Increased SIRT1 Expression Research Articles

Accelerating cancer evolution: a dark side of SIRT1 in genome maintenance

Accelerating cancer evolution: a dark side of SIRT1 in genome maintenance

Abstract 2585: Resveratrol induces autophagy in prostate cancer cells and intervention suppresses the progression of PIN in animals

Abstract Resveratrol (3,4′-trihydroxystilbene), a natural product is present in significant concentrations in red wine, peanuts, walnuts etc. Emerging evidence indicates that resveratrol exerts antitumorigenic activity in various tumor models including prostate. Although multiple biological effects on proliferation, apoptosis and activation of SIRT1 have been demonstrated to be associated with resveratrol-induced activities, the precise mechanism associated with resveratrol-induced SIRT1 activation and cancer cell growth inhibition remains unclear. In addition, whether resveratrol intervention suppresses the development and progression of PIN (Prostatic Intraepithelial Neoplasia) is unknown. In this study, we examined the role of SIRT1 and the underlying mechanism involved in resveratrol-mediated biological effects. Our results show that although resveratrol significantly inhibited proliferation of multiple prostate cell lines including RWPE-1, C42B, PC3 and DU145 at higher concentrations (> 50 µM); however, at lower concentrations (< 25 µM), resveratrol prompted cell proliferation. Interestingly androgen responsive LNCaP cells exhibited resistance to resveratrol-mediated inhibition of cell proliferation. Under these experimental conditions, we also observed significant increase in autophagy without remarkable increase in apoptosis. Given the role of Akt/mTOR signaling pathway as a negative regulator of autophagy, we investigated whether resveratrol induce autophagy via modulation of Akt/mTOR signaling. Our data indicate that resveratrol-induced autophagy was associated with decreased levels of mTORC1 activity and increased SIRT1 expression. Knockdown of SIRT1 enhanced prostate cell proliferation and attenuated resveratrol-induced autophagy. Using prostate specific PTEN−/− mice, we found that dietary administration of resveratrol reduced the incidence of high-grade PIN lesions significantly. These findings implicated an important role for mTORC1/SIRT1 signaling axis in mediating resveratrol-induced upregulation of autophagy and inhibition of apoptosis. [Supported by NIH CA137578 (APK)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2585. doi:1538-7445.AM2012-2585

POLY(ADP‐RIBOSE) POLYMERASE‐2 DEPLETION REDUCES DOXORUBICIN‐INDUCED DAMAGE THROUGH SIRT1 INDUCTION

Doxorubicin (DOX) is used in cytostatic treatments, although it may cause cardiovascular dysfunction. DOX treatment leads to enhanced free radical production that in turn cause DNA strand breakage culminating in poly(ADP‐ribose) polymerase (PARP) activation, mitochondrial and cellular dysfunction. DNA nicks can activate the PARP‐2 enzyme. Depletion of PARP‐2 has been shown to result in a protective phenotype against free radical‐mediated diseases suggesting similar properties in the case of DOX‐induced vascular damage. PARP‐2(+/+) and (−/−) mice and aortic smooth muscle (MOVAS) cells were treated with DOX (25 mg/kg, or 3 μM, respectively). Aortae from PARP‐2(−/−) mice displayed partial protection against DOX toxicity. Depletion of PARP‐2 prevented DOX‐induced mitochondrial dysfunction through SIRT1 activation. Genetic deletion of PARP‐2 resulted in the induction of SIRT1 promoter and consequently increased SIRT1 expression both in aortae and in MOVAS cells. SIRT1 activation enhanced mitochondrial biogenesis. Our data identify PARP‐2 as a mediator of DOX toxicity by regulating vascular SIRT1 activity and mitochondrial biogenesis. Moreover, to our best knowledge this is the first report of SIRT1 as a protective factor in the vasculature upon oxidative stress. This work was supported by TÁMOP 4.2.1./B‐09/1/KONV‐2010‐0007 and 4.2.2‐08/1‐2008‐0019.

