Differences in degradation processes for insulin and its receptor in cultured foetal hepatocytes.

Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.     

Dexamethasone stimulates insulin receptor synthesis in cultured rat hepatocytes

The ability of the glucocorticoid dexamethasone to modulate the insulin receptor was examined directly in primary cultures of hepatocytes prepared from adult male rats. Hepatocytes were cultured in a defined medium in the presence and absence of dexamethasone, 0.1 microM. The exposure of hepatocytes to dexamethasone resulted in a time-dependent (steady state by 32 h) increase in insulin binding in both intact hepatocytes and Triton X-100-soluble extracts (total insulin receptor content). The enhanced insulin binding found in soluble extracts of dexamethasone-treated hepatocytes was the result of an increase in insulin receptor number without a change in receptor affinity. In order to assess the mechanism by which dexamethasone "up-regulates" the insulin receptor, the heavy isotope density-shift technique was used to analyze insulin receptor turnover in control and dexamethasone-treated hepatocytes. Hepatocytes were initially cultured for 32 h in standard culture media containing only "light" (14C, 12C, 1H) amino acids. In hepatocytes exposed to dexamethasone, a 417% increase in insulin binding in Triton X-100-soluble extracts was observed. After 32 h, when steady state binding is achieved in dexamethasone-treated cultures, parallel cultures of hepatocytes incubated in the absence and presence of dexamethasone were washed and subsequently cultured in media containing "heavy" amino acids (15N, 13C, 2H). The time-dependent disappearance of light insulin receptor (receptor degradation) and appearance of heavy insulin receptor (receptor synthesis) were monitored using CsCl gradients to resolve the two density species of receptor. At steady state, the rate of receptor synthesis (k8) was 2.94 and 0.62 fmol of insulin bound h-1 in dexamethasone-treated and control hepatocytes, respectively. In contrast to this large increase in the rate of receptor synthesis observed in dexamethasone-treated cells, the first order rate constant for decay (k d) was the same in dexamethasone-treated (0.074 h-1) and in control (0.077 h-1) hepatocytes. We therefore conclude that glucocorticoid-induced up-regulation of the insulin receptor in the liver is due to stimulation of insulin receptor synthesis.     

Reciprocal changes of insulin and glucagon receptors in primary cultured hepatocytes

The specific [125I]insulin binding to primary cultured hepatocytes was significantly greater than that to freshly isolated hepatocytes. Low affinity insulin binding sites in cultured cells were 6-fold greater in number than those of freshly isolated cells without a significant change in high affinity sites. However, both sensitivity (insulin concentration for half maximum stimulation) and responsiveness (% of increase above the basal level) to insulin for the stimulation of ODC activity were similar for isolated and cultured cells indicating an important role of high affinity sites in the insulin action. On the other hand, the specific [125I]glucagon binding to cultured cells was significantly decreased. Low affinity glucagon binding sites in cultured cells decreased by about 50% in cultured cells without a significant change in high affinity sites. Both sensitivity and responsiveness to glucagon for the stimulation of ketogenesis from palmitate also decreased as compared with those of isolated cells, indicating an important role of low affinity sites in the glucagon action. These results indicate that insulin and glucagon receptors were reciprocally changed in cultured cells, as compared with isolated cells.     

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.     

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.     

Localization of metallothionein in nuclei of growing primary cultured adult rat hepatocytes

In primary cultured adult rat hepatocytes stimulated by epidermal growth factor and insulin, dramatic changes in the subcellular distribution of metallothionein were clarified by indirect immunofluorescence using antisera specific for this protein. Metallothionein was detected only in the cytoplasm of cultured hepatocytes in the G0 and G1 phases, but was concentrated in the cell nuclei in the early S phase. The strongest staining pattern in the nuclei was observed 12 h after stimulation. Subsequently, the intensity of metallothionein staining in the nuclei decreased. These results suggest that primary cultured hepatocytes are suitable for examining the relation between subcellular localization of metallothionein and cell growth.     

Triiodothyronine receptor sites in serum-free cultured hepatocytes from adult rat liver

Nuclear T3 specific binding sites were characterized by Scatchard analyses of L-125I-T3 binding to nuclei extracted from freshly isolated and 1, 2 and 6 day-cultured hepatocytes. The results demonstrate a marked decrease in T3 binding capacity of nuclei extracted from 1 day-cultured cells followed by an almost complete recovery within 6 days. The affinity constant value of nuclear receptor sites is significantly decreased in 1 day-cultured cells with a subsequent partial recovery. The affinity and capacity pattern of nuclear T3 binding sites appears to be in line with the delayed responses of hepatocyte primary cultures to T3.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary Primary monolayer cell cultures of adult rat hepatocytes underwent change in morphology and substantial cell loss between 1 and 3 days postinoculation. Dexamethasone-supplementation (1μM) of the culture medium maintained the polygonal epithelial morphology of the hepatocytes and increased longevity such that over 80% of the cells survived for 3 days and at least 30% for 8 or 9 days. This enhancement of survival was obtained up to 48 hr postinoculation, but the earlier the time of dexamethasone supplementation the greater the effect. Removal of dexamethasone resulted in a decrease in longevity. The positive effect of dexamethasone on longevity was observed following dexamethasone replacement of insulin in supplemented cultures, but the combination of insulin and dexamethasone resulted in poorer survival than with dexamethasone alone. The results are interpreted to indicate that dexamethasone provided a requirement of the in vitro environment for survival and suggest that elaboration of a complex medium is required to maintain hepatocytes in culture. This study was supported by an Alexander Ralston Peacock Memorial Grant for Cancer Research (No. BC-133A) from the American Cancer Society.     

