Wollaston-Hayden Edith E, Harris Ruth B S, Liu Bingqiang, Bridger Robert, Xu Ying, Wells Lance
Complex Carbohydrate Research Center, University of Georgia , Athens, GA , USA ; Department of Biochemistry and Molecular Biology, University of Georgia , Athens, GA , USA.
Department of Physiology, Georgia Health Sciences University , Augusta, GA , USA.
Front Endocrinol (Lausanne). 2015 Jan 22;5:223. doi: 10.3389/fendo.2014.00223. eCollection 2014.
Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-linked β-N-acetylglucosamine (O-GlcNAc) is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify multiple rodent adipocytokines and secreted proteins whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. We have validated the relative levels of several of these factors using immunoblotting. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating secreted protein expression. Here, we show that upon the elevation of O-GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple secreted proteins reflect the modulation observed at the protein level. We validate the transcript levels in male mouse models of diabetes. Using inguinal fat pads from the severely IR db/db mouse model and the mildly IR diet-induced mouse model, we have confirmed that the secreted proteins regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. By comparing the promoters of similarly regulated genes, we determine that Sp1 is a common cis-acting element. Furthermore, we show that the LPL and SPARC promoters are enriched for Sp1 and O-GlcNAc modified proteins during insulin resistance in adipocytes. Thus, the O-GlcNAc modification of proteins bound to promoters, including Sp1, is linked to adipocytokine transcription during insulin resistance.
通过己糖胺生物合成途径通量的增加以及通过O-连接的β-N-乙酰葡糖胺（O-GlcNAc）导致的细胞内蛋白质糖基化相应增加，足以在多个系统中诱导胰岛素抵抗（IR）。此前，我们团队使用鸟枪法蛋白质组学鉴定了多种啮齿动物脂肪细胞因子和分泌蛋白，其水平在通过间接和直接调节O-GlcNAc水平诱导IR时会受到调节。我们已经使用免疫印迹法验证了其中几种因子的相对水平。由于脂肪细胞因子水平主要在转录水平受到调节，并且O-GlcNAc会改变许多转录因子的功能，我们推测关键转录因子上O-GlcNAc水平的升高正在调节分泌蛋白的表达。在此，我们表明，在成熟的3T3-F442a脂肪细胞中O-GlcNAc水平升高并诱导IR后，多种分泌蛋白的转录水平反映了在蛋白质水平观察到的调节情况。我们在雄性糖尿病小鼠模型中验证了转录水平。使用来自严重IR db/db小鼠模型和轻度IR饮食诱导小鼠模型的腹股沟脂肪垫，我们已经证实，在细胞培养中由O-GlcNAc调节的分泌蛋白在整个动物转变为IR时同样受到调节。通过比较类似调节基因的启动子，我们确定Sp1是一个常见的顺式作用元件。此外，我们表明在脂肪细胞胰岛素抵抗期间，LPL和SPARC启动子富含Sp1和O-GlcNAc修饰的蛋白质。因此，与启动子结合的蛋白质（包括Sp1）的O-GlcNAc修饰与胰岛素抵抗期间脂肪细胞因子的转录相关。
