Role of insulin and dexamethasone in the expression of bioactivity in rat hepatocytes cultured in a serum-free defined medium.

Previously, we described primary cultures of rat liver cells in a serum-free multihormone defined medium (Am. J. Physiol. 243: E132-E151, 1982). This cell preparation exhibits marked insulin-dependent syntheses of protein, glycogen and lipids (hereafter collectively referred to as "bioactivity" for brevity). In the present studies, the role of different hormones in the expression of insulin's bioactivity was investigated. Hepatocytes from fasted rats, previously maintained in the multihormone glucagon-supplemented medium, were first cultured in the defined medium without the hormones and, subsequently, tested for bioactivity by replacing the missing hormones singly or in combination. Of all the hormones tested (testosterone, estradiol, thyroxine, glucocorticoid steroid and insulin), only insulin, dexamethasone and thyroxine when present individually in the culture medium, showed slight bioactivity (glycogenesis); however, insulin and dexamethasone, when present together, further increased each bioactivity, protein (1.3-fold), glycogen (8-fold) and lipid (1.6-fold) syntheses. The determination of apparent binding parameters of insulin specific receptors using Scatchard plots showed that insulin exposure caused a decrease in receptor concentration (Ro), dexamethasone exposure caused an increase in affinity (Kd), and, compared to insulin alone, treatment with insulin plus dexamethasone increased receptor concentration with no change in apparent affinity. Insulin induced a consistently small, but statistically significant, increase in the average specific activity of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), with equal distribution between its nonlatent ("readily available") and latent ("cryptic") forms. However, when dexamethasone was present along with insulin, the distribution of GIT was significantly greater in the latent form than in the nonlatent form. Examination by scanning and transmission microscopy showed clear differences, both in the cell surface and intracellular morphology, in the hepatocytes exposed to different hormonal milieu.