Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme-peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

Increased incidence of type 2 diabetes mellitus and obesity has elevated the medical need for new agents to treat these disease states. Resistance to the hormones insulin and leptin are hallmarks of both type 2 diabetes and obesity. Drugs that can ameliorate this resistance should be effective in treating type 2 diabetes and possibly obesity. Protein tyrosine phosphatase 1B (PTP1B) is thought to function as a negative regulator of insulin and leptin signal transduction. This article reviews PTP1B as a novel target for type 2 diabetes, and looks at the challenges in developing small-molecule inhibitors of this phosphatase.

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC ␤ isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC ␣ and ␤ 1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC ␤ selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC ␤ 1 isoform without decreasing PKC ␣ isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E 2 production, and inhibition of Na ϩ -K ϩ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-␤ 1 and extracellular matrix components such as fibronectin and ␣ 1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the ␤ isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na

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SIRT1 is a prominent member of a family of NAD؉ -dependent enzymes and affects a variety of cellular functions ranging from gene silencing, regulation of the cell cycle and apoptosis, to energy homeostasis. In mature adipocytes, SIRT1 triggers lipolysis and loss of fat content. However, the potential effects of SIRT1 on insulin signaling pathways are poorly understood. To assess this, we used RNA interference to knock down SIRT1 in 3T3-L1 adipocytes. SIRT1 depletion inhibited insulin-stimulated glucose uptake and GLUT4 translocation. This was accompanied by increased phosphorylation of JNK and serine phosphorylation of insulin receptor substrate 1 (IRS-1), along with inhibition of insulin signaling steps, such as tyrosine phosphorylation of IRS-1, and phosphorylation of Akt and ERK. In contrast, treatment of cells with specific small molecule SIRT1 activators led to an increase in glucose uptake and insulin signaling as well as a decrease in serine phosphorylation of IRS-1. Moreover, gene expression profiles showed that SIRT1 expression was inversely related to inflammatory gene expression. Finally, we show that treatment of 3T3-L1 adipocytes with a SIRT1 activator attenuated tumor necrosis factor alpha-induced insulin resistance. Taken together, these data indicate that SIRT1 is a positive regulator of insulin signaling at least partially through the anti-inflammatory actions in 3T3-L1 adipocytes.

Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the ␤ isoform, which has been associated with the development of micro-and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKC␤ isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKC␤2 isoform in the myocardium. These mice overexpressed the PKC␤2 isoform transgene by 2-to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKC␤-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKC␤2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.

SIRT3 is a major mitochondrial NAD؉ -dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD ؉ . In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD ؉ . These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.Sirtuins are class III histone deacetylases that couple lysine deacetylation with NAD ϩ hydrolysis and are highly conserved in prokaryotes and eukaryotes (1). Mammals possess seven sirtuins, SIRT1-7, that occupy different subcellular compartments such as the nucleus (SIRT1, -6, -7), cytoplasm (SIRT2), and the mitochondria (SIRT3, -4, and -5) (2). They deacetylate lysines not only on histone substrates (3, 4) but also on nonhistone substrates such as p53 tumor suppressor protein (5), Foxo transcription factors (6, 7), PGC-1␣ (8), ␣-tubulin (9), acetyl-CoA synthetases (10 -12), and glutamate dehydrogenase (13). SIRT4 and SIRT6 have been shown to have ADP-ribosyltransferase activity (14 -16). Sirtuins have been reported to play important roles in gene silencing (17), cell cycle regulation (18,19), metabolism (8, 10 -12, 14, 20 -22), apoptosis (5, 23, 24), the lifespan-extension effects of calorie restriction (25,26), and circadian rhythms (27)(28)(29)(30) (50), and SIRT5 (51). Sirtuins contain a conserved enzymatic core with two domains; that is, a large Rossmann fold domain that binds NAD ϩ and a small domain formed by two insertions of the large domain that binds to a zinc atom. The acetylated peptide substrate binds to the cleft between the two domains. Some of the known structures are apo structures with sirtuin protein alone, whereas others are bound to acetylated peptide substrate and/or NAD ϩ and its analogs. These structures revealed the mechanism of action for the deacetylation activity and substrate specificity.SIRT3 localizes in mitochondria (13, 52-54) and is a major mitochondrial deacetylase. Hyperacetylation of mitochondrial proteins have been observed in SIRT3 knock-out mice (13, 55). Several key enzymes involved in energy production in the mitochondria have been identified as SIRT3 substrates. Acetyl-CoA synthetase 2 (AceCS2) 2 converts acetate into acetyl-CoA in the mitochondria. Deacetyla...