血管紧张素Ⅱ对RIN-m细胞PDX-1 mRNA及蛋白表达的影响

目的 探讨血管紧张素Ⅱ(ATⅡ) 对胰十二指肠同源盒1(PDX-1)表达的影响及其与胰岛素(Ins) 分泌的关系.方法 测定RIN-m细胞在基础、ATⅡ及氯沙坦作用下Ins 分泌量、细胞内Ins 含量及细胞内PDX-1 mRNA 和蛋白的表达水平.结果 在100nmol/L ATⅡ作用下, 葡萄糖刺激后Ins 分泌量、细胞内Ins 含量及PDX-1表达水平均明显降低(P均<0.05).上述作用可被ATⅡ受体阻滞剂氯沙坦部分逆转.结论 抑制PDX-1表达水平可能是ATⅡ影响胰岛β细胞Ins合成及分泌的机制之一.     

游离脂肪酸诱导的胰岛βTC3细胞缺陷与线粒体功能改变的相关性研究

目的通过研究游离脂肪酸(FFA)慢性作用对胰岛细胞解偶联蛋白2(UCP2)mRNA和蛋白表达以及线粒体膜电位的影响,探讨FFA损害胰岛功能的机制。方法将不同浓度油酸作用于胰岛βTC3细胞72h,分别分析基础和葡萄糖刺激的胰岛素分泌量(GSIS),线粒体膜电位,ATP敏感钾电流(IKATP)、UCP2mRNA和蛋白表达;再用油酸和PPARγ配体罗格列酮共同作用,观察UCP2mRNA和蛋白的表达。结果与对照组相比,油酸明显增加2·8mmol/L葡萄糖时的基础胰岛素分泌量,减少16·7mmol/L葡萄糖时的GSIS(P均<0·01),并且呈剂量依赖性。油酸可明显降低线粒体膜电位平均荧光强度(MIF)值,使IKATP外流增加,增加UCP2mRNA和蛋白表达(P均<0·01),呈剂量依赖性;与油酸组相比,油酸 罗格列酮组增加了UCP2mRNA和蛋白表达(P<0·01)。结论在胰岛βTC3细胞中,FFA对胰岛功能的损害与线粒体功能改变有关。FFA可能通过上调PPARγ增加UCP2表达,UCP2的表达与作用增加可导致线粒体膜电位降低,使线粒体功能下降,引起胰岛素分泌减少,产生对胰岛功能的损害作用。     

下调KLF5抑制AngⅡ诱导的心肌细胞肥大机制研究

目的探讨Krüpple样因子5(KLF5)对心肌细胞体积、细胞内总蛋白、氧化损伤及Ca~(2+)水平的影响。方法体外分离培养乳鼠心肌细胞,心肌细胞分为以下几组,对照组:不做任何处理,正常培养。模型组:在细胞培养液中添加100 nm的血管紧张素Ⅱ(AngⅡ)。阴性组:心肌细胞中转染siRNA阴性对照。干扰组:心肌细胞中转染KLF5 siRNA。阴性+AngⅡ组:心肌细胞中转染siRNA阴性对照,并在细胞培养液中添加100 nm的AngⅡ。干扰+AngⅡ组:心肌细胞中转染KLF5 siRNA,并在细胞培养液中添加100 nm的AngⅡ。分别检测KLF5的基因和蛋白表达水平。在心肌细胞内转染KLF5 siRNA,同时用AngⅡ处理,测定心肌细胞的体积和心肌细胞内总蛋白的含量,同时检测细胞内肥大基因心房利钠肽(ANP)mRNA的水平,Western blot方法测定细胞内钙调神经磷酸酶(CaN)蛋白水平,激光共聚焦显微镜检测静息状态下Ca~(2+)水平,二硝基苯肼法测定培养液中乳酸脱氢酶(LDH)水平,硫代巴比妥酸法测定丙二醛(MDA)含量,黄嘌呤法测定超氧化物歧化酶(SOD)含量。结果模型组心肌细胞中KLF5基因和蛋白表达水平高于对照组(P0.05)。干扰组心肌细胞中KLF5水平低于对照组(P0.05)。阴性组和对照组心肌细胞中KLF5水平没有差异(P0.05)。模型组心肌细胞的体积增加,细胞总蛋白含量也增加,细胞内肥大基因ANP mRNA的水平也升高,CaN蛋白表达和静息状态下Ca~(2+)水平上调,细胞内生成的MDA增加,SOD活性降低,细胞培养液中LDH水平升高,与对照组相比,差异有统计学意义(P0.05)。干扰+AngⅡ组心肌细胞体积和蛋白含量下降,细胞内肥大基因ANP mRNA的水平降低,CaN蛋白表达和静息状态下Ca~(2+)水平下降,细胞生成的MDA减少,SOD活性升高,细胞培养液中LDH水平降低,与模型组比较,差异有统计学意义(P0.05)。阴性+AngⅡ组细胞体积、蛋白含量、细胞内肥大基因ANP mRNA、CaN蛋白、静息状态下Ca~(2+)水平、细胞生成的MDA、SOD活性、细胞培养液中LDH水平与模型组比较没有明显差异(P0.05)。结论 AngⅡ诱导心肌细胞表达KLF5,KLF5下调可抑制AngⅡ诱导的心肌细胞肥大,其作用机制可能与心肌细胞氧化损伤和Ca~(2+)水平有关。     

