The relationship between glucagon receptors and metabolic profile in two strains of rats: Wistar-Furth and Sprague-Dawley

We studied glucagon and insulin binding to isolated hepatocyte receptors in Wistar-Furth (WF) and Sprague-Dawley (SD) rats, using 125I-labeled hormones. Hepatocytes from WF rats bound more glucagon than hepatocytes from SD rats. There were no differences in insulin binding. These observations prompted us to investigate other strain differences. Fasting and nonfasting serum glucose, glucagon, insulin, and growth hormone were measured. WF animals had a lower fasting glucose and higher fasting glucagon than SD animals, while SD rats had higher nonfasting insulin levels and a higher hepatic glycogen content. Total hepatic glucose production in response to glucagon (10(-8) M) was greater in WF than in SD rats, while glucagon-stimulated gluconeogenesis from alanine was the same in the two groups of animals. We concluded that the decreased glucagon binding does not play a significant role in the maintenance of serum glucose or in the gluconeogenetic response glucagon, and that neither these responses nor the serum glucagon levels appears to be correlated with the number of glucagon receptors. We conclude further that different animal strains of the same species may differ in their biologic responses.     

Adrenalectomy-induced alterations in glucagon binding and lipolysis in isolated rat adipocytes.

Adipocytes from adrenalectomized rats nearly lost their lipolytic response to glucagon concomitant with a 90% decrease in the number of glucagon receptors per cell. Quantitative analysis of the relation between amount of cell-bound glucagon and hormone-stimulated lipolysis revealed that the ability of the remaining 10% of glucagon receptors to induce lipolysis was not impaired. Binding of the beta-adrenergic antagonist [3H]dihydroalprenolol and maximal lipolysis induced by (-)-isoproterenol, (Bu)2cAMP, 3-isobutyl-1-methylxanthine, and adenosine deaminase were reduced only 10 to 20% after adrenalectomy. Furthermore, glucagon-stimulated cAMP production was greatly decreased in adrenalectomized animals, but isoproterenol-stimulated cAMP production was not. Hydrocortisone replacement in adrenalectomized rats only partially prevented the loss of glucagon receptors and glucagon effects on both cAMP production and lipolysis. These findings suggest that lipolytic cascade distal to hormone receptors was not greatly impaired in adipocytes after adrenalectomy and that the unresponsiveness of these cells to glucagon was mostly due to a marked reduction in the number of glucagon receptors.     

Decreased binding of insulin to its receptors in rats with hormone induced insulin resistance

Insulin and glucagon receptor binding was studied in purified liver membranes from rats made insulin resistant by implantation of an MtT pituitary tumor which secretes growth hormone, prolactin, and ACTH. Insulin binding to its receptors was decreased and correlated with the degree of insulin resistance. In contrast, binding of glucagon to its receptors was unchanged.     

Insulin action and binding in isolated hepatocytes from fasted, streptozotocin-diabetic, and older, spontaneously obese rats

Insulin binding and basal and insulin-stimulated uptake of α-aminoisobutyric acid were measured in isolated hepatocytes from young control rats as well as from older spontaneously obese, 72h-starved, and nonketotic streptozotocin-diabetic rats. Isolated hepatocytes from older spontaneously obese rats are similar to those from younger smaller rats in size, maximal insulin responsiveness, the dose–response relationship for insulin-stimulated aminoisobutyrate uptake, and the number and affinity of insulin receptors. Hepatocytes from 72h-fasted rats have similar numbers of insulin receptors per cell as cells from young control animals, but are significantly smaller, have an enhanced basal rate of aminoisobutyrate uptake, and are insulin resistant with regard to maximal insulin-stimulated uptake of aminoisobutyrate at 0.1mm-aminoisobutyrate. Because of the decreased maximal response to insulin, the concentration of insulin that elicits a half-maximal response of aminoisobutyrate uptake is decreased. Hepatocytes from diabetic animals, like those from starved rats, have significantly greater basal rates of aminoisobutyrate uptake; whereas the maximal absolute insulin response is the same as control cells, the percentage response is smaller. These cells bind significantly more insulin than do control cells. The increase in insulin binding is reflected in a shift to the left of the dose–response curve for insulin-stimulated uptake of aminoisobutyrate. These studies indicate that there is no insulin resistance with regard to uptake of aminoisobutyrate in hepatocytes from older obese rats. Furthermore, the insulin resistance observed in hepatocytes from starved rats occurs despite an increase in the number of receptors per unit surface area and cannot be explained by alterations in the interaction between insulin and its receptor. The enhanced insulin binding per unit surface area, however, is reflected in the shift to the left of the dose–response curve for insulin. This is also true for hepatocytes from diabetic animals, in which insulin binding per cell is increased.     

