G蛋白偶联受体119——治疗糖尿病的新靶点

G蛋白偶联受体119(GPR119)与激动剂结合后,通过cAMP信号转导途径,促进葡萄糖依赖性胰岛素和肠肽激素的分泌,是新一代的治疗2型糖尿病药物靶点。本文对GPR119的组织学分布、生理学作用、内源性配体以及小分子激动剂作一简要的介绍。     

AMG 837: a potent, orally bioavailable GPR40 agonist

The discovery that certain long chain fatty acids potentiate glucose stimulated insulin secretion through the previously orphan receptor GPR40 sparked interest in GPR40 agonists as potential antidiabetic agents. Optimization of a series of β-substituted phenylpropanoic acids led to the identification of (S)-3-(4-((4'-(trifluoromethyl)biphenyl-3-yl)methoxy)phenyl)hex-4-ynoic acid (AMG 837) as a potent GPR40 agonist with a superior pharmacokinetic profile and robust glucose-dependent stimulation of insulin secretion in rodents.     

GPR142 Agonists Stimulate Glucose-Dependent Insulin Secretion via Gq-Dependent Signaling

GPR142 is an islet-enriched G protein-coupled receptor that has been investigated as a novel therapeutic target for the treatment of type 2 diabetes by virtue of its insulin secretagogue activity. However, the signaling pathways downstream of GPR142 and whether its stimulation of insulin release is glucose-dependent remain poorly characterized. In this study, we show that both native and synthetic GPR142 agonists can activate Gq as well as Gi signaling when GPR142 is recombinantly expressed in HEK293 cells. However, in primary pancreatic islets, a native cellular system, the insulin secretagogue activity of GPR142 agonists only requires Gq activation. In addition, our results show that stimulation of insulin secretion by GPR142 in pancreatic islets is strictly glucose-dependent.     

Novel GPR40 agonist AS2575959 exhibits glucose metabolism improvement and synergistic effect with sitagliptin on insulin and incretin secretion

Aims

GPR40 is a free fatty acid receptor that regulates glucose-dependent insulin secretion at pancreatic β-cells and glucagon-like peptide-1 (GLP-1), one of the major incretins, secretion at the endocrine cells of the gastrointestinal tract. We investigated the synergistic effect of AS2575959, a novel GPR40 agonist, in combination with sitagliptin, a major dipeptidyl peptidase-IV (DPP-IV) inhibitor, on glucose-dependent insulin secretion and GLP-1 secretion. In addition, we investigated the chronic effects of AS2575959 on whole-body glucose metabolism.

Main methods

We evaluated acute glucose metabolism on insulin and GLP-1 secretion using an oral glucose tolerance test (OGTT) as well as assessed the chronic glucose metabolism in diabetic ob/ob mice following the repeated administration of AS2575959.

Key findings

We discovered the novel GPR40 agonist sodium [(3S)-6-({4′-[(3S)-3,4-dihydroxybutoxy]-2,2′,6′-trimethyl[1,1′-biphenyl]-3-yl}methoxy)-3H-spiro[1-benzofuran-2,1′-cyclopropan]-3-yl]acetate (AS2575959) and found that the compound influenced glucose-dependent insulin secretion both in vitro pancreas β-cell-derived cells and in vivo mice OGTT. Further, we observed a synergistic effect of AS2575959 and DPP-IV inhibitor on insulin secretion and plasma GLP-1 level. In addition, we discovered the improvement in glucose metabolism on repeated administration of AS2575959.

Significance

To our knowledge, this study is the first to demonstrate the synergistic effect of a GPR40 agonist and DPP-IV inhibitor on the glucose-dependent insulin secretion and GLP-1 concentration increase. These findings suggest that GPR40 agonists may represent a promising therapeutic strategy for the treatment of type 2 diabetes mellitus, particularly when used in combination with DPP-IV inhibitors.     

Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120

Diabetes, a disease in which the body does not produce or use insulin properly, is a serious global health problem. Gut polypeptides secreted in response to food intake, such as glucagon-like peptide-1 (GLP-1), are potent incretin hormones that enhance the glucose-dependent secretion of insulin from pancreatic beta cells. Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules in various cellular processes, including the secretion of gut incretin peptides. Here we show that a G-protein-coupled receptor, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain FFAs. Furthermore, we show that the stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro and in vivo, and increases circulating insulin. Because GLP-1 is the most potent insulinotropic incretin, our results indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes.     

Diphenyl Ether Derivatives as Novel GPR120 Agonists for the Treatment of Type 2 Diabetes Mellitus

Diabetes mellitus (DM) is a serious disease affecting human health. Numerous attempts have been made to develop safe and effective new antidiabetic drugs. Recently, a series of G protein-coupled receptors for free fatty acids (FFAs) have been described and characterized, and small molecule agonists and antagonists of these receptors show considerable promise for managing diabetes and related complications. FFA-activated GPR120 could stimulate the release of glucagon-like peptide-1(GLP-1), which can enhance the glucose-dependent secretion of insulin from pancreatic β cells. GPR120 is a promising target for treating type 2 DM (T2DM). Herein we designed and synthesized a series of novel GPR120 agonists based on the structure of TUG-891, which was the first potent and selective GPR120 agonist. Among the designed compounds, 18 f showed excellent GPR120 activation activity and high selectivity for GPR40 in vitro. Compound 18 f dose-dependently improved glucose tolerance in normal mice, and no hypoglycemic side effects were observed at high dose. In addition, compound 18 f increased insulin release and displayed good antidiabetic effect in diet-induced obese mice. Molecular simulations illustrated that compound 18 f could enter the active site of GPR120 and interact with Arg99. Based on these observations, compound 18 f may be a promising lead compound for the design of novel GPR120 agonists to treat T2DM.     

Omega-3 fatty acids in anti-inflammation (pro-resolution) and GPCRs

Omega-3 fatty acids, such as, DHA and EPA, have well established beneficial effects on human health, but their action mechanisms remain unknown. Recent pharmacological studies have suggested several molecular targets for the anti-inflammatory effects of omega-3 fatty acids, namely, nuclear receptor PPARγ and the G protein-coupled receptor GPR120. Furthermore, the conversions of omega-3 fatty acids to anti-inflammatory and pro-resolving resolvins and protectins and the identifications of putative target GPCRs, ChemR23, BLT?, ALX/FPR2, and GPR32, have drawn great attention. In addition, the pharmacology of omega-3 fatty acids is now under scrutiny. However, questions remain to be answered regarding the in vivo effects of omega-3 fatty acids at the molecular level. In this review, anti-inflammatory effects of omega-3 fatty acids are discussed from the viewpoint of molecular pharmacology, particularly with respect to the above-mentioned GPCRs.     

Identification of a novel GPR119 agonist, AS1269574, with in vitro and in vivo glucose-stimulated insulin secretion

The G protein-coupled receptor 119 (GPR119) is highly expressed in pancreatic β-cells. On activation, this receptor enhances the effect of glucose-stimulated insulin secretion (GSIS) via the elevation of intracellular cAMP concentrations. Although GPR119 agonists represent promising oral antidiabetic agents for the treatment of type 2 diabetes therapy, they suffer from the inability to adequately directly preserve β-cell function. To identify a new structural class of small-molecule GPR119 agonists with both GSIS and the potential to preserve β-cell function, we screened a library of synthetic compounds and identified a candidate molecule, AS1269574, with a 2,4,6-tri-substituted pyrimidine core. Here, we examined the preliminary in vitro and in vivo effects of AS1269574 on insulin secretion and glucose tolerance. AS1269574 had an EC₅₀ value of 2.5 μM in HEK293 cells transiently expressing human GPR119 and enhanced insulin secretion in the mouse pancreatic β-cell line MIN-6 only under high-glucose (16.8 mM) conditions. This contrasted with the action of the sulfonylurea glibenclamide, which also induced insulin secretion under low-glucose conditions (2.8 mM). In in vivo studies, a single administration of AS1269574 to normal mice reduced blood glucose levels after oral glucose loading based on the observed insulin secretion profiles. Significantly, AS1269574 did not affect fed and fasting plasma glucose levels in normal mice. Taken together, these results suggest that AS1269574 represents a novel structural class of small molecule, orally administrable GPR119 agonists with GSIS and promising potential for the treatment of type 2 diabetes.     

