Kinetics of internalization and degradation of asialo-glycoproteins in isolated rat hepatocytes

The rate constants for internalization of surface-bound asialo-orosomucoid by hepatocytes were 0.040 min-1 at 20 degrees C, 0.18 min-1 at 30 degrees C and 0.28 min-1 at 40 degrees C. At 40 degrees C, internalization accounted for most of the increase in cell-associated radioactivity. The activation energy over the temperature range 20 to 40 degrees C was 68 +/- 7 (S.D.) kJ/mol. At 10 degrees C, most of the cell-associated asialo-orosomucoid was bound to the cell surface in a reaction which followed ordinary chemical kinetics. Pre-incubation of hepatocytes with a large concentration of unlabelled asialo-orosomucoid did not influence the uptake of subsequently added 125I-asialofetuin; neither was degradation of 125I-asialo-fetuin affected in this experiment. The fractional rate of degradation (the fraction of cell-associated asialo-fetuin which was degraded per unit time) was constant over a twelve-fold range of intracellular asialo-fetuin concentrations. Increasing the temperature from 20 to 30 degrees C produced approximately a ten-fold increase in the rate of degradation of either asialo-fetuin or asialo-orosomucoid. The average activation energies of degradation over the range 20 to 40 degrees C were 125 kJ/mol for asialo-fetuin and 149 kJ/mol for asialo-orosomucoid; however, the Arrhenius plots were not straight lines over this temperature range.     

Receptor-linked degradation of 125I-insulin is mediated by internalization in isolated rat hepatocytes

When hepatocytes were freshly isolated from rat liver and incubated for various periods of time at 37 degrees C, the media from the incubation, when completely separated from the cells, actively degraded 125I-insulin. THis soluble protease activity was strongly inhibited by bacitracin but was unaffected by the lysosomatropic agent ammonium chloride (NH4Cl). When hepatocytes were incubated with 125I-insulin at 37 degrees C in the presence or absence of 8 mM NH4Cl the ligand initially bound to the plasma membrane and was subsequently internalized as a function of time. When hepatocytes were incubated at 37 degrees C for 30 minutes with 125I-insulin in the presence of bacitracin and NH4Cl or bacitracin alone and the cells were washed, diluted, and the cell-bound radioactivity allowed to dissociate, the percent intact 125I-insulin in the cell pellet and in the incubation media was greater in the presence of NH4Cl at each time point of incubation. Under these same conditions a higher proportion of the cell-associated radioactivity was internalized and a higher proportion was associated with lysosomes. The data suggest that receptor-mediated internalization is required for insulin degradation by the cell, and that this process, at least in part, involves lysosomal enzymes. Furthermore, the data demonstrate that internalization is not blocked by the presence of bacitracin or NH4Cl in the incubation media, but that degradation is inhibited.     

Structural and functional studies on the human interleukin-6 receptor. Binding, cross-linking, internalization, and degradation of interleukin-6 by fibroblasts transfected with human interleukin-6-receptor cDNA

A cDNA coding for the human interleukin-6 receptor (IL-6-R) has been expressed stably in murine NIH/3T3 fibroblasts. Transfected cells exhibited a single class of binding sites for 125I-labeled recombinant human interleukin-6 (125I-rhIL-6) (Kd = 440 pM, 20,000 receptors per cell). Affinity cross-linking of 125I-rhIL-6 to the IL-6-R-expressing NIH/3T3 cells led to the detection of three 125I-rhIL-6-containing protein complexes with molecular masses of 100, 120, and 200 kDa suggesting a complex organization of the IL-6-R in the plasma membrane. IL-6 added to the transfected NIH/3T3 cells exerted growth inhibition. This anti-growth effect was observed by the measurement of cell numbers and ornithine decarboxylase mRNA expression. IL-6-R overexpressing fibroblasts internalized 125I-rhIL-6. Intracellular limited proteolysis of IL-6 could be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible implication of skin fibroblasts in the catabolism of IL-6 is discussed.     

Differential regulation of interleukin-6 receptor and gp130 gene expression in rat hepatocytes.

Interleukin-6 (IL-6) relays an important signal to hepatocytes during the early stages of an acute inflammatory response, causing an alteration in the expression of several major defense proteins. Additional regulation of this signal could occur either by altering the number of IL-6 receptors (IL-6-R) or of the signal transducing protein, gp130. We employed ribonuclease protection assays to measure the expression of IL-6-R and gp130 mRNA in primary rat hepatocytes in response to IL-6, interleukin-1, dexamethasone, and combinations thereof. Dexamethasone increases receptor mRNA levels 2.7-fold above controls but has no detectable effect on that of gp130. Such treatment increased surface expression of IL-6-R from 600 receptors per cell to greater than 6000, without a change in Kd (2.5-4.6 x 10(-10) M). In contrast to the stimulatory effect of the steroid signal, the inflammatory cytokines, individually and together, down-modulated both the mRNA and the cell surface expression of IL-6-R. These findings demonstrate for the first time that a sensitive control system exists between inflammatory mediators and IL-6-R.     

