INS-1E细胞葡萄糖毒性模型的建立

目的:建立胰岛细胞系INS-1E细胞的葡萄糖毒性模型。方法:将INS-1E细胞分别在不同葡萄糖浓度(5.5 mmol/L、16.7mmol/L、25 mmol/L、30 mmol/L)的1640完全培养基中培养不同时间(48 h、72 h、96 h、120 h),分别在不同时间点取细胞进行细胞功能检测,实时荧光定量PCR法检测胰岛素m RNA的表达,ELISA检测葡萄糖刺激的胰岛素的分泌。结果:与对照组相比,高糖浓度(5.5 mmol/L、16.7 mmol/L、25 mmol/L、30 mmol/L)培养基中培养48 h后,INS-1E细胞的胰岛素合成和分泌的功能均增加(P均0.05),随着培养基中葡萄糖浓度的升高以及培养时间的延长,INS-1E细胞胰岛素合成及分泌的功能逐渐下降,当在葡萄糖浓度为30 mmol/L的培养基中培养120 h后,胰岛素m RNA合成及葡萄糖刺激的胰岛素分泌均显著降低(P均0.01)。结论:INS-1E细胞在30 m M的葡萄糖中培养120 h形成稳定的葡萄糖毒性模型。     

GSK-3在阿尔茨海默病样细胞骨架蛋白过度磷酸化中的作用(英)

神经原纤维缠结是阿尔茨海默病（Alzheimer disease, AD）的特征性病理改变.蛋白激酶和蛋白磷酸酯酶失衡可导致骨架蛋白的异常过度磷酸化,而异常过度磷酸化的tau 和神经丝 (neurofilament, NF) 是神经原纤维缠结的组成部分.在众多激酶中,糖原合酶激酶-3（glycogen synthase kinase-3,GSK-3）可能是AD神经退行性变起重要作用.为深入探讨GSK-3在AD样神经退行性变中的作用,以磷酯酰肌醇三磷酸激酶（phosphatidylinositol 3-kinase,PI3K）的特异性抑制剂渥曼青霉素（wortmannin,WT）处理野生型鼠成神经瘤细胞株（wild type mouse neuroblastoma cell lines, N2a wt）,系统观察WT处理N2a wt不同时间点（1 h、3 h、6 h）细胞代谢率、细胞形态、细胞骨架蛋白tau和NF的磷酸化状态改变以及细胞的命运,并分析了GSK-3活性与上述参数改变之间的相关性.结果发现：1 μmol/L WT处理细胞1 h,GSK-3活性与未经WT处理的对照组相比明显增高,并伴有Ser9磷酸化的GSK-3水平的降低; NF磷酸化程度增强,tau在Ser198/Ser199/Ser202位点的磷酸化增强. 1 μmol/L WT处理细胞3 h,GSK-3活性与对照组和处理1 h 组相比明显下降,NF磷酸化程度较1 h降低,但仍高于正常水平.1 μmol/L WT处理细胞6 h,细胞形态、GSK-3活性、Ser9磷酸化形式的GSK-3β的表达、NF磷酸化程度与对照组相比均无明显改变.WT呈剂量依赖性降低细胞代谢率.1 μmol/L WT处理细胞1 h和3 h导致细胞变圆,突起变短甚至消失.1 μmol/L WT处理细胞1 h,用TUNEL法和电子显微镜技术未观察到细胞凋亡.研究结果提示：在N2a细胞中过度激活GSK-3可导致神经细丝和tau蛋白的AD样过度磷酸化,从而引起神经细胞的AD样退行性变.     

