Numerous physiological agents and conditions modulate cellular insulin sensitivity by downregulating or upregulating total cellular insulin receptors. In this study, we examined the effects of replacing complete medium in the absence or presence of insulin on the regulation of insulin-receptor gene expression in cultured human hepatoma cells (HepG2). Failure to replace complete medium resulted in growth arrest of HepG2 cells and a six- to sevenfold increase in insulin-receptor mRNA due to the prolongation of insulin-receptor mRNA half-life. Northern analysis revealed multiple insulin-receptor mRNA species; the largest species (11 kilobases) was disproportionately increased in growth-arrested cells. High concentrations of insulin (500 ng/ml) induced a 33.8% decrease in the abundance of insulin-receptor mRNA (n = 14). At lower concentrations, a trend of inhibition was observed but was not statistically significant. Insulin (500 ng/ml) did not affect insulin-receptor mRNA stability. The effect of conditioned media, insulin, and dexamethasone on insulin-receptor promoter activity was also examined. Various constructs of the 5-flanking region of the insulin-receptor gene were attached immediately upstream to a chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into HepG2 cells via a pBR322-derived plasmid (pCAT). In cells replaced with complete medium, 12 and 118% of the promoter activity was contained within 578 and 877 base pairs, respectively, from the major translational initiation site. Conditioned media from growth-arrested cells in culture for 7 days increased promoter activity approximately twofold in 48 h. However, this increase failed to localize to any specific region on the insulin-receptor promoter. In addition, CAT activity doubled in cells transiently transfected with pCAT containing no 5-flanking region of the insulin-receptor gene, suggesting that conditioned media increased CAT activity posttranscriptionally. Insulin (500 ng/ml) and dexamethasone (10−6 M) had little effect on promoter activity.
Effects of Media Conditions, Insulin, and Dexamethasone on Insulin-Receptor mRNA and Promoter Activity in HepG2 Cells
James R Levy, Geoffrey Krystal, Penny Glickman, Frank Dastvan; Effects of Media Conditions, Insulin, and Dexamethasone on Insulin-Receptor mRNA and Promoter Activity in HepG2 Cells. Diabetes 1 January 1991; 40 (1): 58–65. https://doi.org/10.2337/diab.40.1.58
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