Growth hormone and insulin binding to isolated hepatocytes in the genetically dwarf mouse

The interaction of growth hormone with its specific receptors in dwarf mice was investigated. (1) The interaction of 125I-labeled human growth hormone with isolated mouse liver cells is a specific, time-dependent and saturable process. Hepataocytes of male and female dw/dw mice bound only 10-20% as much growth hormone per unit of cell surface area as those of their litter mates. Scatchard analysis suggested that this decrease in binding was due to a decreased number of receptor sites in th liver cell of the dwarf mouse. (2) In contrast to the marked decrease in growth hormone receptors, the binding of insulin is higher in dwarf mice than in litter mates, at low hormone concentration. (3) Competition and stoichiometric studies indicate that growth hormone and prolactin bind to the same type of binding site in female and male mouse hepatocytes. These results indicate that dwarfism in this animal was associated with a loss in the number of growth hormone binding sites. The decrease in growth hormone receptors and the increase in insulin receptors correlate well with the respective biological activity of these two hormones.     

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

Glucagon binding and lipolytic response in isolated adipocytes from streptozotocin-diabetic rats

Evidence for pre-receptor, receptor and post-receptor glucagon defects was investigated in adipocytes from streptozotocin-diabetic rats. For this purpose male Wistar rats were injected by cardiac puncture with streptozotocin (65 mg/Kg body-weight) or saline solution and sacrificed after 7 and 15 days of drug administration. Increased glucagon levels and increased glucagon degradation in serum together with a decrease in glucagon binding were found in both groups of diabetic rats. The decrease in glucagon binding was related to a decrease in the number of glucagon receptors/cell rather than to a change in receptor affinity. The lipolytic response of glucagon was increased. However, the ability of glucagon to increase basal or theophylline-stimulated cAMP accumulation in the incubation medium of adipocytes from diabetic rats was decreased. Such alterations could represent a counter-regulatory mechanism of the hyperglucagonemia detected in streptozotocin-diabetic rats.     

Changes in the insulin and glucagon receptors in the regenerating liver following intoxication with carbon tetrachloride.

The response of rat liver plasma membrane adenylate cyclase was studied from one to 14 days after a single dose of carbon tetrachloride (CCl₄). The response to glucagon was diminished to a greater extent than that of fluoride and was due to a deficiency in hormone binding. In contrast, insulin binding increased 300% over control; the change was due to increased number of binding sites. The “affinities” of receptors for either hormone were not altered. The tissue levels of adenosine 3′:5′ -monophosphate increased following CCl₄ poisoning reaching a peak precisely when the adenylate cyclase response to glucagon was at its lowest level. These studies present evidence that receptors for pancreatic hormones change differently when liver is damaged and during its regeneration following CCl₄ intoxication. The change in pattern is remarkably similar to changes reported previously in fetal liver development or following partial hepatectomy of adult rat.     

Effects of hypersecretion of growth hormone and prolactin on plasma levels of glucagon and insulin in GH3-cell-tumor-bearing rats, and the influence of bromocriptine treatment

Plasma levels of prolactin, growth hormone, glucagon insulin and glucose were measured in non-treated control rats, bromocriptine-treated control rats and GH3-cell-tumor-bearing rats with and without bromocriptine treatment. Bromocriptine treatment increased plasma levels of glucagon, insulin and glucose in control rats. Tumor-bearing rats had increased body weight and increased plasma levels of prolactin, growth hormone, glucagon, insulin and glucose. Bromocriptine treatment reduced body weight and decreased the plasma levels of prolactin, glucagon and insulin, as compared to non-treated tumor-bearing rats. The drug had no effect on plasma levels of growth hormone and glucose. These results indicate that, in GH3-cell-tumor-bearing rats, prolactin, glucagon and insulin are more sensitive to the action of bromocriptine than growth hormone.     

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.     

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.     