CD36基因缺失改善高脂饮食诱导的糖代谢异常并促进肝脏脂质积聚

本研究旨在探讨在高脂饮食状态下CD36基因缺失对小鼠糖脂代谢的影响及作用机制。根据基因型将小鼠分为野生型小鼠(wild type, WT)及CD36基因敲除(CD36~(-/-))小鼠,给予高脂饮食喂养14周。小鼠腹腔注射葡萄糖(1 g/kg)或胰岛素(5units/kg)进行葡萄糖耐量或胰岛素耐量测试。HE染色观察肝脏脂质变性,全自动生化分析仪测定小鼠血清甘油三酯(triglyceride, TG)、血清游离脂肪酸(free fatty acid, FFA)、天门冬氨酸转氨酶(aspartate aminotransferase, AST)和丙氨酸转氨酶(alanine aminotransferase, ALT)浓度。Real-time PCR和Western blot检测小鼠肝脏、肌肉组织胰岛素信号通路。Real-time PCR检测小鼠原代肝细胞中磷酸烯醇式丙酮酸羧激酶(phosphoenolpyruvate carboxykinase, PEPCK)的mRNA水平,葡萄糖检测试剂盒检测糖异生能力。免疫共沉淀(co-immunoprecipitation, Co-IP)及ELISA检测肌肉胰岛素受体β(insulin receptorβ, IRβ)酪氨酸磷酸化水平。Real-time PCR和免疫荧光染色检测小鼠肌肉葡萄糖转运蛋白4 (glucose transporter 4, GLUT4)的表达和定位。结果显示,在高脂喂养后,CD36~(-/-)小鼠血清FFA、TG、AST及ALT水平较WT小鼠明显升高(P 0.05),CD36~(-/-)小鼠肝脏外观呈脂肪样变性,HE染色结果显示肝脏脂质积聚加重,提示CD36缺失促进脂肪肝的发生。然而,相对于WT小鼠,CD36~(-/-)小鼠的空腹血糖水平降低、糖耐量升高,胰岛素耐量降低(P 0.05),提示在高脂饮食喂养条件下,CD36缺失并不会损害小鼠的糖耐量和胰岛素耐量。与WT小鼠相比,CD36~(-/-)小鼠肝脏IR/IRS/AKT胰岛素信号通路无显著差异,两组小鼠原代肝细胞PEPCK表达水平及糖异生能力均无显著差异。而在CD36~(-/-)小鼠肌肉组织中,Co-IP及ELISA实验显示IRβ酪氨酸磷酸化水平显著升高,p-AKT水平显著升高(P 0.05)。免疫荧光染色实验提示肌肉GLUT4在细胞膜的定位增强,表明CD36~(-/-)小鼠肌肉胰岛素敏感性及葡萄糖利用能力增强。以上结果提示,CD36基因缺失加重高脂饮食诱导的肝脏脂质积聚,对高脂饮食诱导的肝脏糖代谢无显著影响;CD36缺失主要通过提高肌肉组织胰岛素敏感性,促进GLUT4介导的葡萄糖利用以改善高脂饮食诱导的小鼠糖代谢异常。     

1型糖尿病葡萄糖代谢与胰岛素信号传导途径异常导致大鼠海马Alzheimer样tau蛋白过度磷酸化修饰

Tau蛋白过度磷酸化是Alzheimer病(Alzheimer disease, AD)的一个重要病理特征.采用 I 型糖尿病大鼠模型,研究胰岛素信号传导途径及葡萄糖代谢失调对tau蛋白过度磷酸化的形成机制进行探讨.以同龄Wistar大鼠做对照（CTL）,胰腺大部分切除造低胰岛素组（PX）,STZ较大剂量一次性注射造1型糖尿病模型即低胰岛素高血糖组（T1DM）.葡萄糖氧化酶法检测血浆血糖,放免法检测血浆胰岛素,蛋白质印迹分析海马内总tau蛋白及tau蛋白上部分位点（Ser199、Thr212、Ser214、Ser396及Ser422）的磷酸化及神经细胞膜上葡萄糖转运子3（Glucose transport 3,GLUT3）水平.γ-32P-ATP和特异性底物肽检测海马内胰岛素信号传导系统中的关键酶糖原合成酶激酶-3β（Glycogen synthase kinase-3β, GSK-3β）活性.发现3组大鼠海马回总tau蛋白水平无显著差异,但以高血糖、低胰岛素血症为特征的T1DM组在tau蛋白Ser199、Thr212、Ser214、Ser396及Ser422位点上,呈现过度磷酸化状态,以低胰岛素血症为特征而血糖正常的PX组在位点Ser199、Thr212及Ser396上磷酸化程度比CTL组显著上升, 在位点Ser214及 Ser422上的磷酸化程度的改变不显著;T1DM及PX组大鼠海马 GSK-3β活性显著高于CTL组, 而GLUT3水平在T1DM和PX组均降低, 尤以T1DM组降低更显著.研究结果显示,胰岛素水平低下可能通过激活GSK-3β和下调细胞内葡萄糖代谢的双重作用引起脑内tau蛋白过度磷酸化.     

