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

工作概述：转录因子C/EBPalpha是几个脂肪细胞特异性基因的转录正调节因子，这些基因在能量代谢中起作用。然而，关于代谢信号调控其表达的信息很少。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葡萄糖中孵育5-24小时时，C/EBPalpha表达减弱，而在5.7 mM葡萄糖中则没有。核运行转录试验显示C/ ebppha基因的转录抑制，而C/EBPbeta基因的转录抑制不存在。葡萄糖胺是己糖胺途径的产物，在胰岛素处理的细胞中模拟高葡萄糖降低C/EBPalpha表达的能力。木糖醇（戊糖磷酸途径的活化剂）也得到了类似的结果。己糖胺途径代谢物的积累和/或细胞内蛋白糖基化的变化与高葡萄糖、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。这些处理都没有引起细胞内ATP水平的降低。将小鼠C/EBPalpha基因5侧468 bp序列与荧光素酶融合后稳定转染3T3-L1细胞，结果表明这些营养物质也降低了启动子活性。有趣的是，用葡萄糖或氨基葡萄糖治疗大鼠导致附睾脂肪中C/EBPalpha mRNA水平降低，而不是网膜脂肪。综上所述，这些结果表明代谢信号在体外和体内都有助于下调C/EBPalpha的表达。我们计划确定这些营养素用于控制C/EBPalpha基因表达的信号转导途径。将特别强调丝裂原活化蛋白激酶家族成员（ERK， p38和JNK）和磷脂酰肌醇3-激酶。-氨基葡萄糖;信号转导;转录因子;培养中的细胞；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.