Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone-receptor activation.

Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin's "hinge opening" and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone's native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a "smart" insulin analog to mitigate hypoglycemic risk in diabetes therapy.

ß Cell GLP-1R Signaling Alters α Cell Proglucagon Processing after Vertical Sleeve Gastrectomy in Mice.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), causes high rates of type 2 diabetes remission and remarkable increases in postprandial glucagon-like peptide-1 (GLP-1) secretion. GLP-1 plays a critical role in islet function by potentiating glucose-stimulated insulin secretion; however, the mechanisms remain incompletely defined. Therefore, we applied a murine VSG model to an inducible ß cell-specific GLP-1 receptor (GLP-1R) knockout mouse model to investigate the role of the ß cell GLP-1R in islet function. Our data show that loss of ß cell GLP-1R signaling decreases α cell GLP-1 expression after VSG. Furthermore, we find a ß cell GLP-1R-dependent increase in α cell expression of the prohormone convertase required for the production of GLP-1 after VSG. Together, the findings herein reveal two concepts. First, our data support a paracrine role for α cell-derived GLP-1 in the metabolic benefits observed after VSG. Second, we have identified a role for the ß cell GLP-1R as a regulator of α cell proglucagon processing.

Mechanisms to Elevate Endogenous GLP-1 Beyond Injectable GLP-1 Analogs and Metabolic Surgery.

Therapeutic engineering of glucagon-like peptide 1 (GLP-1) has enabled development of new medicines to treat type 2 diabetes. These injectable analogs achieve robust glycemic control by increasing concentrations of "GLP-1 equivalents" (∼50 pmol/L). Similar levels of endogenous GLP-1 occur after gastric bypass surgery, and mechanistic studies indicate glucose lowering by these procedures is driven by GLP-1. Therefore, because of the remarkable signaling and secretory capacity of the GLP-1 system, we sought to discover mechanisms that increase GLP-1 pharmacologically. To study active GLP-1, glucose-dependent insulinotropic polypeptide receptor (Gipr)-deficient mice receiving background dipeptidyl peptidase 4 (DPP4) inhibitor treatment were characterized as a model for evaluating oral agents that increase circulating GLP-1. A somatostatin receptor 5 antagonist, which blunts inhibition of GLP-1 release, and agonists for TGR5 and GPR40, which stimulate GLP-1 secretion, were investigated alone and in combination with the DPP4 inhibitor sitagliptin; these only modestly increased GLP-1 (∼5-30 pmol/L). However, combining molecules to simultaneously intervene at multiple regulatory nodes synergistically elevated active GLP-1 to unprecedented concentrations (∼300-400 pmol/L), drastically reducing glucose in Gipr null and Leprdb/db mice in a GLP-1 receptor-dependent manner. Our studies demonstrate that complementary pathways can be engaged to robustly increase GLP-1 without invasive surgical or injection regimens.

ß-Cell Glucagon-Like Peptide-1 Receptor Contributes to Improved Glucose Tolerance After Vertical Sleeve Gastrectomy.

Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible for this remain incompletely defined. Glucagon-like peptide-1 (GLP-1) is a gut hormone that contributes to the maintenance of glucose homeostasis and is elevated after VSG. VSG-induced increases in postprandial GLP-1 secretion have been proposed to contribute to the glucoregulatory benefits of VSG; however, previous work has been equivocal. In order to test the contribution of enhanced ß-cell GLP-1 receptor (GLP-1R) signaling we used a ß-cell-specific tamoxifen-inducible GLP-1R knockout mouse model. Male ß-cell-specific Glp-1r(ß-cell+/+) wild type (WT) and Glp-1r(ß-cell-/-) knockout (KO) littermates were placed on a high-fat diet for 6 weeks and then switched to high-fat diet supplemented with tamoxifen for the rest of the study. Mice underwent sham or VSG surgery after 2 weeks of tamoxifen diet and were fed ad libitum postoperatively. Mice underwent oral glucose tolerance testing at 3 weeks and were euthanized at 6 weeks after surgery. VSG reduced body weight and food intake independent of genotype. However, glucose tolerance was only improved in VSG WT compared with sham WT, whereas VSG KO had impaired glucose tolerance relative to VSG WT. Augmentation of glucose-stimulated insulin secretion during the oral glucose tolerance test was blunted in VSG KO compared with VSG WT. Therefore, our data suggest that enhanced ß-cell GLP-1R signaling contributes to improved glucose regulation after VSG by promoting increased glucose-stimulated insulin secretion.

