Tanner J W, Leingang K A, Mueckler M M, Glenn K C
Monsanto Co., St. Louis, MO 63198.
Biochem J. 1992 Feb 15;282 ( Pt 1)(Pt 1):99-106. doi: 10.1042/bj2820099.
The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.
利用体外分化的原代大鼠脂肪细胞培养物，研究了生长激素（GH）急性刺激脂肪细胞摄取葡萄糖的细胞机制。从幼鼠腹股沟脂肪垫中通过胶原酶消化分离前脂肪细胞，并在3-异丁基-1-甲基黄嘌呤、胰岛素和地塞米松存在的情况下进行分化。在分化开始后的6天内观察到脂肪细胞形态（即脂质包涵体）的发育。与此表型变化一致的是，通过对总细胞蛋白进行Western免疫印迹测定，甘油醛-3-磷酸脱氢酶（GPDH）活性以及HepG2型（Glut1）和脂肪细胞/肌肉（Glut4）葡萄糖转运异构体的细胞含量增加。年龄匹配的未分化细胞表达Glut1转运体和低水平的GPDH，但既不积累脂质也不表现出可测量的Glut4蛋白表达。在分化开始后的第6天，GH和胰岛素以剂量和时间依赖性方式刺激在无血清条件下培养至少15小时的脂肪细胞摄取2-脱氧[14C]葡萄糖。亚细胞组分的Western印迹分析表明，GH和胰岛素均迅速（20分钟内）刺激Glut1和Glut4蛋白从低密度微粒体组分转运至质膜。利用针对Glut4蛋白C末端区域的抗血清和异硫氰酸荧光素标记的二抗进行的免疫细胞化学实验提供了确证证据。通过共聚焦激光显微镜成像观察细胞，结果与用GH或胰岛素处理后葡萄糖转运体从细胞内区域重新分布到质膜一致。基于这些数据，我们认为GH对脂肪细胞葡萄糖转运的胰岛素样作用涉及Glut1和Glut4蛋白转运至质膜。此外，GH和胰岛素均刺激葡萄糖转运体转运，这可能表明脂肪细胞GH受体和胰岛素受体之间存在共同的细胞信号元件，或者存在多种刺激葡萄糖转运体转运的细胞机制。
