Engineered Glucose Metabolism in Insulin Secreting Cells

胰岛素分泌细胞中的工程葡萄糖代谢

• 批准号: 7989307
• 负责人: CHRISTOPHER B NEWGARD
• 金额: $ 3.68万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-03 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7989307
• 关键词: Acetyl-CoA Carboxylase; Acute; Acyl Coenzyme A; Address; Affect; Amino Acids; Award; Beta Cell; Binding; C-terminal; Cell Line; Cell membrane; Cells; Cytosol; Engineering; Enoyl-CoA Hydratase; Enzymes; Exhibits; Exocytosis; Glucagon; Glucose; Goals; In Vitro; Insulin; Islets of Langerhans; Link; Lipids; Malates; Malonyl Coenzyme A; Mediating; Mediator of activation protein; Metabolic; Mitochondria; Molecular; N-terminal; NADP; Neomycin; Neomycin resistance gene; Nonesterified Fatty Acids; Pathway interactions; Phase; Plasmids; Process; Proinsulin; Proprotein Convertase 2; Publications; Pyruvate; Pyruvate Carboxylase; Pyruvates; Recycling; Regulation; Resistance; Role; Site; Small Interfering RNA; Technology; Testing; Transfection; Translating; acylcarnitine; basal insulin; blood glucose regulation; citrate lyase; dicarboxylate-binding protein; fatty acid oxidation; glucagon-like peptide 1; glucose metabolism; in vivo; insulin granule; insulin secretion; islet; lipid metabolism; malic enzyme; malonyl-CoA decarboxylase; organic acid; overexpression; oxidation; peroxisome; pituitary protein 7B2; receptor; research study; response; voltage

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.

在此功绩奖励扩展应用中，总体目标将继续是对 葡萄糖和其他代谢涉及胰岛素分泌的急性调节的基本机制 燃料。未来五年该项目的具体目的是：1）测试长链酰基Coa GSIS的假设，该假设认为需要与葡萄糖和脂质赛之间的动态联系 用于葡萄糖传感。小型干扰RNA（siRNA）技术将用于调节钥匙的表达 将葡萄糖和脂质代谢的代谢功能的酶和介体中的酶和培养皿中的酶（包括） 柠檬酸裂解酶，乙酰二氧化碳羧化酶1（ACC-1）和GPR40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， 丙酮酸循环13c NMR，有机酸，酰基碳氮，氨基酸和游离的代谢分析 GC/MS和MS/MS的脂肪酸； 2）检验有关丙酮酸回收作用的假设 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 通过在遭受以下β细胞中的全面代谢分析中介导GSI的途径 分子操作：a）丙酮酸羧化酶的抑制和过表达； b）抑制和 PDK-1或PDK-2的过表达； c）胞质或线粒体损害的抑制和过表达 酶； d）抑制二羧酸酯载体（DIG）的抑制和过表达，可介导 从线粒体到细胞质的苹果酸； e）Enoyl COA水合物的抑制和过表达 （ech）; 3）调查两个假设NADPH与调节之间的潜在联系 胰岛素分泌将研究两个假设：a）NADPH通过与 电压门控k（kV）通道的β-亚基，延长了血浆的复极阶段 膜电流； b）丙酮酸循环酶ICDC和MEC对照产生的NADPH 神经内分泌蛋白7B2的表达，促硫蛋白加工酶PC2的调节剂 影响促胰岛素：胰岛素比； 4）翻译有关调节新目标的信息 使用UTMD技术从体外到体内环境的胰岛素分泌。