A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents

Type 2 diabetes is characterized by impaired glucose homeostasis due to defects in insulin secretion, insulin resistance and the incretin response. GPR40 (FFAR1 or FFA1) is a G-protein-coupled receptor (GPCR), primarily expressed in insulin-producing pancreatic β-cells and incretin-producing enteroendocrine cells of the small intestine. Several GPR40 agonists, including AMG 837 and TAK-875, have been disclosed, but no GPR40 synthetic agonists have been reported that engage both the insulinogenic and incretinogenic axes. In this report we provide a molecular explanation and describe the discovery of a unique and potent class of GPR40 full agonists that engages the enteroinsular axis to promote dramatic improvement in glucose control in rodents. GPR40 full agonists AM-1638 and AM-6226 stimulate GLP-1 and GIP secretion from intestinal enteroendocrine cells and increase GSIS from pancreatic islets, leading to enhanced glucose control in the high fat fed, streptozotocin treated and NONcNZO10/LtJ mouse models of type 2 diabetes. The improvement in hyperglycemia by AM-1638 was reduced in the presence of the GLP-1 receptor antagonist Ex(9–39)NH₂.     

Lipid derivatives activate GPR119 and trigger GLP-1 secretion in primary murine L-cells

Aims/hypothesisGlucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid.MethodsGLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter.ResultsL-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells.Conclusions/interpretationGPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.     

The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse

Obesity is typically associated with elevated levels of free fatty acids (FFAs) and is linked to glucose intolerance and type 2 diabetes. FFAs exert divergent effects on insulin secretion from beta cells: acute exposure to FFAs stimulates insulin secretion, whereas chronic exposure impairs insulin secretion. The G protein-coupled receptor GPR40 is selectively expressed in beta cells and is activated by FFAs. We show here that GPR40 mediates both acute and chronic effects of FFAs on insulin secretion and that GPR40 signaling is linked to impaired glucose homeostasis. GPR40-deficient beta cells secrete less insulin in response to FFAs, and loss of GPR40 protects mice from obesity-induced hyperinsulinemia, hepatic steatosis, hypertriglyceridemia, increased hepatic glucose output, hyperglycemia, and glucose intolerance. Conversely, overexpression of GPR40 in beta cells of mice leads to impaired beta cell function, hypoinsulinemia, and diabetes. These results suggest that GPR40 plays an important role in the chain of events linking obesity and type 2 diabetes.     

A Novel Antidiabetic Drug,Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1

Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca²⁺ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca²⁺ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.     

Unsaturated fatty acids induce mesenchymal stem cells to increase secretion of angiogenic mediators

Mesenchymal stem cells (MSC) represent emerging cell-based therapies for diabetes and associated complications. Ongoing clinical trials are using exogenous MSC to treat type 1 and 2 diabetes, cardiovascular disease and non-healing wounds due to diabetes. The majority of these trials are aimed at exploiting the ability of these multipotent mesenchymal stromal cells to release soluble mediators that reduce inflammation and promote both angiogenesis and cell survival at sites of tissue damage. Growing evidence suggests that MSC secretion of soluble factors is dependent on tissue microenvironment. Despite the contribution of fatty acids to the metabolic environment of type 2 diabetes, almost nothing is known about their effects on MSC secretion of growth factors and cytokines. In this study, human bone marrow-derived MSC were exposed to linoleic acid, an omega-6 polyunsaturated fatty acid, or oleic acid, a monounsaturated fatty acid, for seven days in the presence of 5.38 mM glucose. Outcomes measured included MSC proliferation, gene expression, protein secretion and chemotaxis. Linoleic and oleic acids inhibited MSC proliferation and altered MSC expression and secretion of known mediators of angiogenesis. Both unsaturated fatty acids induced MSC to increase secretion of interleukin-6, VEGF and nitric oxide. In addition, linoleic acid but not oleic acid induced MSC to increase production of interleukin-8. Collectively these data suggest that exposure to fatty acids may have functional consequences for MSC therapy. Fatty acids may affect MSC engraftment to injured tissue and MSC secretion of cytokines and growth factors that regulate local cellular responses to injury.     

