Intra-islet α-cell Gs signaling promotes glucagon release.

Glucagon, a hormone released from pancreatic α-cells, is critical for maintaining euglycemia and plays a key role in the pathophysiology of diabetes. To stimulate the development of new classes of therapeutic agents targeting glucagon release, key α-cell signaling pathways that regulate glucagon secretion need to be identified. Here, we focused on the potential importance of α-cell Gs signaling on modulating α-cell function. Studies with α-cell-specific mouse models showed that activation of α-cell Gs signaling causes a marked increase in glucagon secretion. We also found that intra-islet adenosine plays an unexpected autocrine/paracrine role in promoting glucagon release via activation of α-cell Gs-coupled A2A adenosine receptors. Studies with α-cell-specific Gαs knockout mice showed that α-cell Gs also plays an essential role in stimulating the activity of the Gcg gene, thus ensuring proper islet glucagon content. Our data suggest that α-cell enriched Gs-coupled receptors represent potential targets for modulating α-cell function for therapeutic purposes.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Clenbuterol exerts antidiabetic activity through metabolic reprogramming of skeletal muscle cells.

Activation of the sympathetic nervous system causes pronounced metabolic changes that are mediated by multiple adrenergic receptor subtypes. Systemic treatment with ß2-adrenergic receptor agonists results in multiple beneficial metabolic effects, including improved glucose homeostasis. To elucidate the underlying cellular and molecular mechanisms, we chronically treated wild-type mice and several newly developed mutant mouse strains with clenbuterol, a selective ß2-adrenergic receptor agonist. Clenbuterol administration caused pronounced improvements in glucose homeostasis and prevented the metabolic deficits in mouse models of ß-cell dysfunction and insulin resistance. Studies with skeletal muscle-specific mutant mice demonstrated that these metabolic improvements required activation of skeletal muscle ß2-adrenergic receptors and the stimulatory G protein, Gs. Unbiased transcriptomic and metabolomic analyses showed that chronic ß2-adrenergic receptor stimulation caused metabolic reprogramming of skeletal muscle characterized by enhanced glucose utilization. These findings strongly suggest that agents targeting skeletal muscle metabolism by modulating ß2-adrenergic receptor-dependent signaling pathways may prove beneficial as antidiabetic drugs.

ß-Arrestins as Important Regulators of Glucose and Energy Homeostasis.

ß-Arrestin-1 and -2 (also known as arrestin-2 and -3, respectively) are ubiquitously expressed cytoplasmic proteins that dampen signaling through G protein-coupled receptors. However, ß-arrestins can also act as signaling molecules in their own right. To investigate the potential metabolic roles of the two ß-arrestins in modulating glucose and energy homeostasis, recent studies analyzed mutant mice that lacked or overexpressed ß-arrestin-1 and/or -2 in distinct, metabolically important cell types. Metabolic analysis of these mutant mice clearly demonstrated that both ß-arrestins play key roles in regulating the function of most of these cell types, resulting in striking changes in whole-body glucose and/or energy homeostasis. These studies also revealed that ß-arrestin-1 and -2, though structurally closely related, clearly differ in their metabolic roles under physiological and pathophysiological conditions. These new findings should guide the development of novel drugs for the treatment of various metabolic disorders, including type 2 diabetes and obesity.

In vivo metabolic effects after acute activation of skeletal muscle Gs signaling.

OBJECTIVE: The goal of this study was to determine the glucometabolic effects of acute activation of Gs signaling in skeletal muscle (SKM) in vivo and its contribution to whole-body glucose homeostasis. METHODS: To address this question, we studied mice that express a Gs-coupled designer G protein-coupled receptor (Gs-DREADD or GsD) selectively in skeletal muscle. We also identified two Gs-coupled GPCRs that are endogenously expressed by SKM at relatively high levels (ß2-adrenergic receptor and CRF2 receptor) and studied the acute metabolic effects of activating these receptors in vivo by highly selective agonists (clenbuterol and urocortin 2 (UCN2), respectively). RESULTS: Acute stimulation of GsD signaling in SKM impaired glucose tolerance in lean and obese mice by decreasing glucose uptake selectively into SKM. The acute metabolic effects following agonist activation of ß2-adrenergic and, potentially, CRF2 receptors appear primarily mediated by altered insulin release. Clenbuterol injection improved glucose tolerance by increasing insulin secretion in lean mice. In SKM, clenbuterol stimulated glycogen breakdown. UCN2 injection resulted in decreased glucose tolerance associated with lower plasma insulin levels. The acute metabolic effects of UCN2 were not mediated by SKM Gs signaling. CONCLUSIONS: Selective activation of Gs signaling in SKM causes an acute increase in blood glucose levels. However, acute in vivo stimulation of endogenous Gs-coupled receptors enriched in SKM has only a limited impact on whole-body glucose homeostasis, most likely due to the fact that these receptors are also expressed by pancreatic islets where they modulate insulin release.