血管紧张素Ⅱ对人脐静脉内皮细胞p38丝裂素激活蛋白激酶通路的作用

目的探讨血管紧张素Ⅱ(AngⅡ)激活人脐静脉内皮细胞(HUVECs)p38丝裂素激活蛋白激酶(p38MAPK)信号转导通路的作用。方法用含20%胎牛血清的DMEM培养基与CO_2培养箱(5%CO_2+95%空气)培养HUVECs。待细胞生长至80%融合时,无血清培养16h后分组:(1)AngⅡ不同时点观察组:用AngⅡ(终浓度100 nmol/L)分别刺激细胞0、5、10、15、30、45 min和60 min。(2)AngⅡ不同剂量作用组:分别用终浓度为0、10、100、1000 nmol/L和10 000 nmol/L的AngⅡ刺激细胞15 min。(3)AngⅡ+p38MAPK特异性抑制剂SB202190组:在AngⅡ(终浓度100 nmol/L)刺激前30 min,分别将1000 nmol/L和5000 nmol/L(终浓度)的SB202190加入培养基,共同孵育30 min。上述各组作用一定时间后收集细胞,用Western blot方法测定细胞p38MAPK磷酸化表达。结果 AngⅡ(100 nmol/L)可诱导HUVECs p38MAPK磷酸化表达,15～30 min达到高峰,分别升高2.25和2.51倍(P<0.005,n=5),随时间呈峰形变化;AngⅡ呈剂量依赖性诱导HUVECs p38MAPK磷酸化,在AngⅡ100 nmol/L时p38MAPK磷酸化表达即有明显增强,在AngⅡ刺激剂量为1000 nmol/L和10 000 nmol/L时,p38MAPK磷酸化表达分别增加2.03和2.11倍(P<0.005,n=5);p38MAPK特异性抑制剂SB202190可显著抑制HUVECs p38MAPK磷酸化,且呈剂量依赖性,5000 nmol/L SB202190的抑制率为53.9%(P<0.01,n=6)。结论 AngⅡ可激活人脐静脉内皮细胞p38MAPK信号通路。     

