Physiological Signals to MCT Expression

描述（由申请人提供）：乳酸穿梭于间质和脉管系统，是运动过程中分配碳水化合物势能的主要途径。促进乳酸交换发生在细胞、器官和组织之间以及细胞间室之间，通过乳酸、单羧酸转运（MCT）蛋白。MCT的表达和占据的细胞结构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，并与氧化能力有关，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更高。肌肉MCT1蛋白水平可受体内耐力训练和慢性电刺激的影响，通过培养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，我们提出了三个实验来解决L6肌细胞体外培养的具体目标。为了简化对肌肉MCT表达的假定信号的研究，我们从对大鼠和人体内的研究转向对L6肌细胞的体外研究。基于MCT蛋白表达的响应性，在Aim 1下，我们将努力建立一个可能决定MCT1和MCT4表达的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）、H202、ca++、腺嘌呤核苷酸能量电荷（ANEC，通过AICAR）、pH [H v]和乳酸阴离子[La-]。初步结果提示MCTI存在NF-KB信号通路。目前的文献暗示了一个与T3相关的途径控制MCT4的表达。目的1研究将包括通过Western blotting评估假定的调节因子对MCT表达的影响。目的2将是评估肌肉MCT表达受翻译前控制的假设。目标2研究将涉及比较肌肉MCT蛋白水平及其各自mrna水平在受有序水平的假定生理信号培养的肌细胞中。如果蛋白质和信息水平在对假设刺激的反应中是相关的，那么Aim 3将是评估MCT表达在转录水平上受到调节这一假设的可行性。Aim 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。

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.