Gq signaling in α cells is critical for maintaining euglycemia.

Glucagon, a hormone released from pancreatic α cells, plays a key role in maintaining euglycemia. New insights into the signaling pathways that control glucagon secretion may stimulate the development of novel therapeutic agents. In this study, we investigated the potential regulation of α cell function by G proteins of the Gq family. The use of a chemogenetic strategy allowed us to selectively activate Gq signaling in mouse α cells in vitro and in vivo. Acute stimulation of α cell Gq signaling led to elevated plasma glucagon levels, accompanied by increased insulin release and improved glucose tolerance. Moreover, chronic activation of this pathway greatly improved glucose tolerance in obese mice. We also identified an endogenous Gq-coupled receptor (vasopressin 1b receptor; V1bR) that was enriched in mouse and human α cells. Agonist-induced activation of the V1bR strongly stimulated glucagon release in a Gq-dependent fashion. In vivo studies indicated that V1bR-mediated glucagon release played a key role in the counterregulatory hyperglucagonemia under hypoglycemic and glucopenic conditions. These data indicate that α cell Gq signaling represents an important regulator of glucagon secretion, resulting in multiple beneficial metabolic effects. Thus, drugs that target α cell-enriched Gq-coupled receptors may prove useful to restore euglycemia in various pathophysiological conditions.

Adenosine A1 receptor is dispensable for hepatocyte glucose metabolism and insulin sensitivity.

Hepatic insulin resistance (IR) and enhanced hepatic glucose production (HGP) are key features of type 2 diabetes (T2D), contributing to fasting hyperglycemia. Adenosine receptors (ARs) are G protein-coupled and expressed in hepatocytes. Here, we explored the role of hepatic Gi/o-coupled A1AR on insulin resistance and glucose fluxes associated with obesity. We generated a mouse model with hepatocyte-specific deletion of A1AR (A1LΔ/Δ), which was compared with whole body knockout of A1AR or A1AR/A3AR (both Gi-coupled). Selective deletion of hepatic A1AR resulted in a modest improvement in insulin sensitivity. In addition, HFD A1LΔ/Δ mice showed decreased fasting glucose levels. Hyperinsulinemic-euglycemic clamp studies demonstrated enhanced insulin sensitivity with no change in HGP in HFD A1LΔ/Δ mice. Similar to A1LΔ/Δ, fasting blood glucose levels were significantly reduced in whole body A1Δ/Δ and A1Δ/ΔA3Δ/Δ compared to wild-type mice. Taken together, our data support the concept that blocking hepatic A1AR may decrease fasting blood glucose levels without directly affecting hepatocyte glucose metabolism and insulin sensitivity.

ß-Arrestin-1 is required for adaptive ß-cell mass expansion during obesity.

Obesity is the key driver of peripheral insulin resistance, one of the key features of type 2 diabetes (T2D). In insulin-resistant individuals, the expansion of beta-cell mass is able to delay or even prevent the onset of overt T2D. Here, we report that beta-arrestin-1 (barr1), an intracellular protein known to regulate signaling through G protein-coupled receptors, is essential for beta-cell replication and function in insulin-resistant mice maintained on an obesogenic diet. Specifically, insulin-resistant beta-cell-specific barr1 knockout mice display marked reductions in beta-cell mass and the rate of beta-cell proliferation, associated with pronounced impairments in glucose homeostasis. Mechanistic studies suggest that the observed metabolic deficits are due to reduced Pdx1 expression levels caused by beta-cell barr1 deficiency. These findings indicate that strategies aimed at enhancing barr1 activity and/or expression in beta-cells may prove useful to restore proper glucose homeostasis in T2D.

