Asada N, Takahashi Y, Wada M, Naito N, Uchida H, Ikeda M, Honjo M
Pharmaceuticals Group, Life Sciences Laboratory, Central Research Institute, Mitsui Chemicals, Inc, Chiba, Japan.
Mol Cell Endocrinol. 2000 Apr 25;162(1-2):121-9. doi: 10.1016/s0303-7207(00)00202-1.
We have constructed a cell line of 3T3-L1 which can efficiently express human GHR (3T3-L1-hGHR) after differentiation to adipocytes. The expressed hGHR was detected as two bands with approximate molecular sizes of 120K by Western analysis using hGHR specific monoclonal antibody. Maximum lipolytic activity induced by hGH in the 3T3-L1-hGHR was enhanced 10-fold as compared to that in 3T3-L1, suggesting that expressed hGHR is functionally active. Comparative analysis using bGH and hGH revealed that 70% of lipolysis stimulation by 1-10 ng/ml hGH could be attributed to hGHR-mediated response. Analyses on inhibition and phosphorylation of signaling molecules suggested that GH-induced lipolysis stimulation is dependent on gene expression and not mediated through PKA-, PKC-, PLA-, PLC-, nor MAPK-pathway but possibly through JAK-STATs pathway. Duration of STAT5 activation by hGH continued up to 48 h. We also revealed that 22 K hGH isoform, 20K hGH which has been reported as a weaker agonist for GH-induced lipolysis stimulation, possesses equipotent activity and shows stronger action in the presence of hGHBP as compared to 22 K hGH. Taken together we conclude that the hGH-induced lipolysis was not mediated through MAP-, PKA-, PKC-, nor PLA-pathway but might be mediated through STAT pathway and that 20K hGH might show higher lipolytic activity than 22 K hGH in adipose tissue that produces a large amount of GHBP.
我们构建了一种3T3-L1细胞系，该细胞系在分化为脂肪细胞后能够高效表达人GHR（3T3-L1-hGHR）。使用hGHR特异性单克隆抗体通过蛋白质免疫印迹分析检测到表达的hGHR为两条带，其近似分子大小为120K。与3T3-L1相比，hGH在3T3-L1-hGHR中诱导的最大脂解活性增强了10倍，这表明表达的hGHR具有功能活性。使用bGH和hGH的比较分析表明，1-10 ng/ml hGH引起的70%的脂解刺激可归因于hGHR介导的反应。对信号分子的抑制和磷酸化分析表明，GH诱导的脂解刺激依赖于基因表达，不是通过PKA、PKC、PLA、PLC或MAPK途径介导的，而是可能通过JAK-STATs途径介导的。hGH对STAT5的激活持续长达48小时。我们还发现，22K hGH异构体、20K hGH（据报道其作为GH诱导脂解刺激的激动剂较弱）具有同等效力的活性，并且与22K hGH相比，在存在hGHBP的情况下表现出更强的作用。综上所述，我们得出结论，hGH诱导的脂解不是通过MAP、PKA、PKC或PLA途径介导的，而是可能通过STAT途径介导的，并且在产生大量GHBP的脂肪组织中，20K hGH可能比22K hGH表现出更高的脂解活性。
