Lipid signal transduction /oscillatory insulin secretion

描述(由申请人提供)： 胰岛素分泌振荡，可能是由于糖酵解、线粒体能量产生、停滞和脂肪分解的振荡代谢所致。这些振荡在电活动、离子通量以及最终的分泌振荡中产生重要的信号。我们推测FFA可能作为偶联因子参与胰岛代谢信号转导和胰岛素分泌的同步，并提供外周脂肪细胞和胰岛之间的联系。这可能是通过招募贝塔细胞、增加贝塔细胞之间的同步性或增强贝塔细胞对葡萄糖的反应来实现的。我们进一步推测，FFA及其由激素敏感脂肪酶(HSL)释放产生的重要信号调节胰岛素的分泌，以响应燃料刺激和cAMP的升高。我们预测，缺乏PFK-M(磷酸果糖激酶M)或HSL的动物模型将证明糖酵解和脂解途径在胰岛素分泌中的关键重要性。目的1确定游离脂肪酸对葡萄糖诱导的完整胰岛内单个细胞线粒体代谢振荡的影响。内源性FFA作为信号分子的作用将通过监测完整的HSL缺陷胰岛的同步代谢活动来研究。目的2将确定β细胞、胰岛和GSIS中脂肪分配的振荡变化与脂解之间的关系以及FFA对这些参数的影响。使用β细胞和胰岛的共聚焦成像将实时监测荧光FFA在细胞内的分布和运动。此外，刺激细胞释放的游离脂肪酸和甘油将与胰岛素分泌和目标1中的测量结果存在时间相关性。目标3将验证FFA通过直接调节电压门控L型钙通道促进胰岛素分泌的假说。目的4将验证假设，即通过HSL介导的脂肪分解从脂肪细胞振荡释放FFA在胰岛同步分泌胰岛素中起作用。将进行分离的脂肪细胞和胰岛的顺序共灌流。目的5验证长链辅酶A对R细胞中HSL的调节是通过振荡抑制发生的假说。目标6将研究β细胞中HSL缺乏导致GSIS改变的机制。AIM 7将测试完整灌流胰腺的分泌和电同步机制，以及它与胰岛和单细胞研究的关系。

DESCRIPTION (provided by applicant): Insulin secretion oscillates, possibly due to oscillatory metabolism of glycolysis, mitochondrial energy production, anaplerosis and lipolysis. These oscillations generate important signals underlying oscillations in electrical activity, ion fluxes and, ultimately, secretion. We hypothesize that FFA may participate as coupling factors to synchronize islet metabolic signal transduction and insulin secretion and to provide communication between peripheral fat cells and islets. This may be achieved by recruiting beta cells, increasing synchrony among beta cells or enhancing responsiveness of beta-cells to glucose. We further presume that FFA and their release by hormone sensitive lipase (HSL) generate important signals that regulate insulin secretion in response to fuel stimuli and elevation of cAMP. We predict that animal models deficient in PFK-M (phosphofructokinase-M) or HSL will demonstrate the critical importance of glycolytic and lipolytic pathways in insulin secretion. Aim 1 will determine the effect of FFA on glucose-induced mitochondrial metabolic oscillations of individual cells within the intact islet. The role of endogenous FFA as a signaling molecule will be studied by monitoring the synchronized metabolic activity in the intact HSL deficient islet. Aim 2 will determine the relationship between oscillatory changes in lipid partitioning and lipolysis in beta cells and islets and GSIS and the effect of FFA on these parameters. Intracellular distribution and movement in real time of fluorescent FFA will be monitored using confocal imaging of beta cells and islets. In addition FFA and glycerol release from stimulated cells will be temporally correlated with insulin secretion and measurements made in Aim 1. Aim 3 will test the hypothesis that FFA promote secretion via direct modulation of voltage-gated L-type calcium channels. Aim 4 will test the hypothesis that oscillatory FFA release from fat cells via HSL-mediated lipolysis plays a role in synchronized insulin secretion from islets. Sequential co-perifusion of isolated fat cells and pancreatic islets will be undertaken. Aim 5 will test the hypothesis that regulation of HSL in the R-cell occurs via oscillatory inhibition by long chain-CoA. Aim 6 will study the mechanism whereby HSL-deficiency in beta-cell results in altered GSIS. Aim 7 will test mechanisms of secretory and electrical synchronization in the intact perfused pancreas and its relationship to islet and single cell studies.