Metabolic roles of G protein-coupled receptor signaling in obesity and type 2 diabetes.

The incidence of obesity and type 2 diabetes (T2D) has been increasing steadily worldwide. It is estimated that by 2045 more than 800 million people will be suffering from diabetes. Despite the advancements in modern medicine, more effective therapies for treating obesity and T2D are needed. G protein-coupled receptors (GPCRs) have emerged as important drug targets for various chronic diseases, including obesity, T2D, and liver diseases. During the past two decades, many laboratories worldwide focused on understanding the role of GPCR signaling in regulating glucose metabolism and energy homeostasis. The information gained from these studies can guide the development of novel therapeutic agents. In this review, we summarize recent studies providing insights into the role of GPCR signaling in peripheral, metabolically important tissues such as pancreas, liver, skeletal muscle, and adipose tissue, focusing primarily on the use of mutant animal models and human data.

Key Metabolic Functions of ß-Arrestins: Studies with Novel Mouse Models.

ß-Arrestin-1 and -2 are intracellular proteins that are able to inhibit signaling via G protein-coupled receptors (GPCRs). However, both proteins can also modulate cellular functions in a G protein-independent fashion. During the past few years, studies with mutant mice selectivity lacking ß-arrestin-1 and/or -2 in metabolically important cell types have led to novel insights into the mechanisms through which ß-arrestins regulate key metabolic processes in vivo, including whole-body glucose and energy homeostasis. The novel information gained from these studies should inform the development of novel drugs, including ß-arrestin- or G protein-biased GPCR ligands, that could prove useful for the therapy of several important pathophysiological conditions, including type 2 diabetes and obesity.

Beneficial metabolic role of ß-arrestin-1 expressed by AgRP neurons.

ß-Arrestin-1 and ß-arrestin-2 have emerged as important signaling molecules that modulate glucose fluxes in several peripheral tissues. The potential roles of neuronally expressed ß-arrestins in regulating glucose homeostasis remain unknown. We here report that mice lacking ß-arrestin-1 (barr1) selectively in AgRP neurons displayed impaired glucose tolerance and insulin sensitivity when consuming an obesogenic diet, while mice overexpressing barr1 selectively in AgRP neurons were protected against obesity-associated metabolic impairments. Additional physiological, biochemical, and electrophysiological data indicated that the presence of barr1 is essential for insulin-mediated hyperpolarization of AgRP neurons. As a result, barr1 expressed by AgRP neurons regulates efferent neuronal pathways that suppress hepatic glucose production and promote lipolysis in adipose tissue. Mice lacking ß-arrestin-2 (barr2) selectively in AgRP neurons showed no substantial metabolic phenotypes. Our data suggest that agents able to enhance the activity of barr1 in AgRP neurons may prove beneficial as antidiabetic drugs.

ß-arrestin-1 suppresses myogenic reprogramming of brown fat to maintain euglycemia.

A better understanding of the signaling pathways regulating adipocyte function is required for the development of new classes of antidiabetic/obesity drugs. We here report that mice lacking ß-arrestin-1 (barr1), a cytoplasmic and nuclear signaling protein, selectively in adipocytes showed greatly impaired glucose tolerance and insulin sensitivity when consuming an obesogenic diet. In contrast, transgenic mice overexpressing barr1 in adipocytes were protected against the metabolic deficits caused by a high-calorie diet. Barr1 deficiency led to a myogenic reprogramming of brown adipose tissue (BAT), causing elevated plasma myostatin (Mstn) levels, which in turn led to impaired insulin signaling in multiple peripheral tissues. Additional in vivo studies indicated that barr1-mediated suppression of Mstn expression by BAT is required for maintaining euglycemia. These findings convincingly identify barr1 as a critical regulator of BAT function. Strategies aimed at enhancing barr1 activity in BAT may prove beneficial for the treatment of type 2 diabetes.

Adipocyte Gi signaling is essential for maintaining whole-body glucose homeostasis and insulin sensitivity.

