Nuclear Envelope, Lipoprotein Metabolism, and Hepatic Steatosis

肝脏是脂质代谢的主要部位。肝脏脂质代谢异常导致非酒精性脂肪性肝病（NAFLD），这是一种新兴的公共卫生流行病，估计影响约25%的美国人口。NAFLD包括一系列肝脏病理，开始于肝细胞中的脂滴积聚，称为单纯性脂肪变性，其可能进展为非酒精性脂肪性肝炎（NASH）。患有NASH的个体发生后续纤维化、肝硬化和肝细胞癌的风险增加。虽然对单纯性脂肪变性向NASH和纤维化的进展有适当的关注，但也迫切需要进一步了解控制单纯性脂肪变性发展的机制。该提案解决了这一需求，确定了对肝脏脂质代谢的正常调节至关重要的新分子组分，当受损时，会导致严重的脂肪变性。我们在体内和体外的广泛的初步研究新确定的torsinA/lamina相关的多肽1（LAP 1）复合物的内膜的核膜作为一种新的调节肝内脂质代谢，和一个强大的驱动器脂肪变性。扭转蛋白A（A-CKO）或LAP 1（L-CKO）的条件性肝细胞特异性耗竭导致显著的脂肪变性，其可在喂食常规饲料的小鼠中进展为NASH。这些小鼠表现出极低密度脂蛋白（VLDL）甘油三酯（TG）和载脂蛋白B（apo B）的组装和分泌显著减少。然而，这些小鼠中的肝脂肪变性远比在现有的VLDL分泌减少的小鼠模型中或在具有影响VLDL分泌的基因突变的人类中观察到的严重，这表明torsinA/LAP 1复合物丢失的额外影响。这些效应是细胞自主的，因为遗传缺失是肝细胞特异性的，这些小鼠表现出正常的体重，没有胰岛素抵抗的证据。基于这些和额外的初步结果，我们假设破坏torsinA/LAP 1复合物在内核膜引起脂肪变性，损害新合成的脂质转移到下游过程。在目标1中，我们将进行体内，离体和体外研究，以测试这一假设的torsinA/LAP 1复合物的损失损害VLDL的组装和分泌，阻止TG转移到新生的载脂蛋白B。在目标2中，我们将详细检查肝脏脂质代谢，包括L-CKO小鼠中存在的核脂滴（LD）和A-CKO小鼠ER中蓄积的脂质的研究。脂肪酸和TG合成，脂肪酸氧化，磷脂合成，脂滴形成和营业额，以及LD生物合成中的关键蛋白质的研究，将允许识别参与肝脏脂质蓄积的关键途径。在目标3中，我们将通过测试我们是否可以通过过表达torsinA在L-CKO小鼠中预防脂肪变性来定义torsinA和LAP 1之间的上位关系。这些目标的成功完成将涉及新的肝内靶点，以控制VLDL分泌和预防肝脂肪变性。这也将促进理解的作用，核被膜作为一个关键节点的肝脏脂质代谢。

The liver is a major site of lipid metabolism. Abnormalities of hepatic lipid metabolism cause nonalcoholic fatty liver disease (NAFLD), a burgeoning public health epidemic estimated to affect approximately 25% of the U.S. population. NAFLD encompasses a spectrum of liver pathologies, beginning with lipid droplet accumulation in hepatocytes, called simple steatosis, which may progress to nonalcoholic steatohepatitis (NASH). Individuals with NASH are at increased risk for subsequent fibrosis, cirrhosis and hepatocellular carcinoma. Although there is appropriate focus on the progression of simple steatosis to NASH and fibrosis, there is also an urgent need to advance understanding of the mechanisms governing the development of simple steatosis. This proposal addresses this need, identifying novel molecular components that are critical to the normal regulation of hepatic lipid metabolism and, when impaired, cause profound steatosis. Our extensive preliminary studies in vivo and in vitro newly identify the torsinA/lamina-associated polypeptide 1 (LAP1) complex of the inner membrane of the nuclear envelope as a novel regulator of intrahepatic lipid metabolism, and a potent driver of steatosis. Conditional hepatocyte-specific depletion of either torsinA (A-CKO) or LAP1 (L-CKO) causes significant steatosis, which can progress to NASH in mice fed a regular chow diet. These mice demonstrate significant reductions in the assembly and secretion of very low density lipoprotein (VLDL) triglycerides (TG) and apolipoprotein B (apoB). The hepatic steatosis in these mice is far more severe, however, than observed in existing murine models of reduced VLDL secretion or in humans with mutations in genes that affect VLDL secretion, indicating additional effects of loss of the torsinA/LAP1 complex. These effects are cell autonomous as the genetic deletion is hepatocyte-specific, and these mice exhibit normal body mass and no evidence of insulin resistance. Based on these and additional preliminary results, we hypothesize that disruption of the torsinA/LAP1 complex at inner nuclear membrane causes steatosis by impairing the transfer of newly synthesized lipids to downstream processes. In Aim 1, we will conduct in vivo, ex vivo, and in vitro studies to test the hypothesis that loss of the torsinA/LAP1 complex impairs VLDL assembly and secretion by preventing the transfer of TG to nascent apoB. In Aim 2, we will examine hepatic lipid metabolism in detail, including studies of nuclear lipid droplets (LD) present in L-CKO mice and lipid accumulated in the ER in A-CKO mice. Studies of fatty acid and TG synthesis, fatty acid oxidation, phospholipid synthesis, and lipid droplet formation and turnover, as well as examination of key proteins in LD biogenesis, will allow for identification of critical pathways involved in hepatic lipid accumulation. In Aim 3, we will define the epistatic relationship between torsinA and LAP1 in steatosis by testing if we can prevent it in L-CKO mice by overexpressing torsinA. Successful completion of these Aims will implicate new intrahepatic targets to control VLDL secretion and prevent hepatic steatosis. It will also advance understanding of the role of the nuclear envelope as a critical node of liver lipid metabolism.