IMPROVED HEPATOCYTE CULTURE SYSTEM FOR STUDYING THE REGULATION OF GLYCOGEN-SYNTHASE AND THE EFFECTS OF DIABETES

When 3-4-week-old rats (young rats) are used as a source of hepatocyte s, primary culture cells express the adult, differentiated, liver-spec ific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adul t(>150 g rat) hepatocyte cultures. Corresponding synthase enzyme activ ity and mRNA levels decrease over time in culture in adult but not in young hepatocyte cultures. Young rat hepatocytes also have the ability to proliferate in chemically defined medium in the absence of added m itogens. A diabetes-induced increase in total synthase activity has be en demonstrated by our lab and others, using cultured hepatocytes, liv er homogenates, and perfused livers. In the present study, utilizing s ynthase-specific antibody and primary cultures of cells from young nor mal and alloxan diabetic rats, we found that greater total synthase ac tivity in the diabetic cells was associated with higher levels of enzy me protein. Immuneprecipitation of S-35 methionine-labeled freshly pla ted cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are corr espondingly increased in the diabetic relative to normal cells. Chroni c exposure of young, normal hepatocytes to increasing levels of glucos e induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells fr om diabetic animals do not respond by any of these measures to increas ed glucose concentrations. We conclude that this defined primary cultu re system represents a useful model for investigating the regulation o f hepatic glycogen synthase and the defects which occur as a result of diabetes. (C) 1996 Wiley-Liss, Inc.