Resveratrol is a plant polyphenol capable of exerting beneficial metabolic effects which are thought to be mediated in large by the activation of the NAD + -dependent protein deacetylase SIRT1. Although resveratrol has been claimed to be a bona fide SIRT1 activator using a peptide substrate (Fluor de Lys-SIRT1 peptide substrate), recent reports indicate that this finding might be an experimental artifact and need to be clarified. Here, we show that: (i) the Fluor de Lys-SIRT1 peptide is an artificial SIRT1 substrate because in the absence of the covalently linked fluorophore the peptide itself is not a substrate of the enzyme, (ii) resveratrol does not activate SIRT1 in vitro in the presence of either a p53-derived peptide substrate or acetylated PGC1a isolated from cells, and (iii) although SIRT1 deacetylates PGC-1a in both in vitro and cell-based assays, resveratrol did not activate SIRT1 under these conditions. Based on these observations, we conclude that the pharmacological effects of resveratrol in various models are unlikely to be mediated by a direct enhancement of the catalytic activity of the SIRT1 enzyme. In consequence, our data challenge the overall utility of resveratrol as a pharmacological tool to directly activate SIRT1. Key words: Fluor de Lys, resveratrol, SIRT1Abbreviations: AMPK, AMP-activated protein kinase; HPLC, high performance liquid chromatography; NAM, nicotinamide; PGC-1a, peroxisome proliferator-activated receptor-c coactivator-1a; Sir2, silence information regulator 2; SIRTs, sirtuins. Sirtuins constitute the unique family of NAD + -dependent protein deacetylases. Silent information regulator 2 (Sir2) is a sirtuin in budding yeast Saccharomyces cerevisiae and its activity mediates lifespan extension induced by calorie restriction (1). The mammalian Sir2 ortholog, SIRT1, is also induced by calorie restriction and promotes cell survival (2), triggers lipolysis and loss of fat (3), and controls glucose homeostasis (4). The biological effects of SIRT1 are mediated by its ability to deacetylate several important transcriptional factors such as Peroxisome proliferator-activated receptor-c coactivator 1alpha (PGC-1a), p53, and FOXO proteins and consequently regulate their activities (5). PGC-1a, which is activated upon deacetylation by SIRT1, plays an important role in the regulation of mitochondrial function and fatty acid oxidation (4). In contrast, p53 is inactivated once deacetylated by SIRT1 (5), suggesting that SIRT1 may protect cells from apoptosis under conditions of nutrient restriction. Taken together, these findings demonstrate that SIRT1 activity stimulates energy metabolism, improves mitochondrial function and promotes cell survival. Therefore, pharmacological activation of SIRT1 in vivo may provide a new avenue to maintain metabolic homeostasis.Molecular screening of SIRT1 activators led to the identification of plant polyphenols as SIRT1 activators, among which is resveratrol (6). The notion that resveratrol is a SIRT1 activator is consistent with two later pharmacological ...

Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to βKlotho with high affinity and specifically activates signaling from the βKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on βKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.

Edited by Gianni Cesareni Keywords:Fibroblast growth factor-21 b-Klotho Fibroblast growth factor receptor Partial agonist a b s t r a c t Fibroblast growth factor-21 (FGF21) signaling requires the presence of b-Klotho, a co-receptor with a very short cytoplasmic domain. Here we show that FGF21 binds directly to b-Klotho through its Cterminus. Serial C-terminal truncations of FGF21 weakened or even abrogated its interaction with b-Klotho in a Biacore assay, and led to gradual loss of potency in a luciferase reporter assay but with little effect on maximal response. In contrast, serial N-terminal truncations of FGF21 had no impact on b-Klotho binding. Interestingly, several of them exhibited characteristics of partial agonists with minimal effects on potency. These data demonstrate that the C-terminus of FGF21 is critical for binding to b-Klotho and the N-terminus is critical for fibroblast growth factor receptor (FGFR) activation.

Circulating levels of fibroblast growth factor 21 (FGF21), a metabolic regulator of glucose, lipid, and energy homeostasis, are elevated in obese diabetic subjects, raising questions about potential FGF21 resistance. Here we report tissue expression changes in FGF21 and its receptor components, and we describe the target-organ and whole-body responses to FGF21 in ob/ob and diet-induced obese (DIO) mice. Plasma FGF21 concentrations were elevated 8- and 16-fold in DIO and ob/ob mice, respectively, paralleling a dramatic increase in hepatic FGF21 mRNA expression. Concurrently, expression levels of βKlotho, FGF receptor (FGFR)-1c, and FGFR2c were markedly down-regulated in the white adipose tissues (WAT) of ob/ob and DIO mice. However, dose-response curves of recombinant human FGF21 (rhFGF21) stimulation of ERK phosphorylation in the liver and WAT were not right shifted in disease models, although the magnitude of induction in ERK phosphorylation was partially attenuated in DIO mice. Whole-body metabolic responses were preserved in ob/ob and DIO mice, with disease models being more sensitive and responsive than lean mice to the glucose-lowering and weight-loss effects of rhFGF21. Endogenous FGF21 levels, although elevated in diseased mice, were below the half-maximal effective concentrations of rhFGF21, suggesting a state of relative deficiency. Hepatic and WAT FGF21 mRNA expression levels declined after rhFGF21 treatment in the absence of the increased expression levels of βKlotho and FGFR. We conclude that overt FGF21 resistance was not evident in the disease models, and increased hepatic FGF21 expression as a result of local metabolic changes is likely a major cause of elevated circulating FGF21 levels.

Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.