Physiological Signals to MCT Expression

MCT 表达的生理信号

• 批准号: 6865079
• 负责人: GEORGE Austin BROOKS
• 金额: $ 32.35万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-15 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6865079
• 关键词: acidity /alkalinity; adenine nucleotides; biological transport; calcium flux; carbohydrate transport; genetic transcription; hydrogen peroxide; lactates; messenger RNA; muscle cells; muscle proteins; nuclear factor kappa beta; protein isoforms; striated muscles; tissue /cell culture; transport proteins; tumor necrosis factor alpha; western blottings

DESCRIPTION (provided by applicant): Lactate shuttling through the interstitium and vasculature provides a major means of distributing carbohydrate potential energy during exercise. Facilitated lactate exchange occurs between cells organs and tissues as well as between cell compartments by means of lactate, monocarboxylate transport (MCT) proteins. There exist tissue-specific differences in MCT expression and cell domains occupied by MCTs. In mammalian skeletal muscle, two isoforms (MCT1 and MCT4) are expressed. MCT1 exists in the sarcolemma and mitochondria and is associated with oxidative capacity, whereas MCT4 is in sarcolemma only and is higher in fibers with fast myosin isoforms. Muscle MCT1 protein level can be influenced by endurance training and chronic electrical stimulation in vivo, and with cultured L6 myotubes we have been able to affect MCT1 protein levels. Because little is known about the physiological signals affecting MCT expression in vivo, our present goal is to identify the physiological signals affecting expression of MCTs in mammalian skeletal muscle. To achieve our goal, we propose experiments to address three specific aims on L6 myocytes incubated in vitro. To simplify the search to identify the putative signals for muscle MCT expression we move from studies on rats and humans in vivo to studies on L6 myocytes in vitro. Based on responsiveness of MCT protein expression, under Aim 1 we will strive to establish a hierarchy of putative physiological signals determining expression of MCT1 and MCT4. The signals we propose to evaluate are: tumor necrosis factor-a (TNF-a), H202, Ca++, adenine nucleotide energy charge (ANEC, by AICAR), pH [H v] and lactate anion [La-]. Preliminary results implicate an NF-KB signaling pathway for MCTI. Current literature implicates a pathway related to T3 in controlling MCT4 expression. Aim 1 studies will involve an assessment of the effects of putative regulators on MCT expression as evaluated by Western blotting. Aim 2 will be to evaluate the hypothesis that muscle MCT expression is subject to pre-translational control. Aim 2 studies will involve comparisons of the levels of muscle MCT protein levels and their respective mRNAs in cultured myocytes subjected to ordered levels of putative physiological signals. If protein and message levels are correlated in response to putative stimuli, then Aim 3 will be to evaluate viability of the hypothesis that MCT expression is regulated at the level of transcription. Aim 3 studies will involve comparisons of the levels of muscle MCT pre-mRNAs, mRNAs and protein levels. We have the tools to achieve the stated aims.

描述（由申请人提供）：穿过间质和脉管系统的乳酸提供了运动期间分配碳水化合物势能的主要手段。通过乳酸、单羧酸转运 (MCT) 蛋白，促进细胞器官和组织之间以及细胞区室之间的乳酸交换。 MCT 表达和 MCT 占据的细胞域存在组织特异性差异。在哺乳动物骨骼肌中，表达两种亚型（MCT1 和 MCT4）。 MCT1 存在于肌膜和线粒体中，与氧化能力相关，而 MCT4 仅存在于肌膜中，并且在具有快速肌球蛋白亚型的纤维中含量较高。肌肉 MCT1 蛋白水平会受到体内耐力训练和慢性电刺激的影响，并且通过培养的 L6 肌管，我们已经能够影响 MCT1 蛋白水平。由于对体内影响 MCT 表达的生理信号知之甚少，我们目前的目标是确定影响哺乳动物骨骼肌中 MCT 表达的生理信号。为了实现我们的目标，我们提出了实验来解决体外培养的 L6 肌细胞的三个特定目标。为了简化寻找肌肉 MCT 表达的推定信号的过程，我们从对大鼠和人类的体内研究转向对 L6 肌细胞的体外研究。基于 MCT 蛋白表达的反应性，在目标 1 下，我们将努力建立确定 MCT1 和 MCT4 表达的推定生理信号的层次结构。我们建议评估的信号是：肿瘤坏死因子-a (TNF-a)、H2O2、Ca++、腺嘌呤核苷酸能量电荷（ANEC，由 AICAR 提供）、pH [H v] 和乳酸根阴离子 [La-]。初步结果表明 MCTI 存在 NF-KB 信号通路。目前的文献表明与 T3 相关的途径控制 MCT4 表达。目标 1 研究将涉及通过蛋白质印迹评估假定的调节剂对 MCT 表达的影响。目标 2 将评估肌肉 MCT 表达受到翻译前控制的假设。目标 2 研究将涉及比较肌肉 MCT 蛋白水平及其各自在培养的肌细胞中的 mRNA 水平，这些细胞受到有序水平的假定生理信号的影响。如果蛋白质和信息水平与假定的刺激反应相关，那么目标 3 将是评估 MCT 表达在转录水平受到调节的假设的可行性。目标 3 研究将涉及肌肉 MCT 前 mRNA、mRNA 和蛋白质水平的比较。我们拥有实现既定目标的工具。