SIRT1/UCP2通路在脂肪变性HepG2细胞线粒体能量代谢中的调控机制

目的采用油酸诱导Hep G2细胞发生脂变,建立脂肪变性细胞模型,观察SIRT1/UCP2通路在脂肪变性Hep G2细胞能量代谢中的调控机制。方法采用含有2 mmol/L油酸的DMEM培养基诱导Hep G2细胞24 h后,建立脂肪变性Hep G2细胞模型,并设置对照组比较。以油红O染色观察细胞内脂滴形成状况,并用全自动生化仪检测细胞上清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)含量和细胞内甘油三酯(TG)含量;采用流式细胞仪测定线粒体膜电位的改变,采用磷钼酸比色试剂盒测定细胞内三磷酸腺苷(ATP)含量,运用Western印迹法检测各组细胞SIRT1和UCP2蛋白的表达。结果与对照组比较,模型组细胞内橘红色脂滴大量形成,且TG含量明显升高(P0.01),线粒体膜电位及细胞内ATP含量显著降低(P0.05,P0.01),SIRT1蛋白表达均显著降低(P0.05),UCP2蛋白表达显著升高(P0.01)。结论油酸诱导的脂肪变性Hep G2细胞模型可以出现脂质代谢紊乱和线粒体能量代谢失衡,其机制可能与细胞内SIRT1/UCP2通路的激活有关。     

不同糖耐量水平人群胰岛素抵抗与RAAS及MCP-1的相关性研究

目的探讨不同糖耐量人群的肾素-血管紧张素-醛固酮系统(RAAS)、单核细胞趋化因子-1(MCP-1)与胰岛素抵抗的关系。方法纳入新诊断的正常糖耐量组(NGT组,43例)、糖调节受损组(IGR组,41例)及2型糖尿病组(T2DM组,44例)共128例观察对象,用ELISA法测定3组人群肾素、血管紧张素Ⅱ(AngⅡ)、醛固酮的活性及血清MCP-1水平。结果 T2DM组、IGR组MCP-1、胰岛β细胞功能指数(HOMA-β)与NGT组比较,差异均有统计学意义(P0.05),3组人群的胰岛素抵抗指数(HOMA-IR)、AngⅡ比较差异均有统计学意义(P0.05);3组人群肾素、醛固酮、血浆醛固酮/肾素活性(ARR)比较差异无统计学意义(P0.05)。相关性分析显示,MCP-1(r=0.266,P=0.002)、AngⅡ(r=0.27,P=0.002)与HOMA-IR呈正相关,且MCP-1与AngⅡ之间也呈正相关(r=0.485,P=0.000)。结论 AngⅡ、MCP-1与胰岛素抵抗密切相关,其共同参与了2型糖尿病胰岛素抵抗的病情进展。     

血管紧张素Ⅱ对人肝细胞白蛋白和胶原合成的影响

目的 观察血管紧张素Ⅱ(AngⅡ)对人正常肝细胞白蛋白表达及Ⅰ型胶原合成的影响.方法 常规培养人正常肝细胞系HL-7702细胞并分为对照组、AngⅡ刺激组、AngⅡ十依贝沙坦组(共刺激组).采用免疫荧光法和Western印迹法检测各组肝细胞中白蛋白及胶原改变;免疫荧光法观察各组肝细胞中是否有胶原合成;实时定量PCR法定量检测各组肝细胞中Ⅰ型胶原mR-NA表达水平的变化.结果 与对照组相比,经AngⅡ(10-7mol/L)处理72 h后,AngⅡ刺激组肝细胞中白蛋白表达减少(1.41±0.23比0.85±0.11,P=0.000),Ⅰ型胶原mRNA的表达明显升高(1.00±0.08比3.72±0.19,P=0.000),Ⅰ型胶原蛋白合成增加,而经依贝沙坦预处理后,共刺激组肝细胞白蛋白表达较AngⅡ刺激组明显增多(0.85±0.11比1.38±0.32,P=0.000),肝细胞Ⅰ型胶原mRNA表达较AngⅡ刺激组显著下降(3.72±0.19比2.86±0.13,P=0.000),Ⅰ型胶原蛋白合成减少.结论 AngⅡ经Ⅰ型受体诱导人肝细胞表达白蛋白减少,胶原合成增加.     