Novel small molecule glucagon-like peptide-1 receptor agonist stimulates insulin secretion in rodents and from human islets.

OBJECTIVE: The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS: Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS: Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS: These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization.

Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance.

Insulin and insulin-like growth factor 1 (IGF1) are ubiquitous growth factors that regulate proliferation in most mammalian tissues including pancreatic islets. To explore the specificity of insulin receptors in compensatory beta-cell growth, we examined two models of insulin resistance. In the first model, we used liver-specific insulin receptor knockout (LIRKO) mice, which exhibit hyperinsulinemia without developing diabetes due to a compensatory increase in beta-cell mass. LIRKO mice, also lacking functional insulin receptors in beta-cells (beta IRKO/LIRKO), exhibited severe glucose intolerance but failed to develop compensatory islet hyperplasia, together leading to early death. In the second model, we examined the relative significance of insulin versus IGF1 receptors in islet growth by feeding high-fat diets to beta IRKO and beta-cell-specific IGF1 receptor knockout (beta IGFRKO) mice. Although both groups on the high-fat diet developed insulin resistance, beta IRKO, but not beta IGFRKO, mice exhibited poor islet growth consistent with insulin-stimulated phosphorylation, nuclear exclusion of FoxO1, and reduced expression of Pdx-1. Together these data provide direct genetic evidence that insulin/FoxO1/Pdx-1 signaling is one pathway that is crucial for islet compensatory growth response to insulin resistance.

Reduction of low molecular weight protein-tyrosine phosphatase expression improves hyperglycemia and insulin sensitivity in obese mice.

To investigate the role of low molecular weight protein-tyrosine phosphatase (LMW-PTP) in glucose metabolism and insulin action, a specific antisense oligonucleotide (ASO) was used to reduce its expression both in vitro and in vivo. Reduction of LMW-PTP expression with the ASO in cultured mouse hepatocytes and in liver and fat tissues of diet-induced obese (DIO) mice and ob/ob mice led to increased phosphorylation and activity of key insulin signaling intermediates, including insulin receptor-beta subunit, phosphatidylinositol 3-kinase, and Akt in response to insulin stimulation. The ASO-treated DIO and ob/ob animals showed improved insulin sensitivity, which was reflected by a lowering of both plasma insulin and glucose levels and improved glucose and insulin tolerance in DIO mice. The treatment did not decrease body weight or increase metabolic rate. These data demonstrate that LMW-PTP is a key negative regulator of insulin action and a potential novel target for the treatment of insulin resistance and type 2 diabetes.

Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism.

Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC-responsive genes such as PEPCK in the liver and decreased circulating lymphocytes. GCCR ASO treatment completely inhibited the increase in dexamethasone-induced PEPCK expression in the liver without causing any change in the dexamethasone-induced lymphopenia. These studies demonstrate that tissue-selective GCCR antagonism with ASOs may be a viable therapeutic strategy for the treatment of the metabolic syndrome.

Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors.

Uncontrolled hepatic glucose production contributes significantly to hyperglycemia in patients with type 2 diabetes. Hyperglucagonemia is implicated in the etiology of this condition; however, effective therapies to block glucagon signaling and thereby regulate glucose metabolism do not exist. To determine the extent to which blocking glucagon action would reverse hyperglycemia, we targeted the glucagon receptor (GCGR) in rodent models of type 2 diabetes using 2'-methoxyethyl-modified phosphorothioate-antisense oligonucleotide (ASO) inhibitors. Treatment with GCGR ASOs decreased GCGR expression, normalized blood glucose, improved glucose tolerance, and preserved insulin secretion. Importantly, in addition to decreasing expression of cAMP-regulated genes in liver and preventing glucagon-mediated hepatic glucose production, GCGR inhibition increased serum concentrations of active glucagon-like peptide-1 (GLP-1) and insulin levels in pancreatic islets. Together, these studies identify a novel mechanism whereby GCGR inhibitors reverse the diabetes phenotype by the dual action of decreasing hepatic glucose production and improving pancreatic beta cell function.

Lipoatrophic diabetes in Irs1(-/-)/Irs3(-/-) double knockout mice.

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, we generated mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences from Irs1(-/-) mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish the Irs1(-/-)/Irs3(-/-) double knockout mouse as a novel model of lipoatrophic diabetes.