Angiotensin II inhibits hepatic cAMP accumulation induced by glucagon and epinephrine and their metabolic effects

Incubation of isolated hepatocytes containing normal Ca2+ levels with angiotensin II, vasopressin or A23187 caused significant inhibition of the cAMP response to glucagon. Angiotensin II also inhibited cAMP accumulation induced by either glucagon or epinephrine in Ca2+-depleted hepatocytes. When submaximal doses of hormone were employed such that cell cAMP was elevated only 3-4-fold (approximately 2 pmol cAMP/mg wet wt cells) inhibition by angiotensin II was correlated with a decrease in phosphorylase activation. The data demonstrate that inhibition of hepatic cAMP accumulation results in reduced metabolic responses to glucagon and epinephrine and do not support the contention that the hepatic actions of glucagon are independent of cAMP.     

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.     

Alterations of cAMP metabolism and hormone responsiveness of cloned differentiated rat liver cells (RL-PR-C) upon spontaneous transformation

In normal Rat Liver Primary Culture (RL-PR-C) liver cells, cAMP was low prior to confluency, then rose continuously as cells became contact inhibited. In contrast, spontaneously transformed RL-PR-C cells did not become contact inhibited, and cAMP decreased steadily with increasing cell density. Normal cells released large amounts of cAMP into the extracellular fluid at all densities, while transformed cells did not do so at any density. Neither exogenous db-cAMP nor phosphodiesterase inhibitors reversed the uncontrolled growth of transformed cells, nor did conditioned media from contact-inhibited normal cells.While both normal and transformed RL-PR-C hepatocytes produced large amounts of cAMP in response to epinephrine and cholera toxin, transformed cells were much more sensitive to these agents; however, only normal cells responded to glucagon. Although the plasma membrane adenylate cyclase of transformed hepatocytes responded better than did that of normal cells to epinephrine, cholera toxin and fluoride, the basal cyclase activity of transformed cells was only about half that of normal cells. The adenylate cyclase of transformed cells did not respond to glucagon, although the number of glucagon receptors of such cells far exceeded that of normal cells. The V_max of cyclic nucleotide phosphodiesterase of normal hepatocytes was five times that of transformed cells, although the K_m was unchanged.The data indicate that spontaneous transformation of diploid differentiated RL-PR-C hepatocytes leads to cultural hormone receptor and cAMP changes similar to those seen in undifferentiated fibroblasts and other cells transformed by viruses and chemical carcinogens. Although there are significant changes in various parameters of cAMP metabolism upon transformation, decreased cAMP per se does not seem to be responsible for transformation. Furthermore, it is possible that following transformation, these hepatocytes lose some factor necessary for coupling of the glucagon receptor to adenylate cyclase.     

Hypophysectomy does not alter the acinar zonation of gluconeogenesis or the mitochondrial redox state in rat liver.

The biochemical and functional heterogeneity of hepatocytes in different zones of the liver acinus may be related to the concentrations of hormones within the liver acinus. We examined the effects of hypophysectomy, which causes marked changes in plasma hormone levels and in activities of hepatic enzymes that are normally heterogeneously distributed, on the degree of metabolic zonation within the liver acinus. In hypophysectomized rats the activity of alanine aminotransferase was increased, but its normal zonation (predominance in the periportal zone) was preserved. The activity in cultured periportal and perivenous hepatocytes was increased by dexamethasone, but not by glucagon. Periportal hepatocytes from hypophysectomized rats expressed higher rates of gluconeogenesis in culture than did perivenous hepatocytes, irrespective of the absence or presence of dexamethasone, glucagon or insulin. Similar differences in rates of ketogenesis and in the mitochondrial redox state in response to glucagon were observed between periportal and perivenous hepatocytes from hypophysectomized rats as between cell populations from normal rats. Although hypophysectomy causes marked changes in hepatic enzyme activities, it does not alter the degree of zonation of alanine aminotransferase, gluconeogenesis or the mitochondrial redox state within the liver acinus.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Effect of dietary carbohydrates on insulin and glucagon receptors in a new model of noninsulin-dependent diabetes-SHR/N-corpulent rat

