MOLECULAR GENETICS AND PATHOPHYSIOLOGY OF OBESITY

几个实验室最近的工作已经开始， 阐明遗传性肥胖的几种形式的分子基础， 啮齿动物这项研究导致了瘦素的鉴定， 在调节食欲、新陈代谢和体重方面发挥作用。 我们研究了脂肪对瘦素分泌的调节作用， 组织，尤其是瘦素分泌的急性激素调节。 当脂肪组织在体外培养时， 瘦素分泌的时间此外，形态学研究 表明胰岛素改变了瘦素在细胞内的亚细胞定位， 分离的脂肪细胞。这些变化与 解释瘦素分泌除了直接调节 激素调节瘦素转录的作用 基因 有4种已知的人类瘦素受体亚型 (HLR)不同的C端胞质结构域。分开的 实验中，我们获得了编码所有四个的cDNA克隆， 同种型。我们已经用这些试剂研究了细胞内 这些受体的运输，看看是否不同的尾巴引起 受体的差异靶向。我们已经指定了每一个 通过独特的C-末端氨基酸的数量来区分同种型。评选出了媒体 根据瘦素结合位点的分布，没有一种亚型是 在质膜上有效表达。的细胞中 在p53 -67上，只有5%的瘦素结合位点位于p53 - 67上。 质膜;相比之下，约25%的结合位点 在表达β-5，β-15， 或-274。有趣的是，转染了IL-5的细胞表达了4倍的 更多的总结合位点，因此在 质膜比其他亚型。免疫荧光 定位研究表明，所有4种异构体部分共定位 与钙连接蛋白，内质网的标志物。所有4 同种型也与β-COP（高尔基体标记物）部分共定位 在一个不明的点状隔室中发现。虽然所有 受体通过网格蛋白介导的内吞作用内化， 内化率各不相同， 内化速度最快，其次是歼-67、歼-274和歼-5 (in这个命令）。内源性瘦素的降解被抑制， leupeptin，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 可变的程度。其中，274的下调幅度最大 而且似乎比其他人更快到达溶酶体 同种型。值得注意的是，IFN- 274是 介导瘦素的大多数生物学作用，也是瘦素的大多数生物学作用。 对配体诱导的下调敏感。

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.