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: 6535956
• 负责人: WILLIAM WINDER
• 金额: $ 27.64万
• 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/6535956
• 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.

拟议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）的短期调节机制来实现，ACC是负责合成丙二酰辅酶A的酶。 将研究上游信号蛋白、AMP 激活蛋白激酶 (AMPK) 和 AMP 激活蛋白激酶激酶 (AMPKK)。 AMPK 和 AMPKK 也可能参与收缩诱导的葡萄糖摄取刺激以及 GLUT4、己糖激酶和线粒体氧化酶基因表达的控制。 结果将适用于似乎受 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 中的苏氨酸 172（AMPKK 位点）位点的磷酸化状态将与纯化的 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。 将使用体外和原位模型研究磷酸肌酸（AMPK 抑制剂）和 5'-AMP（AMPKK 和 AMPK 激活剂）的作用。 将表征胰岛素对 ACC 的 AMPK 磷酸化位点和 AMPK 的 AMPKK 位点去磷酸化速率的影响。 如果注意到胰岛素的影响，将使用渥曼青霉素和磷酸酶抑制剂研究下游信号传导步骤。 假定的 AMPK 激酶将从骨骼肌中分离出来，并表征其分子量以及磷酸肌酸、5'-AMP 和钙/钙调蛋白的调节。 与 AMPK 类似，AMPKK 是否可以在肌肉收缩过程中被磷酸化激活的问题将被研究。 AMPK/AMPKK 信号系统对耐力训练的适应性将得到进一步表征。 2 型糖尿病和肥胖症是两种相互关联的代谢性疾病，在美国发病率正以惊人的速度增加。 这些实验将提供有关 AMPK 和 AMPKK 调节的新信息，这些关键酶现已成为开发治疗 2 型糖尿病和可能的肥胖症的新药物的目标。 该信息还将为研究某些形式的 2 型糖尿病是由于 AMPK/AMPKK 信号通路缺陷所致的假设提供重要的基础信息。 研究结果将为定期运动预防和治疗 2 型糖尿病和肥胖症提供更多依据。