Screening for bacterial enzymes synthesizing GPR119 agonist in cAMP-responsive cells

G protein-coupled receptor 119(GPR119),encoded by human GPR119 gene,is considered as a novel member of cannabinoid receptors.GPR119 is expressed predominantly in the pancreas and gastrointestinal tract of rodents and humans,as well as in the brain of rodents.GPR119 has also been shown to regulate incretin and insulin hormone secretion.Activation of GPR119 causes a reduction in food intake and lowers gain of body weight in rats[1].Targeting GPR119 with specific agonists has thus been attempted as a novel strategy to treat obesity and diabetes[2–4].In addition,GPR119 also plays an important role in bone metabolism,thus making GPR119 a potential target for bone resorption-associated diseases[5].     

家鸡似G蛋白偶联受体119基因的克隆与组织表达分析

G蛋白偶联受体119(GPR119)是A型视紫红质G蛋白偶联受体家族成员。在哺乳动物中,该基因高表达于胰腺β细胞和PP细胞以及小肠组织的内分泌L细胞,参与胰岛素释放调节。本文以家鸡Gallus gallus小肠cDNA为模板,首次克隆到似GPR119新基因,将其命名为cGPR119b基因。结果显示:cGPR119b基因的cDNA全长954 bp,编码具317个氨基酸的前体蛋白。将家鸡GPR119b前体蛋白氨基酸序列与绿头鸭Anas platyrhynchos、绿海龟Chelonia mydas、西部锦龟Chrysemys picta bellii和火鸡Meleagris gallopavo的序列进行比对,分别具有79.9%、61.0%、62.3%、95.5%的相似度。利用反转录PCR和RACE技术,家鸡GPR119b的5'-UTR亦获分离,该片段678 bp。本研究亦采用实时荧光定量PCR探究cGPR119b基因的组织表达,发现其在大脑、肝脏、肾脏、卵巢、精巢、垂体、胰腺、皮肤、脂肪、肺、十二指肠、空肠、回肠、盲肠、直肠等组织中有表达,其中,在肝脏、肾脏、盲肠和精巢的表达量较高,在胰腺、皮肤、脂肪和肺组织中仅有微量表达。本研究为进一步探究cGPR119b基因在家鸡中的生理功能奠定了基础。     

两个黄酮类化合物对GPR35受体激动活性的研究

GPR35受体是G蛋白偶联受体家族中的孤儿受体,它的很多生物学功能还不是很清楚.寻找发现GPR35受体的配体对于GPR35受体的生物学及药理学研究具有重要意义.本研究利用无标记细胞靶点药理学筛选技术,系统研究了天然黄酮类化合物对GPR35受体的激动活性,新发现了草质素和异补骨脂二氢黄酮具有GPR35受体激动活性,其中草质素EC50=(1.452±0.093)μmol/L,是最具药物开发潜力的一个GPR35受体激动剂.     

孤儿G蛋白偶联受体55在糖尿病性胃轻瘫小鼠发病中的作用

本文旨在探讨孤儿G蛋白偶联受体55(GPR55)在糖尿病性胃轻瘫(diabetic gastroparesis,DG)小鼠发病中的作用。采用链脲菌素(streptozotocin,STZ,65 mg/kg)腹腔注射制备小鼠DG模型。观察造模后小鼠体重及血糖含量变化,同时测定胃电图和酚红排空功能,用放射免疫分析法测定血浆胃动素(motilin,MTL)、胃泌素(gastrin,GAS)、血管活性肠肽(vasoactive intestinal peptide,VIP)、生长抑素(somatostatin,SS)水平,用Real-time PCR和Western blot测定胃组织GPR55表达,用免疫组织化学法观察胃组织GPR55分布,并观察GPR55激动剂溶血磷脂酰肌醇(lysophosphatidylinositol,LPI)对DG的治疗作用。结果显示,STZ组小鼠血糖于STZ注射后第4周末升至(26.6±1.2)mmol/L,体重明显减轻,胃电图慢波振幅下降,胃排空能力明显降低,而腹腔注射LPI可逆转STZ导致的上述变化;STZ小鼠血浆中MTL、GAS明显低于对照组(P0.01),而SS、VIP明显升高(P0.01),LPI可拮抗STZ对四种激素的影响;GPR55存在于正常小鼠胃组织,在DG时其表达明显上调,LPI不影响正常小鼠GPR55表达和分布,但可拮抗STZ对GPR55表达和分布的影响。以上结果提示,GPR55参与调节DG胃运动,可以作为DG治疗的新靶点;GPR55激动剂LPI对STZ导致的DG小鼠有保护作用,该作用机制与GPR55表达的改变、胃肠激素的调整有关。     

