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.

在此功勋奖延期申请中，首要目标将继续是了解 葡萄糖和其他代谢急性调节胰岛素分泌的基本机制 燃料。该项目未来五年的具体目标是：1)测试长链酰基辅酶A GSIS的假说，认为葡萄糖和脂肪代谢之间需要一个完整的联系 用于葡萄糖传感。将使用小干扰RNA(SiRNA)技术来调节Key的表达 在β细胞中连接葡萄糖和脂肪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羧基酶-1(ACC-1)和GPR40受体。全面代谢 将进行分析，包括葡萄糖使用、葡萄糖氧化、葡萄糖转化为脂类、 用~(13)C核磁共振研究丙酮酸循环，以及有机酸、酰肉碱、氨基酸和游离态的代谢 通过GC/MS和MS/MS分析脂肪酸；2)验证关于丙酮酸循环在 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 通过综合代谢分析介导GSIS的β细胞途径(S)，受以下影响 分子操作：a)丙酮酸羧基酶的抑制和过表达；b)抑制和 PDK-1或PDK-2的过度表达；c)胞浆或线粒体苹果酸的抑制和过度表达 酶；d)抑制和过度表达二羧酸载体(DIG)，它介导了 苹果酸从线粒体到胞浆；e)Enoyl CoA水合酶的抑制和过度表达 (ECH)；3)调查关于NADPH和调控之间潜在联系的两个假设 将研究两个假说：a)NADPH通过结合 电压门控性K(Kv)通道的β亚基，延长血浆的复极相 膜电流；b)丙酮酸循环酶ICDC和MEC控制产生的NADPH 因此，胰岛素原加工酶PC2的调节因子神经内分泌蛋白7B2的表达 影响胰岛素原：胰岛素比率；4)翻译有关调节胰岛素原的新靶点的信息 使用UTMD技术从体外到体内的胰岛素分泌。