Determining the Biological Effects of Mitochondrial Acyl Toxicity

项目摘要 在急性代谢应激和慢性代谢疾病，如肥胖和糖尿病，积累 脂肪酸氧化中间代谢产物的毒性一直被怀疑。在这些可能 脂毒性代谢物是长链酰基肉毒碱（LCAC），据称其干扰关键的生理学作用。 这些过程包括胰岛素信号传导、钙稳态和线粒体功能。然而，在这方面， 将这些过程中的缺陷与LCAC积累机械地联系起来是困难的，这是由于缺乏 LCAC累积临床前模型。我们通过开发一种独特的小鼠模型， 通过删除分解代谢LCAC的酶，肉毒碱棕榈酰转移酶-2， 在骨骼肌中（Cpt2Sk-/-）。与LCACs对生物学影响的怀疑作用一致，我们的初步研究结果表明， 数据表明Cpt2Sk-/-肌肉具有减少的力产生和线粒体功能障碍。我们 初步数据还表明，LCAC在氧化肌纤维内大量积累，因此是最常见的。 易受潜在LCAC毒性的影响。虽然我们的Cpt2Sk-/-模型提供了持续升高的LCAC， 生理结果被由于线粒体FAO缺乏引起的能量剥夺所混淆。以减轻 考虑到这个混淆变量，我们将采用三种互补的肌肉小鼠模型， 特异性FAO缺陷：1）Cpt2Sk-/-，其在整个细胞中积累LCAC; 2）肉毒碱酰基肉毒碱移位酶 （CactSk-/-）小鼠，其在线粒体外积累LCAC;和3）酰基-CoA合成酶1（AcsllSk-/-） 完全不积累LCAC的小鼠。在这里，我们将使用这三个模型系统来确定 LCACs对胰岛素信号传导、钙稳态和线粒体功能的影响。结果将首先提供 必要的实验对比，以揭示LCAC对细胞生理学的直接与间接影响。

PROJECT SUMMARY During acute metabolic stress and chronic metabolic disease, such as obesity and diabetes, the accumulation of fatty acid oxidation intermediary metabolites has long been suspected of toxicity. Among these possible lipotoxic metabolites are long-chain acylcarnitines (LCACs), which purportedly interfere with critical physiological processes including insulin signaling, calcium homeostasis, and mitochondrial function. However, mechanistically linking defects in these processes to LCAC accumulation has been difficult due to a lack of LCAC-accumulating pre-clinical models. We overcame this barrier by developing a unique mouse model of LCAC accumulation by deleting the enzyme that catabolizes LCACs, carnitine palmitoyltransferase-2 specifically in skeletal muscle (Cpt2Sk-/-). Consistent with the suspected roles of LCACs effects on biology, our preliminary data demonstrate that Cpt2Sk-/- muscles have reduced force production and mitochondrial dysfunction. Our preliminary data also demonstrate large accumulation of LCACs within oxidative muscle fibers, thus are the most vulnerable to potential LCAC toxicity. While our Cpt2Sk-/- model provides consistently elevated LCACs, the physiological outcomes are confounded by energy deprivation due to mitochondrial FAO deficiency. To mitigate concerns surrounding this confounding variable, we will employ three complimentary mouse models of muscle- specific FAO deficiency: 1) Cpt2Sk-/- that accumulate LCACs across the cell; 2) carnitine acylcarnitine translocase (CactSk-/-) mice that accumulate LCACs outside of the mitochondria; and 3) acyl-CoA synthetase 1 (Acsl1Sk-/-) mice that do not accumulate LCACs at all. Here, we will use this three-model system to determine the role of LCACs on insulin signaling, calcium homeostasis, and mitochondrial function. Results will be the first to provide requisite experimental contrast to unveil direct versus indirect effects of LCACs on cell physiology.