G蛋白偶联受体40研究进展

G蛋白偶联受体40(G protein-coupled receptor 40, GPR40)是一种具有7个跨膜α螺旋结构的G蛋白偶联受体, 主要分布于胰腺细胞和神经系统细胞。它能与机体内游离的中长链脂肪酸结合, 激活细胞内信号通路, 从而调节其生理功能。在胰岛细胞中, GPR40可被游离脂肪酸激活, 促使细胞内钙离子浓度升高, 进而促进胰岛素释放。根据这一机理, 以GPR40为靶点的激动剂类药物相继被开发, 用于糖尿病治疗。GPR40也参与神经发生过程, 但到目前为止其相关机制尚不清楚。文章从基因结构、表达调控、蛋白配体及应用、生理功能等方面详细介绍了GPR40的研究现状, 总结了目前研究中所遇到的问题, 并对未来的研究热点进行展望。     

GPR81激动剂对非酒精性脂肪肝病大鼠胰岛素抵抗的影响

目的: 探讨NOD样受体蛋白3(NLRP3)信号通路对非酒精性脂肪肝病(NAFLD)大鼠胰岛素抵抗的影响及乳酸受体G蛋白偶联受体81(GPR81)激动剂的干预作用。方法: 选择清洁级SD雄性大鼠30只,随机分为3组,对照组、NAFLD组、GPR81激动剂组,每组10只。用高脂饮食建立大鼠非酒精性脂肪肝模型;GPR81激动剂组:在非酒精性脂肪肝模型基础上腹腔注射GPR81特异性乳酸激动剂(50 nmol/L),每周1次,其余两组注射等量的生理盐水,共12周。测定肝生化指标、空腹血糖及胰岛素和肝匀浆中炎症因子的含量,观察各组肝组织病理学形态;Western blot检测肝组织中NLRP3、含CARD结构域的凋亡相关斑点蛋白(ASC)、天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)、胰岛素受体底物-1(IRS-1)、胰岛素受体底物酪氨酸磷酸化(Tyr⁴⁶⁵-IRS-1)、胰岛素受体底物丝氨酸磷酸化(Ser⁶³⁶-IRS-1)、葡萄糖转运蛋白4(GLUT4)的蛋白表达;qRT-PCR法检测肝组织NLRP3、ASC、caspase-1、IRS-1、GLUT4 mRNA表达水平。结果: 与对照组相比,NAFLD组大鼠血清肝生化指标甘油三酯(TG)、丙氨酸转氨酶(ALT)、天门冬氨酸氨基转移酶(AST)、空腹血糖(FPG)、空腹胰岛素(FINS)和胰岛素抵抗指数(HOMA-IR)值均显著升高(P＜0.05);肝组织病理学形态结果表明,NAFLD组大鼠肝组织可见明显的肝脂肪变性,肝细胞有脂肪滴,存在明显的炎性细胞浸润,且NAFLD组肝组织NLRP3、ASC、caspase-1的mRNA和蛋白表达及Ser⁶³⁶-IRS-1的蛋白表达均显著升高,且肝组织及血清中白细胞介素-1β(IL-1β)和白细胞介素-18(IL-18)的含量升高;而IRS-1、GLUT4 的mRNA和蛋白表达Tyr⁴⁶⁵-IRS-1的蛋白表达显著降低(P＜0.05);与NAFLD组相比,GPR81激动剂组上述指标均得到明显改善。结论: NLRP3信号通路活化介导炎症因子产生促进了NAFLD的发生发展,GPR81激动剂可能成为NAFLD潜在的治疗手段。     

孤儿受体GPR52晶体结构揭示配体结合新机制

GPR52受体是一种孤儿G蛋白偶联受体(G protein-coupled receptor, GPCR),在大脑中高度表达,是治疗精神疾病和亨廷顿病的潜在治疗靶点,其内源性配体仍不清楚。由于GPR52与任何已知GPCR结构的相似性很低(20%),无论是同源建模或是结构解析都存在很大困难。缺乏结构信息在很大程度上阻碍了工具配体和药物的发现。上海科技大学生命科学与技术学院i Human研究所徐菲课题组利用X射线晶体学解析了GPR52的三个高分辨率晶体结构——两个无配体结合的APO结构和GPR52结合激动剂小分子的复合物结构。这些结构揭示了GPR52独特的第二个胞外环、新颖的配体结合侧位口袋和特殊扭转的第五个跨膜α螺旋。突变与细胞功能实验验证了研究人员关于GPR52自激活机制的猜想,并提示第二个胞外环对受体的内在激活作用。这些发现为GPR52配体识别的结构基础提供了前所未有的见解,对寻求GPR52内源性配体即脱孤以及理性设计具有不同药理特性的配体化合物具有重要的指导意义。     