血管紧张素Ⅱ和血管紧张素1-7对内皮细胞释放组织纤溶酶原激活物及其抑制剂-1的影响

目的 :研究血管紧张素Ⅱ (AngⅡ )和血管紧张素 1 7[Ang ( 1 7) ]对内皮细胞分泌组织纤溶酶原激活物 (t PA)和纤溶酶原激活物抑制剂 1(PAI 1)的影响。方法 :培养内皮细胞株 (ECV30 4 ) ,分为AngⅡ和Ang ( 1 7)刺激组 ,培养基中AngⅡ和Ang ( 1 7)加至终浓度为 5、10、2 5、5 0、10 0nmol/L ,2 4h后测定上清液中的t PA和PAI 1的含量。结果 :内皮细胞在AngⅡ终浓度为 5 0、10 0nmol/L组刺激 2 4h后 ,其分泌的PAI 1含量较对照组有明显升高 ,差异有统计学意义 (P <0 .0 5 )。而Ang ( 1 7)在同样浓度组却显著降低PAI 1(P <0 .0 5 ) ,AngⅡ和Ang ( 1 7)两组t PA含量差异无统计学意义 ( P >0 .0 5 )。结论 :AngⅡ对内皮细胞株分泌的PAI 1有显著的升高作用 ,而Ang ( 1 7)对PAI 1作用相反。两者对t PA均无显著影响。提示AngⅡ和Ang ( 1 7)对纤溶系统的调节有一定作用     

胰岛素样生长因子1抑制高糖诱导大鼠胃平滑肌细胞内质网应激及其机制的研究

目的探讨高糖状态下胰岛素样生长因子1(IGF-1)对大鼠胃平滑肌细胞内质网应激(ERS)及蛋白激酶Cα(PKCα)/蛋白激酶Cβ1(PKCβ1)-钙离子(Ca~(2+))的影响及意义。方法体外培养原代大鼠胃平滑肌细胞,分为低糖(Con)组、高糖组(HG)、Con+IGF-1组、HG+IGF-1组、HG+PKCβ1抑制剂组(HG+RH)。激光共聚焦显微镜观察各组细胞内Ca~(2+)变化,Western blot法检测葡萄糖调节蛋白78(GRP78)、C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)、PKCα、p-PKCα、PKCβ1及p-PKCβ1的表达。结果与Con组比较,HG组GRP78、CHOP、Ca~(2+)浓度、p-PKCβ1/PKCβ1比值升高(P0.05或P0.01)。与HG组比较,HG+IGF-1、HG+RH组GRP78、CHOP、Ca~(2+)浓度、p-PKCβ1/PKCβ1降低(P0.05或P0.01)。与HG+IGF-1组比较,HG+RH组Ca~(2+)浓度增加(P0.05)。各组p-PKCα/PKCα比较,差异无统计学意义(P0.05)。结论高糖状态下IGF-1通过稳定PKCα活性及降低PKCβ1活性下调细胞内Ca~(2+)水平,抑制大鼠胃平滑肌细胞ERS。     

辛伐他汀抑制大鼠胰岛β细胞胰岛素分泌的机制研究

目的 观察辛伐他汀对大鼠胰岛β细胞葡萄糖刺激的胰岛素分泌(GSIS)的抑制作用及其机制.方法 8周龄健康雄性Wistar大鼠60只,体重250～300 g,饲养1周内分批处死.采用胆管注射胶原酶法提取大鼠胰岛,将新鲜分离或经24 h培养的大鼠胰岛按体积大小分为对照组(胰岛数=60)和辛伐他汀组(胰岛数=60),对照组给予Kreb-Ringer碳酸氢盐缓冲液,辛伐他汀组以100μmol/L辛伐他汀水浴30 min或过夜培养24 h.两组经2.8、5.5、11.1、16.7、25.0 mmol/L葡萄糖刺激后,采用37 ℃水浴法观测胰岛β细胞GSIS变化,选用生物化学发光法测定腺苷三磷酸(ATP)含量.两组间数据比较采用t检验.结果 100 μmol/L辛伐他汀水浴30 min后,在25.0 mmol/L葡萄糖刺激下,胰岛β细胞ATP含量较相应对照组明显下降[(9.2±1.6)vs(12.1±1.9)pmol/胰岛,t=2.97,P＜0.05],GSIS较相应对照组明显减少(2.31±0.38 vs 3.19±0.41,t=3.154,P＜0.05).100μmol/L辛伐他汀过夜培养24 h后,在11.1 mmol/L葡萄糖刺激下,胰岛β细胞ATP含量较相应对照组明显降低[(9.2±1.4)vs(11.9±2.0)pmol/胰岛,t=2.514,P＜0.05];在2.8 mmol/L葡萄糖刺激下,胰岛素分泌即已明显受到抑制(0.28±0.03 vs 0.47±0.05,t=2.460,P＜0.05).辛伐他汀浓度超过30 μmol/L时,可剂量依赖性地抑制GSIS(2.49±0.21 vs 3.17±0.23,t=2.445,P＜0.05).结论 高浓度辛伐他汀可能通过抑制胰岛β细胞ATP生成而抑制GSIS.     