Anti-correlation between longevity gene SirT1 and Notch signaling in ascending aorta biopsies from patients with bicuspid aortic valve disease

About 1-2% of the population present with bicuspid aortic valves (BAV), a defect of the aortic valve resulting in the formation of two leaflets instead of three. This disease leads to an abnormal aorta, altered in strength and size, which in turn is a high risk factor for potentially lethal events such as aortic dissection and aneurysm formation. BAV is inheritable, with a demonstrated association with Notch1, a member of the Notch intercellular signaling pathway that is implicated in various cardiovascular disorders. Sirtuin 1 (SirT1) is a protein deacetylase of the sirtuin family, whose activation appears beneficial for cardiac diseases. A recent study has shown that SirT1 can limit Notch signaling in model systems of vascular growth. If a concomitant dysregulation in Notch and SirT1 signaling pathways can cause the phenotypic form of human BAV is unknown. To address this issue, we analyzed human ascending aorta biopsies from BAV and control patients obtained at the time of cardiac surgery. RNA and proteins were extracted from formalin-fixed and paraffin-embedded specimens, and quantitative real-time PCR and immunoblotting were used to determine the expression of sirtuins and members of the Notch family of proteins. We found a significant increase in SirT1 expression that correlates with a decreased expression of the Notch signaling effectors detected. We put forward the idea that an altered interaction between SirT1 and Notch signaling could participate in BAV pathogenesis and that these molecules could be used as potential clinical markers.

MIGF‐1/JNK1/SirT1 signaling confers protection against oxidative stress in the heart

Oxidative stress contributes to the pathogenesis of aging-associated heart failure. Among various signaling pathways mediating oxidative stress, the NAD(+) -dependent protein deacetylase SirT1 has been implicated in the protection of heart muscle. Expression of a locally acting insulin-like growth factor-1 (IGF-1) propeptide (mIGF-1) helps the heart to recover from infarct and enhances SirT1 expression in cardiomyocytes (CM) in vitro, exerting protection from hypertrophic and oxidative stresses. To study the role of mIGF-1/SirT1 signaling in vivo, we generated cardiac-specific mIGF-1 transgenic mice in which SirT1 was depleted from adult CM in a tamoxifen-inducible and conditional fashion. Analysis of these mice confirmed that mIGF-1-induced SirT1 activity is necessary to protect the heart from paraquat (PQ)-induced oxidative stress and lethality. In cultured CM, mIGF-1 increases SirT1 expression through a c-Jun NH(2)-terminal protein kinase 1 (JNK1)-dependent signaling mechanism. Thus, mIGF-1 protects the heart from oxidative stress via SirT1/JNK1 activity, suggesting new avenues for cardiac therapy during aging and heart failure.

Effects of resveratrol on HepG2 cells as revealed by 1H-NMR based metabolic profiling

Background Resveratrol, a polyphenol found in plant products, has been shown to regulate many cellular processes and to display multiple protective and therapeutic effects. Several in vitro and in vivo studies have demonstrated the influence of resveratrol on multiple intracellular targets that may regulate metabolic homeostasis. Methods We analysed the metabolic modifications induced by resveratrol treatment in a human hepatoblastoma line, HepG2 cells, using a 1H-NMR spectroscopy-based metabolomics approach that allows the simultaneous screening of multiple metabolic pathways. Results Results demonstrated that cells cultured in the presence or absence of resveratrol displayed different metabolic profiles: the treatment induced a decreased utilisation of glucose and amino acids for purposes of energy production and synthesis associated to a decreased release of lactate in the culture medium and an increase in succinate utilisation. At the same time, resveratrol treatment slowed the cell cycle in the S phase without inducing apoptosis, and increased Sirt1 expression, also affecting its intracellular localisation. Conclusions Our results show that the metabolomic analysis of the exometabolome of resveratrol-treated HepG2 cells indicates a metabolic switch from glucose and amino acid utilisation to fat utilisation for the production of energy, and seem in agreement with an effect mediated via AMPK- and Sirt1-activation. General significance NMR-based metabolomics has been applied in a hepatocyte cell culture model in relation to resveratrol treatment; such an approach could be transferred to evaluate the effects of nutritional compounds with health impact.

The Effect of Taurine on Hepatic Steatosis Induced by Thioacetamide in Zebrafish (Danio rerio)