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.     

Conditions affecting primary cell cultures of functional adult rat hepatocytes

Summary The conditions for obtaining representative, primary adult rat hepatocyte cultures were explored. The methods applied included enzymatic liver perfusion which was nondestructive to hepatocytes, the prevention of aggregation of dissociated cells and the selective attachment of viable cells. These procedures yielded a recovery of 50% of the liver cells which gave rise to cultures representing 14% of the total liver cells. The cultures were composed of homogeneous epithelial-like cells cytologically similar to hepatocytes and possessed a number of liver-specific enzymes. There was virtually no cell division initially and most cells died between 24 and 48 hr. Insulin enhanced the attachment of the liver cells, altered their morphology, but did not prolong cell survival. This study was supported by grant no. BC 133 from the American Cancer Society.     

Glucose regulation of insulin receptor affinity in primary cultured adipocytes

Treatment of primary cultured adipocytes with 20 mM glucose resulted in a progressive increase in specific 125I-insulin binding that began almost immediately (no lag period) and culminated in a 60% increase by 24 h. This effect was dose-dependent (glucose ED50 of 4.6 mM) and mediated by an increase in insulin receptor affinity. Moreover, it appears that glucose modulates insulin receptor affinity through de novo protein synthesis rather than through covalent modification of receptors, since cycloheximide selectively inhibited the glucose-induced increase in insulin binding capacity (ED50 of 360 ng/ml) and restored receptor affinity to control values. Importantly, insulin sensitivity of the glucose transport system was increased by glucose treatment (63%) to an extent comparable with the enhancement in receptor affinity, thus indicating a functional coupling between insulin binding and insulin action. When the long term effects of insulin were assessed (24 h), we found that insulin treatment reduced 125I-insulin binding by greater than 60% by down-regulating the number of cell surface receptors in a dose-dependent manner (insulin ED50 of 7.4 ng/ml). On the basis of these studies, we conclude that 1) insulin binding is subject to dual regulation (glucose controls insulin action by enhancing receptor affinity, whereas insulin controls the number of cell surface receptors); and 2) glucose appears to modulate insulin receptor affinity through the rapid biosynthesis of an affinity regulatory protein.     

Biochemical study of cultured hepatocytes obtained from normal adult rats]

A method has been developed for the preparation of hepatocytes that contained 98.6% of that cell type with a yield of 50%. No change was detected in the DNA content per nucleus but losses were noted in RNA and proteins in freshly isolated hepatocytes. The hepatocytes have been kept alive for long periods in suspension or static cultures without any mitoses being observed. However DNA content doubled over a period of two weeks. This level was maintained during the third week. Tritiated thymidine incorportation similarly increased for a period of two weeks and then remained constant. RNA content went down rapidly to one fourth of the original level and remained unchanged thereafter. The protein content did not vary significantly. Incorporation of labelled leucine into proteins has also been measured at various periods of time following isolation of the cells. Even after 62 days in suspension culture, the hepatocytes continued to incorporate leucine into proteins. The incorporation of leucine proceeded through the normal pathway since the presence of t-RNA-bond-leucine was observed. The results reported here suggest that the hepatocytes are arrested in the G2 phase of the cell cycle.     

Effects of insulin and dexamethasone on adenine nucleotide levels in cultured hepatocytes from adult rat

Insulin and dexamethasone, usually added to culture media, play an important role in maintaining the survival of functional hepatocytes. Adenine nucleotide concentrations and energy charge values of cultured hepatocytes were determined to investigate the relationship between the beneficial effects of these hormones and the energy status of the cells. The results indicate that insulin and dexamethasone are essential in maintaining the metabolic competence of cultured hepatocytes and that this correlates with the absolute concentration of ATP rather than with the energy charge.     

Degradation of endogenous proteins and internalized asialofetuin in primary cultured hepatocytes of rats

1. The effects of various metabolic and enzyme inhibitors on the degradation of endogenous proteins and internalized asialofetuin were examined in the primary cultured hepatocytes of rats. 2. The results showed the important physiological role of bestatin-sensitive aminopeptidases in the degradation of endogenous and internalized proteins as well as in the degradation of both long- and short-lived proteins.     

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.     

