Receptor-mediated insulin degradation and insulin-stimulated glycogenesis in cultured foetal hepatocytes.

Insulin-stimulated glycogenesis and insulin degradation were studied simultaneously at 37 degrees C in cultured foetal hepatocytes grown for 2-3 days in the presence of cortisol. Degradation of cell-associated insulin, as measured by trichloroacetic acid precipitation, was significant after 4 min in the presence of 1-3 nM-125I-labelled insulin. This process became maximal (30% of insulin degraded) after 20 min, a time when binding-state conditions were achieved. No insulin-degradative activity was detected in a medium that had been exposed to cells. At steady-state, the appearance of insulin degradation products in the medium was linearly dependent on time (1.5 fmol/min per 10(6) cells at 1nM-125I-labelled insulin). Chloroquine (3-50 microM), bacitracin (0.1-10 mM) and NH4Cl (1-10 mM) inhibited insulin degradation as soon as this became detectable and caused an increase in the association of insulin to hepatocytes after 20 min. Lidocaine and dansylcadaverine had similar effects, whereas N-ethylmaleimide, aprotinin, phenylmethanesulphonyl fluoride and leupeptin were found to be ineffective. Chloroquine, and also bacitracin, at concentrations that inhibited insulin degradation, decreased the insulin-stimulated incorporation of [14C]glucose into glycogen over 2 h. This effect of chloroquine was specific, since it did not modify the basal glycogenesis, or the glycogenic effect of a glucose load in the absence of insulin. It therefore appears that the receptor-mediated insulin degradation (or some associated pathway) is functionally related to the glycogenic effect of insulin in foetal hepatocytes.     

Cell-surface insulin receptor cycling and its implication in the glycogenic response in cultured foetal hepatocytes.

The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.     

Pathways of glycogen synthesis from glucose during the glycogenic response to insulin in cultured foetal hepatocytes.

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.     

Intermediate peptides of insulin degradation in liver and cultured hepatocytes of rats

1. Bestatin, a microbial aminopeptidase inhibitor, induced accumulation of low-molecular weight intermediate peptides of insulin degradation in liver of rats in vivo and in primary cultured rat hepatocytes. However, bestatin did not affect the association and internalization of the hormone into hepatic cells. 2. Results of the HPLC analyses showed that the intermediate peptides of insulin degradation are small ones and specifically accumulate only in the presence of bestatin. 3. The above results, together with those employing other protease inhibitors, show that cytosolic bestatin-sensitive protease(s), trypsin-like protease(s) and thiol protease(s) play an important role in the intracellular degradation process of insulin.     

Role of dexamethasone and insulin on the development of the five urea-cycle enzymes in cultured rat foetal hepatocytes.

The activity changes of the urea-cycle enzymes were monitored in cultured foetal hepatocytes after dexamethasone and insulin treatments. Addition of dexamethasone induced the development of carbamoyl-phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase activities as soon as day 16.5 of gestation. When insulin was added together with dexamethasone, it markedly inhibited the steroid-induced increase in carbamoyl-phosphate synthetase, argininosuccinate synthetase and argininosuccinase activities.     

Glucocorticoid regulation of chloroquine nonsensitive insulin degradation in cultured fetal rat hepatocytes

The influence of cortisol and other culture conditions on insulin degradation by the chloroquine-sensitive pathway and the chloroquine-nonsensitive pathway (CNP) was investigated in fetal rat hepatocytes during 3 days of culture. The proportions of the chloroquine nonsensitive release of 125I-insulin degradation products into the conditioned medium/h increased from the 1st to the 3rd day of culture, i.e. from 19 to 50% by cells grown in the presence of cortisol and from 17 to 82% by those grown in the absence of cortisol. Replacement of the conditioned medium with the respective fresh medium dramatically enhanced cellular insulin degradation by CNP, i.e. from 22 to 58%, and 19 to 85% in cells grown for 2 days in the presence and absence of cortisol, respectively. Thus, the conditioned medium contained some factor(s) that inhibited CNP. Therefore, we used the inhibited insulin and alpha-casein degradation by papain in vitro as an assay to investigate the nature of the putative anti-(insulin) protease. Cycloheximide completely prevented the appearance of anti-papain activity in the medium. Conditioned medium obtained from cells grown in the presence of cortisol contained about 2-fold more anti-papain activity than the medium that was obtained in the absence of the steroid. The release of anti-papain activity also declined with time from 1 to 3 days of culture and showed an inverse relationship with the magnitude of cellular insulin degradation by CNP. The inhibition of papain-mediated insulin degradation by the anti-(insulin) protease was noncompetitive. The anti-(insulin) protease was nondialyzable (up to the 10-kDa exclusion limit) and inactivated by heat treatment at 50 degrees C for 30 min. These results suggest that fetal hepatocytes synthesize and secrete a glucocorticoid-regulated heat-labile low molecular mass (less than 25 kDa) anti-(insulin) protease, which may contribute to the suppression of insulin degradation caused by the enzymes involved in CNP.     

Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes.

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.     

Hormonal regulation of two urea-cycle enzymes in cultured foetal hepatocytes.

Foetal-rat hepatocytes were cultured in primary monolayer culture, and activity changes of argininosuccinate synthetase (ASS, EC 6.3.4.5) and argininosuccinase (ASL, EC 4.3.2.1) were followed under defined hormone conditions. In hormone-free medium, cultured cells maintained the enzyme activities at values equal to those of freshly isolated cells for at least 3 days. Continuous addition of dexamethasone produced the development of the two enzyme activities, but only after the first 20h of culture. Under these conditions, urea production by the foetal hepatocytes was concomitantly increased in the culture medium. Pretreatment with dexamethasone for 20h was sufficient to produce the development of ASL activity within the 2 following days. Introduced alone, glucagon induced an increase of ASL activity, but did not affect the ASS activity. The most powerful stimulation of ASS and ASL could be observed in cultured hepatocytes if glucagon and dexamethasone were added simultaneously or sequentially. These results indicated that the development of the receptor complex for the induction of urea-cycle enzymes appears early before birth and established that glucocorticoids amplify the glucagon stimulation of these enzyme activities during foetal life.     

Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

Differences between glucose and insulin stimulation of glycogenesis in cultured fetal hepatocytes

The glycogenic effects of a glucose load (15 mM) and/or insulin (10 nM) were studied in 18-day-old fetal rat hepatocytes after 2 days of culture when medium contained 4 mM glucose. A glucose load led to a stimulation of [¹⁴C]glucose glycogen labelling (20 min) earlier than with insulin (30–40 min); maximal stimulations were 3-fold after 1 h for the glucose load and 5-fold after 2–3 h for insulin. Simultaneous addition of the two agents produced synergic effects. When insulin was added 4 h after a glucose load (or vice versa), a second glycogenic response was elicited: a further addition of the same glycogenic agent was ineffective. The early glycogenic effects (up to 2 h) also occurred in the presence of 10 μM cycloheximide, with, however, some decrease of insulin stimulation. The contribution of medium glucose to the glycogen formed for 2 days (67% in the absence of glycogenic agent) was clearly enhanced by a glucose load and to a lesser degree by insulin after a 4-h exposure (83 and 71%, respectively). This was accompanied by a related modification of the participation of glucogenic precursors such as fructose and galactose. Thus, acute glycogenic response to glucose and insulin appeared both synergic and independent, and quite different in several aspects in cultured fetal hepatocytes.     

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.     

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose. Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes

Abstract. From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose.
Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.     

Ontogeny of the glucocorticoid receptor and its relationship to tyrosine aminotransferase induction in cultured foetal hepatocytes.

The glucocorticoid receptor activity that can be detected in the liver from 15-day foetal rats would appear to be associated with the haemopoietic cells. In hepatocytes, purified by culture for 1-2 days from 15-day foetal rats, the glucocorticoid receptor activity is low and dexamethasone does not induce the enzyme tyrosine aminotransferase. If culture is continued both receptor activity and steroid responsiveness are acquired. Cultured hepatocytes from 19-day foetal liver contain receptor from the first day of culture and, furthermore, the subsequent level of response to glucocorticoids is directly correlated with the actual receptor concentration. It would appear that the glucocorticoid receptor is not acquired by hepatocytes until after 18 days of gestation. Nevertheless, the fact that bromodeoxyuridine has no effect on the rate of accumulation of receptor in hepatocytes suggests that the differentiative event leading to the subsequent appearance of the receptor has already occurred before day 15 of gestation. However, the acquisition of the receptor would appear to be dependent on mitosis as cytosine arabinoside can inhibit the process.     

Compared roles of glucose, galactose and fructose as glycogen precursors during the acute response to insulin in cultured rat foetal hepatocytes

1. The efficiency of the contribution of hexoses to basal- and stimulated-glycogenesis, when studied in cultured 18 day-old rat foetal hepatocytes in the presence of glucose, was as follows: galactose greater than glucose greater than fructose. 2. Glucose deprivation had opposite effects on the contributions of [14C]galactose (decreased) and [14C]fructose (increased) to glycogenesis, which occurred independently of insulin and were reversed by glucose concentrations as low as 30-100 microM. 3. The stimulation of glycogenesis by insulin measured with [14C]glucose (3.2-fold) was superior to that obtained with either [14C]galactose or [14C]fructose (2.7-fold in both cases), which revealed a specific beneficial effect of insulin on glucose contribution.