Nonalcoholic fatty liver disease is one of the most prevalent forms of chronic liver disease in the Western world. Taurine is a conditionally essential amino acid in humans that may be a promising therapy for treating this disease. To evaluate the effect of taurine on hepatic steatosis induced by thioacetamide in Danio rerio. Animals were divided into four groups: control (20 μl of saline solution), taurine (1,000 mg/kg), thioacetamide (300 mg/kg), and the taurine-thioacetamide group (1,000 + 300 mg/kg). Thioacetamide was injected intraperitoneally three times a week for 2 weeks. The mRNA expression, lipoperoxidation, antioxidant enzymatic activity, and histological analyses were evaluated in the liver and the triglyceride content was assessed in the serum. Thioacetamide injection induced steatosis, as indicated by histological analyses. The lipoperoxidation showed significant lipid damage in the thioacetamide group compared to the taurine-thioacetamide group (p < 0.001). Superoxide dismutase (SOD) activity in the taurine-thioacetamide group (5.95 ± 0.40) was significantly increased compared to the thioacetamide group (4.14 ± 0.18 U SOD/mg of protein) (p < 0.001). The mRNA expression of SIRT1 (0.5-fold) and Adiponectin receptor 2 (0.39-fold) were lower in the thioacetamide group than the control (p < 0.05). TNF-α mRNA expression was 6.4-fold higher in the thioacetamide group than the control (p < 0.05). SIRT1 mRNA expression was 2.6-fold higher in the taurine-thioacetamide group than in the thioacetamide group. Taurine seems to improve hepatic steatosis by reducing oxidative stress and increasing SIRT1 expression.

Peptide Switch Is Essential for Sirt1 Deacetylase Activity

In mammals, the Sirtuins are composed of seven Sir2 orthologs (Sirt1-7) with a conserved deacetylase core that utilizes NAD(+) as a cofactor. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 aa sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an "on switch" for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.

Poly(ADP-ribose) polymerase-2 depletion reduces doxorubicin-induced damage through SIRT1 induction

Doxorubicin (DOX) is widely used in cytostatic treatments, although it may cause cardiovascular dysfunction as a side effect. DOX treatment leads to enhanced free radical production that in turn causes DNA strand breakage culminating in poly(ADP-ribose) polymerase (PARP) activation and mitochondrial and cellular dysfunction. DNA nicks can activate numerous enzymes, such as PARP-2. Depletion of PARP-2 has been shown to result in a protective phenotype against free radical-mediated diseases, suggesting similar properties in the case of DOX-induced vascular damage. PARP-2(+/+) and PARP-2(-/-) mice and aortic smooth muscle (MOVAS) cells were treated with DOX (25 mg/kg or 3 μM, respectively). Aortas were harvested 2-day post-treatment while MOVAS cells were treated with DOX for 7 hours. Aortas from PARP-2(-/-) mice displayed partial protection against DOX toxicity, and the protection depended on the conservation of smooth muscle but not on the conservation of endothelial function. DOX treatment evoked free radical production, DNA breakage and PARP activation. Importantly, depletion of PARP-2 did not quench any of these phenomena, suggesting an alternative mechanism. Depletion of PARP-2 prevented DOX-induced mitochondrial dysfunction through SIRT1 activation. Genetic deletion of PARP-2 resulted in the induction of the SIRT1 promoter and consequently increased SIRT1 expression both in aortas and in MOVAS cells. SIRT1 activation enhanced mitochondrial biogenesis, which provided protection against DOX-induced mitochondrial damage. Our data identify PARP-2 as a mediator of DOX toxicity by regulating vascular SIRT1 activity and mitochondrial biogenesis. Moreover, to the best of our knowledge, this is the first report of SIRT1 as a protective factor in the vasculature upon oxidative stress.

High dose of dietary resveratrol enhances insulin sensitivity in healthy rats but does not lead to metabolite concentrations effective for SIRT1 expression

trans-Resveratrol has been shown to improve insulin sensitivity and to enhance cellular glucose uptake. Evidence from recent studies indicates that these effects depend on SIRT1-pathways. Since ingestion of resveratrol leads to the presence of resveratrol and resveratrol metabolites in the body, we aimed at investigating (i) whether a daily dose of 300 mg resveratrol/kg body weight in healthy male Wistar rats for a period of 8 wk affects the selected parameters of glucose and lipid metabolism and (ii) whether the resulting plasma concentrations of resveratrol metabolites were effective in modulating SIRT1 expression. The dietary dose was based on the results from preceding toxicity studies. The results from the feeding experiment revealed plasma concentrations of resveratrol and its metabolites below 1 μmol/L and showed that fasting glucose and insulin levels were decreased by 35 and 41%, respectively, in the resveratrol group compared with controls. Insulin sensitivity was enhanced by 70%, whereas liver SIRT1 protein expression was not affected. Treatment of HepG2 cells with 10 μM resveratrol (1.49-fold) or its diglucuronides (1.21-fold) increased SIRT1 expression. These results suggest that the improved insulin sensitivity after dietary administration of 300 mg resveratrol/kg body weight does not involve increased protein expression of SIRT1.

