Physiology of Malonyl-CoA in Muscle during Exercise

运动过程中肌肉中丙二酰辅酶A的生理学

• 批准号: 6612643
• 负责人: WILLIAM WINDER
• 金额: $ 20.59万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6612643
• 关键词: acetyl coA carboxylase; adenosine monophosphate; androstane compound; calmodulin; creatine phosphate; electrostimulus; enzyme activity; enzyme mechanism; exercise; fatty acid metabolism; glycogen; insulin; isolation perfusion; laboratory rat; malonyl coA; muscle contraction; muscle metabolism; oxidation; phosphatase inhibitor; phosphorylation; protein kinase; protein purification; striated muscles; western blottings

The principal purpose of the proposed experiments is to elucidate the mechanisms of regulation of malonyl-CoA (inhibitor of fatty acid oxidation) content in skeletal muscle. This will be accomplished by studying the short-term mechanisms of regulation of acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of malonyl-CoA. Upstream signaling proteins, AMP-activated protein kinase (AMPK) and AMP-activated protein kinase kinase (AMPKK) will be investigated. AMPK and AMPKK are also likely involved in contraction-induced stimulation of glucose uptake and control of expression of genes for GLUT4, hexokinase, and mitochondrial oxidative enzymes. Results will be applicable to these other important processes that appear to be regulated by the AMPKK/AMPK signaling system. Phosphorylation states of the AMPK phosphorylation site of muscle ACC and of the threonine 172 (AMPKK site) site in AMPK will be correlated with activity of purified ACC preparations, activity of ACC from electrically stimulated muscle, and activity of ACC in muscle from rats run on the treadmill. Roles of creatine phosphate (AMPK inhibitor) and 5'-AMP (AMPKK and AMPK activator) will be investigated using in vitro and in situ models. The effect of insulin on the rate of dephosphorylation of the AMPK phosphorylation site of ACC and the AMPKK site of AMPK will characterized. If effects of insulin are noted, the downstream signaling steps will be investigated, using wortmannin and phosphatase inhibitors. A putative AMPK kinase will be isolated from skeletal muscle and characterized with respect to molecular weight and regulation by creatine phosphate, 5'-AMP, and calcium/calmodulin. The question of whether AMPKK can be activated by phosphorylation during muscle contraction similarly to AMPK will be investigated. Adaptations of the AMPK/AMPKK signaling system to endurance training will be further characterized. Type 2 diabetes mellitus and obesity are two interrelated metabolic diseases that are increasing in incidence in the United States at alarming rates. These experiments will provide new information on regulation of AMPK and AMPKK, key enzymes that have now become targets for development of new pharmaceuticals for treatment of Type 2 diabetes and possibly obesity. This information will also provide essential basic information for investigation of the hypothesis that some forms of type 2 diabetes are due to defects in the AMPK/AMPKK signaling pathway. The results will provide additional rationale for the use of regular exercise in preventing and treating type 2 diabetes and obesity.

本实验的主要目的是阐明骨骼肌中丙二酰辅酶a（脂肪酸氧化抑制剂）含量的调节机制。这将通过研究乙酰辅酶a羧化酶（ACC）的短期调节机制来完成，ACC是一种负责合成丙二酰辅酶a的酶。上游信号蛋白，amp活化蛋白激酶（AMPK）和amp活化蛋白激酶（AMPKK）将被研究。AMPK和AMPKK也可能参与收缩诱导的葡萄糖摄取刺激和控制GLUT4、己糖激酶和线粒体氧化酶基因的表达。结果将适用于这些似乎由AMPKK/AMPK信号系统调节的其他重要过程。肌肉ACC的AMPK磷酸化位点和AMPK中苏氨酸172 （AMPKK位点）位点的磷酸化状态将与纯化ACC制剂的活性、电刺激肌肉的ACC活性以及在跑步机上跑步的大鼠肌肉中的ACC活性相关。磷酸肌酸（AMPK抑制剂）和5'-AMP （AMPKK和AMPK激活剂）的作用将通过体外和原位模型进行研究。胰岛素对ACC的AMPK磷酸化位点和AMPK的AMPKK位点去磷酸化速率的影响将被表征。如果注意到胰岛素的作用，下游信号步骤将被研究，使用wortmannin和磷酸酶抑制剂。一个假定的AMPK激酶将从骨骼肌中分离出来，并表征其分子量和磷酸肌酸、5'-AMP和钙/钙调蛋白的调节。AMPKK是否可以像AMPK一样在肌肉收缩过程中被磷酸化激活，这个问题将被研究。AMPK/AMPKK信号系统对耐力训练的适应性将进一步表征。2型糖尿病和肥胖症是两种相互关联的代谢性疾病，它们在美国的发病率正以惊人的速度增长。这些实验将提供关于AMPK和AMPKK调控的新信息，这两种关键酶现在已经成为开发治疗2型糖尿病和可能的肥胖的新药的靶点。这一信息也将为研究某些形式的2型糖尿病是由于AMPK/AMPKK信号通路缺陷引起的假设提供必要的基础信息。研究结果将为定期锻炼预防和治疗2型糖尿病和肥胖症提供额外的理论依据。