GAS6/AXL信号通路失活对小鼠能量代谢的影响

目的研究小鼠生长停滞特异蛋白6(growth arrest-specific gene 6,GAS6)信号通路的失活对维持机体能量代谢稳态的影响。方法以GAS6主要受体Axl基因敲除(Axl-/-)小鼠及其野生型(Axl+/+)小鼠为研究对象,比较两组动物基础血糖值、血脂四项指标、脂肪系数及骨骼肌中糖代谢信号蛋白PI3K、AKT与葡萄糖转运蛋白4(glucose transporter 4,GLUT4)基因表达水平及其蛋白磷酸化水平间的差异;同时检测人工诱发2型糖尿病(type2 diabetes mellitus,T2DM)造模前后小鼠血清GAS6蛋白水平与T2DM模型成模率的改变之间是否存在相关性。结果 Axl-/-小鼠血脂出现异常波动,且脂肪沉积率显著高于野生型小鼠(P0.01)。Axl-/-小鼠血糖调节能力受损,其空腹血糖值显著高于野生型,骨骼肌Glut4的mRNA水平升高,而PI3K、AKT和GLUT4蛋白的磷酸化水平均略有下降。经人工诱发T2DM后,Axl+/+和Axl-/-小鼠血浆中GAS6浓度均显著低于各自对照组,且Axl-/-小鼠T2DM模型的成模率是Axl+/+小鼠的2倍(P0.01)。结论 GAS6/AXL信号通路的激活在一定程度上降低血糖和抑制脂肪沉积。     

膳食因素和运动对脑内葡萄糖转运蛋白Glut1、Glut4基因表达的影响

目的：探讨运动和膳食因素对小鼠脑内葡萄糖转运蛋白Glut1和Glut4基因表达的影响。方法：ICR小鼠分组施以高脂膳食、限量摄食、运动、高脂膳食＋运动等不同实验干预60d,RT-PCR法测定大脑Glut1、Glut4 mRNA水平。结果：与正常对照组比较,运动组和限食组小鼠脑组织Glut1和Glut4基因表达显著增高,高脂膳食组小鼠Glut4基因表达显著降低而Glut1表达水平无显著改变。结论：限食和运动增强脑内胰岛素反应型Glut4和非胰岛素反应型Glut1基因的表达,高脂膳食降低脑内胰岛素反应型Glut4基因的表达。     

罗格列酮改善胰岛素抵抗大鼠海马Alzheimer病样tau蛋白磷酸化水平

Tau蛋白过度磷酸化是Alzheimer病(AD)发病的关键事件.由于2型糖尿病是AD的风险因子,并且胰岛素抵抗是2型糖尿病的特征,检测了胰岛素抵抗大鼠大脑海马tau蛋白磷酸化水平,以及运用胰岛素增敏剂罗格列酮(TZD)后磷酸化的变化,发现胰岛素抵抗组大鼠海马tau蛋白呈过度磷酸化改变,但运用TZD后,tau蛋白的磷酸化状态有所恢复.由于糖原合成激酶-3β(GSK-3β)位于胰岛素信号转导途径中,并且是tau蛋白的重要磷酸激酶,研究检测罗格列酮干预前后GSK-3β活性,发现均升高.研究结果表明,肥胖时胰岛素抵抗导致细胞内胰岛素信号转导途径中,GSK-3β活性上调可能是引起大鼠海马内tau蛋白过度磷酸化的一个重要原因;虽然TZD可抑制tau蛋白的过度磷酸化,但可能不是通过下调GSK-3β活性的途径.     

