In Vivo Regulation of Protein Turnover by Hormones

描述（由申请方提供）：初步证据表明，T2D患者中发生了对胰岛素应答的肌肉线粒体（mito）功能障碍。胰岛素与氨基酸（AA）联合对肌肉线粒体功能具有重要的调节作用。T2D患者中肌肉对胰岛素的mito反应是否由于胰岛素抵抗而降低，或者胰岛素抵抗是否导致mito功能障碍，仍有待确定。为了理解T2 D患者中线粒体功能障碍的潜在机制，我们开发了同时测量多种肌肉线粒体蛋白的合成速率、编码线粒体和转录因子蛋白的基因的转录水平以及来自人类肌肉活检样品的ATP产生的方法。目前的提案将确定T2D患者与非糖尿病患者相比，在胰岛素水平相似的情况下，肌肉ATP产生和蛋白质合成是否发生改变。该缺陷的潜在机制将通过使用我们开发的定制基因阵列比较转录水平、信号蛋白的磷酸化以及胰岛素和AA在调节T2D患者和非糖尿病患者的肌肉线粒体蛋白合成和ATP产生中的相互作用来研究。研究将采用胰岛素、胰高血糖素和GH的生长抑素钳夹法进行控制，并使用不同剂量的胰岛素和必需氨基酸混合物。我们将进一步研究药理学胰岛素增敏剂是否能增强T2D患者的肌肉线粒体功能。为了明确胰岛素和高血糖症在调节肌肉线粒体功能中的作用，我们将在胰岛素缺乏（高血糖症）和胰岛素替代（高血糖和正常血糖水平）期间对T1D患者进行研究。上一个资助期的结果也表明，AA是内脏蛋白质合成的主要调节剂，体外研究表明AA对基因转录和蛋白质合成的选择性作用。我们开发了同时测量多种血浆蛋白（其中大部分来自肝脏）的分数合成率的方法。这些蛋白质具有关键功能，包括在大血管疾病的病理生理学中的潜在作用。拟议的研究将检查胰岛素和AA的作用，以及它们在调节具有良好功能的血浆蛋白合成速率方面的相互作用。拟议的研究将首次提供有关AA和胰岛素在调节特定肌肉线粒体和血浆蛋白合成中的作用的信息。将确定胰岛素和AA浓度变化以及胰岛素抵抗对蛋白质合成的潜在功能影响。此外，这些研究将有望为T2D患者的病理生理学、肌肉胰岛素抵抗和线粒体功能提供新的见解。

DESCRIPTION (provided by applicant): Preliminary evidence indicates that muscle mitochondrial (mito) dysfunction in response to insulin occurs in T2D patients. Insulin in association with amino acids (AA) has an important regulatory role on muscle mito function. It remains to be determined whether muscle mito response to insulin is reduced in T2D patients because of insulin resistance or if insulin resistance causes mito dysfunction. To understand the underlying mechanism of mito dysfunction in T2 D patients we developed methods to simultaneously measure synthesis rates of multiple muscle mito proteins, transcript levels of genes encoding mito and transcription factors proteins and ATP production from needle muscle biopsy samples in human. The current proposal will determine whether muscle ATP production and protein synthesis is altered in T2D patients in comparison with non-diabetic people while on similar insulin levels. The underlying mechanism of this defect will be investigated by comparing transcript levels using custom gene arrays that we have developed, phosphorylation of signaling proteins and interaction of insulin and AA in regulating muscle mito protein synthesis and ATP production in T2D patients and non-diabetic people. Studies will be controlled with a somatostatin clamp of insulin, glucagon, and GH with varying dose of insulin and essential amino acid mixtures. We will further investigate whether pharmacological insulin sensitizers enhance muscle mito function in people with T2D. In order to clearly define the roles of insulin and hyperglycemia in regulating muscle mito functions, we will perform studies in T1D patients during insulin deficiency (hyperglycemia) and insulin replacement with high and normal glucose levels. Results from previous funding period also demonstrated that AA are predominant regulators of splanchnic protein synthesis and in vitro studies demonstrated selective effects of AA on gene transcripts and protein synthesis. We developed methods to simultaneously measure fractional synthesis rates of multiple plasma proteins (most of which originate from the liver). These proteins have critical functions including underlying roles in the pathophysiology of macrovascular disease. The proposed studies will examine the roles of insulin and AA and their interaction in regulating synthesis rates of plasma proteins with well-established functions. The proposed studies will for the first time provide information on the roles of AA and insulin in regulating synthesis of specific muscle mito and plasma proteins. The potential functional impact of changes in insulin and AA concentrations and insulin resistance on protein synthesis will be determined. In addition, the studies will hopefully provide novel insights on pathophysiology muscle insulin resistance and mito functions in T2D patients.