| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high-carbohydrate diet. A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified. We cloned a basic helix-loop-helix protein, enhancer of split-and hairy-related protein-2 (SHARP-2), as the cDNA of ChoRE-binding proteins. Northern blot analysis revealed that the levels of SHARP-2 mRNA increase, when a high-carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats. In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA and the increase was blocked by wortmannin and LY294002. In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin. Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway. Next, we examined the issue of whether the expression of SHARP-2 mRNA is regu
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lated by insulin in other insulin-responsive cells, such as 3T3-L1 adipocytes and L6 myotubes. We found that SHARP-2 is also an insulin-inducible transcription factor in these cells and cyclic AMP also increased the level of SHARP-2 mRNA in both cells in an additive manner.
Then, the influence of the SHARP-2 transcriptional repressor on the expression of rat phosphoenolpyruvate carboxykinase (PEPCK) gene was examined. The adenovirus-mediated overexpression of SHARP-2 in H4IIE cells and primary cultured rat hepatocytes led to a decrease in the levels of PEPCK mRNA. Finally, when a SHARP-2 expression plasmid was transiently transfected with various reporter plasmids into MH1C1 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.
We also reported regulation of expression of the rat SHARP-2 gene by gonadotropins and molecular cloning and characterization of transcriptional regulatory mechanism of the gene. Less
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