Minghan Wang

Resveratrol is not a direct activator of SIRT1 enzyme activity.

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 PGC‐1α isolated from cells, and (iii) although SIRT1 deacetylates PGC‐1α 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.

FGF21 N- and C-termini play different roles in receptor interaction and activation.

MINT‐6799907, MINT‐6799922: FGF21 (uniprotkb: Q9NSA1) binds (MI:0407) to β‐Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)

Treating Diabetes and Obesity with an FGF21-Mimetic Antibody Activating the βKlotho/FGFR1c Receptor Complex

A monoclonal antibody mimic of FGF21 exerts beneficial metabolic effects in obese monkeys. A Metabolic Mimic Losing weight typically requires exercise and a healthy diet. Managing diabetes similarly relies on diet and exercise but also includes insulin therapy. Now, both diabetes and obesity could be treated together by targeting the fibroblast growth factor 21 (FGF21) pathway. Foltz and colleagues show that an antibody mimic of FGF21 works to regulate glucose and insulin homeostasis, leading to weight loss and glucose tolerance in monkeys. The authors first engineered the FGF21-mimetic monoclonal antibody, which they termed “mimAb1.” This antibody was able to activate human and monkey FGF receptor 1c (FGFR1c)/βKlotho signaling similar to its native counterpart, FGF21. In vivo in obese cynomolgus monkeys, mimAb1 treatment led to a decrease in body weight and body mass index (BMI)—a decrease that was maintained for 9 weeks after the second round of treatment. These beneficial effects on metabolism were seen only initially with FGF21, before animals regained weight. Animals treated with mimAb1 also showed a decrease in fasting and fed plasma insulin levels, suggesting an improvement in insulin sensitivity, as well as a reduction in plasma triglyceride and glucose levels. Native FGF21 is difficult to develop as a therapeutic for diabetes and obesity; efforts to date have fallen short. mimAb1 recreates all of the beneficial metabolic effects of FGF21 as measured but is easier to manufacture, has prolonged pharmacokinetics, and has been engineered with high specificity. This mimAb1 will need additional safety and toxicity testing for translation, but early efficacy data in nonhuman primates suggest that this antibody is on its way to helping treat patients with diet-induced obesity and diabetes. 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.

Lack of Overt FGF21 Resistance in Two Mouse Models of Obesity and Insulin Resistance

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.

Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

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.

Discovery of a Potent, Orally Active 11β-Hydroxysteroid Dehydrogenase Type 1 Inhibitor for Clinical Study: Identification of (S)-2-((1S,2S,4R)-Bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221)

Thiazolones with an exo-norbornylamine at the 2-position and an isopropyl group on the 5-position are potent 11beta-HSD1 inhibitors. However, the C-5 center was prone to epimerization in vitro and in vivo, forming a less potent diastereomer. A methyl group was added to the C-5 position to eliminate epimerization, leading to the discovery of (S)-2-((1S,2S,4R)-bicyclo[2.2.1]heptan-2-ylamino)-5-isopropyl-5-methylthiazol-4(5H)-one (AMG 221). This compound decreased fed blood glucose and insulin levels and reduced body weight in diet-induced obesity mice.

Blockade of Glucocorticoid Excess at the Tissue Level : Inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1 as a Therapy for Type 2 Diabetes

Glucocorticoids are stress hormones with regulatory effects on carbohydrate, protein, and lipid metabolism. There are two forms of glucocorticoids in humans: the active cortisol (corticosterone in rodents) and inactive cortisone (11-dehydrocorticosterone in rodents). The physiological actions of glucocorticoids are mediated by glucocorticoid receptor (GR) which, upon binding to its natural ligand cortisol, is activated and regulates diverse physiological events. GR is a nuclear receptor ubiquitously expressed in tissues and triggers biological effects through transcriptional activation or suppression of target genes (Figure 1). Cortisol is synthesized in the adrenal glands as part of adrenal steroidogenesis that also involves the production of mineralocorticoids and androgens. Cortisol is secreted in a relatively high level at 10-20 mg/day. Cortisol biosynthesis is tightly controlled by adrenocorticotropic hormone (ACTH), a peptide hormone secreted from the anterior pituitary and is itself regulated by the hypothalamic peptide corticotrophin-releasing hormone (CRH). Circulating cortisol regulates its own biosynthesis by sending negative feedback signals to the pituitary and hypothalamus. Together, this neuroendocrine feedback circuit constitutes the hypothalamic-pituitary-adrenal (HPA) axis. The HPA activity is stimulated by physical or psychological stress and varies throughout the 24 h cycle. As a result, the circulating cortisol undergoes circadian rhythm, reaching its peak concentration of ∼800 nmol/L in the morning and nadir of ∼200 nmol/L at midnight in humans. About 96% of the circulating cortisol is protein-bound with 6% to albumin and 90% to corticosteroid binding globulin (CBG). Circulating CBG levels are approximately 700 nmol/L and regulated by estrogens and disease conditions. Free cortisol dictates glucocorticoid action. It is thought that CBG may serve to restrict access of cortisol to target tissues and regulate its bioavailability and metabolic clearance. CBG may also serve as a carrier for cortisol facilitating transport of cortisol in blood to certain tissues. In contrast, the inactive cortisone is in a free unbound form and its plasma concentration remains steady at approximately 100 nmol/L throughout the day. The metabolism of both cortisol and cortisone occurs in liver involving the A-ring reductases and several other enzymes, and the principal metabolites are tetrahydrocortisone (THE) and 5Rand 5 -tetrahydrocortisol (5Rand 5 -THF) (Figure 2). Another aspect of the regulation of glucocorticoid production involves two 11 -hydroxysteroid dehydrogenase (11 -HSD) isozymes that interconvert cortisone and cortisol (Figure 2). 11 -HSD1 is a reductase in vivo converting cortisone to cortisol and amplifies glucocorticoid action in a tissue-specific manner. In contrast, its isozyme 11 HSD2 acts as a dehydrogenase and catalyzes the opposite reaction, converting cortisol to cortisone. 11 -HSD1 is predominantly expressed in liver, adipose, placenta, and brain. 11 -HSD2 is primarily expressed in kidney and functions as the main source of cortisone production. Together, glucocorticoid homeostasis is maintained by the HPA axis and the activities of the 11 -HSD enzymes. The metabolic syndrome is a cluster of metabolic abnormalities including central obesity, insulin resistance, atherogenic * To whom correspondence should be addressed. Telephone: 805-4475598. Fax: 805-499-0953. E-mail: mwang@amgen.com. † Department of Medicinal Chemistry. ‡ Department of Metabolic Disorders. a Abbreviations: 11 -HSD1 or -2, 11 -hydroxysteroid dehydrogenase type 1 or 2; CBG, corticosteroid binding globulin; CBX, carbenoxolone; CRH, corticotrophin-releasing hormone; GA, glycyrrhetinic acid; GR, glucocorticoid receptor; HPA axis, the hypothalamic-pituitary-adrenal axis; THE, tetrahydrocortisone; THF, tetrahydrocortisol.  Copyright 2008 by the American Chemical Society

