Physiological Signals to MCT Expression

描述（由申请人提供）：乳酸盐穿梭于动脉和血管系统，是运动期间分配碳水化合物势能的主要方式。促进乳酸交换发生在细胞器官和组织之间，以及细胞隔室之间，通过乳酸单羧酸转运（MCT）蛋白。MCT表达和MCT占据的细胞结构域存在组织特异性差异。在哺乳动物骨骼肌中，表达两种亚型（MCT 1和MCT 4）。MCT 1存在于肌膜和线粒体中，与氧化能力相关，而MCT 4仅存在于肌膜中，在具有快速肌球蛋白亚型的纤维中含量较高。肌肉MCT 1蛋白水平可以受到耐力训练和体内慢性电刺激的影响，并且利用培养的L 6肌管，我们已经能够影响MCT 1蛋白水平。由于对体内影响MCT表达的生理信号知之甚少，我们目前的目标是鉴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，以解决三个特定的目标，在体外培养的L 6肌细胞。为了简化搜索，以确定肌肉MCT表达的假定信号，我们从大鼠和人体体内研究转移到体外L 6肌细胞研究。基于MCT蛋白表达的响应性，在目标1下，我们将努力建立一个确定MCT 1和MCT 4表达的假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-α（TNF-α）、H2 O2、Ca++、腺嘌呤核苷酸能荷（ANEC，AICAR）、pH [Hv]和乳酸根阴离子[La-]。初步结果暗示MCTI的NF-κ B信号通路。目前的文献暗示了与T3相关的控制MCT 4表达的途径。目的1研究将涉及通过Western印迹法评估推定调节剂对MCT表达的影响。目的2将评估肌肉MCT表达受翻译前控制的假设。目的2研究将涉及肌肉MCT蛋白水平及其各自的mRNA在培养的肌细胞中的水平进行有序水平的假定生理信号的比较。如果蛋白质和信息水平在对假定刺激的反应中相关，则目标3将是评价MCT表达在转录水平上受到调节的假设的可行性。目的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.