The Role of Leptin in Liver Fibrogenesis

描述（由申请人提供）：非酒精性脂肪性肝炎或非酒精性脂肪肝（NAFL）是一个日益严重的临床问题，可能是新认识到的隐源性肝硬化的病因。瘦素是一种由肥胖基因转录产生的16千道尔顿蛋白质，是一种与体重和饱腹感控制相关的激素。NAFL，沿着肥胖、II型糖尿病和高脂血症，是以高循环浓度的激素瘦素为特征的病症。这项提案的总体目标是证明瘦素作为肝纤维化（肝硬化的前兆）的新介质的生物学后果。初步数据表明，瘦素在肝脏的主要胶原蛋白产生细胞，肝星状细胞（HSC）中具有促纤维化活性。体内数据表明，瘦素也需要四氯化碳（CCl 4）诱导的肝纤维化瘦，但不肥胖，小鼠。初步的数据和目前的建议符合本实验室的长期目标：了解慢性肝纤维化的基本机制。在该提议中，假设瘦素是活化的肝星状细胞中的促纤维化细胞因子，并通过磷酸化信号转导和转录激活因子（pSTAT）增强AP-1与α 2（I）胶原启动子的结合来增加α 2（I）胶原表达。三个目标概述了测试这一假设。首先，（a）通过检查负责纤维化肝脏中细胞外基质（ECM）产生增加的基因，进一步将瘦素表征为新的促纤维化细胞因子;和（B）阐明负责瘦素对HSC中β 2（I）胶原表达的影响的特异性信号转导途径。其次是（a）表征瘦素相关的α 2（I）胶原蛋白mRNA稳定性;（B）通过使用缺失突变体和定点诱变，鉴定受瘦素信号传导影响的人α 2（I）胶原蛋白启动子沿线的特定顺式作用元件，所述顺式作用元件沿着引起胶原蛋白基因表达增加;和（c）通过DNA酶I保护分析和电泳迁移率变动分析鉴定与瘦素改变的胶原基因表达相关的特异性转录因子。第三，利用肥胖的啮齿动物模型及其瘦的同窝仔，以确定暴露于CC 14的野生型小鼠中肝纤维化可能需要瘦素的机制，但在相同处理的db/db或ob/ob小鼠中不需要瘦素;以及在使用fa/fa或Zucker糖尿病脂肪（ZDF）大鼠及其瘦的同窝仔的胆总管结扎（CBDL）损伤模型中是否需要瘦素。

DESCRIPTION (provided by applicant): Non-alcoholic steatohepatitis or non-alcoholic fatty liver (NAFL) is a growing clinical problem that may account for a newly recognized etiology for cryptogenic cirrhosis. Leptin, a 16-kilodalton protein resulting from the transcription of the obese gene, is a hormone associated with weight and satiety control. NAFL, along with obesity, type II diabetes mellitus, and hyperlipidemia, are conditions characterized by high circulating concentrations of the hormone leptin. The overall goal of this proposal is to demonstrate the biological consequences of leptin as a novel mediator of liver fibrosis, the precursor to cirrhosis. Preliminary data indicate leptin has profibrogenic activity in the principal collagen producing cells of the liver, hepatic stellate cells (HSCs). In vivo data indicate that leptin is also required for liver fibrosis induced by carbon tetrachloride (CCl4) in lean, but not obese, mice. The preliminary data and the current proposal meet the long-term objectives of this laboratory: to understand basic mechanisms underlying chronic liver fibrosis. In this proposal, it is hypothesized that leptin is a profibrogenic cytokine in activated hepatic stellate cells and increases (2(I) collagen expression by phosphorylated signal transduction and activator of transcription (pSTAT) enhancing AP-1 binding to the alpha2(I) collagen promoter. Three aims are outlined to test this hypothesis. First to (a) further characterize leptin as a novel profibrogenic cytokine by examining genes responsible for increased extracellular matrix (ECM) production in fibrotic liver; and (b) to elucidate the specific signal transduction pathway(s) responsible for the effect of leptin on (2(I) collagen expression in HSCs. Second to (a) characterize leptin-associated alpha2(I) collagen mRNA stabilization; to (b) identify specific cis-acting elements along the human alpha2(I) collagen promoter affected by leptin signaling that are responsible for increased collagen gene expression by employing deletion mutant and site-directed mutagenesis; and (c) to identify, by DNase I protection analysis and electrophoretic mobility shift assay, specific transcription factors associated with leptin altered collagen gene expression. Third, to exploit rodent animal models of obesity, and their lean littermates, to determine the mechanisms by which leptin may be required for liver fibrosis in wild-type mice exposed to CC14 but not in identically treated db/db or ob/ob mice; and whether leptin is required in a common bile duct ligation (CBDL) injury model using fa/fa, or Zucker Diabetic Fatty (ZDF) rats and their lean littermates.