Nuclear Envelope, Lipoprotein Metabolism, and Hepatic Steatosis

肝脏是脂肪代谢的主要部位。肝脂代谢异常会导致非酒精性脂肪性肝病(NAFLD)，这是一种迅速发展的公共卫生流行病，估计影响大约25%的美国人口。NAFLD包括一系列的肝脏病理，首先是肝细胞中的脂滴聚集，称为单纯性脂肪变性，这可能进展为非酒精性脂肪性肝炎(NASH)。患有NASH的个体患上随后的纤维化、肝硬变和肝细胞癌的风险增加。尽管人们对单纯性脂肪变性向NASH和纤维化的进展给予了适当的关注，但也迫切需要促进对单纯性脂肪变性发生机制的了解。这项建议解决了这一需求，确定了对肝脏脂肪代谢的正常调节至关重要的新分子成分，当受损时，会导致严重的脂肪变性。我们在体内和体外进行的广泛的初步研究新发现，核膜内膜的TorsinA/LAP1复合体是一种新的肝内脂肪代谢调节因子，也是脂肪变性的有力驱动因素。肝细胞特异性的TorsinA(A-CKO)或LAP1(L-CKO)的条件性耗竭会导致显著的脂肪变性，在喂食常规饮食的小鼠中可能进展为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(ApoB)的组装和分泌显着减少。然而，与现有的极低密度脂蛋白分泌减少的小鼠模型或影响极低密度脂蛋白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复合体丢失的额外影响。这些影响是细胞自主的，因为基因缺失是肝细胞特有的，这些小鼠表现出正常的体重，没有胰岛素抵抗的证据。基于这些和其他初步结果，我们假设内核膜TorsinA/LAP1复合体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。在目标1中，我们将进行体内、体外和体外研究，以验证TorsinA/LAP1复合体的丢失通过阻止TG转移到新生apoB而损害VLDL组装和分泌的假设。在目标2中，我们将详细研究肝脏的脂质代谢，包括L-CKO小鼠体内存在的核脂滴(LD)和A-CKO小鼠内质网中积累的脂质。对脂肪酸和甘油三酯合成、脂肪酸氧化、磷脂合成、脂滴形成和周转的研究，以及对LD生物发生中关键蛋白质的检测，将有助于确定参与肝脏脂质积累的关键途径。在目标3中，我们将通过测试是否可以通过过度表达TorsinA来预防L-CKO小鼠的脂肪变性，来确定TorsinA和LAP1在脂肪变性中的上位关系。这些目标的成功实现将涉及控制极低密度脂蛋白分泌和预防肝脏脂肪变性的新的肝内靶点。它还将促进对核膜作为肝脏脂质代谢的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.