James Mu

Chronic Inhibition of Dipeptidyl Peptidase-4 With a Sitagliptin Analog Preserves Pancreatic β-Cell Mass and Function in a Rodent Model of Type 2 Diabetes

Inhibitors of dipeptidyl peptidase-4 (DPP-4), a key regulator of the actions of incretin hormones, exert antihyperglycemic effects in type 2 diabetic patients. A major unanswered question concerns the potential ability of DPP-4 inhibition to have beneficial disease-modifying effects, specifically to attenuate loss of pancreatic β-cell mass and function. Here, we investigated the effects of a potent and selective DPP-4 inhibitor, an analog of sitagliptin (des-fluoro-sitagliptin), on glycemic control and pancreatic β-cell mass and function in a mouse model with defects in insulin sensitivity and secretion, namely high-fat diet (HFD) streptozotocin (STZ)-induced diabetic mice. Significant and dose-dependent correction of postprandial and fasting hyperglycemia, HbA1c, and plasma triglyceride and free fatty acid levels were observed in HFD/STZ mice following 2–3 months of chronic therapy. Treatment with des-fluoro-sitagliptin dose dependently increased the number of insulin-positive β-cells in islets, leading to the normalization of β-cell mass and β-cell–to–α-cell ratio. In addition, treatment of mice with des-fluoro-sitagliptin, but not glipizide, significantly increased islet insulin content and improved glucose-stimulated insulin secretion in isolated islets. These findings suggest that DPP-4 inhibitors may offer long-lasting efficacy in the treatment of type 2 diabetes by modifying the courses of the disease.

Adipose Fibroblast Growth Factor 21 Is Up-Regulated by Peroxisome Proliferator-Activated Receptor γ and Altered Metabolic States

Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) γ agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPARγ agonists. Of the 379 genes robustly regulated by two structurally distinct PPARγ agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPARγ agonists and in liver by PPARα agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPARγ agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPARγ agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPARγ agonists.

Inhibition of DPP-4 with sitagliptin improves glycemic control and restores islet cell mass and function in a rodent model of type 2 diabetes.

Inhibition of dipeptidyl peptidase-4 (DPP-4) activity has been shown to improve glycemic control in patients with type 2 diabetes by prolonging and potentiating the actions of incretin hormones. This study is designed to determine the effects of the DPP-4 inhibitor sitagliptin on improving islet function in a mouse model of insulin resistance and insulin secretion defects. ICR mice were pre-treated with high fat diet and a low dose of streptozotocin to induce insulin resistance and impaired insulin secretion, respectively. Diabetic mice were treated with sitagliptin or the sulfonylurea agent glipizide as admixture to high fat diet for ten weeks. Sustained reduction of blood glucose, HbA(1c), circulating glucagon and improvement in oral glucose tolerance were observed in mice treated with sitagliptin. In contrast, glipizide improved glycemic control only during the early weeks and to a lesser degree compared to sitagliptin, and had no effect on circulating glucagon levels or glucose tolerance. The improvement in glycemic control in sitagliptin-treated mice was associated with a significant increase in glucose-dependent insulin secretion in both perfused pancreas and isolated islets. Importantly, in contrast to the lack of effect by glipizide, sitagliptin significantly restored beta and alpha cell mass as well as alpha/beta cell ratio. These data indicate that DPP-4 inhibition by sitagliptin provided better overall improvement of glycemic control compared to glipizide in the high fat diet/streptozotocin induced diabetic mouse model. The ability of sitagliptin to enhance islet cell function may offer insight into the potential for disease modification.

FGF21 Analogs of Sustained Action Enabled by Orthogonal Biosynthesis Demonstrate Enhanced Antidiabetic Pharmacology in Rodents

Fibroblast growth factor 21 (FGF21) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF21 analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF21 for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF21 ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF21 analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF21 at a specific and preferred site confers superior metabolic pharmacology.