糖尿病大鼠脑GSK-3与PP-2A失调诱导tau蛋白过度磷酸化

探讨胰岛素缺乏的糖尿病大鼠皮层糖原合酶激酶-3(GSK-3)及蛋白磷酯酶-2A(PP-2A)变化及其对tau蛋白磷酸化的作用.用链脲佐菌素(streptozotocin,STZ)建立胰岛素缺乏的糖尿病大鼠模型,用放射性配体结合实验检测了GSK-3和PP-2A的活性,蛋白质印迹检测了tau蛋白的磷酸化水平及PP-2A的表达.结果提示:在糖尿病大鼠皮层,GSK-3活性升高,PP-2A活性及表达降低,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化.应用GSK-3的选择性抑制剂Li2CO3后,GSK-3活性降低,PP-2A活性及表达恢复,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化水平降低.研究提示:糖尿病大鼠皮层GSK-3升高可能抑制PP-2A的活性,升高的GSK-3和降低的PP-2A协同促进tau蛋白的磷酸化.     

十子代平方水煎液对原代骨骼肌细胞胰岛素抵抗模型的影响

本实验观察十子代平方对原代骨骼肌细胞胰岛素抵抗模型的影响,探讨其改善胰岛素抵抗的作用机制。对原代骨骼肌细胞应用5×10-7mol/L胰岛素干预12 h建立胰岛素抵抗模型,应用十子代平方高、中、低浓度(400、100、25μg/m L)(SZDP-H、SZDP-M、SZDP-L)对造模成功的骨骼肌细胞进行干预,同时另设正常组、模型组,吡格列酮组(40μmol/L)作对照,药物干预24 h后用葡萄糖氧化酶法测定骨骼肌细胞上清液葡萄糖剩余量,采用Western-blot方法测定该方药物干预后骨骼肌细胞AKT、GSK-3β蛋白的表达。实验结果表明十子代平方可以改善骨骼肌细胞胰岛素抵抗模型的葡萄糖代谢,增加AKT和磷酸化位点Ser473蛋白表达,降低GSK-3β蛋白表达,增加其磷酸化位点Ser9蛋白表达。十子代平方可能通过调节AKT/GSK3β通路的机制改善胰岛素抵抗模型骨骼肌细胞的葡萄糖代谢。     

肥胖及2型糖尿病大鼠Alzheimer病样Tau蛋白过度磷酸化修饰及机制探讨

Tau蛋白异常过度磷酸化修饰在阿尔茨海默病(Alzheimerdisease,AD)发病机理中起非常重要的作用,而2型糖尿病是AD的风险因素之一.采用蛋白质印迹研究2型糖尿病及单纯肥胖大鼠脑中海马回tau蛋白磷酸化程度,发现在这两种大鼠模型中海马tau蛋白在多个位点上都呈现过度磷酸化状态.同时,胰岛素信号传导系统中的关键酶糖原合成激酶-3β(glycogensynthasekinase-3β,GSK-3β)活性在这两种大鼠模型的海马回中明显增高,经脑立体定位法向大鼠海马回注射GSK-3β抑制剂氯化锂(LiCl),可阻止2型糖尿病及肥胖大鼠模型中的GSK-3β激活,但仅阻止单纯肥胖大鼠海马回tau蛋白过度磷酸化.另外,海马神经细胞膜上胰岛素受体β亚基水平在两种实验模型中显著下降.研究结果表明,2型糖尿病及肥胖可能通过增高胰岛素抵抗,从而导致GSK-3β激活和tau蛋白的过度磷酸化来提高AD的发病风险.2型糖尿病脑中低下的葡萄糖代谢也可能在tau蛋白的过度磷酸化起一定作用.     

