Analysis of regulation of gene expression of an insulin-inducible transcription factor and its action mechanism

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We identified a basic helix-loop-helix protein, enhancer of split-and hairy-related protein-2 (SHARP-2), as an insulin-inducible transcriptional repressor in the rat liver. In this study, the influence of the SHARP-2 transcriptional repressor on the expression of an insulin-regulatable rat phosphoenolpyruvate carboxykinase (PEPCK) gene, was examined. The adenovirus-mediated overexpression of SHARP-2 in insulin responsive. H4IIE rat hepatoma cells and primary cultured rat hepatocytes led to a decrease in the levels of PEPCK mRNA. In addition, when a SHARP-2 expression plasmid was transiently transfected with various reporter plasmids into MH_1C_1 cells, the promoter activity of a PEPCK reporter plasmid was specifically decreased. Based on these findings, we concluded that SHARP-2 is a potential repressor of PEPCK gene expression. Then, to investigate the insulin responsive element (IRE) of the rat SHARP-2 gene promoter, SHARP-2/ firefly luciferase reporter plasmids were transfected into H4IIE cells. In the presence or absence of insulin, their promoter activities were determined and compared. However, there was no IREs up to 10 kb upstream of the transcription initiation sites of this gene. On the other hand, adiponectin secreted from adipocytes lower blood glucose level by transcriptional repression of hepatic PEPCK gene via an AMP-activated protein kinase (AMPK) pathway. To determine an issue of whether AMPK affects the expression of the rat SHARP-2 gene, the levels of SHARP-2 mRNA were determined in H4IIE cells treated with AICAR, a chemical AMPK activator. The levels were rapidly and temporally increased as well as insulin treatment. Thus, the result suggests that AMPK induces the expression of the rat SHARP-2 gene.