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，可能是新发现的隐源性肝硬化病因。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有关的激素。NAFL与肥胖、2型糖尿病和高脂血症一样，都是以高循环浓度的瘦素激素为特征的。该建议的总体目标是证明瘦素作为肝纤维化（肝硬化的前体）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生成细胞，肝星状细胞（hsc）中具有促纤维化活性。体内数据表明，瘦素也是四氯化碳（CCl4）诱导的瘦小鼠肝纤维化所必需的，而不是肥胖小鼠。初步数据和目前的建议符合该实验室的长期目标：了解慢性肝纤维化的基本机制。本研究假设瘦素是活化的肝星状细胞中的促纤维化细胞因子，通过磷酸化的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，从而增加α 2(I)胶原的表达。为了验证这一假设，概述了三个目标。首先(a)通过检测纤维化肝脏中负责细胞外基质（ECM）产生增加的基因，进一步表征瘦素作为一种新型促纤维化细胞因子；(b)阐明瘦素影响hsc中(2(I)胶原表达的具体信号转导途径。第二(a)表征瘦素相关的α 2(I)胶原mRNA稳定性；(b)通过缺失突变和定点突变，确定受瘦素信号影响的人α 2(I)胶原启动子沿线的特定顺式作用元件，这些元件负责胶原基因表达的增加；(c)通过DNase I保护分析和电泳迁移转移试验，确定与瘦素改变胶原基因表达相关的特定转录因子。第三，利用啮齿类动物肥胖模型及其瘦仔鼠，确定暴露于CC14的野生型小鼠肝纤维化可能需要瘦素的机制，而相同处理的db/db或ob/ob小鼠则不需要瘦素；以及在使用fa/fa或Zucker糖尿病脂肪（ZDF）大鼠及其瘦仔鼠的胆总管结扎（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.