Protein degradation in cultured fetal hepatocytes. Absence of an inhibitory effect of insulin.

The role of insulin to regulate protein turnover in fetal liver was investigated using primary cultures of fetal-rat hepatocytes. The basal rate of protein degradation (in the presence of insulin and amino acids) was the same in cultured fetal and adult hepatocytes (2.48 +/- 0.16 versus 2.46 +/- 0.06% of total protein degraded/h respectively). Incubation of cells in an unsupplemented media (without insulin or amino acids) resulted in a deprivation-induced increase in degradation in cells from both groups (P less than 0.05). Rates of proteolysis could be returned to their respective basal values by the addition of amino acids at 5 times their normal plasma concentrations. In adult cells, addition of insulin alone significantly inhibited protein degradation (P less than 0.05), whereas, in contrast, insulin was without effect on protein degradation in fetal hepatocytes. Both fetal and adults cells responded to dibutyryl cyclic AMP with an increase in protein degradation above that seen in the no-additions group. Results of experiments in which the effect of inhibitors of protein degradation (chloroquine, NH4Cl, amino acids and dinitrophenol) were tested suggested that lysosomes were responsible for 20-30% of total protein degradation in fetal hepatocytes. Impaired insulin processing in fetal hepatocytes was examined as a possible cause of the insulin-resistance in these cells. As determined by h.p.l.c. analysis, the same pattern of initial degradation products of insulin was found in fetal hepatocytes as had previously been found in adult hepatocytes. Incubation of cells with various doses of chloroquine resulted in an increase in cell-associated 125I-insulin and a decrease in insulin degradation in both fetal and adult cells. At the highest dose of chloroquine tested (500 microM), a slightly greater increase in insulin binding and a decrease in insulin degradation were observed in fetal cells as compared with adult cells. Rates of insulin internalization were also compared between fetal and adult cells. A 30% slower rate of insulin internalization was observed in fetal cells, as compared with adult cells. It was concluded that the absence of an effect of insulin on protein degradation in fetal hepatocytes is not the result of a major difference in insulin internalization and processing between fetal and adult hepatocytes.

Insulin-responsive cultured foetal-rat hepatocytes. Their preparation and characterization.

An improved non-perfusion method for the preparation of cultured foetal-rat hepatocytes is described. Digestion of the liver with collagenase and deoxyribonuclease I gave yields of 40 X 10(6) hepatocytes/g of liver. The plating efficiency of hepatocytes in medium with 10 microM-cortisol was 50%. Cell morphology and metabolism were maintained through 3 days of monolayer culture, with minimal contamination by haematopoietic cells or fibroblasts. The cultured cells bound and degraded 125I-insulin in a time- and dose-dependent manner. The estimated ED50 for competitive binding at 37 degrees C was 1.1 nM. Curvilinear Scatchard plots were observed, with estimates of 16 500 high-affinity sites (Kd = 813 pM) and 53 000 low-affinity sites (Kd = 23 nM) per cell. The cultured cells demonstrated a glycogenic response to insulin, with an estimated ED50 of 120 pM. The degree of glycogenic response to insulin varied with time in culture: 500% above basal on day 1, 200% on day 2, and only 150% on day 3. Cultured foetal cells also exhibited a time-dependent uptake of 2-aminoisobutyric acid, which, in contrast with previous reports with adult cells, was not stimulated by the presence of 10 nM-insulin. Cultured foetal hepatocytes may provide an interesting model with which to study the relationship between insulin-receptor binding and insulin action.

Combination chemotherapy of L1210 leukemia with 1-beta-D-arabinofuranosylcytosine and 5-azacytidine.

1-beta-D-Arabinofuranosylcytosine (cytarabine; ara-C) and 5-azacytidine (5-azaCR), cytosine nucleoside antimetabolites with different mechanisms of action, are both effective in the treatment of human leukemia, and the clinical use of these two agents in combination has been suggested. We have studied the therapeutic effect in L1210 leukemic mice of single i.p. doses of ara-C and 5-azaCR in combination. Therapeutic effects observed depended markedly on the sequence and time interval between the doses of each agent. Antagonism was observed when both agents were administered simultaneously. The optimal therapeutic effect was observed when 5-azaCR was administered after ara-C at a time when tumor DNA synthesis had maximally recovered after the ara-C dose. The dose-interval effect and correlation with recovery of DNA synthesis capacity were also observed in studies in vitro in which the survival of L1210 cells in culture was examined. ara-C was shown to inhibit the incorporation of [4-14C]-5-azaCR-derived radioactivity into DNA of L1210 cells in culture, and the therapeutic effects observed are interpreted in terms of these latter results and the mechanisms of action of the two agents.