The Glucagon Receptor Is Involved in Mediating the Body Weight-Lowering Effects of Oxyntomodulin

Oxyntomodulin (OXM) is a peptide secreted postprandially from the L‐cells of the gut that has a weak affinity for both the glucagon‐like peptide‐1 receptor (GLP1R) and the glucagon receptor (GCGR). Peripheral administration of OXM in humans and rodents causes weight loss reducing food intake and increasing energy expenditure. It has been suggested that OXM modulates energy intake solely through GLP1R agonism. Because glucagon decreases food intake in rodents and humans, we examined whether activation of the GCGR is involved in the body weight‐lowering effects of OXM. We identified an equipotent GLP1R‐selective peptide agonist that differs from OXM by only one residue (Q3→E, OXMQ3E), but has no significant GCGR agonist activity in vitro and ∼100‐fold reduced ability to stimulate liver glycogenolysis. Chronic treatment of obese mice with OXM and OXMQ3E demonstrated that OXM exhibits superior weight loss and lipid‐lowering efficacy, and antihyperglycemic activity that is comparable to the corresponding GLP1R‐selective agonist. Studies in Glp1r−/− mice and coadministration of OXM and a GCGR antagonist revealed that the antiobesity effect of OXM requires activation of both GLP1R and GCGR. Our data provide new insight into the mechanism of action of OXM and suggest that activation of GCGR is involved in the body weight‐lowering action of OXM.

Chronic treatment with a glucagon receptor antagonist lowers glucose and moderately raises circulating glucagon and glucagon-like peptide 1 without severe alpha cell hypertrophy in diet-induced obese mice.

Aims/hypothesisAntagonism of the glucagon receptor (GCGR) represents a potential approach for treating diabetes. Cpd-A, a potent and selective GCGR antagonist (GRA) was studied in preclinical models to assess its effects on alpha cells.MethodsStudies were conducted with Cpd-A to examine the effects on glucose-lowering efficacy, its effects in combination with a dipeptidyl peptidase-4 (DPP-4) inhibitor, and the extent and reversibility of alpha cell hypertrophy associated with GCGR antagonism in mouse models.ResultsChronic treatment with Cpd-A resulted in effective and sustained glucose lowering in mouse models in which endogenous murine Gcgr was replaced with human GCGR (hGCGR). Treatment with Cpd-A also led to stable, moderate elevations in both glucagon and glucagon-like peptide 1 (GLP-1) levels, which were completely reversible and not associated with a hyperglycaemic overshoot following termination of treatment. When combined with a DPP-4 inhibitor, Cpd-A led to additional improvement of glycaemic control correlated with elevated active GLP-1 levels after glucose challenge. In contrast to Gcgr-knockout mice in which alpha cell hypertrophy was detected, chronic treatment with Cpd-A in obese hGCGR mice did not result in gross morphological changes in pancreatic tissue.Conclusions/interpretationA GRA lowered glucose effectively in diabetic models without significant alpha cell hypertrophy during or following chronic treatment. Treatment with a GRA may represent an effective approach for glycaemic control in patients with type 2 diabetes, which could be further enhanced when combined with DPP-4 inhibitors.

PANIC-ATTAC: A Mouse Model for Inducible and Reversible β-Cell Ablation

OBJECTIVE—Islet transplantations have been performed clinically, but their practical applications are limited. An extensive effort has been made toward the identification of pancreatic β-cell stem cells that has yielded many insights to date, yet targeted reconstitution of β-cell mass remains elusive. Here, we present a mouse model for inducible and reversible ablation of pancreatic β-cells named the PANIC-ATTAC (pancreatic islet β-cell apoptosis through targeted activation of caspase 8) mouse. RESEARCH DESIGN AND METHODS—We efficiently induce β-cell death through apoptosis and concomitant hyperglycemia by administration of a chemical dimerizer to the transgenic mice. In contrast to animals administered streptozotocin, the diabetes phenotype and β-cell loss are fully reversible in the PANIC-ATTAC mice, and we find significant β-cell recovery with normalization of glucose levels after 2 months. RESULTS—The rate of recovery can be enhanced by various pharmacological interventions with agents acting on the glucagon-like peptide 1 axis and agonists of peroxisome proliferator–activated receptor-γ. During recovery, we find an increased population of GLUT2+/insulin− cells in the islets of PANIC-ATTAC mice, which may represent a novel pool of potential β-cell precursors. CONCLUSIONS—The PANIC-ATTAC mouse may be used as an animal model of inducible and reversible β-cell ablation and therefore has applications in many areas of diabetes research that include identification of β-cell precursors, evaluation of glucotoxicity effects in diabetes, and examination of pharmacological interventions.

Anti-diabetic efficacy and impact on amino acid metabolism of GRA1, a novel small-molecule glucagon receptor antagonist.

Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.

A glucose-responsive insulin therapy protects animals against hypoglycemia

Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.

Engineering Glucose Responsiveness Into Insulin

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a “smart” insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).