GLUCOSE SIGNALING AND REGULATION OF GENE EXPRESSION IN 3T3-L1 ADIPOCYTES

工作总结：转录因子C/EBPalpha是几个脂肪细胞特异性基因的正向转录调节器，这些基因在能量代谢中发挥作用。然而，关于代谢信号调节其表达的信息很少。在最近发表的一篇论文中，我们报道了当3T3-L1脂肪细胞在24 mM的葡萄糖中孵育时，暴露于胰岛素5-24小时会减弱C/EBPalpha的表达，但在5.7 mM的葡萄糖中孵育不会。核连续转录实验显示C/EBPalpha基因的转录抑制，但C/EBPbeta基因的转录抑制。氨基葡萄糖是氨基己糖途径的产物，它模拟了高糖降低胰岛素处理细胞中C/EBPalpha表达的能力。磷酸戊糖途径的激活剂木糖醇也得到了类似的结果。氨基己糖途径代谢产物的积累和/或细胞内蛋白糖基化的变化与高糖、氨基葡萄糖或木糖醇下调C/EBPalpha表达的能力之间没有相关性。所有这些治疗都没有导致细胞内ATP水平的下降。用融合于荧光素酶的小鼠C/EBPalpha基因5侧翼的468-bp序列稳定地转染3T3-L1细胞，结果表明这些营养素也降低了启动子的活性。有趣的是，用葡萄糖或氨基葡萄糖治疗大鼠可以降低附睾脂肪中C/EBPalpha的mRNA水平，但不能降低大网膜脂肪的水平。综上所述，这些结果表明，代谢信号在体外和体内都下调了C/EBPalpha的表达。我们计划确定这些营养物质用来控制C/EBPalpha基因表达的信号转导途径。将特别强调有丝分裂原激活蛋白激酶家族(ERK、p38和JNK)和磷脂酰肌醇3-激酶的成员。氨基葡萄糖；信号转导；转录因子；培养细胞；脂肪组织；葡萄糖代谢。

Summary of work: The transcription factor C/EBPalpha is a positive modulator of transcription for several adipocyte-specific genes which play a role in energy metabolism. However, there is little information available regarding regulation of its expression by metabolic signals. In a recent published paper, we reported that exposure to insulin for 5-24 h attenuated C/EBPalpha expression when 3T3-L1 adipocytes were incubated in 24 mM glucose, but not in 5.7 mM glucose. Nuclear run-on transcription assays indicated a transcriptional repression of C/EBPalpha gene, but not that of C/EBPbeta. Glucosamine, a product of the hexosamine pathway, mimicked the ability of high glucose to reduce C/EBPalpha expression in insulin-treated cells. Similar results were obtained with xylitol, an activator of the pentose phosphate pathway. There was no correlation between the accumulation of hexosamine pathway metabolites and/or changes in intracellular protein glycosylation with the ability of high glucose, glucosamine, or xylitol to down-regulate C/EBPalpha expression. None of these treatments caused a reduction in intracellular ATP levels. Stable transfection of 3T3-L1 cells with the 5-flanking 468-bp sequence of the mouse C/EBPalpha gene fused to luciferase demonstrated that promoter activity was also reduced by these nutrients. Of interest, treatment of rats with glucose or glucosamine led to a reduction in C/EBPalpha mRNA levels in epididymal, but not omental, fat. Taken together, these results suggest that metabolic signals serve to down-regulate C/EBPalpha expression both in vitro and in vivo. We plan to determine the signal transduction pathways used by these nutrients to control expression of the C/EBPalpha gene. Special emphasis will be given toward members of the mitogen-activated protein kinase family (ERK, p38 and JNK) and phosphatidylinositol 3-kinase. - Glucosamine; Signal transduction; Transcription factors; Cells in culture; Adipose tissue; Glucose metabolism.