Identification and characterization of novel gene involved in insulin action

为了更好地了解胰岛素代谢活动中涉及的细胞内信号，我试图识别有助于胰岛素活动的新的信号分子。我首先尝试用金属螯合亲和层析法纯化新型的胰岛素诱导的磷酸化蛋白。尽管GA-螯合亲和层析柱能够高效地吸附磷酸化蛋白质，但用这种方法我们不能在3T3-L1脂肪细胞中鉴定到任何响应胰岛素的新的磷酸化蛋白质。作为第二种方法，通过使用基因芯片分析，我接下来试图识别由胰岛素诱导表达的新基因。我们分析了正常小鼠、注射链脲佐菌素的糖尿病小鼠以及在肝脏中特异表达显性PI 3-激酶负突变(ΔP85)的小鼠在快速喂养周期中肝脏基因表达的变化。我们挑选了一些基因，这些基因在快速喂养周期中表达上调，在STZ小鼠中通过胰岛素注射而表达上调，并受到ΔP85表达的下调。在这些基因中，STRA13是一种基本的螺旋-环-螺旋转录因子，在原代培养的肝细胞中被发现在蛋白质和mRNA水平上被胰岛素上调。胰岛素诱导的Stra13基因的表达可被PI 3-激酶的抑制剂抑制，而在无胰岛素的情况下，该激酶的活性形式可诱导其表达，表明Stra13基因是胰岛素诱导的基因。我们还鉴定了另外几个转录因子，它们的表达被胰岛素抑制，或被糖皮质激素或cAMP诱导。这些转录因子可能参与肝脏的代谢调节。

For better understanding of intracellular signaling involved in insulin's metabolic actions, I have attempted to identify novel signaling molecules that contribute to insulin action. I first tried to purify novel insulin-induced phosphorylated proteins with the use of metal-chelating affinity chromatography. Although Ga-chelating affinity column was found to absorb phosphorylated proteins with high efficiency ; we could not identify any novel protein that is phosphorylated in 3T3-L1 adipocytes in response to insulin with this method. As a second approach, with the use of Gene Chip analysis, I next attempted to identify novel genes of which expression is induced by insulin. We analyzed the alteration of hepatic gene expression during a fast-feeding cycle in normal mice, in insuhn-administered STZ-diabetes mice, and in mice that express a dominant negative mutant of PI 3-kinase (Δp85) specifically in the liver. We picked up genes of which expression are upregulated in a fast-feeding cycle and by insulin administration in STZ mice and are downregulated by Δp85 expression. Among such genes, Stra13, a basic helix-loop-helix transcription factor, was found to be upregulated by insulin in primary cultured hepatocyte both in protein and mRNAlevels. Insulin-induced expression of Stra13 was attenuated by an inhibitor of PI 3-kinase, and an active form of the kinase induced its expression in the absence of insulin, indicating that Stra13 is an insulin-induced gene. We also identified several more transcription factors whose expression is suppressed by insulin or induced by glucocorticoid or cAMP. These transcription factors may be involved in metabolic regulation in the liver.