Regulation of Liver CPT-I during Diabetic Ketosis

描述(申请人提供)：肝脏在生理和病理条件下发挥核心作用，从碳水化合物转变为以脂肪酸为基础的新陈代谢。肉毒碱-L棕榈酰转移酶(CPT-I)是激素通过cAMP信号系统介导的线粒体脂肪酸氧化过程中的关键调控步骤。丙二酰辅酶A抑制CPT-I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敏感性的机制尚未被发现。我们发现：1)CPT-I的调节涉及CPT-I蛋白的磷酸化和去磷酸化的共价修饰，2)50%的线粒体CPT-I定位于接触部位，3)在糖尿病酮症酸中毒时CPT-I在接触部位的定位增强，4)糖尿病酮症酸中毒大鼠肝脏线粒体接触部位CPT-I的抑制动力学明显不同于胰岛素治疗的糖尿病大鼠。我们的假设是，在导致肝脏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，需要更多的丙二酰辅酶A才能产生相同程度的抑制，但CPT-I的速度不变。我们假设接触部位作为参与CPT-I调节的蛋白激酶和蛋白磷酸酶的对接结构域。CPT-I的磷酸化/去磷酸化调控发生在主要存在磷酸化CPT-I的接触部位。目的1测定免疫沉淀CPT-I消化后的磷酸化肽的氨基酸序列。目的2确定CPT-I磷酸化的蛋白激酶(S)和去磷酸化的磷酸酶(S)。这些研究将解决CPT-I在分离的肝细胞中磷酸化的功能后果。目的3研究糖尿病酮症酸中毒大鼠肝脏CPT-I动力学与磷酸化的关系。目的探讨接触部位是否提供蛋白激酶(S)和磷酸酶(S)以及肝脏CPT-I亚型的对接结构域。在AIM中，将测定不同代谢状态下肝脏中丙二酰辅酶A的含量。拟议的研究将在分子和生化水平上建立CPT-I的磷酸化循环，以及它如何与肝脏中CPT-I的动力学有关。

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.