Reactive oxygen species and uncoupling protein 2 in pancreatic β-cell function

Growing evidence indicates that reactive oxygen species (ROS) are not just deleterious by-products of respiratory metabolism in mitochondria, but can be essential elements for many biological responses, including in pancreatic β-cells. ROS can be a 'second-messenger signal' in response to hormone/receptor activation that serves as part of the 'code' to trigger the ultimate biological response, or it can be a 'protective signal' to increase the levels of antioxidant enzymes and small molecules to scavenge ROS, thus restoring cellular redox homeostasis. In pancreatic β-cells evidence is emerging that acute and transient glucose-dependent ROS contributes to normal glucose-stimulated insulin secretion (GSIS). However, chronic and persistent elevation of ROS, resulting from inflammation or excessive metabolic fuels such as glucose and fatty acids, may elevate antioxidant enzymes such that they blunt ROS and redox signalling, thus impairing β-cell function. An interesting mitochondrial protein whose main function appears to be the control of ROS is uncoupling protein 2 (UCP2). Despite continuing investigation of the exact mechanism by which UCP2 is 'activated', it is clear that UCP2 levels and/or activity impact the efficacy of GSIS in pancreatic islets. This review will focus on the paradoxical roles of ROS in pancreatic β-cell function and the regulatory role of UCP2 in ROS signalling and GSIS.     

β−cell adaptation/dysfunction in an animal model of dyslipidemia and insulin resistance induced by the chronic administration of a sucrose-rich diet

Glucose stimulated insulin secretion (GSIS) was different in rats chronically fed a sucrose-rich diet (SRD) for 3 or 30 wk. This work proposes possible mechanisms underlying insulin secretion changes from β-cell throughout these feeding periods. In isolated islets of rats fed the SRD or a control diet (CD) we examined: 1- the glucokinase and hexokinase activities and their protein mass expression; 2- pyruvate dehydrogenase activity; 3- uncoupling protein 2 (UCP2) and peroxisome proliferators-activated receptor γ (PPAR γ) protein mass expression. At 3 wk on diet the SRD-fed rats showed: a marked increase in the first peak of GSIS; increased glucokinase protein mass expression without changes in glucokinase and hexokinase activities; increased PPARγ protein mass expression without changes in the UCP2 protein mass expression. No changes in either glucose oxidation and triglyceride content within the β-cell were observed. After 30 wk of feeding, a significant decrease of both glucokinase activity and its protein mass expression was accompanied by altered glucose oxidation, a triglyceride increase within the β-cell and a significant increase of PPARγ and UCP2 protein mass expression. Moreover GSIS depicted an absence of the first peak with an increase in the second phase. Finally, the SRD chronic administration altered GSIS by different mechanisms depending on the time on diet. At an early stage, the increased protein mass expression of the glucokinase and a fatty acid cooperative effect inducing PPARγ expression seem to be the mechanisms involved. At a late stage, glucolipotoxicity appears to be the cellular mechanism contributing to progressive β-cell dysfunction.     

