Effect of insulin on glucagon binding to isolated rat epididymal adipocytes

Effect of insulin on glucagon binding to rat epididymal adipocytes was studied in vitro. [125I]iodoglucagon binding to isolated adipocytes was increased by preincubation of the cells with insulin. Maximal increase was observed with 7 X 10(-10) M insulin. In Scatchard analysis, [125I]iodoglucagon competition data generated one binding site with a single affinity for glucagon binding in the cells pretreated with buffer alone. Pretreatment of the cells with insulin increased the affinity without changes in the number of binding sites. [125I]iodoglucagon binding to isolated adipocytes was not affected by pretreatment of the cells with luteinizing hormone, follicle-stimulating hormone, growth hormone, or with prolactin. These results suggest that insulin stimulates glucagon binding to adipocytes.     

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.     

Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.     

Interactions of glucagon and glucagon analogs with isolated canine hepatocytes

We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 10(6)-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites.     

A quantitative analysis of glucagon binding to isolated intact neonatal and adult rat hepatocytes on the basis of two different binding models

The interaction of glucagon with specific receptors has been studied in isolated intact neonatal and adult rat hepatocytes. The hormone binding measured directly with ¹²⁵I-labelled glucagon was saturable and reversible. The ¹²⁵I-labelled glucagon binding was inhibited by unlabelled homologous hormone at concentrations ranging from 0.5 nM to 50 μM. Two different binding models were assumed to analyse the binding data by a nonlinear least-squares procedure: (I) a single class of independent sites and (II) two classes of independent sites. The comparison of the fitted theoretical curves reveals that both binding models are in fact compatible with these data. Adult hepatocytes have a considerably higher affinity for glucagon than neonatal hepatocytes; the binding capacity of neonatal liver cells from 1–7-days-old rats proved to be markedly reduced compared with the cells from adult rats. The glucagon-induced intracellular cyclic AMP production was measured at various hormone concentrations under conditions identical to those for the determination of extracellular hormone binding. The correlation of both parameters indicates a direct connection between receptor-occupancy and adenylate cyclase stimulation. These results suggest that a decrease receptor concentration in neonatal hepatocytes is responsible for the decreased cyclic AMP production.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Beta-adrenergic receptors and stimulatory effects of (-) isoproterenol on testosterone production in fetal mouse Leydig cells

Beta-adrenergic receptors were characterized in freshly excised fetal mouse testis using the radioiodinated antagonist iodocyanopindolol (ICYP). [125I]-CYP bound to a single class of high affinity sites with a KD value of 42.2 +/- 7.0 pM. Adrenergic agonists competed for ICYP binding sites with the following order of potency: (-)isoproterenol greater than (-)epinephrine much greater than (-)norepinephrine which is typical for a beta 2-adrenergic receptor. A selective beta 2-receptor antagonist ICI 118-551 showed an approximately 200 fold higher affinity than the beta 1-selective compound, betaxolol. The beta-adrenergic agonist (-)isoproterenol did not or slightly affect testosterone production by freshly isolated fetal Leydig cells. The ability of fetal Leydig cells to respond to (-)isoproterenol increased during culture. This change in responsiveness was not accompanied either by modification of the number of binding sites or by change in the binding affinity. Taken together these data suggest that i) the stimulatory effect of (-)isoproterenol on testosterone production by cultured fetal Leydig cells is mediated through beta 2-adrenergic receptors ii), the inability of freshly Leydig cells to respond to catecholamines is probably due to post receptor events.     

Effect of Escherichia coli lipopolysaccharide on the glucagon and insulin binding to isolated rat hepatocytes

Summary Number and affinity constant of low affinity binding sites of insulin and glucagon to isolated hepatocytes decreased when the cells were incubated with Escherichia coli 0111:B4 lipopolysaccharide. This effect agrees with a non-specific binding of lipopolysaccharide to hepatocytes, similar to the well-recognized non-specific binding of albumin. Also, binding of different lectins to their glycoprotein receptors did not affect the [¹⁴C]lipopolysaccharide interaction with the cell membrane surface. Endotoxin depresses gluconeogenesis from lactate when the precursor was incubated with the cells for short time intervals. The longer the preincubation interval with lipopolysaccharide, the higher the inhibition of gluconeogenesis in the absence and in the presence of glucagon.The effect of endotoxin was also studied on the glucagon-induced synthesis of cyclic AMP and the glucagon binding. Levels of cyclic AMP and hormone binding decreased with increasing both endotoxin concentrations and preincubation intervals at which cells were in contact with endotoxin.     

Characterization of angiotensin II receptor subtypes in rat liver

Radioligand binding studies identified two classes of 125I-angiotensin II-binding sites in rat liver membranes. High affinity binding sites (Kd = 0.35 +/- 0.13 nM, N = 372 +/- 69 fmol/mg of protein) were inactivated by dithiothreitol (0.1-10 mM) without any apparent change in low affinity binding sites (Kd = 3.1 +/- 0.8 nM, N = 658 +/- 112 fmol/mg of protein). Dithiothreitol inactivation was readily reversible but could be made permanent by alkylation of membrane proteins with iodoacetamide. Angiotensin II stimulation of glycogen phosphorylase in isolated rat hepatocytes (maximal stimulation 780%, EC50 = 0.4 nM) was completely inhibited by 10 mM dithiothreitol, a concentration which also abolished high affinity site binding; phosphorylase stimulation by glucagon and norepinephrine under these conditions was unaltered. Angiotensin II inhibition of glucagon-stimulated adenylate cyclase activity in hepatocytes required higher angiotensin II concentrations (EC50 = 3 nM) than phosphorylase stimulation and was not affected by dithiothreitol. Fractional occupancy of high affinity binding sites by 125I-angiotensin II correlated closely with angiotensin II-mediated phosphorylase stimulation, whereas occupancy of low affinity sites paralleled inhibition of adenylate cyclase activity. These data indicate that the physiologic effects of angiotensin II in rat liver are mediated by two distinct receptors, apparently not interconvertible, and provide the first evidence for angiotensin II receptor subtypes with differing biochemical features and mechanisms of action.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.     

