Engineered Glucose Metabolism in Insulin Secreting Cells

在这个优秀奖的扩展应用程序中，首要目标将继续是理解 参与葡萄糖和其他代谢产物对胰岛素分泌的急性调节的基本机制 燃料.本项目未来五年的具体目标是：1）检测长链酰基辅酶A GSIS假说，认为葡萄糖和脂质代谢之间需要一个完整的联系 用于葡萄糖感测。小干扰RNA（siRNA）技术将用于调节关键基因的表达， 在β细胞中连接葡萄糖和脂质代谢的代谢功能的酶和介质，包括 柠檬酸裂解酶、乙酰辅酶A羧化酶-1（ACC-1）和GPR 40受体。综合代谢 将进行分析，包括葡萄糖使用，葡萄糖氧化，葡萄糖转化为脂质， 通过13 C NMR测定丙酮酸循环，以及有机酸、酰基肉毒碱、氨基酸和游离氨基酸的代谢谱 通过GC/MS和MS/MS分析脂肪酸; 2）为了验证关于丙酮酸再循环在 调节葡萄糖刺激的胰岛素分泌。我们试图确定特定的丙酮酸循环 通过β-细胞中的综合代谢分析介导GSIS的途径， 分子操作：a）抑制和过度表达丙酮酸羧化酶; B）抑制和 PDK-1或PDK-2的过表达; c）细胞溶质或线粒体苹果酸的抑制和过表达; d）抑制和过度表达二羧酸载体（DIG），其介导 e）抑制和过度表达烯酰CoA水合酶 (ECH)3）探讨NADPH与细胞凋亡调控之间的两种可能联系： 将研究两种假设：a）NADPH通过结合胰岛素分泌调节胰岛素分泌。 电压门控性K（Kv）通道的β亚单位，延长血浆的复极期 膜电流; B）丙酮酸循环酶ICDc和MEc对照产生的NADPH 表达神经内分泌蛋白7 B2，胰岛素原加工酶PC 2的调节剂，从而 影响胰岛素原：胰岛素比率; 4）翻译关于调节胰岛素的新靶点的信息， 使用UTMD技术从体外到体内环境的胰岛素分泌。

In this Merit Award extension application, the overarching goal will continue to be the understanding of fundamental mechanisms involved in acute regulation of insulin secretion by glucose and other metabolic fuels. The specific aims of the project for the next five years are: 1) To test the long-chain acylCoA hypothesis of GSIS, which holds that anintact link betweenglucose andlipidmetabolism is required for glucose sensing. Small interfering RNA (siRNA) technology will be used to modulate expression of key enzymes and mediators of metabolic function that link glucose and lipid metabolism in beta-cells, including citrate lyase, acetyl CoA carboxylase-1 (ACC-1), and the GPR40 receptor. Comprehensive metabolic analysis will be conducted, including glucose usage, glucose oxidation, glucose conversion into lipids, pyruvate cycling by 13C NMR, and metabolic profiling of organic acids, acylcarnitines, amino acids, and free fatty acids by GC/MS and MS/MS; 2) To test a hypothesis concerning the role of pyruvate recycling in regulation of glucose-stimulated insulin secretion. We seek to identify the specific pyruvate cycling pathway(s) that mediates GSIS via comprehensive metabolic analysis in beta-cells subjected to the following molecular manipulations: a) suppression and overexpression of pyruvate carboxylase; b) suppression and overexpression of PDK-1 or PDK-2; c) suppression and overexpression of cytosolic or mitochondrial malic enzymes; d) suppression and overexpression of the dicarboxylate carrier (DIG), that mediates transport of malate from the mitochondria to the cytosol; e) suppression and overexpression of enoyl CoA hydratase (ECH); 3) To investigate two hypothesesabout the potential link between NADPH and regulation of insulin secretion Two hypotheses will be investigated: a) NADPH regulates insulin secretion by binding to the beta-subunit of voltage-gated K (Kv) channels, prolonging the repolarization phase of the plasma membrane current; b) NADPH produced by the pyruvate cycling enzymes ICDc and MEc controls expression of neuroendocrine protein 7B2, a regulator of the proinsulin processing enzyme PC2, thereby affecting proinsulin:insulin ratios; 4) To translate information about new targets for regulation of insulin secretion from the in vitro to the in vivo setting using UTMD technology.