MOLECULAR GENETICS AND PATHOPHYSIOLOGY OF OBESITY

最近几个实验室的工作已经开始阐明啮齿动物中几种遗传性肥胖形式的分子基础。这项研究导致了瘦素的鉴定，瘦素是一种在调节食欲、新陈代谢和体重方面发挥作用的肽。我们研究了脂肪组织对瘦素分泌的调节，特别是瘦素分泌的急性激素调节。当脂肪组织在体外培养时，胰岛素增加瘦素的分泌速率。此外，形态学研究表明，胰岛素改变了瘦素在分离的脂肪细胞中的亚细胞定位。这些变化是一致的解释，瘦素分泌的调节直接除了激素的作用，以调节转录的leptin gene.There是4个已知的同种型的人瘦素受体（leptin receptor，RXR）具有不同的C-末端胞质域。在单独的实验中，我们获得了编码所有四种亚型的cDNA克隆。我们已经使用这些试剂来研究这些受体的细胞内运输，以观察不同的尾部是否引起受体的差异靶向。我们通过独特的C-末端氨基酸的数量来指定每个同种型。根据瘦素结合位点的分布判断，没有一种亚型在质膜上有效表达。在表达α-67的细胞中，只有5%的总瘦素结合位点位于质膜上;相反，在表达α-5、α-15或α- 274的细胞中，约25%的结合位点位于质膜上。有趣的是，与其他同种型相比，转染了β-5的细胞表达了4倍多的总结合位点，因此在质膜上具有更多的结合位点。免疫荧光定位研究表明，所有4种亚型部分共定位与钙连接蛋白，内质网的标志物。所有4种亚型也与β-COP（高尔基体标记物）部分共定位，并在未识别的点状隔室中观察到。虽然所有受体均通过网格蛋白介导的内吞作用内化，但内化速率不同，其中α-15内化最快，其次是α-67、α-274和α-5（按此顺序）。leupeptin可抑制内化瘦素的降解，表明瘦素在溶酶体中降解。过夜暴露于瘦素下调所有4种亚型，但在不同程度上。α-274表现出最大的下调，并且似乎比其他同种型更快地到达溶酶体。值得注意的是，瘦素-274是介导瘦素的大部分生物学作用的同种型，并且也是最易受配体诱导的下调的。- 瘦素，肥胖，下调，瘦素受体，蛋白运输，内吞

Recent work from several laboratories has begun to elucidate the molecular basis of several forms of genetic obesity in rodents. This research led to the identification of leptin, a peptide that has a role in regulating appetite, metabolism, and body weight. We have investigated the regulation of leptin secretion by adipose tissue, especially the acute hormonal regulation of leptin secretion. When adipose tissue is incubated in vitro, insulin increases the rate at which leptin is secreted. Furthermore, morphologic studies suggest that insulin alters the subcellular localization of leptin in isolated adipocytes. These changes are consistent with the interpretation that leptin secretion is regulated directly in addition to the action of hormones to regulate transcription of the leptin gene.There are 4 known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains. In separate experiments, we have obtained cDNA clones encoding all four isoforms. We have used these reagents to study the intracellular trafficking of these receptors to see if the different tails caused differential targeting of the receptors. We have designated each isoform by the number of unique C-terminal amino acids. As judged by the distribution of leptin binding sites, none of the isoforms were efficiently expressed at the plasma membrane. In cells expressing HLR-67, only 5% of the total leptin binding sites were located at the plasma membrane; in contrast, about 25% of the binding sites were at the plasma membrane in cells expressing HLR-5,-15, or- 274. Interestingly, HLR-5 transfected cells expressed 4-fold more total binding sites and thus had more binding sites at the plasma membrane than the other isoforms. Immunofluorescent localization studies showed that all 4 isoforms partially co-localized with calnexin, a marker of the endoplasmic reticulum. All 4 isoforms also partially co-localized with beta-COP (a golgi marker) and were seen in an unidentified punctate compartment. While all the receptors were internalized via clathrin mediated endocytosis, the internalization rates were different, with HLR-15 being internalized the fastest followed by HLR- 67, HLR-274, and HLR-5 (in that order). Degradation of internalized leptin was inhibited by leupeptin, indicating that leptin was degraded in lysosomes. Overnight exposure to leptin down-regulated all 4 isoforms, but to a variable extent. HLR-274 displayed the greatest down- regulation and also appeared to reach lysosomes more quickly than the other isoforms. It is noteworthy that HLR-274 is the isoform that mediates most of the biological actions of leptin, and is also most susceptible to ligand-induced down-regulation. - leptin, obesity, down- regulation, leptin receptor, protein trafficking, endocytosis