Uehara T, Hoshino S, Ui M, Tokumitsu Y, Nomura Y
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Biochim Biophys Acta. 1994 Nov 10;1224(2):302-10. doi: 10.1016/0167-4889(94)90204-6.
Insulin/dexamethasone/methylisobutylxanthine (hormones/IBMX) induce 3T3-L1 fibroblasts to differentiate into adipocytes. Our previous study suggested that pertussis toxin (IAP)-sensitive GTP-binding protein(s) (G-protein) is involved in the process of differentiation by hormones/IBMX, accompanied by c-fos induction. Northern blotting indicated that among the IAP-sensitive G-proteins, the levels of Gi2 alpha, Go alpha, and Gi3 alpha mRNA were decreased, increased and unchanged, respectively. Gi1 alpha was undetectable and IAP attenuated the decrease in Gi2 alpha mRNA level but did not affect the change in Go alpha mRNA level during the adipocyte differentiation. These results indicate that IAP-sensitive Gi2 alpha mRNA level is decreased during adipocyte differentiation. A combination of phosphatidylinositol-specific phospholipase C (PI-PLC) and IBMX induced c-fos expression in 3T3-L1 fibroblasts similar to that induced with hormones/IBMX. c-fos induced by both stimulators was also diminished by anti-inositolglycan antibody or anti-PI-PLC antiserum. Insulin stimulated the release of inositolproteoglycan and diacylglycerol from 3T3-L1 fibroblasts, which was suppressed by IAP treatment. These findings suggested that one of the pathways of adipocyte differentiation induced by hormones/IBMX occurs via the inositolglycan-specific PI-PLC cascade coupled to IAP-sensitive G-protein(s). Both activation of glycerophosphate dehydrogenase and stimulation of insulin-dependent 2-deoxyglucose uptake induced by hormones/IBMX were enhanced in protein kinase C-depleted cells exposed to phorbol 12-myristate 13-acetate (PMA), and attenuated in IAP-treated cells. The level of a 32P-labeled 52 kDa protein in plasma membrane fractions immunoprecipitated by anti-PI-PLC antiserum was increased by PMA stimulation, abolished in PMA-treated cells, and increased in IAP-treated cells. These findings suggest that protein kinase C phosphorylates PI-PLC, resulting in a decrease in PI-PLC activity related to the signal transduction pathway of adipocyte differentiation of 3T3-L1 fibroblasts.
胰岛素/地塞米松/甲基异丁基黄嘌呤（激素/异丁基甲基黄嘌呤）可诱导3T3-L1成纤维细胞分化为脂肪细胞。我们之前的研究表明，百日咳毒素（IAP）敏感的GTP结合蛋白（G蛋白）参与了激素/异丁基甲基黄嘌呤诱导的分化过程，并伴有c-fos的诱导。Northern印迹法表明，在IAP敏感的G蛋白中，Gi2α、Goα和Gi3α mRNA的水平分别降低、升高和不变。未检测到Gi1α，且IAP减弱了脂肪细胞分化过程中Gi2α mRNA水平的降低，但不影响Goα mRNA水平的变化。这些结果表明，在脂肪细胞分化过程中，IAP敏感的Gi2α mRNA水平降低。磷脂酰肌醇特异性磷脂酶C（PI-PLC）与异丁基甲基黄嘌呤的组合在3T3-L1成纤维细胞中诱导c-fos表达，类似于激素/异丁基甲基黄嘌呤诱导的表达。两种刺激物诱导的c-fos也被抗肌醇聚糖抗体或抗PI-PLC抗血清所减弱。胰岛素刺激3T3-L1成纤维细胞释放肌醇蛋白聚糖和二酰基甘油，IAP处理可抑制这种释放。这些发现表明，激素/异丁基甲基黄嘌呤诱导的脂肪细胞分化途径之一是通过与IAP敏感的G蛋白偶联的肌醇聚糖特异性PI-PLC级联反应。在暴露于佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）的蛋白激酶C缺失细胞中，激素/异丁基甲基黄嘌呤诱导的甘油磷酸脱氢酶的激活和胰岛素依赖性2-脱氧葡萄糖摄取的刺激均增强，而在IAP处理的细胞中则减弱。抗PI-PLC抗血清免疫沉淀的质膜组分中32P标记的52 kDa蛋白水平在PMA刺激下升高，在PMA处理的细胞中消失，在IAP处理的细胞中升高。这些发现表明，蛋白激酶C使PI-PLC磷酸化，导致与3T3-L1成纤维细胞脂肪细胞分化信号转导途径相关的PI-PLC活性降低。
