Preparation of extended in vitro cultures of bovine hepatocytes that are hormonally responsive.

Hepatocytes isolated from male dairy calves were used in monolayer culture or in suspension culture to determine their suitability for the study of hormonal regulation of hepatic gluconeogenesis. The rate of gluconeogenesis (nanomoles of 2.5 mM [2-14C]propionate incorporated into glucose.microgram of DNA-1.hour-1) was higher for monolayers than for suspension cultures. Gluconeogenesis and ureagenesis (nanomoles of urea 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 gluconeogenesis from propionate in monolayers but was without effect on suspension cultures. Actinomycin D (800 nM) and cycloheximide (200 microM) abolished glucagon stimulation of gluconeogenesis, suggesting that glucagon acts to mediate gene expression. Prolonged exposure (45 h) of monolayers to insulin (1,000 nM) decreased basal gluconeogenic rates but did not affect glucagon-stimulated gluconeogenesis. Prior incubation with glucose or valerate did not affect gluconeogenesis. Cells can be successfully maintained in serum-free media for 41 h at the expense of diminished basal gluconeogenic activity. Culture of bovine hepatocytes as monolayers provides a useful tool for the study of chronic and acute hormonal regulation of specific liver functions in the bovine.