Glucose homeostasis, insulin secretion, and islet phospholipids in mice that overexpress iPLA2beta in pancreatic beta-cells and in iPLA2beta-null mice

Studies with genetically modified insulinoma cells suggest that group VIA phospholipase A(2) (iPLA(2)beta) participates in amplifying glucose-induced insulin secretion. INS-1 insulinoma cells that overexpress iPLA(2)beta, for example, exhibit amplified insulin-secretory responses to glucose and cAMP-elevating agents. To determine whether similar effects occur in whole animals, we prepared transgenic (TG) mice in which the rat insulin 1 promoter (RIP) drives iPLA(2)beta overexpression, and two characterized TG mouse lines exhibit similar phenotypes. Their pancreatic islet iPLA(2)beta expression is increased severalfold, as reflected by quantitative PCR of iPLA(2)beta mRNA, immunoblotting of iPLA(2)beta protein, and iPLA(2)beta enzymatic activity. Immunofluorescence microscopic studies of pancreatic sections confirm iPLA(2)beta overexpression in RIP-iPLA(2)beta-TG islet beta-cells without obviously perturbed islet morphology. Male RIP-iPLA(2)beta-TG mice exhibit lower blood glucose and higher plasma insulin concentrations than wild-type (WT) mice when fasting and develop lower blood glucose levels in glucose tolerance tests, but WT and TG blood glucose levels do not differ in insulin tolerance tests. Islets from male RIP-iPLA(2)beta-TG mice exhibit greater amplification of glucose-induced insulin secretion by a cAMP-elevating agent than WT islets. In contrast, islets from male iPLA(2)beta-null mice exhibit blunted insulin secretion, and those mice have impaired glucose tolerance. Arachidonate incorporation into and the phospholipid composition of RIP-iPLA(2)beta-TG islets are normal, but they exhibit reduced Kv2.1 delayed rectifier current and prolonged glucose-induced action potentials and elevations of cytosolic Ca(2+) concentration that suggest a molecular mechanism for the physiological role of iPLA(2)beta to amplify insulin secretion.     

Glut4 is upregulated despite decreased insulin signaling during prolonged fasting in northern elephant seal pups

Postprandial cellular glucose uptake is dependent on an insulin-signaling cascade in muscle and adipose tissue, resulting in the translocation of the insulin-dependent glucose transporter 4 (Glut4) into the plasma membrane. Additionally, extended food deprivation is characterized by suppressed insulin signaling and decreased Glut4 expression. Northern elephant seals are adapted to prolonged fasts characterized by high levels of plasma glucose. To address the hypothesis that the fasting-induced decrease in insulin is associated with reduced insulin signaling in prolonged fasted seals, we compared the adipose protein levels of the cellular insulin-signaling pathway, Glut4 and plasma glucose, insulin, cortisol, and adiponectin concentrations between Early (n = 9; 2-3 wks postweaning) and Late (n = 8; 6-8 wks postweaning) fasted seals. Plasma adiponectin (230 ± 13 vs. 177 ± 11 ng/ml), insulin (2.7 ± 0.4 vs. 1.0 ± 0.1 μU/ml), and glucose (9.8 ± 0.5 vs. 8.0 ± 0.3 mM) decreased, while cortisol (124 ± 6 vs. 257 ± 30 nM) doubled with fasting. Glut4 increased (31%) with fasting despite the significant decreases in the cellular content of phosphatidylinositol 3-kinase as well as phosphorylated insulin receptor, insulin receptor substrate-1, and Akt2. Increased Glut4 may have contributed to the decrease in plasma glucose, but the decrease in insulin and insulin signaling suggests that Glut4 is not insulin-dependent in adipose tissue during prolonged fasting in elephant seals. The reduction of plasma glucose independent of insulin may make these animals an ideal model for the study of insulin resistance.     