Understanding the physical interactions in the FGF21/FGFR/β-Klotho complex: structural requirements and implications in FGF21 signaling.

The endocrine fibroblast growth factor 21 (FGF21) requires both fibroblast growth factor receptor (FGFR) and β‐Klotho for signaling. In this study, we sought to understand the inter‐molecular physical interactions in the FGF21/FGFR/β‐Klotho complex by deleting key regions in FGFR1c or FGF21. Deletion of the D1 and the D1‐D2 linker (the D1/linker region) from FGFR1c led to β‐Klotho‐independent receptor activation by FGF21, suggesting that there may be a direct interaction between FGF21 and the D1/linker region‐deficient FGFR1c. Consistent with this, the extracellular portion of FGFR1c lacking the D1/linker region blocked FGF21 action in a reporter assay, presumably by binding to and sequestering FGF21 from acting on cell surface receptor complex. In addition, the D1/linker region‐deficient FGFR1c had enhanced interaction with β‐Klotho. Further, we demonstrated that deletion of the D1/linker region enhanced the formation of the FGF21/β‐Klotho/FGFR1c ternary complex in both Biacore and asymmetrical flow field flow fractionation studies. Finally, we found that the N‐terminus of FGF21 is involved in the interaction with FGFR1c and FGF21/β‐Klotho/FGFR1c ternary complex formation. Taken together, our data suggest that the D1/linker region regulates both the FGF21/FGFR1c and FGFR1c/β‐Klotho interaction, and a direct interaction of FGF21 with FGFR1c may be an important step in receptor‐mediated FGF21 signaling.

Characterization of a FGF19 variant with altered receptor specificity revealed a central role for FGFR1c in the regulation of glucose metabolism.

Diabetes and associated metabolic conditions have reached pandemic proportions worldwide, and there is a clear unmet medical need for new therapies that are both effective and safe. FGF19 and FGF21 are distinctive members of the FGF family that function as endocrine hormones. Both have potent effects on normalizing glucose, lipid, and energy homeostasis, and therefore, represent attractive potential next generation therapies for combating the growing epidemics of type 2 diabetes and obesity. The mechanism responsible for these impressive metabolic effects remains unknown. While both FGF19 and FGF21 can activate FGFRs 1c, 2c, and 3c in the presence of co-receptor βKlotho in vitro, which receptor is responsible for the metabolic activities observed in vivo remains unknown. Here we have generated a variant of FGF19, FGF19-7, that has altered receptor specificity with a strong bias toward FGFR1c. We show that FGF19-7 is equally efficacious as wild type FGF19 in regulating glucose, lipid, and energy metabolism in both diet-induced obesity and leptin-deficient mouse models. These results are the first direct demonstration of the central role of the βKlotho/FGFR1c receptor complex in glucose and lipid regulation, and also strongly suggest that activation of this receptor complex alone might be sufficient to achieve all the metabolic functions of endocrine FGF molecules.

Antidiabetic effects of 11β-HSD1 inhibition in a mouse model of combined diabetes, dyslipidaemia and atherosclerosis

Aim:  11 β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is considered to contribute to the aetiology of the metabolic syndrome, and specific inhibitors have begun to emerge as treatments for insulin resistance and other facets of the syndrome, including atherosclerosis. Given the role of glucocorticoids and 11β‐HSD1 in the anti‐inflammatory response and the involvement of inflammation in the development of atherosclerosis, 11β‐HSD1 inhibition may exacerbate atherosclerosis. Our aim was to investigate in vivo the effects of a specific 11β‐HSD1 inhibitor (2922) on atherosclerosis while assessing glucose homeostasis.