Yue-Sheng Li

Fibroblast Growth Factor 21 Reverses Hepatic Steatosis, Increases Energy Expenditure, and Improves Insulin Sensitivity in Diet-Induced Obese Mice

OBJECTIVE—Fibroblast growth factor 21 (FGF21) has emerged as an important metabolic regulator of glucose and lipid metabolism. The aims of the current study are to evaluate the role of FGF21 in energy metabolism and to provide mechanistic insights into its glucose and lipid-lowering effects in a high-fat diet–induced obesity (DIO) model. RESEARCH DESIGN AND METHODS—DIO or normal lean mice were treated with vehicle or recombinant murine FGF21. Metabolic parameters including body weight, glucose, and lipid levels were monitored, and hepatic gene expression was analyzed. Energy metabolism and insulin sensitivity were assessed using indirect calorimetry and hyperinsulinemic-euglycemic clamp techniques. RESULTS—FGF21 dose dependently reduced body weight and whole-body fat mass in DIO mice due to marked increases in total energy expenditure and physical activity levels. FGF21 also reduced blood glucose, insulin, and lipid levels and reversed hepatic steatosis. The profound reduction of hepatic triglyceride levels was associated with FGF21 inhibition of nuclear sterol regulatory element binding protein-1 and the expression of a wide array of genes involved in fatty acid and triglyceride synthesis. FGF21 also dramatically improved hepatic and peripheral insulin sensitivity in both lean and DIO mice independently of reduction in body weight and adiposity. CONCLUSIONS—FGF21 corrects multiple metabolic disorders in DIO mice and has the potential to become a powerful therapeutic to treat hepatic steatosis, obesity, and type 2 diabetes.

Acute glucose-lowering and insulin-sensitizing action of FGF21 in insulin-resistant mouse models—association with liver and adipose tissue effects

Recombinant fibroblast growth factor (FGF)21 has antihyperglycemic, antihyperlipidemic, and antiobesity effects in diabetic rodent and monkey models. Previous studies were confined to measuring steady-state effects of FGF21 following subchronic or chronic administration. The present study focuses on the kinetics of biological actions of FGF21 following a single injection and on the associated physiological and cellular mechanisms underlying FGF21 actions. We show that FGF21 resulted in rapid decline of blood glucose levels and immediate improvement of glucose tolerance and insulin sensitivity in two animal models of insulin resistance (ob/ob and DIO mice). In ob/ob mice, FGF21 led to a 40-60% decrease in blood glucose, insulin, and amylin levels within 1 h after injection, and the maximal effects were sustained for more than 6 h despite the 1- to 2-h half-life of FGF21. In DIO mice, FGF21 reduced fasting blood glucose and insulin levels and improved glucose tolerance and insulin sensitivity within 3 h of treatment. The acute improvement of glucose metabolism was associated with a 30% reduction of hepatic glucose production and an increase in peripheral glucose turnover. FGF21 appeared to have no direct effect on ex vivo pancreatic islet insulin or glucagon secretion. However, it rapidly induced typical FGF signaling in liver and adipose tissues and in several hepatoma-derived cell lines and differentiated adipocytes. FGF21 was able to inhibit glucose release from H4IIE hepatoma cells and stimulate glucose uptake in 3T3-L1 adipocytes. We conclude that the acute glucose-lowering and insulin-sensitizing effects of FGF21 are potentially associated with its metabolic actions in liver and adipose tissues.

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)

Co-receptor Requirements for Fibroblast Growth Factor-19 Signaling

FGF19 is a unique member of the fibroblast growth factor (FGF) family of secreted proteins that regulates bile acid homeostasis and metabolic state in an endocrine fashion. Here we investigate the cell surface receptors required for signaling by FGF19. We show that βKlotho, a single-pass transmembrane protein highly expressed in liver and fat, induced ERK1/2 phosphorylation in response to FGF19 treatment and significantly increased the interactions between FGF19 and FGFR4. Interestingly, our results show that αKlotho, another Klotho family protein related to βKlotho, also induced ERK1/2 phosphorylation in response to FGF19 treatment and increased FGF19-FGFR4 interactions in vitro, similar to the effects of βKlotho. In addition, heparin further enhanced the effects of both αKlotho and βKlotho in FGF19 signaling and interaction experiments. These results suggest that a functional FGF19 receptor may consist of FGF receptor (FGFR) and heparan sulfate complexed with either αKlotho or βKlotho.