A new congenic strain of rat, the SHR/N-corpulent, provides a good model for noninsulin-dependent diabetes and was used in the present study. Corpulent rats as compared to their lean littermates are obese, hyperlipidemic, and severely hyperinsulinemic, and show an age-dependent loss of glucose tolerance. Mild fasting hyperglycemia is seen only in corpulent rats fed sucrose. Since dietary sucrose is more lipogenic than starch and since insulin and glucagon are involved in lipid and carbohydrate metabolism, we studied the effect of the type of dietary carbohydrate on insulin and glucagon levels and their receptors in lean and corpulent SHR/N rats. A significant phenotypic effect was observed (corpulent greater than lean) on plasma levels of triglyceride, cholesterol, and insulin. Dietary sucrose increased these parameters in corpulent rats but not in lean rats. Insulin and glucagon binding to liver plasma membranes was lower in corpulent rats than in lean; decreases were due to fewer receptors without a significant change in affinity. Thus, in corpulent rats, in addition to hyperinsulinemia, fewer glucagon receptors and their failure to be regulated by plasma glucagon levels appear to contribute to the hyperlipidemia. Furthermore, the hyperglycemia observed in sucrose-fed corpulent rats may be due to extreme resistance to insulin despite lower plasma glucagon and fewer glucagon receptors.     

Binding and action of glucagon in isolated adipocytes from cortisol-treated rats

Evidence for pre-receptor, receptor and post-receptor glucagon defects was investigated in adipocytes from cortisol-treated rats. A decrease in glucagon binding due to a decreased number of receptors was observed. No changes in receptor affinity were detected. Both, the lipolytic response of glucagon and the ability of glucagon to increase basal and theophylline-stimulated cAMP accumulation remained unaltered. Moreover, a hyperglucagonemia accompanied by an increase in glucagon degradation in the serum of cortisol-treated rats was observed. Such alterations could represent a new mechanism by which glucocorticoids exert their biological actions.     

Regulatory relation between insulin receptor and its functional responses in primary cultured hepatocytes of adult rats

The relation between changes of insulin receptor and various metabolic responses were studied in adult rat hepatocytes in primary culture. In cells cultured for 3 h without insulin, the number of high affinity sites and the dissociation constant (Kd) of insulin receptor, determined from a Scatchard plot, were 1.05 x 10(5) sites/cell and 1.5 x 10(-9) M, respectively. The receptor number increased 2-fold, but the Kd value remained constant during 2-days culture in insulin-free medium (up-regulation). Addition of dexamethasone (Dex), growth hormone, glucagon or triiodothyronine did not change the number of insulin receptors or the Kd value. In contrast, 1-day culture in insulin (1 x 10(-7) M) medium decreased the receptor number by half (down-regulation) without change of the Kd value. Short-term responses of glycogenesis, amino acid transport and lipogenesis by insulin increased as the receptor number increased. In these cases, the sensitivity to insulin (Ka: half dose for the maximum response) did not change in cells with different receptor numbers, but the maximum response changed. These results show that hepatocytes, unlike adipocytes, do not have spare receptors of insulin. During down-regulation, the receptor number decreased by only half, but the insulin responses were lost almost completely. The receptor number returned to the normal level after culture in insulin-free medium for 12 h, but recovery of the responses took longer, suggesting that for the insulin response not only change of receptor number, but also other regulatory mechanisms for post-receptor processes, such as desensitization, are involved.     

Insulin receptors on erythrocytes and hepatocytes from streptozotocin induced diabetic rats

Insulin receptors on hepatocytes and erythrocytes were studied in rats two and eight weeks after the injection of streptozotocin (50 mg/kg) to see if erythrocyte insulin receptors change parallel with hepatocyte insulin receptors in response to hypoinsulinemia. Insulin binding to hepatocytes increased two (14.0 +/- 2.5% v.s. 7.7 +/- 0.7%; P less than 0.025) and eight weeks (15.9 +/- 1.9% v.s. 6.6 +/- 1.1%; P less than 0.005) after the streptozotocin injection. Scatchard analysis revealed that this increase was due to a rise in both the receptor concentration and affinity. The number of receptors was comparable in the two- and eight-week-streptozotocin rats while the increase in the affinity was more pronounced in the latter group. Insulin binding to the erythrocytes was also increased in both two- (5.0 +/- 0.7% v.s. 4.2 +/- 0.6%) and eight-week- (4.3 +/- 0.6% v.s. 2.7 +/- 1.2%) streptozotocin rats. This increase was due to a rise in the receptor concentration rather than the affinity. However, compared to hepatocytes, these changes were inconsistent and statistically not significant. Furthermore, no correlation was obtained between the binding and plasma insulin concentration. These results indicate that insulin receptors on rat erythrocytes are less sensitive to a change in the plasma insulin concentration and do not always reflect accurately the receptor state on hepatocytes.