title: Improved hepatocyte culture system for studying the regulation of glycogen synthase and the effects of diabetes.
abstract: When 3-4-week-old rats (young rats) are used as a source of hepatocytes, primary culture cells express the adult, differentiated, liver-specific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adult (> 150 g rat) hepatocyte cultures. Corresponding synthase enzyme activity 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 mitogens. A diabetes-induced increase in total synthase activity has been demonstrated by our lab and others, using cultured hepatocytes, liver homogenates, and perfused livers. In the present study, utilizing synthase-specific antibody and primary cultures of cells from young normal and alloxan diabetic rats, we found that greater total synthase activity in the diabetic cells was associated with higher levels of enzyme protein. Immuneprecipitation of 35S methionine-labeled freshly plated cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are correspondingly increased in the diabetic relative to normal cells. Chronic exposure of young, normal hepatocytes to increasing levels of glucose induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells from diabetic animals do not respond by any of these measures to increased glucose concentrations. We conclude that this defined primary culture system represents a useful model for investigating the regulation of hepatic glycogen synthase and the defects which occur as a result of diabetes.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655, USA.
authors.forename: M
authors.initials: M
authors.lastname: Van Auken
authors.forename: J
authors.initials: J
authors.lastname: Rulfs
authors.forename: M A
authors.initials: MA
authors.lastname: Buckholt
authors.forename: A K
authors.initials: AK
authors.lastname: Garnache
authors.forename: T B
authors.initials: TB
authors.lastname: Miller
mesh_terms: D000367:Age Factors; D000496:Alloxan / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D000918:Antibody Specificity; D015153:Blotting, Western; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology / Q000201:enzymology; D003470:Culture Media; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000494:pharmacology; D006006:Glycogen Synthase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008934:Mitogens / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012260:Ribonucleases
chemical_list: D003470:Culture Media; D008934:Mitogens; D012333:RNA, Messenger; D000496:Alloxan; D006006:Glycogen Synthase; D012260:Ribonucleases; D005947:Glucose