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转换为基于脂肪酸的代谢，在生理和病理条件中发挥核心作用。肉毒碱棕榈酰转移酶-I（CPT-I）是肉毒碱诱导的线粒体脂肪酸氧化变化的关键调节步骤，其通过cAMP信号系统介导。丙二酰辅酶A抑制CPT-I活性，代表脂肪酸氧化的控制点。CPT-I在空腹和糖尿病中丙二酰辅酶A敏感性显著降低的机制尚未发现。我们已经表明1）CPT-I的调节涉及CPT-I蛋白通过磷酸化和去磷酸化的共价修饰，2）50%的线粒体CPT-I定位于接触位点，3）CPT-I在接触位点的定位在糖尿病酮症酸中毒中增强，（4）糖尿病酮症酸中毒大鼠肝线粒体接触部位CPT-Ⅰ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，在导致肝蛋白激酶活化的激素环境中，CPT-1被磷酸化，导致对丙二酰辅酶A抑制的抗性;因此，需要更多的丙二酰辅酶A来实现相同程度的抑制，但CPT-1的速度没有变化。我们推测，接触网站作为蛋白激酶和蛋白磷酸酶参与CPT-I调节的对接域。CPT-I的基于磷酸化/去磷酸化的调节发生在磷酸化CPT-I主要存在的接触位点。目的1是确定免疫沉淀的CPT-Ⅰ消化后磷酸化肽的氨基酸序列。目的2是鉴定CPT-I的磷酸化激酶和去磷酸化磷酸酶。这些研究将解决CPT-I磷酸化在分离的肝细胞中的功能后果。目的3探讨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4探讨接触位点是否为CPT-Ⅰ的激酶和磷酸酶以及肝同种型提供对接结构域。目的5将测定各种代谢状态下肝脏的丙二酰辅酶A含量。拟议的研究将在分子和生物化学水平上建立CPT-I的磷酸化循环以及它如何与CPT-I在肝脏中的动力学相关。

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.