Physiological Signals to MCT Expression

MCT 表达的生理信号

• 批准号: 7019197
• 负责人: GEORGE Austin BROOKS
• 金额: $ 31.75万
• 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/7019197
• 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）：乳酸穿梭于整个间质和脉管系统提供了在运动过程中分配碳水化合物势能的主要手段。通过乳酸，单羧酸盐转运（MCT）蛋白质，促进的乳酸交换发生在细胞器官和组织之间以及细胞室之间。 MCT表达和MCT占据的细胞结构域存在组织特异性差异。在哺乳动物的骨骼肌中，表达了两种同工型（MCT1和MCT4）。 MCT1存在于肌膜和线粒体中，与氧化能力有关，而MCT4仅在肌膜中，并且在具有快速肌球蛋白同工型的纤维中较高。肌肉MCT1蛋白水平可以受到体内耐力训练和慢性电刺激的影响，并且在培养的L6肌管中，我们能够影响MCT1蛋白水平。由于对影响体内MCT表达的生理信号知之甚少，因此我们目前的目标是确定影响MAMMALIAN骨骼肌表达的生理信号。为了实现我们的目标，我们提出实验，以解决体外孵育的L6肌细胞的三个特定目标。为了简化搜索以确定肌肉MCT表达的假定信号，我们从对体内大鼠和人类的研究转变为体外L6肌细胞的研究。基于MCT蛋白表达的响应能力，在AIM 1下，我们将努力建立决定MCT1和MCT4表达的假定生理信号的层次结构。我们提出的评估信号是：肿瘤坏死因子-A（TNF-A），H202，Ca ++，腺嘌呤核苷酸能量电荷（ANEC，AICAR，AICAR），pH [H V]和乳酸阴离子[LA-]。初步结果暗示MCTI的NF-KB信号通路。当前文献暗示了与T3有关的途径在控制MCT4表达中。 AIM 1研究将涉及对推定调节剂对MCT表达的影响的评估，并通过蛋白质印迹评估。目标2将是评估肌肉MCT表达受到翻译前控制的假设。 AIM 2研究将涉及比较培养的肌细胞中肌肉MCT蛋白水平及其各自的mRNA的比较，受到了假定的生理信号的有序水平。如果蛋白质和信息水平与推定的刺激相关，则目标3将是评估MCT表达在转录水平下受调节的假设的生存能力。 AIM 3研究将涉及比较肌肉MCT前MRNA，mRNA和蛋白质水平的水平。我们有实现既定目标的工具。