Roles of SIRT1 in High Glucose-induced Endothelial Impairment: Association with Diabetic Atherosclerosis

Background and Aims We undertook this study to investigate the roles of SIRT1 in high glucose-induced endothelial impairment and their association with diabetic atherosclerosis. Methods Otsuka Long-Evans Tokushima Fatty (OLETF) rats and nondiabetic rats of the same genetic background were included. Real-time PCR was used to detect SIRT1 mRNA expression in abdominal aorta at week 42. To further investigate the roles of SIRT1 on the function of endothelial cells in high glucose, human endothelial cells were treated with SIRT1 activator resveratrol for 24 h before being cultured in high glucose medium for 48 h. Results Along with the early manifestation of atherosclerosis, SIRT1 mRNA level in OLETF group was significantly lower than that in control group ( p <0.05). Compared with control cells, high glucose decreased nitric oxide (NO) secretion, but resveratrol treatment increased the expression of SIRT1 and the secretion of NO. After interfering with the expression of SIRT1 using SIRT1 siRNA, the effects of resveratrol on NO secretion were impaired. SIRT1 also counteracted the other pro-atherosclerotic effects of high glucose including the upregulating roles of high glucose on the expression of E-selectin mRNA and the downregulating roles of high glucose on the expression of endothelial nitric oxide synthase. Conclusions Decreased expression of SIRT1 in artery may be involved in the initiation and development of diabetic atherosclerosis. Increasing SIRT1 expression may hold great promise in the prevention and therapy of atherosclerosis in diabetic patients.

PARP-2 Regulates SIRT1 Expression and Whole-Body Energy Expenditure

SIRT1 is a NAD(+)-dependent enzyme that affects metabolism by deacetylating key transcriptional regulators of energy expenditure. Here, we tested whether deletion of PARP-2, an alternative NAD(+)-consuming enzyme, impacts on NAD(+) bioavailability and SIRT1 activity. Our results indicate that PARP-2 deficiency increases SIRT1 activity in cultured myotubes. However, this increase was not due to changes in NAD(+) levels, but to an increase in SIRT1 expression, as PARP-2 acts as a direct negative regulator of the SIRT1 promoter. PARP-2 deletion in mice increases SIRT1 levels, promotes energy expenditure, and increases mitochondrial content. Furthermore, PARP-2(-/-) mice were protected against diet-induced obesity. Despite being insulin sensitized, PARP-2(-/-) mice were glucose intolerant due to a defective pancreatic function. Hence, while inhibition of PARP activity promotes oxidative metabolism through SIRT1 activation, the use of PARP inhibitors for metabolic purposes will require further understanding of the specific functions of different PARP family members.

Whey protein isolate protects against diet-induced obesity and fatty liver formation

We investigated the potential of a novel whey protein isolate (WPI) rich in lactoperoxidase, lactoferrin, growth factors and immunoglobulins, in the prevention and treatment of diet-induced obesity in C57Bl/6J mice. Compared with casein, WPI dose-dependently enhanced weight loss and decreased body fat content during energy restriction. WPI decreased hepatic S6 ribosomal protein phosphorylation and increased SIRT3 expression. WPI did not influence energy intake, apparent fat digestibility, or adipose tissue inflammation. Compared with casein, WPI dose-dependently prevented weight regain and protected against the development of fatty liver formation during ad libitum feeding after weight loss. WPI also modestly improved glucose tolerance. It was concluded that compared with casein, a diet based on WPI as protein source significantly improved outcome of weight loss and subsequent weight regain in high-fat-fed C57Bl/6J mice. The effects are mediated, at least partly, via inhibition of mTOR nutrient sensing pathway and activation of SIRT3 in the liver.

