Regulation of Liver CPT-I during Diabetic Ketosis

描述（由申请人提供）：肝脏通过从碳水化合物到脂肪酸的代谢转换，在生理和病理条件中起着核心作用。肉碱棕榈酰基转移酶- 1 （CPT-I）是通过cAMP信号系统介导的激素诱导线粒体脂肪酸氧化变化的关键调控步骤。丙二酰辅酶a抑制cpt - 1活性，是脂肪酸氧化的控制点。CPT-I在空腹和糖尿病中显著降低丙二酰辅酶a敏感性的机制尚未发现。我们发现1)CPT-I的调控涉及通过磷酸化和去磷酸化对CPT-I蛋白的共价修饰，2)50%的线粒体CPT-I定位于接触位点，3)CPT-I在接触位点的定位在糖尿病酮症酸中毒中得到增强，4)糖尿病酮症酸中毒大鼠的CPT-I在肝脏线粒体接触位点的抑制动力学与胰岛素治疗的糖尿病大鼠明显不同。我们的假设是，在导致肝蛋白激酶激活的激素环境中，CPT-I被磷酸化，导致对丙二酰辅酶a抑制的抵抗；因此，需要更多的丙二醇辅酶a来达到相同程度的抑制，但不改变cpt - 1的速度。我们假设接触位点作为参与CPT-I调控的蛋白激酶和蛋白磷酸酶的对接域。基于磷酸化/去磷酸化的CPT-I调控发生在磷酸化CPT-I主要存在的接触位点。目的1是确定免疫沉淀cpt - 1消化后磷酸化肽的氨基酸序列。目的2是确定用于磷酸化的激酶和用于去磷酸化cpt - 1的磷酸酶。这些研究将解决cpt - 1磷酸化在分离肝细胞中的功能后果。目的3将研究糖尿病酮症酸中毒大鼠肝脏中CPT-I动力学与磷酸化的关系。目的4探讨接触位点是否为激酶和磷酸酶以及CPT-I的肝异构体提供对接域。在Aim 5中，肝脏在各种代谢状态下的丙二酰辅酶a含量将被确定。拟开展的研究将在分子和生化水平上确定CPT-I的磷酸化周期及其与肝脏中CPT-I动力学的关系。

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.