Fully Human Monoclonal Antibodies Antagonizing the Glucagon Receptor Improve Glucose Homeostasis in Mice and Monkeys

Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for reducing excess hepatic glucose production in patients with type 2 diabetes. Despite extensive efforts, there is currently no human therapeutic that directly inhibits the glucagon/glucagon receptor pathway. We undertook a novel approach by generating high-affinity human monoclonal antibodies (mAbs) to the human glucagon receptor (GCGR) that display potent antagonistic activity in vitro and in vivo. A single injection of a lead antibody, mAb B, at 3 mg/kg, normalized blood glucose levels in ob/ob mice for 8 days. In addition, a single injection of mAb B dose-dependently lowered fasting blood glucose levels without inducing hypoglycemia and improved glucose tolerance in normal C57BL/6 mice. In normal cynomolgus monkeys, a single injection improved glucose tolerance while increasing glucagon and active glucagon-like peptide-1 levels. Thus, the anti-GCGR mAb could represent an effective new therapeutic for the treatment of type 2 diabetes.

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes.

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

Inhibition of lipolysis may contribute to the acute regulation of plasma FFA and glucose by FGF21 in ob/ob mice

FGF21 is a unique member of the fibroblast growth factors (FGFs) and a novel hormone that regulates glucose, lipid, and energy homeostasis. The beneficial effects of FGF21 reported thus far have mostly been from chronic treatments. In order to better understand the mechanism for FGF21 action, we evaluated the acute effects of FGF21 in vivo and in vitro. Here we report that a single injection of FGF21 acutely reduced plasma free fatty acid levels similar to its acute effects on plasma glucose in ob/ob mice. In vitro, FGF21 inhibited lipolysis in adipocytes during a short treatment and reduced total lipase activity. These results demonstrate the potential importance of adipocyte lipolysis to the observed acute improvements in plasma parameters.

A Novel Fc-FGF21 With Improved Resistance to Proteolysis, Increased Affinity Toward β-Klotho, and Enhanced Efficacy in Mice and Cynomolgus Monkeys

Fibroblast growth factor (FGF) 21 is a natural hormone that modulates glucose, lipid, and energy metabolism. Previously, we engineered an Fc fusion FGF21 variant with two mutations, Fc-FGF21(RG), to extend the half-life and reduce aggregation and in vivo degradation of FGF21. We now describe a new variant developed to reduce the extreme C-terminal degradation and improve the binding affinity to β-Klotho. We demonstrate, by introducing one additional mutation located at the C terminus of FGF21 (A180E), that the new molecule, Fc-FGF21(RGE), has gained many improved attributes. Compared with Fc-FGF21(RG), Fc-FGF21(RGE) has similar in vitro potency, preserves β-Klotho dependency, and maintains FGF receptor selectivity and cross-species reactivity. In vivo, Fc-FGF21(RGE) showed reduced susceptibility to extreme C-terminal degradation and increased plasma levels of the bioactive intact molecule. The circulating half-life of intact Fc-FGF21(RGE) increased twofold compared with that of Fc-FGF21(RG) in mice and cynomolgus monkeys. Additionally, Fc-FGF21(RGE) exhibited threefold to fivefold enhanced binding affinity to coreceptor β-Klotho across mouse, cynomolgus monkey, and human species. In obese and diabetic mouse and cynomolgus monkey models, Fc-FGF21(RGE) demonstrated greater efficacies to Fc-FGF21(RG), resulting in larger and more sustained improvements in multiple metabolic parameters. No increased immunogenicity was observed with Fc-FGF21(RGE). The superior biophysical, pharmacokinetic, and pharmacodynamic properties, as well as the positive metabolic effects across species, suggest that further clinical development of Fc-FGF21(RGE) as a metabolic therapy for diabetic and/or obese patients may be warranted.