Grunfeld C, Jones D S
Endocrinology. 1983 Nov;113(5):1763-70. doi: 10.1210/endo-113-5-1763.
3T3-L1 fibroblasts and fat cells have been extensively used to study the development of insulin-stimulated glucose and lipid metabolism during adipocyte differentiation in vitro. In this paper we explore the ability of insulin to stimulate amino acid uptake in 3T3-L1 cells using the nonmetabolizable amino acid analog methylaminoisobutyric acid (MAIB). In differentiated 3T3-L1 fat cells, a 12-h preincubation with insulin was required for maximal stimulation of MAIB uptake. In contrast, in the undifferentiated fibroblasts, insulin stimulation peaked between 6 and 8 h and then declined significantly. Maximal stimulation of MAIB uptake in the differentiated fat cell exceeded that in the fibroblast phenotype. This increased ability of insulin to stimulate MAIB uptake in fat cells appeared within the first day after removal of the differentiation medium. 3T3-L1 fat cells were 30 times more sensitive to the effects of insulin on MAIB transport than the undifferentiated fibroblasts. These findings are consistent with previous data on insulin-stimulated deoxyglucose uptake, in that the increased sensitivity to insulin with differentiation is more than can be accounted for by the increase in receptor number. The activity of porcine proinsulin indicates that this stimulation reflects the known characteristics of the insulin receptor. The stimulation of MAIB uptake by insulin in 3T3-L1 fat cells was blocked by inhibitors of protein synthesis (cycloheximide and puromycin) and mRNA synthesis (actinomycin D). Colchicine, an inhibitor of microtubule function, showed little inhibition of insulin-stimulated MAIB uptake. Insulin stimulation of MAIB uptake was greater when 3T3-L1 cells were preincubated with insulin in the absence of essential amino acids. Basal transport in 3T3-L1 cells was not influenced by the presence or absence of amino acids. Thus, amino acid deprivation appears specifically to enhance the ability of insulin to stimulate amino acid transport in cultured adipocytes.
3T3-L1成纤维细胞和脂肪细胞已被广泛用于体外研究脂肪细胞分化过程中胰岛素刺激的葡萄糖和脂质代谢的发展。在本文中，我们使用不可代谢的氨基酸类似物甲基氨基异丁酸（MAIB）来探究胰岛素刺激3T3-L1细胞摄取氨基酸的能力。在分化的3T3-L1脂肪细胞中，需要用胰岛素预孵育12小时才能最大程度地刺激MAIB摄取。相比之下，在未分化的成纤维细胞中，胰岛素刺激在6至8小时达到峰值，然后显著下降。分化的脂肪细胞中MAIB摄取的最大刺激超过了成纤维细胞表型中的刺激。胰岛素刺激脂肪细胞摄取MAIB的这种增强能力在去除分化培养基后的第一天内就出现了。3T3-L1脂肪细胞对胰岛素对MAIB转运的影响的敏感性是未分化成纤维细胞的30倍。这些发现与先前关于胰岛素刺激的脱氧葡萄糖摄取的数据一致，即随着分化对胰岛素的敏感性增加，超过了受体数量增加所能解释的范围。猪胰岛素原的活性表明这种刺激反映了胰岛素受体的已知特征。胰岛素对3T3-L1脂肪细胞中MAIB摄取的刺激被蛋白质合成抑制剂（环己酰亚胺和嘌呤霉素）和mRNA合成抑制剂（放线菌素D）阻断。秋水仙碱是微管功能的抑制剂，对胰岛素刺激的MAIB摄取几乎没有抑制作用。当3T3-L1细胞在无必需氨基酸的情况下用胰岛素预孵育时，胰岛素对MAIB摄取的刺激更大。3T3-L1细胞中的基础转运不受氨基酸存在与否的影响。因此，氨基酸剥夺似乎特异性地增强了胰岛素刺激培养的脂肪细胞中氨基酸转运的能力。
