Mechanisms and therapeutic potential of blocking the mitochondrial Mg2+ channel Mrs2 in obesity and NAFLD

摘要 肝脏葡萄糖和脂肪代谢异常是几种普遍的代谢紊乱的特征，包括 肥胖、2型糖尿病(T2 DM)和非酒精性脂肪性肝病(NAFLD)。肝脏血糖升高 代谢产物直接导致T2 DM患者的高血糖，而肝脏脂肪变性是其定义 非酒精性脂肪肝的特点。2型糖尿病和非酒精性脂肪肝患病率的增加对公众健康构成了重大威胁， 迫切需要针对潜在病理过程的治疗策略。虽然钙离子 代谢性疾病中的信号正在浮现，镁离子动力学在线粒体上的潜在交互作用 人们对生物能量学的了解要少得多，它是当前提案的重点。在初步研究中，我们 使用CRISPR/Cas9建立了线粒体镁离子转运体MRS2的全局KO模型，并研究了 对肝脏新陈代谢的影响(Daw Et Al Cell 2020)。有趣的是，mrs2的缺失会损害mMg2+的摄取和 在体内防止肝脏脂肪变性。这与线粒体呼吸能力的增加有关。 肝细胞和明显增强的白色脂肪组织的褐变，表明 能源支出。使用创新的、无偏见的RNA-SEQ，动物生理学研究，分子，细胞生物学， 生物化学和生物物理方法，我们已经确定了连接MRS-2介导的mMg2+的候选途径 WD对线粒体摄取OXPHOS和脂肪酸氧化的抑制作用。值得注意的是，海拔 在mrs2KO小鼠中，从头脂肪生成前体柠檬酸盐被钝化，从而控制饮食诱导的肥胖。基座 根据这些初步数据，我们假设mrs2介导的mmg2+摄取是 疾病进展过程中肝脏线粒体生物能衰竭，以及减轻过量mMg2+的作用 使用小分子阻断剂阻断mrs2通道活性可阻止NAFLD的发展 通过恢复信号和线粒体生物能量学。目前提案的总体目标是 确定由mMg2+动力学控制的分子信号及其对生物能量学和 线粒体质量控制，并描述将mrs2与关键肝脏代谢联系起来的分子机制 代谢性疾病中调节失调的通路。使用体外和体内一体化的组合 我们将进行机制研究，以确定细胞内镁离子信号中断的影响 对线粒体功能、肝脏脂肪和葡萄糖代谢、全身能量平衡以及 非酒精性脂肪肝的进展。鉴于这些初步调查结果，在提案的具体目标1中，我们将采用我们的 独特的小鼠模型研究mRs2和mMg2+摄取对肝细胞线粒体功能的影响 并将确定mMg2+摄取受损对肝脏和全身糖脂代谢的影响。 在特定的目标2中，我们将研究柠檬酸在Hif-1α依赖的信号转导中的作用，该信号在mrs2KO中改变， 最终恢复线粒体的能量学和质量控制。最后，在具体的目标3中，我们将使用这两个 遗传和药物干预以评估MRS2在NAFLD模型中的意义。

ABSTRACT Aberrant hepatic glucose and lipid metabolism is a feature of several prevalent metabolic disorders, including obesity, type 2 diabetes (T2DM) and nonalcoholic fatty liver disease (NAFLD). Increased hepatic glucose production directly contributes to hyperglycemia in patients with T2DM, and hepatic steatosis is the defining feature of NAFLD. The increased prevalence of T2DM and NAFLD present a significant threat to public health, and treatment strategies that address the underlying pathological processes are urgently needed. Although Ca2+ signaling in metabolic disease is emerging, the potential reciprocal role of Mg2+ dynamics on mitochondrial bioenergetics is much less understood and is the focus of the current proposal. In preliminary studies, we generated a global KO model of the mitochondrial Mg2+ transporter Mrs2 using CRISPR/Cas9 and examined the impact on hepatic metabolism (Daw et al Cell 2020). Interestingly, loss of Mrs2 impairs mMg2+ uptake and prevents hepatic steatosis in vivo. This is associated with increased mitochondrial respiratory capacity in hepatocytes and markedly enhanced browning of white adipose tissue, indicative of increased capacity for energy expenditure. Using innovative, unbiased RNA-seq, animal physiology studies, molecular, cell biological, biochemistry and biophysical approaches, we have identified candidate pathways linking Mrs2-mediated mMg2+ uptake to mitochondrial suppression of OXPHOS and fatty acid oxidation under WD. Remarkably, the elevation of de novo lipogenesis precursor citrate is blunted in Mrs2 KO mice and thus controls diet induced obesity. Based on these preliminary data, we hypothesize that Mrs2-mediated mMg2+ uptake is a key determinant of mitochondrial bioenergetic failure in the liver during disease progression, and that mitigating excess mMg2+ uptake using a small molecule blocker of the Mrs2 channel activity will prevent the development of NAFLD through restoration of signaling and mitochondrial bioenergetics. The overall aim of the current proposal is to identify molecular signals that are controlled by mMg2+ dynamics, and their impact on bioenergetics and mitochondrial quality control, and to describe the molecular mechanisms linking Mrs2 to key hepatic metabolic pathways that are dysregulated in metabolic diseases. Using a combination of integrative in vitro and in vivo approaches, we will perform mechanistic studies to determine the impact of disrupted intracellular Mg2+ signals on mitochondrial function, hepatic lipid and glucose metabolism, whole-body energy homeostasis, and the progression of NAFLD. Given these preliminary findings, in Specific Aim 1 of the proposal we will employ our unique mouse model to examine the role of Mrs2 and mMg2+ uptake on mitochondrial function in hepatocytes and will determine the impact of impaired mMg2+ uptake on hepatic and whole-body glucose and lipid metabolism. In Specific Aim 2, we will investigate the role of citrate in Hif-1α dependent signaling that is altered in Mrs2 KO, which ultimately restores mitochondrial energetics and quality control. Finally, in Specific Aim 3, we will use both genetic and pharmacologic interventions to evaluate the significance of MRS2 in NAFLD models.