Nuclear Envelope, Lipoprotein Metabolism, and Hepatic Steatosis

肝脏是脂质代谢的主要部位。肝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，从肝细胞脂滴积聚开始，称为单纯性脂肪变性，可发展为非酒精性脂肪性肝炎（NASH）。NASH患者随后发生纤维化、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正常调节至关重要的新分子成分，当受损时，会导致深度脂肪变性。我们在体内和体外进行了广泛的初步研究，新发现核膜内膜的torsinA/lamina-associated polypeptide 1 （LAP1）复合体是肝内脂质代谢的一种新的调节剂，也是脂肪变性的一种强有力的驱动因素。条件性肝细胞特异性torsinA （a - cko）或LAP1 （L-CKO）的缺失会导致显著的脂肪变性，在喂食常规食物的小鼠中可发展为NASH。这些小鼠显示极低密度脂蛋白（VLDL）甘油三酯（TG）和载脂蛋白B （apoB）的组装和分泌显著减少。然而，与现有的VLDL分泌减少的小鼠模型或影响VLDL分泌的基因突变的人类相比，这些小鼠的肝脏脂肪变性要严重得多，这表明torsinA/LAP1复合物缺失的额外影响。这些影响是细胞自主的，因为基因缺失是肝细胞特异性的，这些小鼠表现出正常的体重，没有胰岛素抵抗的迹象。基于这些和其他初步结果，我们假设核膜torsinA/LAP1复合物的破坏通过损害新合成的脂质向下游过程的转移而导致脂肪变性。在Aim 1中，我们将进行体内、离体和体外研究，以验证torsinA/LAP1复合物的缺失通过阻止TG向新生apoB转移而损害VLDL组装和分泌的假设。在Aim 2中，我们将详细检查肝脏脂质代谢，包括研究L-CKO小鼠中存在的核脂滴（LD）和A-CKO小鼠内质网中积累的脂质。脂肪酸和TG合成、脂肪酸氧化、磷脂合成、脂滴形成和转化的研究，以及LD生物发生过程中关键蛋白的检测，将有助于识别肝脏脂质积累的关键途径。在Aim 3中，我们将通过测试是否可以通过过表达torsinA来预防L-CKO小鼠的脂肪变性，从而确定torsinA和LAP1在脂肪变性中的地位关系。这些目标的成功完成将意味着新的肝内靶点控制VLDL分泌和防止肝脂肪变性。它也将促进对核包膜作为肝脂质代谢关键节点的作用的理解。

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.