Norepinephrine stimulates interleukin-6 mRNA expression in primary cultured rat hepatocytes

Non-invasive immobilization stress causes an increase in the plasma interleukin (IL)-6 level accompanied by increased IL-6 mRNA expression and IL-6 immunoactivity in the liver [Biochem. Biophys. Res. Commun. (1997) 238, 707-711]. In the present study, using rat primary cultured hepatocytes and non-parenchymal liver cells, the effect of norepinephrine (NE) on IL-6 mRNA expression was determined. IL-6 mRNA expression in hepatocytes, but not in non-parenchymal liver cells, increased when the cells were treated with NE. The stimulatory effect of NE was inhibited by the combined use of alpha- and beta-adrenergic antagonists. IL-6 mRNA expression in hepatocytes also increased on incubation with the culture medium of non-parenchymal liver cells treated with NE. The effect of the medium was blocked by an IL-1 receptor antagonist. Moreover, exogenous IL-1beta stimulated IL-6 mRNA expression in hepatocytes. IL-1beta was present in the medium of non-parenchymal liver cells and increased with NE-treatment. These results suggest that NE released from sympathetic nerve terminals during stress can directly increase IL-6 mRNA expression in hepatocytes and indirectly through IL-1beta production from non-parenchymal liver cells.     

Autophagic degradation of peroxisomes in isolated rat hepatocytes.

Degradation of the peroxisomal enzymes fatty acyl-CoA oxidase and catalase was studied in hepatocytes isolated from rats treated with clofibrate and from control rats. Hepatocytes were incubated in the absence of amino acids in order to ensure maximal flux through the autophagic pathway and in the presence of cycloheximide to inhibit protein synthesis. (1) Degradation of the two peroxisomal enzymes in hepatocytes from clofibrate-fed rats, but not in hepatocytes from control rats, was much faster than that of other intracellular enzymes. This increased degradation of the peroxisomal enzymes was almost completely prevented by 3-methyladenine, an inhibitor of macroautophagic sequestration. (2) The increased degradation of the peroxisomal enzymes was also inhibited by a long-chain (C16:0) and a very-long-chain (C26:0) fatty acid, but not by C12:0, a medium-chain fatty acid, or by C8:0, a short-chain fatty acid. These results provide direct evidence for the proposal that autophagic sequestration can be highly selective [(1987) Exp. Mol. Pathol. 46, 114-122]. It is concluded that preferential autophagy of peroxisomes is prevented when these organelles are supplied with their fatty acid substrates.     

Quantitative studies of the rate of insulin internalization in isolated rat hepatocytes.

We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.     

Intracellular localization and degradation of asialofetuin in isolated rat hepatocytes.

Analysis by isopycnic and differential centrifuging of the intracellular distribution of radioactivity following uptake of 125I-labelled asialofetuin by isolated rat hepatocytes showed that during incubations up to 1 h, most of the radioactivity was associated with structures which had a subcellular distribution pattern different from both the lysosomes and the plasma membrane. The latter two organelles were followed by means of enzyme markers. Ca2+ is necessary for the binding of asialofetuin to the plasma membrane, and it was also possible to differentiate between asialofetuin bound to the plasma membrane and that contained in intracellular structures by removing Ca2+ from the medium (by EGTA). Such experiments showed that asialofetuin became rapidly internalized. Practically all the labelled protein was located intracellularly in cells that had been incubated with asialofetuin for more than 30 min. When incubations were carried out for more than 1 h a peak appeared in the radioactivity distribution in the same place as the peak of activity of lysosomal marker enzymes. However, degradation of asialofetuin takes place in the lysosomes and this starts before the labelled protein can be found in the lysosomal fractions. Our data suggest that the rate-determining step in the cellular handling of asialofetuin is the transport of endocytized protein from the endocytic vesicles to the lysosomes.     

Uptake and degradation of asialo-fetuin by isolated rat hepatocytes.

125I-labelled asialo-fetuin was taken up by isolated rat hepatocytes by a saturable process. Half maximum uptake was seen at about 3 . 10(-8) M asialo-fetuin. Rate of uptake of asialo-fetuin exceeded rate of degradation at all concentrations of asialo-fetuin tested. Degradation of asialo-fetuin, as indicated by release of acid-soluble radioactivity from the cells, was inhibited by NH4Cl and chloroquine. The intracellular distribution of labelled asialo-fetuin was studied by differential and density gradient centrifuging. The distribution curves for radioactivity indicated that asialo-fetuin was present in lysosomes about 1 h after the uptake had started. Chloroquine and ammonium ions seemed to inhibit the uptake of asialo-fetuin into the lysosomes, possibly by interfering with the fusion between phagosomes and lysosomes.     

