Regulation of Muscle ATP Synthase Beta Subunit Metabolism in Obesity

描述(申请人提供)：肥胖与骨骼肌中三磷酸腺苷(ATP)周转减少有关。这种情况会导致不利的结果，从体力活动能力下降，到与细胞功能相关的生理机制的激活受损，再到氧化应激增加和胰岛素抵抗的发展。在肥胖和胰岛素抵抗的个体中，肌肉线粒体中ATP合成酶的β亚基(β-F1-ATPase)的丰度降低。β-F1-ATPase是三磷酸腺苷合成酶的催化部位，是三磷酸腺苷合成的限速成分。我们认为肥胖时肌肉β-F1-ATPase合成减少。由于传统的气相色谱-质谱法对骨骼肌中发现的少量蛋白质不能用来测量单个蛋白质的稳定同位素富集度，我们开发了一种利用高效液相-电喷雾串联质谱仪定量体内标记的肌肉β-F1-ATPase的同位素富集量的方法，它是基于对肌肉中一种独特的多肽-F1-ATPase的同位素富集量的定量。使用这种方法，我们将检验肥胖者肌肉-F1-ATPase合成率降低的假设。我们还打算研究增加血浆氨基酸浓度以及运动对肥胖者和非肥胖者骨骼肌中β-F1-ATPase合成率的影响。肌肉-F1-ATPase合成是这项建议的主要终点，将通过在肥胖和非肥胖受试者静脉内持续输注D9-亮氨酸，并通过测量D9-亮氨酸对-F1-ATPase多肽的富集量来确定。将测试以下条件：生理盐水注入(对照)、氨基酸注入、有氧运动以及有氧运动与氨基酸注入相结合。这些研究将首次确定人体内肌肉F1-ATPase的合成速度，以及已知的促进肌肉蛋白质合成代谢的干预措施如何改变它。总体而言，这些研究的结果将有助于更好地理解肥胖者和非肥胖者骨骼肌中β-F1-ATPase丰度的调节机制。此外，他们将提供科学知识，为生活方式干预提供基础，以改善肥胖者的肌肉ATP周转。

DESCRIPTION (provided by applicant): Obesity is associated with reduced adenosine triphosphate (ATP) turnover in skeletal muscle. This condition results in unfavorable outcomes, proposed to range from decreased capacity for physical activity, to impaired activation of physiological mechanisms associated with cell function, to increased oxidative stress and the development of insulin resistance. The abundance of the beta subunit of the ATP synthase (¿ -F1-ATPase) in muscle mitochondria is decreased in obese, insulin-resistant individuals. ¿ -F1- ATPase makes up the catalytic site of the ATP synthase, and it is a rate-limiting component of ATP synthesis. We propose that muscle ¿ -F1-ATPase synthesis is reduced in obesity. Because measurement of stable isotopic enrichment of individual proteins is not practical using traditional gas chromatography-mass spectrometry approaches for proteins that are found in small amounts in skeletal muscle, we have developed an approach to quantify the isotopic enrichment of in vivo labeled muscle ¿ -F1-ATPase using HPLC-ESI-MS/MS. It is based on the quantification of the isotopic enrichment of a unique peptide of muscle ¿ -F1-ATPase. Using this approach we will test the hypothesis that the rate of muscle ¿ -F1-ATPase synthesis is reduced in obese individuals. We also intend to investigate the effects of increased plasma amino acid concentrations as well as exercise on stimulating the synthesis rate of ¿ -F1-ATPase in skeletal muscle of both obese and non-obese individuals. Muscle ¿ -F1-ATPase synthesis, which is the main end-point of this proposal, will be determined using an intravenous constant infusion of d9-leucine in obese and non-obese subjects, and by measuring the d9-leucine enrichment of a ¿ -F1-ATPase peptide. The following conditions will be tested: saline infusion (control), amino acid infusion, aerobic exercise, and a combination of aerobic exercise with amino acid infusion. These studies will for the first time determinate the rate of synthesis of muscle ¿ -F1-ATPase in humans in vivo, and how it is altered by interventions known to promote muscle protein anabolism. Overall, the results of these studies will lead to better understanding of the mechanisms regulating the abundance of ¿ -F1-ATPase in skeletal muscle in both obese and non-obese individuals. Further, they will provide scientific knowledge to base lifestyle interventions to improve muscle ATP turnover in obese individuals.