Reed B C, Shade D, Alperovich F, Vang M
Department of Biochemistry and Molecular Biology, LSU Medical Center, Shreveport 71130.
Arch Biochem Biophys. 1990 Jun;279(2):261-74. doi: 10.1016/0003-9861(90)90490-p.
A glucose transporter cDNA (GLUT) clone was isolated from mouse 3T3-L1 adipocytes and sequenced. The nucleotide and deduced amino acid sequences were, respectively, 95 and 99% homologous to those of the rat brain transporter. The mouse cDNA and a polyclonal antibody recognizing the corresponding in vitro translation product were used to compare changes in transporter mRNA and protein levels during differentiation, glucose starvation, and chronic insulin exposure of 3T3-L1 preadipocytes. The respective cellular content of transporter mRNA and protein were increased 6.6- and 7.8-fold during differentiation, and 3.8- and 2.5-fold from chronic insulin exposure of differentiated adipocytes. Glucose starvation increased transporter mRNA and protein levels 2.2- and 3.5-fold in undifferentiated preadipocytes and 1.8- and 3.1-fold in differentiated adipocytes. Starvation of undifferentiated cells completely converted the native transporter to an incompletely glycosylated form, while increasing basal transport rates 4.5-fold. Either full glycosylation is not required to produce a functionally active transporter, or starvation causes a unique predifferentiation induction of the normally absent "responsive" transporter. The changes in transporter protein expression elicited by differentiation were attributed primarily to increased rates of transporter synthesis, while the disproportionate changes in mRNA and protein expression from chronic insulin treatment and starvation suggested these conditions increase synthesis and decrease turnover rates in regulating transporter protein expression. Although chronic insulin exposure and glucose starvation each raised the expression of transporter protein greater than 3-fold and basal transport rates 2.5- to 4.5-fold, no significant increase in the insulin responsiveness of 3T3-L1 preadipocytes or differentiated adipocytes was observed. Thus, the changes in the transporter mRNA and protein expression observed in this study were most consistent with their being associated with the regulated expression of a basal or low level insulin-responsive transporter.
从小鼠3T3-L1脂肪细胞中分离出一个葡萄糖转运体cDNA（GLUT）克隆并进行测序。其核苷酸序列和推导的氨基酸序列与大鼠脑转运体的相应序列分别具有95%和99%的同源性。利用小鼠cDNA和识别相应体外翻译产物的多克隆抗体，比较了3T3-L1前脂肪细胞在分化、葡萄糖饥饿和长期胰岛素暴露过程中转运体mRNA和蛋白质水平的变化。在分化过程中，转运体mRNA和蛋白质的细胞含量分别增加了6.6倍和7.8倍，在分化的脂肪细胞长期暴露于胰岛素后分别增加了3.8倍和2.5倍。葡萄糖饥饿使未分化前脂肪细胞中的转运体mRNA和蛋白质水平分别增加2.2倍和3.5倍，在分化的脂肪细胞中分别增加1.8倍和3.1倍。未分化细胞饥饿完全将天然转运体转化为不完全糖基化形式，同时基础转运速率增加4.5倍。要么产生功能活性转运体不需要完全糖基化，要么饥饿导致正常情况下不存在的“反应性”转运体的独特预分化诱导。分化引起的转运体蛋白表达变化主要归因于转运体合成速率的增加，而长期胰岛素处理和饥饿导致的mRNA和蛋白质表达的不成比例变化表明，这些条件在调节转运体蛋白表达时增加了合成并降低了周转率。尽管长期胰岛素暴露和葡萄糖饥饿均使转运体蛋白表达增加超过3倍，基础转运速率增加2.5至4.5倍，但未观察到3T3-L1前脂肪细胞或分化脂肪细胞的胰岛素反应性有显著增加。因此，本研究中观察到的转运体mRNA和蛋白质表达变化与其与基础或低水平胰岛素反应性转运体的调控表达相关最为一致。
