Regulation of Liver CPT-I during Diabetic Ketosis

糖尿病酮症期间肝脏 CPT-I 的调节

• 批准号: 7093174
• 负责人: Charles Leslie Hoppel
• 金额: $ 27.16万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-11 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7093174
• 关键词: carnitine palmitoyltransferase 1; chemical kinetics; diabetic acidosis; electrospray ionization mass spectrometry; enzyme activity; enzyme induction /repression; enzyme mechanism; fatty acid metabolism; high performance liquid chromatography; hormone regulation /control mechanism; immunoprecipitation; laboratory rabbit; laboratory rat; liver metabolism; malonyl coA; mitochondria; multiple sclerosis; phosphoprotein phosphatase; phosphorylation; posttranslational modifications; protein kinase; protein sequence

DESCRIPTION (provided by applicant): The liver plays a central role in physiological and pathological conditions by switching from a carbohydrate to fatty acid-based metabolism. Carnitine palmitoyltransferase-l (CPT-I) is the key regulated step in the hormone-induced changes in mitochondrial fatty acid oxidation mediated via the cAMP signaling system. Malonyl-CoA inhibits CPT-I activity and represents the control point for fatty acid oxidation. The mechanism for the dramatically decreased malonyl-CoA sensitivity of CPT-I in fasting and diabetes has not been uncovered. We have shown 1) regulation of CPT-I involves the covalent modification of the CPT-I protein by phosphorylation and dephosphorylation, 2) 50 percent of mitochondrial CPT-I localizes with contact sites, 3) the localization of CPT-I in contact sites is enhanced in diabetic ketoacidosis, 4) inhibition kinetics of CPT-I in liver mitochondrial contact sites from diabetic ketoacidotic rats differs markedly from insulin-treated diabetic rats. Our hypothesis is that in the hormonal milieu resulting in the activation of hepatic protein kinases, CPT-I is phosphorylated leading to resistance to malonyl-CoA inhibition; thus more malonyl-CoA is required to effect the same degree of inhibition, but without a change in the velocity of CPT-I. We hypothesize that contact sites serve as docking domains for protein kinases and protein phosphatases involved in CPT-I regulation. The phosphorylation / dephosphorylation-based regulation of CPT-I occurs in contact sites where the phosphorylated CPT-I predominantly exists. Aim 1 is to determine the amino acid sequence of the phosphorylated peptide following digestion of the immunoprecipitated CPT-I. Aim 2 is to identify the kinase(s) for phosphorylation and the phosphatase(s) for dephosphorylation of CPT-I. The studies will address the functional consequences of CPT-I phosphorylation in isolated hepatocytes. Aim 3 will examine the relationship between CPT-I kinetics and phosphorylation in liver of diabetic ketoacidotic rats. Aim 4 approaches whether contact sites provide docking domains for kinase(s) and phosphatase(s), as well as, for liver isoform of CPT-I. In Aim 5 malonyl-CoA content of the liver will be determined under the various metabolic states. The proposed studies will establish at the molecular and biochemical level the phosphorylation cycle of CPT-I and how it relates to the kinetics of CPT-I in liver.

描述（由申请人提供）：肝脏通过从碳水化合物转换为基于脂肪酸的代谢，在生理和病理条件下起着核心作用。 Carnitine Palmitoyltransferase-L（CPT-I）是激素诱导的通过cAMP信号系统介导的线粒体脂肪酸氧化变化的关键调节步骤。 Malonyl-COA抑制CPT-I活性，代表脂肪酸氧化的控制点。尚未发现CPT-I在禁食和糖尿病中大大降低丙二酰-COA敏感性的机制。 We have shown 1) regulation of CPT-I involves the covalent modification of the CPT-I protein by phosphorylation and dephosphorylation, 2) 50 percent of mitochondrial CPT-I localizes with contact sites, 3) the localization of CPT-I in contact sites is enhanced in diabetic ketoacidosis, 4) inhibition kinetics of CPT-I in liver mitochondrial contact来自糖尿病性酮酸酸性大鼠的部位与胰岛素治疗的糖尿病大鼠明显不同。我们的假设是，在激活肝蛋白激酶的激素环境中，CPT-I被磷酸化，从而导致对丙二酰-COA抑制的抗性。因此，需要更多的malonyl-COA才能实现相同程度的抑制作用，但不会改变CPT-I的速度。我们假设接触位点是参与CPT-I调节的蛋白激酶和蛋白质磷酸酶的对接结构域。 CPT-I的基于磷酸化 /基于去磷酸化的调节发生在磷酸化的CPT-I主要存在的接触部位。 AIM 1是在消化免疫沉淀的CPT-I消化后确定磷酸化肽的氨基酸序列。 AIM 2是鉴定用于磷酸化的激酶和磷酸酶的磷酸化酶，以进行CPT-I的去磷酸化。这些研究将解决分离的肝细胞中CPT-I磷酸化的功能后果。 AIM 3将检查糖尿病性酮症酸酸大鼠肝脏中CPT-I动力学与磷酸化之间的关系。 AIM 4接触位点是否为激酶和磷酸酶以及CPT-I的肝同工型提供对接结构域。在AIM 5中，将在各种代谢状态下确定肝脏含量。拟议的研究将在分子和生化水平建立CPT-I的磷酸化周期及其与肝脏中CPT-I的动力学的关系。