Effects of insulin on total RNA, poly(A)+ RNA, and mRNA in primary cultures of rat hepatocytes

Abstract

The purpose of this study was to examine mechanisms involved in the regulation of protein synthesis in primary cultures of rat hepatocytes. Hepatocytes were maintained in a chemically defined serum-free medium in the presence or absence of insulin. The rate of protein synthesis in hepatocytes deprived of insulin between days 2 and 5 of culture was reduced to 67% of the rate observed in insulin-maintained controls. The decrease in protein synthetic rate was accompanied by a proportional fall in the content of both total RNA and poly(A)+RNA, suggesting that the capacity for protein synthesis was reduced in the absence of insulin. Both total RNA and poly(A)+ RNA contents and the protein synthetic rate were returned to control values after 3 days of insulin resupplementation. In addition, the effect of insulin on the expression of specific mRNAs was assessed by in vitro translation of total RNA followed by two-dimensional gel analysis of radiolabeled translation products. Only 13 of the greater than 150 spots discernible on the two-dimensional gels were altered in response to insulin. The mRNAs that were altered include examples of repression and stimulation of expression in response to insulin deprivation. Thus, in isolated rat hepatocytes, insulin regulates the capacity of both overall protein synthesis as well as the capacity for the synthesis of specific proteins.