Growth factors and gene expression in cultured rat hepatocytes

Background/Aim: The aim of this study was to evaluate the effect of replication on function variables in cultured hepatocytes. Methods: Isolated rat hepatocytes were cultured in HCM medium and plated on collagen-coated dishes at cell densities from 0.2 • 105 (subconfluent) to 1.0 • 105 • cm−2 (confluent) with and without addition of hepatocyte growth factor, epidermal growth factor and insulin-like growth factor-I. The synthesis rate was measured for DNA, albumin, urea, and glucose together with mRNA levels (Northern blots) for albumin, urea cycle enzymes, and acute phase and “housekeeping” proteins. Results: In subconfluent culture the synthesis of DNA and urea was higher (118% and 112%, respectively), and of albumin and glucose lower (40% and 67%, respectively) than in confluent culture. The mRNA levels of carbamoylphosphate synthase, argininosuccinate synthetase, argininosuccinate lyase, arginase, α2-macroglobulin, β-fibrinogen, and albumin were lower (23%, 58%, 77%, 33%, 12%, 50%, and 51%, respectively) in subconfluent culture compared with confluent culture. Relatively increased levels were found for β-actin (109%) and α-tubulin (136%). In subconfluent culture hepatocyte growth factor increased the DNA synthesis rate 6-fold, epidermal growth factor 3-fold, and insulin-like growth factor-I 2-fold; that of albumin, urea and glucose was not increased significantly. In confluent culture the effect of growth factors on synthesis rates was not significant, and the growth factors had little influence on mRNA levels. Conclusions: Hepatocytes produce urea at the same rate in subconfluent as in confluent culture in spite of a lower mRNA level of urea cycle enzymes. Hepatocyte growth factor and epidermal growth factor increase DNA synthesis markedly in subconfluent culture only, without significantly changing the ratio between subconfluent and confluent culture of other variables. This suggests that active replication is not an important cause of the relatively low liver-specific function of hepatocytes in subconfluent culture.