Understanding basic regulation of mitochondrial bioenergetics and adaptations to exercise

了解线粒体生物能量学的基本调节和运动适应

• 批准号: RGPIN-2014-03656
• 负责人: Holloway, Graham
• 金额: $ 2.48万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=653773
• 关键词: Understanding; basic; regulation; mitochondrial; bioenergetics

Within muscle, mitochondria influence several processes, including metabolic homeostasis, apoptosis and redox balance. However, the regulation of mitochondria remains poorly elucidated. Therefore, a better understanding of these dynamic structures could provide insight into many cellular processes. By incorporating molecular, biochemical and physiological approaches my long-term goals are to determine a) mechanisms regulating mitochondrial bioenergetics, b) mitochondrial biogenesis, and c) understand how these effect substrate utilization in skeletal muscle.**My specific hypotheses and short-term plans are:*1. Regulation of mitochondrial FAT/CD36 translocation: I have previously shown that exercise redistributes FAT/CD36 to mitochondrial membranes, however, the molecular basis for this subcellular trafficking remains unknown. I hypothesize that the C-terminal YCACR motif of FAT/CD36 and AMP activated protein kinase (AMPK) signaling are both required for this cellular process. I will transfect wild type and C-terminal mutants (C-terminal amino acids deleted) into the skeletal muscle of FAT/CD36 knock out mice. Thereafter I will provide metabolic challenges (eg. sciatic nerve stimulated contraction) and determine the ability of FAT/CD36 to accumulate on mitochondrial membranes and the functional consequence on rates of fatty acid oxidation/respiration. Similar experiments in wild type and AMPK kinase dead mice will determine the necessity of AMPK signaling in mediating mitochondrial FAT/CD36 translocation.**2. Regulation of carnitine palmitoyltransferase-I (CPTI) malonyl-CoA (M-CoA) kinetics: I have previously shown that exercise augments the ability of M-CoA to inhibit CPTI, but the molecular basis for this remains unknown. To ascertain direct regulation of CPTI, substrate kinetics (PCoA and M-CoA) will be performed and CPTI will be immunoprecipitated and examined for redox modifications and acetylation status before and after exercise in humans. In addition, CPTI substrate kinetics will be determined various ways in the presence and absence of the cytoskeletal network (specifically ß-tubulin) to determine the ability of the cytoskeletal network to regulate CPTI.**3. The metabolic role of the mitochondrial reticulum: I have previously shown that over-expressing mitofusin-2 (MFN2) independently does not influence mitochondrial bioenergetics. I therefore hypothesize that the primary metabolic role of MFN2 is to anchor mitochondria to the sarcoplasmic reticulum, creating an efficient micro domain for ADP transport from SERCA to mitochondria. Therefore, I will over-and-under express MFN2 in rat skeletal muscle and subsequently determine a) muscle fatigue rates, b) intracellular calcium levels during contraction, and c) calcium stimulated mitochondrial respiration kinetics. **4. Regulation of mitochondrial biogenesis: Scarce information exists regarding post-transcriptional mechanisms that influence mitochondrial biogenesis. In eukaryotic cells a variety of RNA binding proteins (RBPs) have been identified which regulate mRNA stability. The role of these RBPs in mammalian muscle remains unknown. Therefore, I will over-express HuR (stabilizer), CUB-BP1 and AUF1 (both destabilizers) alone and in combination in rat muscle to determine the effect on mRNA profiles, whole muscle metabolism, mitochondrial content/respiratory capacity, and responses to acute and chronic muscle contraction.**SIGNIFICANCE; All required methodologies have been established in my laboratory. The proposed studies will provide fundamental knowledge on diverse cellular processes, and therefore, HQP within my laboratory will be comprehensively trained.

在肌肉中，线粒体影响多种过程，包括代谢稳态、细胞凋亡和氧化还原平衡，但线粒体的调节仍不清楚，因此，通过结合分子、生化和氧化还原作用，更好地了解这些动态结构可以提供对许多细胞过程的深入了解。生理学方法 我的长期目标是确定 a) 调节线粒体生物能量的机制，b) 线粒体生物发生，以及 c) 这些如何影响骨骼肌中的底物利用。**我的具体假设和短期计划了解是：*1。规定线粒体 FAT/CD36 易位的影响：我之前已经表明，运动会将 FAT/CD36 重新分配到线粒体膜，但是，这种亚细胞运输的分子基础仍然未知，我认为 FAT/CD36 的 C 端 YCACR 基序和 AMP 激活蛋白。该细胞过程需要激酶 (AMPK) 信号传导，我将把野生型和 C 端突变体（C 端氨基酸删除）转染到骨骼肌中。 FAT/CD36 敲除小鼠。此后我将提供代谢挑战（例如坐骨神经刺激收缩）并确定 FAT/CD36 在线粒体膜上积累的能力以及对脂肪酸氧化/呼吸速率的功能影响。野生型和 AMPK 激酶死亡小鼠将确定 AMPK 信号传导在介导线粒体 FAT/CD36 易位中的必要性。 **2。丙二酰辅酶 A (M-CoA) 动力学：我之前已经证明运动增强了 M-CoA 抑制 CPTI 的能力，但其分子基础仍不清楚。为了确定 CPTI 的直接调节、底物动力学（PCoA 和 M-CoA）。将进行 CoA) CoA) 并对 CPTI 进行免疫沉淀并检查人体运动前后的氧化还原修饰和乙酰化状态。此外，还将确定各种 CPTI 底物动力学。在细胞骨架网络（特别是β-微管蛋白）存在和不存在的情况下确定细胞骨架网络调节 CPTI 的能力。 **3. 线粒体网的代谢作用：我之前已经证明，过度表达线粒体融合蛋白- 2 (MFN2) 独立地不影响线粒体生物能，因此我发现 MFN2 的主要代谢作用是将线粒体锚定到肌浆网，从而产生肌浆网。因此，我将在大鼠骨骼肌中过量表达 MFN2，然后确定 a) 肌肉疲劳率，b) 收缩过程中的细胞内钙水平，以及 c) 钙刺激的线粒体。 **4. 线粒体生物发生的调节：有关影响线粒体生物发生的转录后机制的信息很少。这些 RBP 在哺乳动物肌肉中的作用仍然未知，因此，我将在大鼠肌肉中单独和组合过度表达 HuR（稳定剂）、CUB-BP1 和 AUF1，以确定对 mRNA 谱的影响。整个肌肉代谢、线粒体含量/呼吸能力以及对急性和慢性肌肉收缩的反应。**意义；所有必需的方法都已在我的实验室中建立，拟议的研究将提供有关不同细胞过程的基础知识，并且因此，我实验室的HQP将受到全面的培训。