPhos-DLBCLs were less sensitive to tenovin-6 than BCR-type DLBCLs, we hypothesized that, given the feedback loop between SIRT1 and HSP90, simultaneous inhibition of HSP90 would increase the cellular sensitivity to tenovin-6. Consistent with this, we found that combination of these two compounds exhibited synergistic activity in OxPhos-DLBCL. Taken together, our data demonstrate that SIRT1 and HSP90alpha are mutually linked and involved in the pathogenesis of OxPhos-DLBCL. Targeting SIRT1-HSP90alpha loop with combinatorial use of SIRT1 and HSP90 inhibitors might be a promising treatment strategy in OxPhos-DLBCLs. DisclosuresWarzocha:BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

A low-salt diet increases the expression of renal sirtuin 1 through activation of the ghrelin receptor in rats.

Previous studies have shown that sirtuin 1 (Sirt1) is renoprotective; however, details regarding its distribution and functions in the kidney remain unknown. Here, we demonstrated that Sirt1 was mainly expressed in the tubulointerstitial cells of normal rat kidneys and was co-localized with aquaporin 2, indicating it may be involved in water/salt regulation. Renal Sirt1 expression increased in the non-glomerular cytoplasmic portion of the kidney after a 24-h fast, but no significant changes in Sirt1 expression occurred after water loading (50 mL/kg) or 24-h water deprivation. After consuming a low-salt (0.075%) or 60% calorie restriction diet for 7 days, Sirt1 expression in the rat kidney was significantly increased, whereas a high-salt (8%) diet did not change the level of Sirt1 expression. The low-salt diet also increased Sirt1 expression in the heart, muscle, brain, and fat tissues. The increased Sirt1 that was observed in rats on a low-salt diet was associated with increased ghrelin expression in the distal nephron, with both molecules exhibiting similar distribution patterns. An in vitro experiment suggested that ghrelin increases Sirt1 expression in cortical collecting duct cells by activating ghrelin receptors. Our study indicates that this ‘ghrelin-Sirt1 system’ may participate in regulating sodium reabsorption in the distal nephron.

Overexpression of SIRT1 in rat skeletal muscle does not alter glucose induced insulin resistance.

SIRT1 is a NAD+-dependent deacetylase thought to regulate cellular metabolic pathways in response to alterations in nutrient flux. In the current study we investigated whether acute changes in SIRT1 expression affect markers of muscle mitochondrial content and also determined whether SIRT1 influenced muscle insulin resistance induced by acute glucose oversupply. In male Wistar rats either SIRT1 or a deacetylase inactive mutant form (H363Y) was electroprated into the tibialis cranialis (TC) muscle. The other leg was electroporated with an empty control vector. One week later, glucose was infused and hyperglycaemia was maintained at ~11mM. After 5 hours, 11mM glucose induced significant insulin resistance in skeletal muscle. Interestingly, overexpression of either SIRT1 or SIRT1 (H363Y) for 1 week did not change markers of mitochondrial content or function. SIRT1 or SIRT1 (H363Y) overexpression had no effect on the reduction in glucose uptake and glycogen synthesis in muscle in response to hyperglycemia. Therefore we conclude that acute increases in SIRT1 protein have little impact on mitochondrial content and that overexpressing SIRT1 does not prevent the development of insulin resistance during hyperglycaemia.

A Redox-resistant Sirtuin-1 Mutant Protects against Hepatic Metabolic and Oxidant Stress

Sirtuin-1 (SirT1), a member of the NAD(+)-dependent class III histone deacetylase family, is inactivated in vitro by oxidation of critical cysteine thiols. In a model of metabolic syndrome, SirT1 activation attenuated apoptosis of hepatocytes and improved liver function including lipid metabolism. We show in SirT1-overexpressing HepG2 cells that oxidants (nitrosocysteine and hydrogen peroxide) or metabolic stress (high palmitate and high glucose) inactivated SirT1 by reversible oxidative post-translational modifications (OPTMs) on three cysteines. Mutating these oxidation-sensitive cysteines to serine preserved SirT1 activity and abolished reversible OPTMs. Overexpressed mutant SirT1 maintained deacetylase activity and attenuated proapoptotic signaling, whereas overexpressed wild type SirT1 was less protective in metabolically or oxidant-stressed cells. To prove that OPTMs of SirT1 are glutathione (GSH) adducts, glutaredoxin-1 was overexpressed to remove this modification. Glutaredoxin-1 overexpression maintained endogenous SirT1 activity and prevented proapoptotic signaling in metabolically stressed HepG2 cells. The in vivo significance of oxidative inactivation of SirT1 was investigated in livers of high fat diet-fed C57/B6J mice. SirT1 deacetylase activity was decreased in the absence of changes in SirT1 expression and associated with a marked increase in OPTMs. These results indicate that glutathione adducts on specific SirT1 thiols may be responsible for dysfunctional SirT1 associated with liver disease in metabolic syndrome.