Glucagon-stimulated but not isoproterenol-stimulated glucose formation inhibition by interleukin-6 in primary cultured rat hepatocytes.

During prolonged sepsis, impairment of glucose supply by the liver leads to hypoglycemia. Our aim was to investigate whether proinflammatory cytokine interleukin-6, a major mediator of the hepatic acute phase reaction, could contribute to this impairment by inhibiting hepatic glucose production stimulated by glucagon or isoproterenol in rat hepatocytes. Interleukin-6 inhibited the stimulation of glucose formation from glycogen by glucagon but not by isoproterenol in cultured rat hepatocytes. This was confirmed in the perfused rat liver. In cultured hepatocytes, the increase in cyclic adenosine-3',5'-monophosphate formation by glucagon was inhibited by interleukin-6, which was probably due to attenuation of glucagon binding to the glucagon receptor. The increase in cyclic adenosine-3',5'-monophosphate stimulated by isoproterenol was not affected by interleukin-6. However, the cytokine inhibited both expression of the key gluconeogenic control enzyme, phosphoenolpyruvate carboxykinase, stimulated by glucagon and isoproterenol. Thus, while increased glucose demand during the acute-phase reaction might initially be accomplished by catecholamine-mediated stimulation of glucose formation from glycogen, inhibition of gluconeogenesis by interleukin-6 may contribute to the impairment of glucose homeostasis during the prolonged acute phase reaction.     

Binding and degradation of 125I-insulin by rat hepatocytes.

The binding and the velocity of degradation of 125I-insulin in the absence or presence of varying concentrations of native procline insulin were studied using isolated rat hepatocytes. At insulin concentrations ranging from 5 X 10(-11) to 10(-6) M, insulin degradation velocity showed a first order dependence on the total concentration of insulin bound at steady state. The overall reaction had an apparent rate constant of 0.030 +/- 0.011 min-1. Furthermore, the degradation of a given amount of 125I-insulin bound to cells was more rapid and extensive than the degradation of the same amount of insulin which had been newly exposed to fresh cells. Mid pretreatment of isolated hepatocytes with trypsin or chymotrypsin at concentrations of 5 to 20 mug/ml depressed to the same degree the amount of 125-I-insulin bound at steady state and the 125I-insulin degradation velocity. Peptide or protein hormones unrelated to insulin, including the oxidized A and B chains of insulin, failed to depress the amount of insulin bound or the velocity of insulin degradation when present at concentrations of 10-5 or 10-6 M. Over a wide range of concentrations, various synthetic insulin analogues and naturally occurring insulins depressed to the same degree the amount of 125I-insulin bound at steady state and the 125I-insulin degradation velocity. These observations suggest that insulin bound to hepatocyte plasma membranes is the substrate for insulin degradation by the liver.     

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.

Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.     

Accelerated phospholipid degradation in anoxic rat hepatocytes

Accelerated degradation of membrane phospholipids characterizes the reaction of rat liver and myocardial cells to ischemia. A similar disturbance in phospholipid metabolism was sought in anoxic hepatocytes. Primary cultures of adult rat hepatocytes were made anoxic by evacuation of the CO₂O₂ atmosphere with N₂. The resulting loss of ATP was reversible upon reoxygenation after periods of anoxia up to 2 h. With 3–4 h of anoxia, the cells were incapable of regenerating ATP levels. Loss of viability was also indicated by the inability of over 90% of the cells after 3–4 h to exclude trypan blue. The baseline rate of turnover of [¹⁴C]-ethanolamine or glycerol prelabeled phospholipids was then established. A constant rate of turnover was found for, at least, the first 3 days the cells were in culture. No loss of total phospholipid occurred during this time. Anoxia induced very significant differences in the fate of prelabeled phospholipids. With [¹⁴C]-ethanolamine there was a 30% loss of total cellular radioactivity within 4 h. Total phospholipids determined as lipid phosphate decreased by 20%. This depletion of cellular phospholipids was paralleled by an accumulation of hydrophilic degradation products in the culture medium. Phosphorylethanolamine accounted for 50% of these, with equal amounts of glycerophosphorylethanolamine and ethanolamine the other 50%. A similar accumulation in the medium occurred with [¹⁴C]-glycerol- and [¹⁴C]choline-prelabeled phospholipids. The accelerated degradation of phospholipid was accompanied by evidence of membrane dysfunction as shown by the loss of 50% of the glucose 6-phosphatase activity in whole cell homogenates. The results of these studies establish that anoxia induces in cultured rat hepatocytes a similar disturbance to phospholipid metabolism as does ischemia of the same cells in the intact animal. This implies that the deprivation of oxygen per se determines the characteristic reaction of cells to ischemia. This conclusion allows further analysis of the effects of O₂ deprivation on cultured hepatocytes as a new experimental model with which to further explore the effects of ischemia on cells.     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

Inhibition of protein degradation in isolated rat hepatocytes

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [³H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [³H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [³H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.     

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.