PREPARATION OF EXTENDED IN-VITRO CULTURES OF BOVINE HEPATOCYTES THAT ARE HORMONALLY RESPONSIVE

Hepatocytes isolated from male dairy calves were used in monolayer cul ture or in suspension culture to determine their suitability for the s tudy of hormonal regulation of hepatic gluconeogenesis. The rate of gl uconeogenesis (nanomoles of 2.5 nM [2-C-14]propionate incorporated int o glucose microgram of DNA-1-hour-1) was higher for monolayers than fo r suspension cultures. Gluconeogenesis and ureagenesis (nanomoles of u rea N formed.microgram of DNA-1-3 hours-1) were similar in monolayers cultured for 24 and 48 h but declined by 120 h. Ureagenesis was barely detectable in suspension cultures. Glucagon (10 nM) increased glucone ogenesis from propionate in monolayers but was without effect on suspe nsion cultures. Actinomycin D (800 nM) and cycloheximide (200 muM) abo lished glucagon stimulation of gluconeogenesis, suggesting that glucag on acts to mediate gene expression. Prolonged exposure (45 h) of monol ayers to insulin (1,000 nM) decreased basal gluconeogenic rates but di d not affect glucagon-stimulated gluconeogenesis. Prior incubation wit h glucose or valerate did not affect gluconeogenesis. Cells can be suc cessfully maintained in serum-free media for 41 h at the expense of di minished basal gluconeogenic activity. Culture of bovine hepatocytes a s monolayers provides a useful tool for the study of chronic and acute hormonal regulation of specific liver functions in the bovine.