Metabolic energy sensors (AMPK and SIRT1), protein carbonylation, and cardiac failure as biomarkers of thermal stress in an intertidal limpet: linking energetic allocation with environmental temperature during aerial emersion

The effects of heat stress on organisms are manifested at the levels of organ function, metabolic activity, protein stability and gene expression. Here, we examined effects of high temperature on the intertidal limpet Cellana toreuma to determine how the temperatures at which (1) organ failure (cardiac function), (2) irreversible protein damage (carbonylation) and (3) expression of genes encoding proteins involved in molecular chaperoning (hsp70 and hsp90) and metabolic regulation (ampk and sirt1) occur compare with field temperatures, which commonly exceed 30°C and can reach 46°C. Heart failure, indexed by the Arrhenius break temperature, occurred at 34.3°C. Protein carbonylation rose significantly at 38°C. Genes for heat shock proteins HSP70 (hsp70) and HSP90 (hsp90), for two subunits of AMP-activated protein kinase (AMPK) (ampkα and ampkβ) and for histone/protein deacetylase SIRT1 (sirt1) all showed increased expression at 30°C. Temperatures of maximal expression differed among genes, as did temperatures at which upregulation ceased. Expression patterns for ampk and sirt1 indicate that heat stress influenced cellular energy homeostasis; above ~30°C, upregulation of ATP-generating pathways is suggested by elevated expression of genes for ampk; an altered balance between reliance on carbohydrate and lipid fuels is indicated by changes in expression of sirt1. These results show that C. toreuma commonly experiences temperatures that induce expression of genes associated with the stress response (hsp70 and hsp90) and regulation of energy metabolism (ampk and sirt1). At high temperatures, there is likely to be a shift away from anabolic processes such as growth to catabolic processes, to provide energy for coping with stress-induced damage, notably to proteins.

Effects of Exercise Training Duration on SIRT1 and eNOS Expression in Rat Aortas

Previous studies demonstrate that exercise training improves endothelial function and provides protection against numerous cardiovascular diseases. SIRT1, a NAD‐dependent deacetylase, has recently been shown to increase eNOS activity and alter endothelium dependent relaxation. The purpose of this study is to examine SIRT1 and eNOS expression in rat aortas during the course of an exercise training program. 8–9 month‐old, male, Wistar rats were divided into four groups Sedentary (SED, n= 11), 1 day (1DY, n=12), 2 week (2WK, n=10), 6 week (6WK, n=8). Animals ran on a motor‐driven treadmill at 15 m/min, 15% grade for 40 min (1D) or increased up to 1 h at the end of 2 weeks (2WK) and sustained for an additional 4 weeks (6WK). Aorta SIRT1, eNOS, and GAPDH expression were evaluated by Western blot. Although there was a trend towards exercise‐induced increase in SIRT1 and eNOS expression, the results of this study did not indicate any significant differences in SIRT1 and eNOS expression at the various time points. Although previous studies have shown that vascular reactivity is altered during the course of an exercise training program including altered responses to SIRT1 inhibition, these data suggest that neither changes in SIRT1 expression or eNOS expression appear to play a role. Therefore, other mechanisms are likely involved in the exercise‐induced changes in vascular reactivity and altered responses to SIRT1 inhibition.

Changes in SIRT1 Expression and Its Downstream Pathways in Age-Related Cataract in Humans

Purpose: SIRT1, the most well-known sirtuin family (class III histone deacetylases) member, is involved in many age-related diseases. However, no study has demonstrated its relationship with age-related cataract (ARC). This study was to investigate the expression of SIRT1 in human lens epithelial cells, and to observe the changes in SIRT1 expression and its downstream P53 and forkhead box class O (FOXO) proteins at the onset of ARC.Methods: The anterior lens capsule specimens from 360 normal human donor eyes (age 19–91 years) were divided into three groups: group A of young lens (younger than age 49 without cataract), group B of old but normal lens (older than age 50 without cataract), and group C of ARC lens (older than age 50 with ARC). Real-time quantitative reverse transcription of SIRT1 mRNA; Western blot analysis by anti-SIRT1, anti-p53, anti-p53 (acetyl K379), anti-FOXO3a, anti-FOXO4, anti-p27kip1, anti-p130, and anti-Bim; immunofluorescence of SIRT1; and TUNEL assay were performed in each group.Results: SIRT1 expression was significantly decreased in group B compared with group A, but increased in group C compared with group B. P53 expression increased with age and topped in group C; however, there was a decrease in active acetyl-P53 expression in group C compared with group B. The expression of both FOXO3a and FOXO4 decreased with age, but in group C, their expression level is equivalent to group A. Accordingly, the downstream p27kip1 and p130 showed the similar changes among three groups. In contrast, the expression of Bim was lowest in the ARC lens, and TUNEL assay showed significantly increased apoptosis incidence in group C.Conclusions: The expression of SIRT1 increased in ARC in humans. Its downstream p53 was inhibited, and FOXO pathway was activated, indicating that SIRT1 may play a protective role in ARC formation.

Myocardial SIRT1 expression following endurance and resistance exercise training in young and old rats

Previous studies indicate a link between caloric restriction, SIRT1 expression, and longevity. It has been suggested that the metabolic stress of exercise may also alter SIRT1 expression. Therefore, we evaluated the effects of two different modes of exercise training on myocardial SIRT1 protein expression in young and old rats. In the first study, young (8 mo) and old (24 mo) Fisher 344 rats were subjected to a progressive treadmill training program for 10 wks ending at 60 min/day, 15 m/min, 0% grade. Myocardial SIRT1 expression was not significantly different (P>0.05) between young and old rats. However, endurance training resulted in a significant (P<0.05) decrease in myocardial SIRT1 protein expression. In the second study, young (5 mo) and old (22 mo) Fisher 344 rats were resistance trained by ladder climbing for 8 wks. Animals began by lifting 50% body weight and ended the training protocol lifting 276±6% body weight (young) and 141±12% body weight (old). Again, we observed no effect of age on myocardial SIRT1 expression. However, in contrast to endurance training, resistance training resulted in no significant (P>0.05) change in SIRT1 expression. The results of this study demonstrate that exercise‐induced changes in myocardial SIRT1 expression are dependent on the mode of training.Supported by Borgenicht Program for Aging Studies and Exercise Science (MBH), AHA SDG 0430157N (MBH), and the NIA (MRD).