FoxO1 Mediates an Autofeedback Loop Regulating SIRT1 Expression

Forkhead transcription factor FoxO1 and the NAD(+)-dependent histone deacetylase SIRT1 are evolutionarily conserved regulators of the development of aging, oxidative stress resistance, insulin resistance, and metabolism in species ranging from invertebrates to mammals. SIRT1 deacetylates FoxO1 and enables activation of FoxO1 transcription in multiple systems. The functional consequences of the interactions between FoxO1 and SIRT1 remain incompletely understood. Here, we demonstrate that the 1.5-kb rat sirt1 promoter region contains a cluster of five putative FoxO1 core binding repeat motifs (5×IRS-1) and a forkhead-like consensus binding site (FKHD-L). Luciferase promoter assays demonstrate that FoxO1 directly activates SIRT1 promoter activity and that both the IRS-1 and FKHD-L enable FoxO1-dependent SIRT1 transcription. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that FoxO1 binds to the IRS-1 and FKHD-L sites of the SIRT1 promoter. Consistently, FoxO1 overexpression increases SIRT1 expression, and FoxO1 depletion by siRNA reduces SIRT1 expression at both the messenger RNA and protein levels in vascular smooth muscle cells and HEK293 cells. Thus, endogenous FoxO1 is a positive transcriptional regulator of SIRT1. Conversely, SIRT1 promotes FoxO1-driven SIRT1 autotranscription through interacting with and deacetylating FoxO1. Moreover, resveratrol, a plant polyphenol activator of SIRT1, increases FoxO1-dependent SIRT1 transcription activity and thus induces its expression. These findings suggest that positive feedback mechanisms regulate FoxO1-dependent SIRT1 transcription and indicate a previously unappreciated function for FoxO1. This signaling network may coordinate multiple pathways acting upon immune, inflammatory, regenerative, and metabolic processes.

Induction of Sirt1 by Mechanical Stretch of Skeletal Muscle through the Early Response Factor EGR1 Triggers an Antioxidative Response

Mechanical loading of muscles by intrinsic muscle activity or passive stretch leads to an increase in the production of reactive oxygen species. The NAD-dependent protein deacetylase SIRT1 is involved in the protection against oxidative stress by enhancing FOXO-driven Sod2 transcription. In this report, we unravel a mechanism triggered by mechanical stretch of skeletal muscle cells that leads to an EGR1-dependent transcriptional activation of the Sirt1 gene. The resulting transient increase in SIRT1 expression generates an antioxidative response that contributes to reactive oxygen species scavenging.

Moderate Caloric Restriction Suppresses microRNAs miR-696 and miR-199a in a Voluntary Running Wheel Model

The transcriptional coactivator PGC-1α is central to skeletal muscle adaptation to increased physical activity. SIRT1, an energy sensing deacetylase, and important regulator of transcription, plays a role in the benefits of caloric restriction. SIRT1 is also a regulator of PGC-1αin skeletal muscle. MicroRNAs (miRNAs) are short noncoding RNA molecules involved in post-transcriptional regulation of gene expression by binding to target mRNA. Expression levels of miRNAs can be altered by physiological stimuli. miR-696 and miR-199a are reported to target PGC-1αand SIRT1, respectively. Neither expression of miR-696 nor miR-199a has been studied in the adaptation of skeletal muscle to physical activity and moderate caloric restriction (CR). PURPOSE: We tested the hypothesis that moderate CR in physically active mice would suppress expression of miR-696 and miR-199a. METHODS: Male C57BL/6J mice (9 wks old) were individually housed and assigned to one of two groups: ad libitum feeding (AL, n=7) or moderate CR (n=6). The AL mice had free access to food and a running wheel for 23 days. The moderate CR mice had free access to a running wheel for 23 days and free access to food for the initial 8 days, and then moderate CR for 15 days. The moderate CR protocol was implemented to achieve a 20% reduction in body mass within 3 days. Body mass reduction was then maintained for the remainder of the experiment. Running distance was measured daily. Total RNA was isolated from the plantaris (PLA). Total RNA and microRNA were reverse transcribed in separate reactions to measure expression levels of COXI, PGC-1α, and SIRT1, or miR-696, miR-199a. The relative copy number of mitochondrial (mt) DNA was measured in total DNA (nuclear and mitochondrial) extracted from PLA. RESULTS: The moderate CR group required a 26% decrease in food intake to achieve the 20% decrease in body mass. During the 15 days of moderate CR mice ran 10.5 km/day while the AL mice ran 6.9 km/day (p<0.05). mtDNA copy number and COXI expression were significantly (p<0.05) greater, 27% and 64% respectively, in moderate CR mice, while PGC-1αexpression was not affected (p>0.05). However, miR-696 expression was suppressed (70%, p<0.05) in moderate CR mice. Moderate CR resulted in increased SIRT1 expression (66%, p<0.05) and conversely, decreased miR-199a expression (58%, p<0.05). CONCLUSION: Compared to wheel running alone, moderate CR significantly increased running wheel distance, COX1 and SIRT1 mRNA expression, mtDNA copy number, and suppresses microRNAs miR-696 and miR-199a in physically active mice. These data suggest that moderate CR may regulate mitochondrial biogenesis through post-transcriptional mechanisms.