Adipocyte dysfunction links obesity to insulin resistance and type 2 diabetes. Adipocyte function is regulated by receptor-mediated activation of heterotrimeric G proteins. Little is known about the potential in vivo metabolic roles of Gi-type G proteins expressed by adipocytes, primarily due to the lack of suitable animal models. To address this question, we generated mice lacking functional Gi proteins selectively in adipocytes. Here we report that these mutant mice displayed significantly impaired glucose tolerance and reduced insulin sensitivity when maintained on an obesogenic diet. In contrast, using a chemogenetic strategy, we demonstrated that activation of Gi signaling selectively in adipocytes greatly improved glucose homeostasis and insulin signaling. We also elucidated the cellular mechanisms underlying the observed metabolic phenotypes. Our data support the concept that adipocyte Gi signaling is essential for maintaining euglycemia. Drug-mediated activation of adipocyte Gi signaling may prove beneficial for restoring proper glucose homeostasis in type 2 diabetes.

Novel metabolic role for BDNF in pancreatic ß-cell insulin secretion.

BDNF signaling in hypothalamic circuitries regulates mammalian food intake. However, whether BDNF exerts metabolic effects on peripheral organs is currently unknown. Here, we show that the BDNF receptor TrkB.T1 is expressed by pancreatic ß-cells where it regulates insulin release. Mice lacking TrkB.T1 show impaired glucose tolerance and insulin secretion. ß-cell BDNF-TrkB.T1 signaling triggers calcium release from intracellular stores, increasing glucose-induced insulin secretion. Additionally, BDNF is secreted by skeletal muscle and muscle-specific BDNF knockout phenocopies the ß-cell TrkB.T1 deletion metabolic impairments. The finding that BDNF is also secreted by differentiated human muscle cells and induces insulin secretion in human islets via TrkB.T1 identifies a new regulatory function of BDNF on metabolism that is independent of CNS activity. Our data suggest that muscle-derived BDNF may be a key factor mediating increased glucose metabolism in response to exercise, with implications for the treatment of diabetes and related metabolic diseases.

ß-Cell-intrinsic ß-arrestin 1 signaling enhances sulfonylurea-induced insulin secretion.

Beta-arrestin-1 and -2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic beta-cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating beta-cell function and whole body glucose homeostasis. Initially, we inactivated the Barr1 gene in beta-cells of adult mice (beta-barr1-KO mice). Beta-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, two widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and co-immunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in beta-cells may prove useful for the development of efficacious antidiabetic drugs.

Intra-islet glucagon signaling is critical for maintaining glucose homeostasis.

Glucagon, a hormone released from pancreatic alpha-cells, plays a key role in maintaining proper glucose homeostasis and has been implicated in the pathophysiology of diabetes. In vitro studies suggest that intra-islet glucagon can modulate the function of pancreatic beta-cells. However, because of the lack of suitable experimental tools, the in vivo physiological role of this intra-islet cross-talk has remained elusive. To address this issue, we generated a novel mouse model that selectively expressed an inhibitory designer G protein-coupled receptor (Gi DREADD) in α-cells only. Drug-induced activation of this inhibitory designer receptor almost completely shut off glucagon secretion in vivo, resulting in significantly impaired insulin secretion, hyperglycemia, and glucose intolerance. Additional studies with mouse and human islets indicated that intra-islet glucagon stimulates insulin release primarily by activating ß-cell GLP-1 receptors. These new findings strongly suggest that intra-islet glucagon signaling is essential for maintaining proper glucose homeostasis in vivo. Our work may pave the way toward the development of novel classes of antidiabetic drugs that act by modulating intra-islet cross-talk between α- and ß-cells.

Sympathetic innervation is essential for metabolic homeostasis and pancreatic beta cell function in adult rats.