β-Cell adaptation/dysfunction in an animal model of dyslipidemia and insulin resistance induced by the chronic administration of a sucrose-rich diet

Glucose stimulated insulin secretion (GSIS) was different in rats chronically fed a sucrose-rich diet (SRD) for 3 or 30 wk. This work proposes possible mechanisms underlying insulin secretion changes from β-cell throughout these feeding periods. In isolated islets of rats fed the SRD or a control diet (CD) we examined: 1- the glucokinase and hexokinase activities and their protein mass expression; 2- pyruvate dehydrogenase activity; 3- uncoupling protein 2 (UCP2) and peroxisome proliferators-activated receptor γ (PPAR γ) protein mass expression. At 3 wk on diet the SRD-fed rats showed: a marked increase in the first peak of GSIS; increased glucokinase protein mass expression without changes in glucokinase and hexokinase activities; increased PPARγ protein mass expression without changes in the UCP2 protein mass expression. No changes in either glucose oxidation and triglyceride content within the β-cell were observed. After 30 wk of feeding, a significant decrease of both glucokinase activity and its protein mass expression was accompanied by altered glucose oxidation, a triglyceride increase within the β-cell and a significant increase of PPARγ and UCP2 protein mass expression. Moreover GSIS depicted an absence of the first peak with an increase in the second phase. Finally, the SRD chronic administration altered GSIS by different mechanisms depending on the time on diet. At an early stage, the increased protein mass expression of the glucokinase and a fatty acid cooperative effect inducing PPARγ expression seem to be the mechanisms involved. At a late stage, glucolipotoxicity appears to be the cellular mechanism contributing to progressive β-cell dysfunction.     

The role of peroxidation of mitochondrial membrane phospholipids in pancreatic β -cell failure

Type 2 diabetes (T2D) is characterized by peripheral insulin resistance and pancreatic islet β-cell failure. Accumulating evidence indicates that mitochondrial dysfunction is a central contributor to β-cell failure in the pathogenesis of T2D. This review focuses on mechanisms whereby reactive oxygen species (ROS) produced by β-cell in response to metabolic stress affect mitochondrial structure and function and lead to β-cell failure. Specifically, ROS oxidize mitochondrial membrane phospholipids such as cardiolipin, which impairs membrane integrity and leads to cytochrome c release and apoptosis. In addition, ROS activate UCP2 via peroxidation of the mitochondrial membrane phospholipids, which results in proton leak leading to reduced ATP synthesis and content in β-cells - critical parameters in the regulation of glucose-stimulated insulin secretion. Group VIA Phospholipase A2 (iPLA2β) appears to be a component of a mechanism for repairing mitochondrial phospholipids that contain oxidized fatty acid substituents, and genetic or acquired iPLA2β-deficiency increases β-cell mitochondrial susceptibility to injury from ROS and predisposes to development of T2D. Interventions that attenuate the adverse effects of ROS on β-cell mitochondrial phospholipids may prevent or retard the development of T2D.     

一氧化氮和血管紧张素Ⅱ在原发性高血压中的作用

目的：探讨一氧化氮（NO）和血管紧张素Ⅱ（AngⅡ）与原发性高血压（EH）的关系。方法：分别选择EH合并左室肥厚（LVH）患者（EH＋LVH组）、EH无LVH患者（EH组）及正常人（正常对照组）各30例,检测NO、AngⅡ水平,并进行组间比较分析。结果：（1）EH＋LVH组NO水平明显低于EH组、正常对照组[（37.24±11.27）μmol/L比（51.79±20.04）μmol/L比（80.25±20.58）μmol/L],且EH组NO水平明显低于正常对照组（P均〈0.01）;EH＋LVH组AngⅡ水平明显高于EH组、正常对照组[（198.37±93.54）ng/L比（139.87±56.39）ng/L比（57.34±18.85）ng/L],且EH组AngⅡ水平明显高于正常对照组（P〈0.05～〈0.01）;（2）直线相关分析显示收缩压、舒张压与NO呈负相关（r=-0.448、P=0.000;r=-0.249,P=0.018）,与AngⅡ呈正相关（r=0.491,P=0.000;r=0.265,P=0.012）,NO与AngⅡ呈负相关（r=-0.555,P=0.000）。结论：一氧化氮、血管紧张素Ⅱ参与了原发性高血压发病的病理生理过程。     