《美国科学院院刊》:科学家发现减肥植物的有效成分及机制

<正>中国科学院上海药物研究所谢欣课题组与中国科学院上海有机化学研究所俞飚课题组合作,发现了减肥植物蝴蝶亚仙人掌的有效成分及减肥机制。研究论文于9月22日在线发表于《美国科学院院刊》上。有文献报道,蝴蝶亚仙人掌中的一个化合物P57是其抑制食欲的主要活性成分,但其作用机制并不清楚。研究团队通过大量化合物筛选发现一个提取自蝴蝶亚仙人掌的化合物Gordonoside F,而不是P57,可以特异性激活与代谢密切相关的G蛋白偶联受体GPR119。Gordonoside F通过激活GPR119刺激胰高血糖素样肽-1(GLP-1)和胰岛素的分泌,并抑制小鼠食欲。     

神经介素S——一种新的食欲抑制肽

神经介素S（neuromedin S,NMS）是一种由36个氨基酸组成的新的神经肽。Takanori Ida等在2005年Endocrinology上报道在大鼠脑中发现NMS,主要在视交叉神经核上表达,是G蛋白偶联受体FM-3／GPR66和FM-4／TGR—l的配体,这些受体分别是神经介素U（neuromedin U,NMU）1型和2型。他们研究发现NMS是一种新的食欲调节肽。给大鼠侧脑室内注射NMS后,NMS可以剂量依赖性地降低大鼠夜间12小时的食物和水的摄入量．其摄含抑制作用较相同剂量的NMU的抑制作用强,持续时间久,     

阿必鲁肽：一种超长效Ⅱ型糖尿病治疗新药

阿必鲁肽(Albiglutide)为新型GLP-1受体激动剂,可通过与胰岛β细胞表面的GLP-1受体结合,激活由细胞膜偶联G蛋白调控的胞内信号传导系统,修复及促进胰岛β细胞功能,增加胰岛素的分泌,从而降低血糖浓度及糖化血红蛋白(Hb A1c)水平,是治疗Ⅱ型糖尿病的理想药物。目前开发的各种长效GLP-1受体激动剂成为近二十年糖尿病药物的研究热点。本文对一种极具市场潜力的新型GLP-1受体激动剂阿必鲁肽(Albiglutide)的分子结构、作用机制、剂量研究、药代动力学、药效学以及副作用等临床试验研究进展进行综述。     

Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled receptor

A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.     

Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice

G protein-coupled receptor (GPR) 119 is highly expressed in pancreatic β-cells and enhances the effect of glucose-stimulated insulin secretion (GSIS) on activation. The development of an oral GPR119 agonist that specifically targets the first phase of GSIS represents a promising strategy for the treatment of type 2 diabetes. In the present study, we evaluated the therapeutic potential of a novel small molecule GPR119 agonist, AS1535907, which was modified from the previously identified 2,4,6-tri-substituted pyrimidine core agonist AS1269574. AS1535907 displayed an EC₅₀ value of 4.8 μM in HEK293 cells stably expressing human GPR119 and stimulated insulin secretion in rat islets only under high-glucose (16.8 mM) conditions. In isolated perfused pancreata from normal rats, AS1535907 enhanced the first phase of insulin secretion at 16.8 mM glucose, but had no effect at 2.8 mM glucose. In contrast, the sulfonylurea glibenclamide predominantly induced insulin release in the second phase at 16.8 mM glucose and also markedly stimulated insulin secretion at 2.8 mM glucose. In in vivo studies, a single 10 μM administration of AS1535907 to diabetic db/db mice reduced blood glucose levels due to the rapid secretion of insulin secretion following oral glucose loading. These results demonstrate that GPR119 agonist AS1535907 has the ability to stimulate the first phase of GSIS, which is important for preventing the development of postprandial hypoglycemia. In conclusion, the GPR119 agonist AS1535907 induces a more rapid and physiological pattern of insulin release than glibenclamide, and represents a novel strategy for the treatment of type 2 diabetes.     

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.     