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro.

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 microM) increased specific [125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 microM) increased specific [125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in Bmax to 119% by cortisol (0.5 microM, 48 h) and to 194% by dexamethasone (0.5 microM, 48 h), and no change in Kd for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Phorbol esters inhibit alpha 1-adrenergic effects and decrease the affinity of liver cell alpha 1-adrenergic receptors for (-)-epinephrine

4 beta-Phorbol 12-myristate 13-acetate (PMA) modified the metabolic actions of three calcium-dependent hormones in different ways. The stimulations of glycogenolysis ureogenesis and phosphatidylinositol labeling produced by alpha 1-adrenergic agonist was blocked by the phorbol ester. In contrast, PMA slightly increased the stimulation of ureogenesis produced by low concentration of angiotensin II without modifying the maximal response. No effect of PMA was observed on the stimulation of ureogenesis induced by vasopressin. The stimulation of phosphatidylinositol labeling induced by vasopressin was decreased by PMA, whereas that induced by angiotensin II was not affected. In intact freshly isolated hepatocytes, [3H]prazosin binds with high affinity to a site which displays the characteristics of alpha 1-adrenergic receptor. Competitive inhibition studies with (-)-epinephrine reveal two different sites for this agonist: a high affinity site (Kd 9 nM) and a low affinity site (Kd 2 microM). In the presence of phorbol esters, (-)-epinephrine binding data now show the presence of a single class of low affinity sites, with similar affinity to those present in control cells. Thus, the inhibition of hepatocyte alpha 1-adrenergic action by PMA may be related to the loss of high affinity binding sites caused by the tumor promoter.     

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.     

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5°C, cell surface [¹²⁵I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37°C, due to a decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [³⁵S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37°C after 2 h at 42.5°C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [¹⁴C]glucose incorporation into glycogen measured at 37°C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4—fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress. © 1995 Wiley-Liss, Inc.     

Insulin and glucagon binding to isolated hepatocytes of Egyptian sand rats (Psammomys obesus): evidence for an insulin receptor defect

To determine whether a defect in insulin binding could contribute to insulin resistance in Egyptian sand rats (Psammomys obesus), insulin binding to isolated hepatocytes from euglycemic sand rats was compared to that of normal Sprague-Dawley rats (Rattus norvegicus). Because of its potential importance in glucoregulation, glucagon binding to hepatocytes from these species was also measured. Hepatocytes of sand rats exhibit an almost complete lack of insulin receptors compared to hepatocytes from Sprague-Dawley rats, whereas there are numerous high affinity glucagon binding sites on sand rat hepatocytes. The lack of insulin binding to sand rat tissues is sufficient to entirely explain the insulin resistance seen in this species. Glucagon may be primarily responsible for glucose homeostasis in Psammomys obesus.     

Lack of insulin effect on its own receptors in fetal rat hepatocytes

To determine the effect of insulin on its receptor concentrations in hepatocytes of fetal and adult rats, these cells were preincubated in the presence or absence of insulin. The reduced [125I]-insulin binding observed in adult hepatocytes was dependent on the concentration of insulin and on the duration of exposure, while in fetal hepatocytes insulin did not induce any reduction in insulin binding. In contrast, glucagon receptors were unaffected by preincubation with insulin. The modifications observed in insulin binding were accounted for by changes in receptor concentrations rather than any change in receptor affinity for the hormone. Studies on the kinetic properties of the insulin receptors of fetuses and adult rats revealed that association and dissociation rates were undistinguishable. These results indicate an absence of insulin receptor down-regulation in the fetus, which could favour anabolic processes during intrauterine life.     

Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.

To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.     

Glucagon binding by isolated chick hepatocytes

Studies have been made on the binding of 125I-glucagon by isolated chick hepatocytes. It was shown that pH and temperature dependence of the binding does not differ from that in rat hepatocytes. Optimum binding was observed at pH 7.6, the rate of binding being higher at 37 degrees C as compared to that at 20 degrees C, although the binding capacity increased with the decrease in the temperature. Unlabeled glucagon was able to compete with 125I-glucagon at the binding sites. Scatchard plot was found to be curvilinear revealing two classes of the binding sites with Kd values 10(-9) and 10(-7) M at temperatures 20 and 37 degrees C correspondingly. Earlier studies revealed in rats the binding sites of a sole class with Kd value 10(-9) M. Preincubation of cells with native glucagon results in changes of labeled glucagon binding, the effect being proportional to the concentration of native glucagon. Preincubation effect was observed at 37 degrees C, being absent at 20 degrees C; the effect was due to the decrease in the number of both high and low affinity binding sites. The presence of down-regulation of glucagon receptors in chick hepatocytes is suggested.