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Uncoupling between the insulin-receptor cycle and the cellular degradation of the hormone in cultured foetal hepatocytes. Effect of drugs and temperature that inhibit insulin degradation.

ABSTRACT: Sequential changes in the numbers of cell-surface receptors induced by a transitory exposure to insulin in cultured 18-day foetal-rat hepatocytes were investigated in the presence of drugs and at a temperature of 22 degrees C, which inhibit cellular insulin degradation. Chloroquine (70 microM) and monensin (3 microM) did not greatly change the initial rate of internalization of cell-surface receptor sites after exposure to 10 nM-insulin, but led to a steady state after 20 min, which represented 40% of the initial binding, compared with 5 min and 60% in the absence of the drug. Moreover, these drugs strongly decreased the proportion of receptor sites recovered at the cell surface after subsequent removal of the hormone. They were ineffective when insulin was not present. The removal of monensin together with the hormone allowed partial restoration of cell-surface receptor sites and degradation of cell-associated insulin to start again at the initial speed, indicating a reversible effect of the drug. During this phase, the drug concentration-dependence for the two effects showed that receptor recycling was restored with concentrations of monensin not as low as for insulin degradation. The effect of vinblastine (50-100 microM) was similar to that of chloroquine and monensin, whereas no modification in the internalization and recovery processes was observed in the presence of bacitracin concentrations (1-3 mM) that inhibit insulin degradation by 70%. A temperature of 22 degrees C did not prevent the receptor internalization, but had a slowing effect on the recycling process, which appeared to vary in experiments where insulin degradation remained inhibited. The present study shows that the process of insulin degradation mediated by receptor endocytosis is not a prerequisite for insulin-receptor recycling in cultured foetal hepatocytes.

SUBMITTER: Soubigou P

PROVIDER: S-EPMC1148318 | biostudies-other | 1987 Sep

REPOSITORIES: biostudies-other

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Project description:The pathways of glycogen synthesis from glucose were studied using double-isotope procedures in 18-day cultured foetal-rat hepatocytes in which glycogenesis is strongly stimulated by insulin. When the medium containing 4 mM-glucose was supplemented with [2-3H,U-14C]glucose or [3-3H,U-14C]glucose, the ratios of 3H/14C in glycogen relative to that in glucose were 0.23 +/- 0.04 (n = 6) and 0.63 +/- 0.09 (n = 8) respectively after 2 h. This indicates that more than 75% of glucose was first metabolized to fructose 6-phosphate, whereas 40% reached the step of the triose phosphates prior to incorporation into glycogen. The stimulatory effect of 10 nM-insulin on glycogenesis (4-fold) was accompanied by a significant increase in the (3H/14C in glycogen)/(3H/14C in glucose) ratio with 3H in the C-2 position (0.29 +/- 0.05, n = 6, P less than 0.001) or in the C-3 position (0.68 +/- 0.09, n = 8, P less than 0.01) of glucose, whereas the effect of a 12 mM-glucose load (3.5-fold) did not alter these ratios. Fructose (4 mM) displaced [U-14C]glucose during labelling of glycogen in the presence and absence of insulin by 50 and 20% respectively, and produced under both conditions a similar increase (45%) in the (3H/14C in glycogen)/(3H/14C in glucose) ratio when 3H was in the C-2 position. 3-Mercaptopicolinate (1 mM), an inhibitor of gluconeogenesis from lactate/pyruvate, further decreased the already poor labelling of glycogen from [U-14C]alanine, whereas it increased both glycogen content and incorporation of label from [U-14C]serine and [U-14C]glucose with no effect on the relative 3H/14C ratios in glycogen and glucose with 3H in the C-3 position of glucose. These results indicate that an alternative pathway in addition to direct glucose incorporation is involved in glycogen synthesis in cultured foetal hepatocytes, but that insulin preferentially favours the classical direct route. The alternative foetal pathway does not require gluconeogenesis from pyruvate-derived metabolites, contrary to the situation in the adult liver.

Project description: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.

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Uncoupling between the insulin-receptor cycle and the cellular degradation of the hormone in cultured foetal hepatocytes. Effect of drugs and temperature that inhibit insulin degradation.

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