Role of carnitine acetyltransferase in mitochondrial function and insulin action

描述（由申请人提供）：本项目的长期目标是探索与脂质诱导的胰岛素抵抗相关的机制。一个共同的主题是，线粒体功能在这些条件下受损。在最近的研究中，我们发现慢性高脂肪喂养导致线粒体呼吸能力降低，此时不完全氧化加速，因此意味着氧化和三羧酸循环活动之间的断开。我们假设，线粒体基质酶肉毒碱乙酰转移酶（CrAT），通过其行动允许出口过剩的不完全氧化中间体从线粒体，在维持骨骼肌线粒体功能下的时期，过量的脂质负担的关键作用。在骨骼肌细胞培养模型（L 6和C2 C12细胞系）中使用腺病毒介导的CrAT沉默，以及在小鼠中使用肌肉特异性靶向CrAT沉默（腺病毒注射或电穿孔），我们将探索CrAT在线粒体底物处理中的作用。将采用经典的功能测定和基于质谱的代谢分析来确定siCrAT治疗是否增加对脂质诱导的线粒体功能障碍的易感性，并随后使骨骼肌倾向于发展胰岛素抵抗。如果观察到表型，我们将探索CrAT是否调节线粒体蛋白乙酰化和/或氧化应激，以评估这种酶在响应高脂质环境调节线粒体功能中的作用背后的潜在机制。这些实验的结果将提供有价值的见解CrAT在脂质诱导的线粒体功能障碍和胰岛素抵抗的作用。 公共卫生相关性：肥胖、胰岛素抵抗和糖尿病的发病率正以惊人的速度增加，并且与脂质代谢和线粒体功能的失调密切相关。CrAT是一种位于线粒体基质内的酶，将短链酰基辅酶A转化为短链酰基肉毒碱，然后可以从线粒体输出，从而提供在脂质过量条件下维持线粒体功能的机制。拟议的实验将大大提高我们的理解CrAT在线粒体底物处理的调节中的作用，以及建立这种酶在减轻肥胖，胰岛素抵抗和糖尿病的潜在作用。

DESCRIPTION (provided by applicant): The long-term goal of this project is to explore mechanisms linked to lipid-induced insulin resistance. A common theme emerging is that mitochondrial function is impaired in these conditions. In recent studies we discovered chronic high fat feeding resulted in diminished mitochondrial respiratory capacity at a time when incomplete ¿-oxidation was accelerated, thus implying a disconnect between ¿-oxidation and tricarboxylic acid cycle activity. We hypothesize that the mitochondrial matrix enzyme carnitine acetyltransferase (CrAT), through its action allowing the export of excess incomplete ¿-oxidative intermediates from the mitochondria, plays a critical role in maintaining skeletal muscle mitochondrial function under periods of excess lipid burden. Using adenoviral-mediated silencing of CrAT in skeletal muscle cell culture models (L6 and C2C12 cell lines), as well as muscle specific targeted silencing of CrAT (adenoviral injection or electroporation) in mice, we will explore the role of CrAT in mitochondrial substrate handling. Classic functional assays and mass spectrometry-based metabolic profiling will be employed to determine whether siCrAT treatment increases susceptibility to lipid-induced mitochondrial dysfunction and subsequently predisposes skeletal muscle toward the development of insulin resistance. Provided a phenotype is observed we will explore whether CrAT modulates mitochondrial protein acetylation &/or oxidative stress to assess potential mechanisms behind the role of this enzyme in regulating mitochondrial function in response to a high lipid environment. Results from these experiments will provide valuable insights into the role of CrAT in lipid-induced mitochondrial dysfunction and insulin resistance. PUBLIC HEALTH RELEVANCE: The incidence of obesity, insulin resistance, and diabetes are increasing at alarming rates and are closely associated with dysregulation of lipid metabolism and mitochondrial function. CrAT, an enzyme localized within the mitochondrial matrix, converts short chain acyl-CoAs to short chain acylcarnitines which can then be exported from the mitochondria, thereby providing a mechanism to maintain mitochondrial function under conditions of lipid excess. The proposed experiments will greatly enhance our understanding of the role of CrAT in the regulation of mitochondrial substrate handling, as well as establishing a potential role of this enzyme in the alleviation of obesity, insulin resistance, and diabetes.