Synthesis of barbituric acid derivatives and their effect on survival of functional hepatocytes from adult rats in primary culture

Summary Ten barbituric acid (BA) derivatives were synthesized and tested for their potency for supporting survival of functional hepatocytes from adult rats in primary culture. Of the 10 BA derivatives, 7 compounds (C-2, 3, 4, 5, 6, 9, and 10) efficiently supported hepatocyte survival for at least 2 wks in primary culture. Especially C-5, 6, and 9 showed excellent efficiency for such action. The optimum concentrations of the BA derivatives for observing the morphological and biochemical effects differed from each other. The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium. The morphologic features of hepatocytes surviving in the presence of the BA derivatives resembled those of hepatocytes 24 h after inoculation. The surviving hepatocytes secreted remarkably large amounts of albumin into the culture media. Tyrosine aminotransferase (TAT) activity was higher in the 1-wk-old cultures treated with C-5, 6, and 9 than in the freshly isolated hepatocytes. The addition of dexamethasone (10 μM) caused a 1.7 to 2.1-fold induction in TAT activity. The basal levels of TAT activity and the induction rates increased in the cultures treated with C-5 and 6 from Week 1 to 2 of primary culture.     

Insulin induces specific interaction between insulin receptor and protein kinase C delta in primary cultured skeletal muscle

Certain protein kinase C (PKC) isoforms, in particular PKCs beta II, delta, and zeta, are activated by insulin stimulation. In primary cultures of skeletal muscle, PKCs beta II and zeta, but not PKC delta, are activated via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. The purpose of this study was to investigate the possibility that PKC delta may be activated upstream of PI3K by direct interaction with insulin receptor (IR). Experiments were done on primary cultures of newborn rat skeletal muscle, age 5--6 days in vitro. The time course of insulin-induced activation of PKC delta closely paralleled that of IR. Insulin stimulation caused a selective coprecipitation of PKC delta with IR, and these IR immunoprecipitates from insulin-stimulated cells displayed a striking induction of PKC activity due specifically to PKC delta. To examine the involvement of PKC delta in the IR signaling cascade, we used recombinant adenovirus constructs of wild-type (W.T.) or dominant negative (D.N.) PKC delta. Overexpression of W.T.PKC delta induced PKC delta activity and coassociation of PKC delta and IR without addition of insulin. Overexpression of D.N.PKC delta abrogated insulin- induced coassociation of PKC delta and IR. Insulin-induced tyrosine phosphorylation of IR was greatly attenuated in cells overexpressing W.T.PKC delta, whereas in myotubes overexpressing D.N.PKC delta, tyrosine phosphorylation occurred without addition of insulin and was sustained longer than that in control myotubes. In control myotubes IR displayed a low level of serine phosphorylation, which was increased by insulin stimulation. In cells overexpressing W.T.PKC delta, serine phosphorylation was strikingly high under basal conditions and did not increase after insulin stimulation. In contrast, in cells overexpressing D.N.PKC delta, the level of serine phosphorylation was lower than that in nonoverexpressing cells and did not change notably after addition of insulin. Overexpression of W.T.PKC delta caused IR to localize mainly in the internal membrane fractions, and blockade of PKC delta abrogated insulin-induced IR internalization. We conclude that PKC delta is involved in regulation of IR activity and routing, and this regulation may be important in subsequent steps in the IR signaling cascade.     

Modulation of functional activities in cultured rat hepatocytes

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.     

Stimulation of DNA synthesis by heated human serum in primary cultured normal adult rat hepatocytes

In primary culture of normal adult rat hepatocytes, human serum heated at 56°C for 30 min stimulated dose-dependently [³H]thymidine incorporation into trichloroacetic acid insoluble fraction of the cells, most of which was solubilized into hot trichloroacetic acid solution. The solubilized fraction was reduced when hydroxyurea was added to the culture. The heated serum also increased dose-dependently protein synthesis and cell viability determined from morphological findings. These results suggest that human serum has heat-stable factors stimulating DNA synthesis and maintaining cell viability of cultured rat hepatocytes.     

Fructose utilization and altered cytochrome P-450 in cultured hepatocytes from adult rats

Cultured adult rat hepatocytes incubated in media containing fructose exhibit increased levels of cytochrome P-450, relative to cells incubated with equimolar glucose, and the effect of fructose is proportional to its concentration between 2 and 10 mM. For investigating the mechanism of the effect of fructose on cytochrome P-450 in cultured cells, [U-¹⁴C]fructose or [U-¹⁴C]-glucose were added to the incubation medium, and their uptake and utilization were compared. While the uptake kinetics of the two hexoses were similar, the rate of phosphorylation of fructose was more than 10-fold that of glucose. Similarly, the appearance of fructose carbon in metabolic pools, as well as its conversion to CO₂ and cellular glycerolipid, was increased. The latter finding suggested that fructose might alter cytochrome P-450 by stimulating glycerolipid synthesis, since the stability of the cytochrome is lipid-dependent. However, the changes in glycerolipid formation failed to parallel changes in the level of cytochrome P-450 in fructose-treated cells. Moreover, the relative distribution of ¹⁴C into specific lipids was similar for both hexoses, suggesting that an increased carbon flux in cells incubated with fructose did not directly impose a qualitative change in cellular lipid synthesis. We conclude that the fructose-mediated alteration of cytochrome P-450 in cultured rat hepatocytes reflects a process other than increased incorporation of fructose carbon into metabolic pools.