AS1907417, a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes

G-protein-coupled receptor (GPR) 119 is involved in glucose-stimulated insulin secretion (GSIS) and represents a promising target for the treatment of type 2 diabetes as it is highly expressed in pancreatic β-cells. Although a number of oral GPR119 agonists have been developed, their inability to adequately directly preserve β-cell function limits their effectiveness. Here, we evaluated the therapeutic potential of a novel small-molecule GPR119 agonist, AS1907417, which represents a modified form of a 2,4,6-tri-substituted pyrimidine core agonist, AS1269574, we previously identified. The exposure of HEK293 cells expressing human GPR119, NIT-1 cells expressing human insulin promoter, and the pancreatic β-cell line MIN-6-B1 to AS1907417, enhanced intracellular cAMP, GSIS, and human insulin promoter activity, respectively. In in vivo experiments involving fasted normal mice, a single dose of AS1907417 improved glucose tolerance, but did not affect plasma glucose or insulin levels. Twice-daily doses of AS1907417 for 4 weeks in diabetic db/db, aged db/db mice, ob/ob mice, and Zucker diabetic fatty rats reduced hemoglobin A1c levels by 1.6%, 0.8%, 1.5%, and 0.9%, respectively. In db/db mice, AS1907417 improved plasma glucose, plasma insulin, pancreatic insulin content, lipid profiles, and increased pancreatic insulin and pancreatic and duodenal homeobox 1 (PDX-1) mRNA levels. These data demonstrate that novel GPR119 agonist AS1907417 not only effectively controls glucose levels, but also preserves pancreatic β-cell function. We therefore propose that AS1907417 represents a new type of antihyperglycemic agent with promising potential for the effective treatment of type 2 diabetes.     

Discovery and biological evaluation of novel 4-amino-2-phenylpyrimidine derivatives as potent and orally active GPR119 agonists

Novel 4-amino-2-phenylpyrimidine derivatives were synthesized and evaluated as GPR119 agonists. Optimization of the substituents on the phenyl ring at the 2-position and the amino group at the 4-position led to the identification of 3,4-dihalogenated and 2,4,5-trihalogenated phenyl derivatives showing potent GPR119 agonistic activity. The advanced analog (2R)-3-{[2-(4-chloro-2,5-difluorophenyl)-6-ethylpyrimidin-4-yl]amino}propane-1,2-diol (24g) was found to improve glucose tolerance at 1mg/kg po in mice and to show excellent pharmacokinetic profiles in mice and monkeys. Compound 24g also showed an excellent antidiabetic effect in diabetic kk/Ay mice after one week of single daily treatment. These results demonstrate that novel GPR119 agonist 24g improves glucose tolerance not only by enhancing glucose-dependent insulin secretion but also by preserving pancreatic β-cell function.     

Acute effects of unsaturated fatty acids in splanchnic artery occlusion shock

Diets enriched with omega-3 unsaturated fatty acids are associated with decreased hypercholesterolemia and decreased risk of ischemic and atherosclerotic diseases. We studied the acute intravascular effects of some of these unsaturated fatty acids (i.e., eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) along with omega-6 unsaturated fatty acids, (i.e., linoleic and linolenic acid) in splanchnic artery occlusion (SAO) shock in rats. Anesthetized rats subjected to total occlusion of the celiac and superior mesenteric arteries for 40 minutes followed by reperfusion usually resulted in a fatal outcome 90-120 minutes after releasing the clamps. SAO shock rats treated with the omega-3 unsaturated fatty acid, EPA, exhibited an improved survival time and rate (p less than 0.05 from vehicle) compared to those receiving only vehicle (i.e., 50% ethanol). EPA and DHA treated SAO rats also exhibited lower plasma activities of the lysosomal protease, cathepsin D, free amino-nitrogen compounds, and the cardiotoxic peptide, myocardial depressant factor. These results indicate that omega-3 unsaturated fatty acids, especially EPA, have some acute beneficial effects in SAO shock in rats.     