5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist and enhances glucose-dependent insulin secretion

GPR119 is one of the G-protein-coupled receptors expressed in pancreatic β-cells and intestinal endocrine cells. Since agonists to GPR119 stimulate glucose-dependent insulin secretion, GPR119 agonists are anticipated to promote anti-diabetic effects and control of glucose homeostasis. Here, we reported that an omega-3 unsaturated fatty acid metabolite, 5-hydroxy-eicosapentaenoic acid (5-HEPE), was a potent agonist for GPR119 and enhanced glucose-dependent insulin secretion. 5-HEPE stimulated cAMP accumulation in mouse MIN6 insulinoma cells and human HuTu80 intestinal adenocarcinoma cells. These effects were blunted by GPR119-specific siRNA. Recombinant GPR119 also responded to 5-HEPE as well as authentic agonists. Several previous reports have indicated the beneficial biological effects of omega-3 unsaturated fatty acids, and epidemiological studies have suggested that these fatty acids plays a protective role against diabetes. However, the molecular pharmacology and receptor identifications of omega-3 unsaturated fatty acids and their metabolites have not yet been well investigated. It is hoped that our findings will encourage novel investigations into the molecular relationships between omega-3 fatty acids and diabetes.     

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.     

Oleoyl-lysophosphatidylinositol enhances glucagon-like peptide-1 secretion from enteroendocrine L-cells through GPR119

The gastrointestinal tract is increasingly viewed as critical in controlling glucose metabolism, because of its role in secreting multiple glucoregulatory hormones, such as glucagon like peptide-1 (GLP-1). Here we investigate the molecular pathways behind the GLP-1- and insulin-secreting capabilities of a novel GPR119 agonist, Oleoyl-lysophosphatidylinositol (Oleoyl-LPI). Oleoyl-LPI is the only LPI species able to potently stimulate the release of GLP-1 in vitro, from murine and human L-cells, and ex-vivo from murine colonic primary cell preparations. Here we show that Oleoyl-LPI mediates GLP-1 secretion through GPR119 as this activity is ablated in cells lacking GPR119 and in colonic primary cell preparation from GPR119^?/? mice. Similarly, Oleoyl-LPI-mediated insulin secretion is impaired in islets isolated from GPR119^?/? mice. On the other hand, GLP-1 secretion is not impaired in cells lacking GPR55 in vitro or in colonic primary cell preparation from GPR55^?/? mice. We therefore conclude that GPR119 is the Oleoyl-LPI receptor, upstream of ERK1/2 and cAMP/PKA/CREB pathways, where primarily ERK1/2 is required for GLP-1 secretion, while CREB activation appears dispensable.     

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.     

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.     

Simvastatin Impairs Insulin Secretion by Multiple Mechanisms in MIN6 Cells

Statins are widely used in the treatment of hypercholesterolemia and are efficient in the prevention of cardiovascular disease. Molecular mechanisms explaining statin-induced impairment in insulin secretion remain largely unknown. In the current study, we show that simvastatin decreased glucose-stimulated insulin secretion in mouse pancreatic MIN6 β-cells by 59% and 79% (p<0.01) at glucose concentration of 5.5 mmol/l and 16.7 mmol/l, respectively, compared to control, whereas pravastatin did not impair insulin secretion. Simvastatin induced decrease in insulin secretion occurred through multiple targets. In addition to its established effects on ATP-sensitive potassium channels (p = 0.004) and voltage-gated calcium channels (p = 0.004), simvastatin suppressed insulin secretion stimulated by muscarinic M3 or GPR40 receptor agonists (Tak875 by 33%, p = 0.002; GW9508 by 77%, p = 0.01) at glucose level of 5.5 mmol/l, and inhibited calcium release from the endoplasmic reticulum. Impaired insulin secretion caused by simvastatin treatment were efficiently restored by GPR119 or GLP-1 receptor stimulation and by direct activation of cAMP-dependent signaling pathways with forskolin. The effects of simvastatin treatment on insulin secretion were not affected by the presence of hyperglycemia. Our observation of the opposite effects of simvastatin and pravastatin on glucose-stimulated insulin secretion is in agreement with previous reports showing that simvastatin, but not pravastatin, was associated with increased risk of incident diabetes.     

一个潜在的糖尿病新靶标——GPR40

G蛋白偶联受体40(GPR40)是典型的七次跨膜受体,在游离脂肪酸的刺激下,它能起到放大葡萄糖刺激的胰岛素分泌效应,是一种潜在的治疗糖尿病药物的靶标。另外,GPR40还被认为和一些神经类疾病以及某些癌症有关。本文着重叙述了游离脂肪酸经由GPR40放大葡萄糖刺激的胰岛素分泌机制,同时也介绍了GPR40的其他一些生理功能。