Glucagon-like peptide-1 enhances glucokinase activity in pancreatic β-cells through the association of Epac2 with Rim2 and Rab3A

Glucokinase (GK), which phosphorylates D-glucose, is a major glucose sensor in β-cells for glucose-stimulated insulin secretion (GSIS) and is a promising new drug target for type 2 diabetes (T2D). In T2D, pancreatic β-cells exhibit defective glucose sensitivity, which leads to impaired GSIS. Although glucagon-like peptide-1-(7-36)-amide (GLP-1) is known to enhance β-cell glucose sensitivity, the effect of GLP-1 on GK activity is still unknown. The present study demonstrated that GLP-1 pretreatment for 30 min significantly enhanced GK activity in a glucose-dependent manner, with a lower Michaelis-Menten constant (K(m)) but unchanged maximal velocity (V(max)). Thus, GLP-1 acutely enhanced cellular glucose uptake, mitochondrial membrane potential, and cellular ATP levels in response to glucose in rat INS-1 and native β-cells. This effect of GLP-1 occurred via its G protein-coupled receptor pathway in a cAMP-dependent but protein kinase A-independent manner with evidence of exchange protein activated by cAMP (Epac) involvement. Silencing Epac2, interacting molecule of the small G protein Rab3 (Rim2), or Ras-associated protein Rab3A (Rab3A) significantly blocked the effect of GLP-1. These results suggested that GLP-1 can further potentiate GSIS by enhancing GK activity through the signaling of Epac2 to Rim2 and Rab3A, which is the similar pathway for GLP-1 to potentiate Ca(2+)-dependent insulin granule exocytosis. The present finding may also be an important mechanism of GLP-1 for recovery of GSIS in T2D.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1^-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1^-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1^-/- islets and in Bmal1 knockdown cells, as compared with controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1^-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1^-/- islets. This defect in GSIS occurs as a result of increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2 in isolated islets leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1^-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.     

Loss of Bmal1 leads to uncoupling and impaired glucose-stimulated insulin secretion in β-cells

The circadian clock has been shown to regulate metabolic homeostasis. Mice with a deletion of Bmal1, a key component of the core molecular clock, develop hyperglycemia and hypoinsulinemia, suggesting β-cell dysfunction. However, the underlying mechanisms are not fully known. In this study, we investigated the mechanisms underlying the regulation of β-cell function by Bmal1. We studied β-cell function in global Bmal1-/- mice, in vivo and in isolated islets ex vivo, as well as in rat insulinoma cell lines with shRNA-mediated Bmal1 knockdown. Global Bmal1-/- mice develop diabetes secondary to a significant impairment in glucose-stimulated insulin secretion (GSIS). There is a blunting of GSIS in both isolated Bmal1-/- islets and in Bmal1 knockdown cells, as compared to controls, suggesting that this is secondary to a loss of cell-autonomous effect of Bmal1. In contrast to previous studies, in these Bmal1-/- mice on a C57Bl/6 background, the loss of stimulated insulin secretion, interestingly, is with glucose but not to other depolarizing secretagogues, suggesting that events downstream of membrane depolarization are largely normal in Bmal1-/- islets. This defect in GSIS occurs as a result increased mitochondrial uncoupling with consequent impairment of glucose-induced mitochondrial potential generation and ATP synthesis, due to an upregulation of Ucp2. Inhibition of Ucp2, in isolated islets, leads to a rescue of the glucose-induced ATP production and insulin secretion in Bmal1-/- islets. Thus, Bmal1 regulates mitochondrial energy metabolism to maintain normal GSIS and its disruption leads to diabetes due to a loss of GSIS.