SIRT1 Regulates UV-Induced DNA Repair through Deacetylating XPA

SIRT1, a NAD(+)-dependent histone deacetylase, plays crucial roles in multiple biological processes including gene transcription, cellular metabolism, stress response, and tumorigenesis. Xeroderma pigmentosum group A (XPA) is a core nucleotide excision repair (NER) factor essential for NER process. Here we show that SIRT1 plays an important role in the regulation of NER pathway. Downregulation of SIRT1 significantly sensitizes cells to UV irradiation. SIRT1 interacts with XPA, and the interaction is enhanced after UV irradiation. XPA can be acetylated at lysines 63 and 67. SIRT1 deacetylates XPA both in vitro and in cells. Importantly, SIRT1-mediated deacetylation of XPA is required for optimal NER pathway since XPA-deficient cells complemented with XPA-K6367Q, which mimics hyperacetylated XPA, display significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Furthermore, SIRT1-mediated XPA deacetylation enhances its interaction with RPA32. Our results demonstrate that SIRT1 regulates NER pathway through modulation of XPA acetylation status.

Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney

Mitochondrial oxidative damage is a basic mechanism of aging, and multiple studies demonstrate that this process is attenuated by calorie restriction (CR). However, the molecular mechanism that underlies the beneficial effect of CR on mitochondrial dysfunction is unclear. Here, we investigated in mice the mechanisms underlying CR-mediated protection against hypoxia in aged kidney, with a special focus on the role of the NAD-dependent deacetylase sirtuin 1 (Sirt1), which is linked to CR-related longevity in model organisms, on mitochondrial autophagy. Adult-onset and long-term CR in mice promoted increased Sirt1 expression in aged kidney and attenuated hypoxia-associated mitochondrial and renal damage by enhancing BCL2/adenovirus E1B 19-kDa interacting protein 3-dependent (Bnip3-dependent) autophagy. Culture of primary renal proximal tubular cells (PTCs) in serum from CR mice promoted Sirt1-mediated forkhead box O3 (Foxo3) deacetylation. This activity was essential for expression of Bnip3 and p27Kip1 and for subsequent autophagy and cell survival of PTCs under hypoxia. Furthermore, the kidneys of aged Sirt1+/- mice were resistant to CR-mediated improvement in the accumulation of damaged mitochondria under hypoxia. These data highlight the role of the Sirt1-Foxo3 axis in cellular adaptation to hypoxia, delineate a molecular mechanism of the CR-mediated antiaging effect, and could potentially direct the design of new therapies for age- and hypoxia-related tissue damage.

Acute exercise reverses aged-induced impairments in insulin signaling in rodent skeletal muscle

The insulin resistance associated with aging is improved by exercise, but the molecular mechanisms of this improvement are not fully understood. We investigated whether the improvement in insulin action, associated with acute exercise in old rats is dependent on the modulation of pIRS-1Ser307, JNK, IkBα and PTP-1B. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were killed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the reduction in glucose disappearance rate (Kitt), observed in aged rats, was restored at 16 h after exercise. Aging led to an increase in Ser307 phosphorylation of IRS-1, and this was reversed by exercise in the skeletal muscle, in parallel with a reduction in pJNK and IkBα degradation. Moreover, aging induced an increase in the expression of PTP-1B and attenuated insulin signaling in the muscle of rats, a phenomenon that was reversed by exercise. Interestingly, the decrease in PTP-1B expression in the muscle of exercised old rats was accompanied by an increase in SIRT1 expression. These results provide new insights into the mechanisms by which exercise restores insulin sensitivity during aging.

C/EBPα regulates SIRT1 expression during adipogenesis

SIRT1 plays an important role in adipogenesis, but how SIRT1 is regulated in adipogenesis is largely unknown. In this study, we show that both SIRT1 protein and mRNA levels were increased along with CCAAT/enhancer-binding protein alpha (C/EBPalpha) during adipocyte differentiation. C/EBPalpha, but not C/EBPalphap30, activated SIRT1 promoter in both HeLa cells and 3T3-L1 preadipocytes. Furthermore, C/EBPalpha upregulated SIRT1 mRNA and protein levels in HeLa cells and increased SIRT1 expression in a p53-independent manner in Soas2 cells. In preadipocytes, ectopic expression of C/EBPalpha upregulated SIRT1 protein level and knockdown of C/EBPalpha led to the decrease of SIRT1 protein level. Moreover, by promoter deletion analysis, gel shift assay and chromatin immunoprecipitation, we found that C/EBPalpha bound to the SIRT1 promoter at a consensus C/EBPalpha binding site. These data demonstrate that C/EBPalpha regulates SIRT1 expression during adipogenesis by directly binding to the SIRT1 promoter.