Obesity is associated with an imbalance in the activity of the autonomic nervous system (ANS), specifically in the organs involved in energy metabolism. The pancreatic islets are richly innervated by the ANS, which tunes the insulin release due to changes in energy demand. Therefore, changes in the sympathetic input that reach the pancreas can lead to metabolic dysfunctions. To evaluate the role of the sympathetic ends that innervate the pancreas, 60-day-old male Wistar rats were subjected to sympathectomy (SYM) or were sham-operated (SO). At 120 day-old SYM rats exhibited an increase in body weight, fat pads and metabolic dysfunctions. Decreases in the HOMA-IR and reductions in insulin release were observed both in vivo and in vitro. Furthermore, the SYM rats exhibited altered pancreatic islet function in both muscarinic and adrenergic assays and exhibited high protein expression of the alpha-2 adrenergic receptor (α2AR). Because α2AR has been linked to type 2 diabetes, these findings demonstrate the clinical implications of this study.

Glibenclamide treatment blocks metabolic dysfunctions and improves vagal activity in monosodium glutamate-obese male rats.

BACKGROUND/AIMS: Autonomic nervous system imbalance is associated with metabolic diseases, including diabetes. Glibenclamide is an antidiabetic drug that acts by stimulating insulin secretion from pancreatic beta cells and is widely used in the treatment of type 2 diabetes. Since there is scarce data concerning autonomic nervous system activity and diabetes, the aim of this work was to test whether glibenclamide can improve autonomic nervous system activity and muscarinic acetylcholine receptor function in pre-diabetic obese male rats. METHODS: Pre-diabetes was induced by treatment with monosodium L-glutamate in neonatal rats. The monosodium L-glutamate group was treated with glibenclamide (2 mg/kg body weight /day) from weaning to 100 days of age, and the control group was treated with water. Body weight, food intake, Lee index, fasting glucose, insulin levels, homeostasis model assessment of insulin resistance, omeostasis model assessment of ß-cell function, and fat tissue accumulation were measured. The vagus and sympathetic nerve electrical activity were recorded. Insulin secretion was measured in isolated islets challenged with glucose, acetylcholine, and the selective muscarinic acetylcholine receptor antagonists by radioimmunoassay technique. RESULTS: Glibenclamide treatment prevented the onset of obesity and diminished the retroperitoneal (18%) and epididymal (25%) fat pad tissues. In addition, the glibenclamide treatment also reduced the parasympathetic activity by 28% and glycemia by 20% in monosodium L-glutamate-treated rats. The insulinotropic effect and unaltered cholinergic actions in islets from monosodium L-glutamate groups were increased. CONCLUSION: Early glibenclamide treatment prevents monosodium L-glutamate-induced obesity onset by balancing autonomic nervous system activity.

HPA axis and vagus nervous function are involved in impaired insulin secretion of MSG-obese rats.

Neuroendocrine dysfunctions such as the hyperactivity of the vagus nerve and hypothalamus-pituitary-adrenal (HPA) axis greatly contribute to obesity and hyperinsulinemia; however, little is known about these dysfunctions in the pancreatic ß-cells of obese individuals. We used a hypothalamic-obesity model obtained by neonatal treatment with monosodium l-glutamate (MSG) to induce obesity. To assess the role of the HPA axis and vagal tonus in the genesis of hypercorticosteronemia and hyperinsulinemia in an adult MSG-obese rat model, bilateral adrenalectomy (ADX) and subdiaphragmatic vagotomy (VAG) alone or combined surgeries (ADX-VAG) were performed. To study glucose-induced insulin secretion (GIIS) and the cholinergic insulinotropic process, pancreatic islets were incubated with different glucose concentrations with or without oxotremorine-M, a selective agonist of the M3 muscarinic acetylcholine receptor (M3AChR) subtype. Protein expression of M3AChR in pancreatic islets, corticosteronemia, and vagus nerve activity was also evaluated. Surgeries reduced 80% of the body weight gain. Fasting glucose and insulin were reduced both by ADX and ADX-VAG, whereas VAG was only associated with hyperglycemia. The serum insulin post-glucose stimulation was lower in all animals that underwent an operation. Vagal activity was decreased by 50% in ADX rats. In the highest glucose concentration, both surgeries reduced GIIS by 50%, whereas ADX-VAG decreased by 70%. Additionally, M3AChR activity was recovered by the individual surgeries. M3AChR protein expression was reduced by ADX. Both the adrenal gland and vagus nerve contribute to the hyperinsulinemia in the MSG model, although adrenal is more crucial as it appears to modulate parasympathetic activity and M3AChR expression in obesity.