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.     

L-亮氨酸对克隆的胰岛β细胞株INS-1E细胞分泌胰岛素的葡萄糖依赖性研究

目的:探讨L-亮氨酸对克隆的胰岛β细胞株INS-1E细胞分泌胰岛素的刺激作用及其葡萄糖依赖性。方法:INS-1E细胞经传代培养2 d后,在Krebs-Ringer缓冲液中37℃培养箱预培养30 min,再用含有不同浓度葡萄糖和不同浓度L-亮氨酸的改良Krebs-Ringer缓冲液培养60 min,然后留取上清液进行胰岛素测定。结果:L-亮氨酸在0.1~10 mmol.L-1范围不增加16.7mmol.L-1葡萄糖刺激的INS-1E细胞的胰岛素分泌,仅20 mmol.L-1的L-亮氨酸促进葡萄糖诱导的胰岛素分泌;10 mmol.L-1L-亮氨酸在1.1、3.3、6.7 mmol.L-1葡萄糖存在的情况下促进INS-1E细胞的胰岛素分泌,而在11.1、16.7、25 mmol.L-1葡萄糖存在的情况下无促进胰岛素分泌的作用。结论:本研究显示在无刺激胰岛素分泌的葡萄糖浓度条件下,10 mmol.L-1L-亮氨酸即显示了刺激INS-1E细胞分泌胰岛素的作用,在较高葡萄糖的条件下,10 mmol.L-1L-亮氨酸的作用减弱或消失。     

Different secretory response of pancreatic islets and insulin secreting cell lines INS-1 and INS-1E to osmotic stimuli

Objective of this study was to characterize osmotically-induced insulin secretion in two tumor cell lines. We compared response of freshly isolated rat pancreatic islets and INS-1 and INS-1E tumor cell lines to high glucose, 30 % hypotonic medium and 20 % hypertonic medium. In Ca(2+)-containing medium glucose induced insulin release in all three cell types. Hypotonicity induced insulin secretion from islets and INS-1 cells but not from INS-1E cells, in which secretion was inhibited despite similar increase in cell volume in both cell types. GdCl(3) (100 micromol/l) did not affect insulin response from INS-1E cells to hypotonic challenge. Hypertonic medium inhibited glucose-induced insulin secretion from islets but not from tumor cells. Noradrenaline (1 micromol/l) inhibited glucose-induced but not swelling-induced insulin secretion from INS-1 cells. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity while that of INS-1 cells was partially inhibited. Functioning glucose-induced insulin secretion is not sufficient prerequisite for hypotonicity-induced response in INS-1E cells suggesting that swelling-induced exocytosis is not essential step in the mechanism mediating glucose-induced insulin secretion. Both cell lines are resistant to inhibitory effect of hyperosmolarity on glucose-induced insulin secretion. Response of INS-1E cells to hypotonicity is inhibited by the presence of Ca(2+) in medium.     

Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.     

Glucose regulates expression of the nerve growth factor (NGF) receptors TrkA and p75NTR in rat islets and INS-1E beta-cells

BACKGROUND: The function and survival of pancreatic beta-cells strongly depend on glucose concentration and on autocrine secretion of peptide growth factors. NGF and its specific receptors TrkA and p75NTR play a pivotal role in islet survival and glucose-dependent insulin secretion. We therefore investigated whether or not glucose concentration influences expression of TrkA and p75NTR in rat islets and in INS-1E beta-cells at the mRNA and protein level (INS-1E). METHODS: Gene expression of the NGF receptors TrkA and p75NTR but also of the metabolic gene liver-type pyruvate kinase (L-PK) and the neurotrophin receptors TrkB and TrkC was studied by semi-quantitative PCR and by real-time PCR in islets and INS-1E beta-cells. RESULTS: In rat islets, high glucose exposure (25 mmol/l) increased gene expression of TrkA, p75NTR and L-PK. Expression of TrkA, p75NTR and L-PK reflected insulin secretion at the respective glucose concentration. In rat INS-1E insulinoma cells, expression of L-PK and p75NTR was suppressed by low glucose as in the islets, while expression of TrkA was strongly increased by low glucose levels and thus was regulated differently than in islets. Expression of TrkB and TrkC was not regulated by glucose concentration at all. TrkA protein was regulated in the same fashion as its mRNA expression, while p75NTR protein was not significantly regulated within 24 h. CONCLUSION: Glucose interacts with gene expression of TrkA and p75NTR that are strongly involved in beta-cell growth and glucose-dependent insulin secretion. The fact that TrkA expression is regulated the opposite way in islets and in INS-1E beta-cells might reflect their specific grade of differentiation and tendency to proliferate.     

