Primary induction of vitellogenin synthesis in monolayer cultures of amphibian hepatocytes.

Direct induction of vitellogenin production in cultured male amphibian hepatocytes by estradiol-17 beta has been accomplished. Liver cells were isolated from adult male bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium containing insulin and estradiol. Vitellogenin production was measured by direct immunoprecipitation from radioactively labeled secreted protein with a specific antiserum against vitellogenin. Significant quantities of vitellogenin were detected in the exported protein on the second day of hormone treatment. Vitellogenin production increased with duration of culture in the presence of estradiol until by the eighth day approximately 90% of secreted protein was vitellogenin. This response is largely comparable to that obtainable in vivo. Indirect immunofluorescence microscopy was used to identify cells synthesizing vitellogenin in response to estradiol. An increase in cytoplasmic fluorescence could be seen in cells throughout the cultures, with increasing time in the presence of estradiol. By the sixth day of treatment, the majority of cells showed significant fluorescence labeling. The results suggest that studies on the mechanisms underlying the primary activation of the vitellogenin gene may now be conducted under well defined conditions in a monolayer liver cell culture system.

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes.

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.