Eric Hawkins

FGF-21 as a novel metabolic regulator

Diabetes mellitus is a major health concern, affecting more than 5% of the population. Here we describe a potential novel therapeutic agent for this disease, FGF-21, which was discovered to be a potent regulator of glucose uptake in mouse 3T3-L1 and primary human adipocytes. FGF-21-transgenic mice were viable and resistant to diet-induced obesity. Therapeutic administration of FGF-21 reduced plasma glucose and triglycerides to near normal levels in both ob/ob and db/db mice. These effects persisted for at least 24 hours following the cessation of FGF-21 administration. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic or healthy animals or when overexpressed in transgenic mice. Thus, we conclude that FGF-21, which we have identified as a novel metabolic factor, exhibits the therapeutic characteristics necessary for an effective treatment of diabetes.

Absence of Glucagon and Insulin Action Reveals a Role for the GLP-1 Receptor in Endogenous Glucose Production

The absence of insulin results in oscillating hyperglycemia and ketoacidosis in type 1 diabetes. Remarkably, mice genetically deficient in the glucagon receptor (Gcgr) are refractory to the pathophysiological symptoms of insulin deficiency, and therefore, studies interrogating this unique model may uncover metabolic regulatory mechanisms that are independent of insulin. A significant feature of Gcgr-null mice is the high circulating concentrations of GLP-1. Hence, the objective of this report was to investigate potential noninsulinotropic roles of GLP-1 in mice where GCGR signaling is inactivated. For these studies, pancreatic β-cells were chemically destroyed by streptozotocin (STZ) in Gcgr−/−:Glp-1r−/− mice and in Glp-1r−/− animals that were subsequently treated with a high-affinity GCGR antagonist antibody that recapitulates the physiological state of Gcgr ablation. Loss of GLP-1 action substantially worsened nonfasting glucose concentrations and glucose tolerance in mice deficient in, and undergoing pharmacological inhibition of, the GCGR. Further, lack of the Glp-1r in STZ-treated Gcgr−/− mice elevated rates of endogenous glucose production, likely accounting for the differences in glucose homeostasis. These results support the emerging hypothesis that non–β-cell actions of GLP-1 analogs may improve metabolic control in patients with insulinopenic diabetes.

Genetic ablation of myelin protein zero-like 3 in mice increases energy expenditure, improves glycemic control, and reduces hepatic lipid synthesis.

Obesity continues to be a global health problem, and thus it is imperative that new pathways regulating energy balance be identified. Recently, it was reported: (Hayashi K, Cao T, Passmore H, Jourdan-Le Saux C, Fogelgren B, Khan S, Hornstra I, Kim Y, Hayashi M, Csiszar K. J Invest Dermatol 123: 864-871, 2004) that mice carrying a missense mutation in myelin protein zero-like 3 (Mpzl3rc) have reduced body weight. To determine how Mpzl3 controls energy balance in vivo, we generated mice deficient in myelin protein zero-like 3 (Mpzl3-KO). Interestingly, KO mice were hyperphagic yet had reduced body weight and fat mass. Moreover, KO mice were highly resistant to body weight and fat mass gain after exposure to a high-fat, energy-dense diet. These effects on body weight and adiposity were driven, in part, by a pronounced increase in whole body energy expenditure levels in KO mice. KO mice also had reduced blood glucose levels during an intraperitoneal glucose challenge and significant reductions in circulating insulin levels suggesting an increase in insulin sensitivity. In addition, there was an overall increase in oxidative capacity and contractile force in skeletal muscle isolated from KO mice. Hepatic triglyceride levels were reduced by 92% in livers of KO mice, in part due to a reduction in de novo lipid synthesis. Interestingly, Mpzl3 mRNA expression in liver was increased in diet-induced obese mice. Moreover, KO mice exhibited an increase in insulin-stimulated Akt signaling in the liver, further demonstrating that Mpzl3 can regulate insulin sensitivity in this tissue. We have determined that Mpzl3 has a novel physiological role in controlling body weight regulation, energy expenditure, glycemic control, and hepatic triglyceride synthesis in mice.

Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase

To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.

Insulin-XTEN® Exhibits a Size-Dependent Alteration in Tissue Action in Rats

To optimize the action of exogenously administered insulin, we employed XTEN® technology to create insulins with variably sized XTEN amino acid polymers. Recombinant fusions of XTEN polymers linked to insulin lispro with an A21G mutation were prepared in various amino acid lengths. Insulin-XTEN molecules demonstrated 15-fold lower potency in binding and receptor phosphorylation than insulin lispro but did not differ from each other. These insulin-XTEN molecules were equally effective in lowering blood glucose at a 100nmol/kg dose in diabetic Sprague-Dawley rats. Furthermore, the larger insulin-XTEN molecules had a longer duration of glucose lowering. Insulin-XTENs were compared to insulin lispro in rat euglycemic clamp studies, using insulin doses that would elicit steady plasma insulin concentrations and equivalent increases in glucose infusion rate. Insulin-mediated suppression of endogenous glucose production was not significantly different among any of the administered insulins. However, plasma free fatty acids and soleus muscle glucose uptake were significantly decreased in an XTEN size-dependent manner when compared to insulin lispro. Additional studies demonstrated equal hepatic pAkt accumulation in rats treated with insulin lispro or any of the insulin-XTENs, but revealed a significant XTEN size-dependent reduction in skeletal muscle pAkt in rats administered insulin-XTENs compared to insulin lispro. These data suggest a possible XTEN size-dependent regulation of insulin action and that the differing sizes of the XTEN polymer may convey preferential tissue action. In conclusion, XTEN technology may permit “tuning” of the glucodynamic effects of the insulin, leading to an enhanced time extension and improved hepatic and peripheral pharmacodynamic action that could more closely mimic the action of endogenously secreted insulin into the portal circulation. Disclosure M.E. Christe: Employee; Self; Eli Lilly and Company. D. Konkol: None. J. Friedrich: None. J. Jacobs: None. E. Hawkins: Employee; Self; Eli Lilly and Company. J. Moyers: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. C. Zhang: Employee; Self; Eli Lilly and Company. S.D. Kahl: Employee; Self; Eli Lilly and Company. H.E. Baker: None. A.L. Cox: None. R.J. Hansen: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. A. Sperry: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. M. Michael: Employee; Self; Eli Lilly and Company. Stock/Shareholder; Self; Eli Lilly and Company. Employee; Spouse/Partner; Eli Lilly and Company. Stock/Shareholder; Spouse/Partner; Eli Lilly and Company. V. Schellenberger: None. D. Baldwin: None. J.M. Beals: Employee; Self; Eli Lilly and Company. A. Korytko: None.