Relative hypoglycemia and hyperinsulinemia in mice with heterozygous lipoprotein lipase (LPL) deficiency. Islet LPL regulates insulin secretion.

Lipoprotein lipase (LPL) provides tissues with fatty acids, which have complex effects on glucose utilization and insulin secretion. To determine if LPL has direct effects on glucose metabolism, we studied mice with heterozygous LPL deficiency (LPL+/-). LPL+/- mice had mean fasting glucose values that were up to 39 mg/dl lower than LPL+/+ littermates. Despite having lower glucose levels, LPL+/- mice had fasting insulin levels that were twice those of +/+ mice. Hyperinsulinemic clamp experiments showed no effect of genotype on basal or insulin-stimulated glucose utilization. LPL message was detected in mouse islets, INS-1 cells (a rat insulinoma cell line), and human islets. LPL enzyme activity was detected in the media from both mouse and human islets incubated in vitro. In mice, +/- islets expressed half the enzyme activity of +/+ islets. Islets isolated from +/+ mice secreted less insulin in vitro than +/- and -/- islets, suggesting that LPL suppresses insulin secretion. To test this notion directly, LPL enzyme activity was manipulated in INS-1 cells. INS-1 cells treated with an adeno-associated virus expressing human LPL had more LPL enzyme activity and secreted less insulin than adeno-associated virus-beta-galactosidase-treated cells. INS-1 cells transfected with an antisense LPL oligonucleotide had less LPL enzyme activity and secreted more insulin than cells transfected with a control oligonucleotide. These data suggest that islet LPL is a novel regulator of insulin secretion. They further suggest that genetically determined levels of LPL play a role in establishing glucose levels in mice.     

Inhibition of Ca2+-independent phospholipase A2 results in insufficient insulin secretion and impaired glucose tolerance

Islet Ca2+-independent phospholipase A2 (iPLA2) is postulated to mediate insulin secretion by releasing arachidonic acid in response to insulin secretagogues. However, the significance of iPLA2 signaling in insulin secretion in vivo remains unexplored. Here we investigated the physiological role of iPLA2 in beta-cell lines, isolated islets, and mice. We showed that small interfering RNA-specific silencing of iPLA2 expression in INS-1 cells significantly reduced insulin-secretory responses of INS-1 cells to glucose. Immunohistochemical analysis revealed that mouse islet cells expressed significantly higher levels of iPLA2 than pancreatic exocrine acinar cells. Bromoenol lactone (BEL), a selective inhibitor of iPLA2, inhibited glucose-stimulated insulin secretion from isolated mouse islets; this inhibition was overcome by exogenous arachidonic acid. We also showed that iv BEL administration to mice resulted in sustained hyperglycemia and reduced insulin levels during glucose tolerance tests. Clamp experiments demonstrated that the impaired glucose tolerance was due to insufficient insulin secretion rather than decreased insulin sensitivity. Short-term administration of BEL to mice had no effect on fasting glucose levels and caused no apparent pathological changes of islets in pancreas sections. These results unambiguously demonstrate that iPLA2 signaling plays an important role in glucose-stimulated insulin secretion under physiological conditions.