Finding a target for resveratrol

Despite resveratrol's well-documented health benefits, its mechanism of action remains controversial. In particular, the direct molecular target of resveratrol has been elusive. Park et?al. now show that resveratrol directly inhibits cAMP-dependent phosphodiesterases, triggering a cascade of events that converge on the important energy-sensing metabolic regulators AMPK, SIRT1, and PGC-1α.     

The controversial links among calorie restriction, SIRT1, and resveratrol

It has been widely known that slow metabolism induced by calorie restriction (CR) can extend the life span of model organisms though the underlying mechanism remains poorly understood. Accumulated evidence suggests that SIRT1 may be actively involved in CR-induced signaling pathways. As a putative activator of SIRT1, resveratrol, known for the French paradox, can partially mimic the physiological effects of CR. While the deacetylase activity of SIRT1 is important for the beneficial effects of resveratrol, resveratrol-induced SIRT1 activation has recently been challenged by the observations that resveratrol could not induce SIRT1-mediated deacetylation of native substrates in vitro. To resolve the discrepancy of resveratrol-induced activation of SIRT1 deacetylase activity between the in vitro and in vivo assays, a model of indirect SIRT1 activation by resveratrol is proposed. In this review, we will discuss the emerging roles of SIRT1 and resveratrol in CR and focus on debate over the links between SIRT1 and resveratrol.     

SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function

Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD(+) levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.     

Resveratrol prevents dexamethasone-induced expression of the muscle atrophy-related ubiquitin ligases atrogin-1 and MuRF1 in cultured myotubes through a SIRT1-dependent mechanism

Resveratrol (3,5,4'-trihydroxystilbene) has been ascribed multiple beneficial biological effects but the influence of resveratrol on glucocorticoid-induced muscle atrophy is not known. We examined the effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression, FOXO1 acetylation, protein degradation and atrophy in cultured L6 myotubes. In addition, the role of the deacetylase SIRT1 in the effects of resveratrol was determined by transfecting myotubes with SIRT1 siRNA. The catabolic effects of dexamethasone were prevented by resveratrol and the protective effects of resveratrol on dexamethasone-induced atrogin-1 and MuRF1 expression were abolished in myotubes transfected with SIRT1 siRNA. Results suggest that resveratrol can prevent glucocorticoid-induced muscle wasting and that this effect is at least in part SIRT1-dependent.     

Resveratrol Inhibits Proliferation and Promotes Apoptosis of Neuroblastoma Cells: Role of Sirtuin 1

Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-β. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.     

白藜芦醇通过促进SIRT1表达，抑制NF-κB和TNF-α表达抵抗牛磺胆酸诱导的胎盘合体滋养细胞HTR-8的炎症损伤

摘要 已有研究证明,沉默信息调节子1(silent information regulator , SIRT1）和核因子κB(nuclear factor-κB, NF-κB）参与多种炎性反应调节;SIRT1激活剂——白藜芦醇（resveratrol）可通过依赖或不依赖于SIRT1的途径抑制炎症反应。然而,SIRT1及白藜芦醇在妊娠期肝内胆汁瘀积症（intrahepatic cholestasis of pregnancy, ICP）炎性反应中的作用在国内外尚未见报道。本研究证明,白藜芦醇可通过上调SIRT1表达,下调NF-κB及肿瘤坏死因子α（tumor necrosis factor-alpha, TNF-α）表达保护牛磺胆酸 （taurocholic acid, TCA）引起的胎盘合体滋养细胞HTR-8炎性损伤。免疫组化及Western印迹显示,SIRT1蛋白在正常胎盘组织的表达水平明显高于妊娠期ICP胎盘组织,而NF-κB蛋白表达在正常胎盘组织的表达低于ICP胎盘组织。Western印迹揭示,SIRT1蛋白在HTR-8细胞中的表达水平随暴露的TCA浓度增高而降低;相反,NF-κB和TNF-α蛋白质水平随TCA浓度增高而升高。采用白藜芦醇处理HTR-8细胞,该差异可被逆转;且白藜芦醇这一作用可被Ex-527（一种SIRT1 抑制剂）阻断。上述结果证明,在ICP胎盘合体滋养细胞炎性反应中SIRT1表达下调,而NF-κB表达上调;其可能的机制是ICP高浓度胆汁酸干扰胎盘合体滋养细胞SIRT1-NF-κB通路,诱导炎症反应。上述结果还提示,SIRT1激动剂——白藜芦醇可通过诱导SIRT1表达,抑制NF-κB及TNF-α表达,保护牛磺胆酸诱导的胎盘合体滋养细胞的炎性损伤。本研究为白藜芦醇临床治疗（妊娠期）肝内胆汁瘀积症提供了新的证据。至于白藜芦醇是否可直接抑制NF-κB的表达还有待进一步探讨。     

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

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.     

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

Mechanism of human SIRT1 activation by resveratrol

The NAD+-dependent protein deacetylase family, Sir2 (or sirtuins), is important for many cellular processes including gene silencing, regulation of p53, fatty acid metabolism, cell cycle regulation, and life span extension. Resveratrol, a polyphenol found in wines and thought to harbor major health benefits, was reported to be an activator of Sir2 enzymes in vivo and in vitro. In addition, resveratrol was shown to increase life span in three model organisms through a Sir2-dependent pathway. Here, we investigated the molecular basis for Sir2 activation by resveratrol. Among the three enzymes tested (yeast Sir2, human SIRT1, and human SIRT2), only SIRT1 exhibited significant enzyme activation ( approximately 8-fold) using the commercially available Fluor de Lys kit (BioMol). To examine the requirements for resveratrol activation of SIRT1, we synthesized three p53 acetylpeptide substrates either lacking a fluorophore or containing a 7-amino-4-methylcoumarin (p53-AMC) or rhodamine 110 (p53-R110). Although SIRT1 activation was independent of the acetylpeptide sequence, resveratrol activation was completely dependent on the presence of a covalently attached fluorophore. Substrate competition studies indicated that the fluorophore decreased the binding affinity of the peptide, and, in the presence of resveratrol, fluorophore-containing substrates bound more tightly to SIRT1. Using available crystal structures, a model of SIRT1 bound to p53-AMC peptide was constructed. Without resveratrol, the coumarin of p53-AMC peptide is solvent-exposed and makes no significant contacts with SIRT1. We propose that binding of resveratrol to SIRT1 promotes a conformational change that better accommodates the attached coumarin group.     

SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling

Accumulating evidence suggests that neurodegeneration induced by pathogenic proteins depends on contributions from surrounding glia. Here we demonstrate that NF-kappaB signaling in microglia is critically involved in neuronal death induced by amyloid-beta (Abeta) peptides, which are widely presumed to cause Alzheimer disease. Constitutive inhibition of NF-kappaB signaling in microglia by expression of the nondegradable IkappaBalpha superrepressor blocked neurotoxicity, indicating a pivotal role for microglial NF-kappaB signaling in mediating Abeta toxicity. Stimulation of microglia with Abeta increased acetylation of RelA/p65 at lysine 310, which regulates the NF-kappaB pathway. Overexpression of SIRT1 deacetylase and the addition of the SIRT1 agonist resveratrol markedly reduced NF-kappaB signaling stimulated by Abeta and had strong neuroprotective effects. Our results support a glial loop hypothesis by demonstrating a critical role for microglial NF-kappaB signaling in Abeta-dependent neurodegeneration. They also implicate SIRT1 in this pathway and highlight the therapeutic potential of resveratrol and other sirtuin-activating compounds in Alzheimer disease.     

Resveratrol‐enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol‐induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol‐mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long‐term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol‐induced down‐regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H₂O₂ increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1‐dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.     

Resveratrol activates SIRT1 in a Lamin A-dependent manner

Human sirtuin1 (SIRT1), the closest homolog of the yeast sir2 protein, functions as an NAD+-dependent histone and non-histone protein deacetylase in several cellular processes, like energy metabolism, stress responses, aging, etc. In our recent study, we have shown that lamin A (a major nuclear matrix protein) directly binds with and activates SIRT1. Resveratrol, a natural phenol, has long been known as an activator of SIRT1. However, resveratrol’s direct activation of SIRT1 has been refuted several times. In our study, we have provided a mechanistic explanation to this question, and have shown that resveratrol activates SIRT1 by increasing its binding with lamin A, thus aiding in the nuclear matrix (NM) localization of SIRT1. We have also shown that rescue of adult stem cell (ASC) decline in laminopathy-based premature aging mice by resveratrol is SIRT1-dependent. Further, resveratrol’s ameliorating effects on progeria and its capacity to extend lifespan in progeria mice has been established. Here we have summarized these findings and their probable implications on other aspects, like chromatin remodeling, stem cell therapy, DNA damage responses, etc.     

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.     

Interplay among BRCA1, SIRT1, and Survivin during BRCA1-associated tumorigenesis

Germline mutations of BRCA1 predispose women to breast and ovarian cancers. However, the downstream mediators of BRCA1 function in tumor suppression remain elusive. We found that human BRCA1-associated breast cancers have lower levels of SIRT1 than their normal controls. We further demonstrated that mammary tumors from Brca1 mutant mice have low levels of Sirt1 and high levels of Survivin, which is reversed by induced expression of Brca1. BRCA1 binds to the SIRT1 promoter and increases SIRT1 expression, which in turn inhibits Survivin by changing the epigenetic modification of histone H3. Absence of SIRT1 blocks the regulation of Survivin by BRCA1. Furthermore, we demonstrated that activation of Sirt1 and inhibition of Survivin expression by resveratrol elicit a more profound inhibitory effect on Brca1 mutant cancer cells than on Brca1-wild-type cancer cells both in vitro and in vivo. These findings suggest that resveratrol treatment serves as an excellent strategy for targeted therapy for BRCA1-associated breast cancer.     

Resveratrol alleviates alcoholic fatty liver in mice

Alcoholic fatty liver is associated with inhibition of sirtuin 1 (SIRT1) and AMP-activated kinase (AMPK), two critical signaling molecules regulating the pathways of hepatic lipid metabolism in animals. Resveratrol, a dietary polyphenol, has been identified as a potent activator for both SIRT1 and AMPK. In the present study, we have carried out in vivo animal experiments that test the ability of resveratrol to reverse the inhibitory effects of chronic ethanol feeding on hepatic SIRT1-AMPK signaling system and to prevent the development of alcoholic liver steatosis. Resveratrol treatment increased SIRT1 expression levels and stimulated AMPK activity in livers of ethanol-fed mice. The resveratrol-mediated increase in activities of SIRT1 and AMPK was associated with suppression of sterol regulatory element binding protein 1 (SREBP-1) and activation of peroxisome proliferator-activated receptor gamma coactivator alpha (PGC-1alpha). In parallel, in ethanol-fed mice, resveratrol administration markedly increased circulating adiponectin levels and enhanced mRNA expression of hepatic adiponectin receptors (AdipoR1/R2). In conclusion, resveratrol treatment led to reduced lipid synthesis and increased rates of fatty acid oxidation and prevented alcoholic liver steatosis. The protective action of resveratrol is in whole or in part mediated through the upregulation of a SIRT1-AMPK signaling system in the livers of ethanol-fed mice. Our study suggests that resveratrol may serve as a promising agent for preventing or treating human alcoholic fatty liver disease.