Alteration of the Glucagon Axis in GPR120 (FFAR4) Knockout Mice: A ROLE FOR GPR120 IN GLUCAGON SECRETION

GPR40 (FFAR1) and GPR120 (FFAR4) are G-protein-coupled receptors (GPCRs) that are activated by long chain fatty acids (LCFAs). GPR40 is expressed at high levels in islets and mediates the ability of LCFAs to potentiate glucose-stimulated insulin secretion (GSIS). GPR120 is expressed at high levels in colon, adipose, and pituitary, and at more modest levels in pancreatic islets. The role of GPR120 in islets has not been explored extensively. Here, we confirm that saturated (e.g. palmitic acid) and unsaturated (e.g. docosahexaenoic acid (DHA)) LCFAs engage GPR120 and demonstrate that palmitate- and DHA-potentiated glucagon secretion are greatly reduced in isolated GPR120 KO islets. Remarkably, LCFA potentiated glucagon secretion is similarly reduced in GPR40 KO islets. Compensatory changes in mRNA expression of GPR120 in GPR40 KO islets, and vice versa, do not explain that LCFA potentiated glucagon secretion seemingly involves both receptors. LCFA-potentiated GSIS remains intact in GPR120 KO islets. Consistent with previous reports, GPR120 KO mice are hyperglycemic and glucose intolerant; however, our KO mice display evidence of a hyperactive counter-regulatory response rather than insulin resistance during insulin tolerance tests. An arginine stimulation test and a glucagon challenge confirmed both increases in glucagon secretion and liver glucagon sensitivity in GPR120 KO mice relative to WT mice. Our findings demonstrate that GPR120 is a nutrient sensor that is activated endogenously by both saturated and unsaturated long chain fatty acids and that an altered glucagon axis likely contributes to the impaired glucose homeostasis observed in GPR120 KO mice.     

Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells

Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic β cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic β cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic β cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.     

Free fatty acids administered into the colon promote the secretion of glucagon-like peptide-1 and insulin

We examined whether free fatty acids (FFAs) promote glucagon-like peptide-1 (GLP-1) secretion when administered into the intestinal tract. We found that an unsaturated long-chain FFA, alpha-linolenic acid (alpha-LA), resulted in increased plasma GLP-1 and insulin levels when administered into the colon. Such stimulatory effects were not apparent with either vehicle or a saturated middle-chain FFA, octanoic acid (OA). Concomitant with GLP-1 secretion, the administration of alpha-LA, but not vehicle or OA, also resulted in a significant increase in the population of pERK positive cells within the GLP-1 positive cells of the colonic mucosa. Moreover, colonic administration of alpha-LA into normal C3H/He mice caused a reduction in plasma glucose levels, as well as in type 2 diabetic model NSY mice. Our results indicate that the in vivo colonic administration of alpha-LA promotes secretion of incretin GLP-1 by activating the ERK pathway in L-cells and thereby enhances the secretion of insulin.     

Phenylalanine derivatives as GPR142 agonists for the treatment of Type II diabetes

GPR142 is a novel GPCR that is predominantly expressed in pancreatic β-cells. GPR142 agonists potentiate glucose-dependent insulin secretion, and therefore can reduce the risk of hypoglycemia. Optimization of our lead pyridinone-phenylalanine series led to a proof-of-concept compound 22, which showed in vivo efficacy in mice with dose-dependent increase in insulin secretion and a decrease in glucose levels.