MOLECULAR GENETICS AND PATHOPHYSIOLOGY OF OBESITY

几个实验室最近的工作已经开始阐明啮齿动物几种形式的遗传性肥胖的分子基础。这项研究确定了瘦素，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，胰岛素会增加瘦素分泌的速度。此外，形态学研究表明胰岛素改变了离体脂肪细胞中瘦素的亚细胞定位。这些变化与瘦素分泌在激素作用下直接调节瘦素基因转录的解释一致。人类瘦素受体（HLR）有4种已知的同种异构体，具有不同的c端细胞质结构域。在单独的实验中，我们获得了编码所有四种同种异构体的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，看看不同的尾部是否引起受体的不同靶向。我们通过独特的c端氨基酸的数量来指定每个异构体。从瘦素结合位点的分布来看，没有一个亚型在质膜上有效表达。在表达HLR-67的细胞中，只有5%的瘦素结合位点位于质膜上；相比之下，在表达HLR-5、-15或- 274的细胞中，约25%的结合位点位于质膜上。有趣的是，转染HLR-5的细胞表达的总结合位点比其他亚型多4倍，因此在质膜上有更多的结合位点。免疫荧光定位研究表明，所有4种异构体都与内质网标记物钙连联素部分共定位。所有4种异构体也与β - cop（一种高尔基体标记物）部分共定位，并在未确定的点状室中可见。虽然所有受体都通过网格蛋白介导的内吞作用被内化，但内化速度不同，以HLR-15内化速度最快，其次是HLR- 67、HLR-274和HLR-5（依次）。内化瘦素的降解被瘦素抑制，表明瘦素在溶酶体中被降解。整夜暴露于瘦素中，所有4种亚型均下调，但程度不同。HLR-274表现出最大的下调，并且似乎比其他同工型更快地到达溶酶体。值得注意的是，HLR-274是介导瘦素大部分生物作用的亚型，也是最容易受到配体诱导下调的亚型。-瘦素，肥胖，下调，瘦素受体，蛋白质运输，内吞作用

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