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变的病因。瘦素是一种16千道尔顿的蛋白质，由肥胖基因转录而成，是一种与体重和饱腹感控制相关的激素。NAFL与肥胖、II型糖尿病和高脂血症一起，都是以循环中高浓度的激素瘦素为特征的疾病。这项提案的总体目标是证明瘦素作为一种新的肝纤维化介质的生物学后果，肝纤维化是肝硬变的先兆。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(HSCs)中具有促纤维化活性。体内数据表明，瘦素在四氯化碳(CCl4)诱导的瘦小但不肥胖的小鼠肝纤维化中也是必需的。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。在该方案中，假设瘦素是激活的肝星状细胞中的促纤维化细胞因子，并通过磷酸化的信号转导和转录激活因子(PSTAT)促进AP-1与α2(I)胶原启动子的结合而增加(2(I)胶原的表达。为了检验这一假说，我们提出了三个目标。首先，(A)通过检测导致纤维化肝脏细胞外基质产生增加的基因，进一步确定瘦素是一种新的促纤维化细胞因子；(B)阐明瘦素对肝星状细胞(2)胶原表达的影响的特定信号转导途径(S)。第二，(A)鉴定瘦素相关α2(I)胶原mRNA的稳定性；(B)确定人α2(I)胶原启动子受瘦素信号影响的特定顺式作用元件，这些元件通过缺失突变和定点突变导致胶原基因表达增加；以及(C)通过DNase I保护分析和凝胶迁移率改变分析鉴定与瘦素相关的特定转录因子改变胶原基因表达。第三，利用肥胖的啮齿动物模型和它们的瘦子，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)大鼠及其瘦子的胆总管结扎(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.