Metabolic effects and cyclic AMP levels produced by glucagon, (1-Nα-trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes

[1-N^α-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.     

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.     

Flux through glycine cleavage system in isolated hepatocytes: effects of glucagon, cAMP, and calcium

The hepatic glycine cleavage system (GCS) is the principal route for the metabolism of glycine in mammals. Flux through the GCS in isolated rat hepatocytes was stimulated by about 100% by glucagon (10(-7) M), forskolin (10(-4) M), and dibutyryl cAMP (10(-4) M). The stimulation of flux through the GCS by these agents was accompanied by marked elevation of cellular cAMP levels. A significant correlation was observed between increased cellular cAMP levels induced by glucagon and stimulation of flux through the GCS by glucagon. Exclusion of calcium from the incubation medium reduced the basal flux by 38%, but did not affect the degree of stimulation of flux through the GCS by glucagon. A single intraperitoneal injection of glucagon to rats prior to isolation of hepatocytes resulted in a 76% stimulation of flux through the GCS. These hepatocytes with stimulated flux through the GCS showed reduced sensitivity for further stimulation by glucagon. Half-maximal stimulation of flux through the GCS occurred at 3.8 +/- 1.1 and 8.5 +/- 1.4 nM glucagon in hepatocytes isolated from control and glucagon-injected rats, respectively. We conclude that cAMP is involved in the regulation of flux through the GCS by glucagon.     

Modulation by thyroid status of cyclic AMP-dependent and Ca2+-dependent mechanisms of hormone action in rat liver cells. Possible involvement of two different transduction mechanisms in alpha 1-adrenergic action

The actions of hormones which are associated to cAMP-dependent and calcium-dependent mechanisms of signal transduction were studied in hepatocytes obtained from rats with different thyroid states. In cells from euthyroid and hyperthyroid rats, the metabolic actions of epinephrine were mediated mainly through alpha 1-adrenoceptors; beta-adrenoceptors seem to be functionally unimportant. In contrast, both alpha 1- and beta-adrenoceptors mediate the actions of epinephrine in hepatocytes from hypothyroid animals. Phosphatidylinositol labeling was strongly stimulated by epinephrine, vasopressin and angiotensin II in cells from eu-, hyper- or hypothyroid rats. However, metabolic responsiveness to vasopressin and angiotensin II was markedly impaired in the hypothyroid state. The glycogenolytic response to the calcium ionophore A-23187 was also impaired, suggesting that hepatocytes from hypothyroid rats are less sensitive to calcium signalling. The persistence of alpha 1-adrenergic responsiveness in the hypothyroid state suggests that the mechanism of signal transduction for alpha 1-adrenergic amines is not identical to that of the vasopressor peptides. alpha 1-Adrenergic stimulation of cyclic AMP accumulation was not detected in cells from hypothyroid rats. These data suggest that factors besides calcium and besides cAMP are probably involved in alpha 1-adrenergic actions. Metabolic responses to glucagon and to the cAMP analogue dibutyryl cAMP were not markedly changed during hypothyroidism, although cAMP accumulation produced by glucagon and beta-adrenergic agonists was enhanced. In hyperthyroidism, cell responsiveness to epinephrine, vasopressin, angiotensin II and glucagon was decreased, but sensitivity to cAMP was not markedly altered. The factors involved in this hyposensitivity to hormones during hyperthyroidism are unclear.     

Effects of glucagon and an analogue of cAMP on tyrosine aminotransferase in isolated chick embryo hepatocytes.

Hepatocytes were isolated from 17-day-old chick embryos by the use of collagenase. Glucagon and dibutyryl cAMP (bt2cAMP), individually or in combination, stimulated tyrosine aminotransferase (TAT) activity and synthesis in the isolated hepatocytes; maximal stimulation occurred 4 h after exposure of hepatocytes to the inducers. The stimulatory effects produced by glucagon and bt2cAMP were abolished by treatment of hepatocytes with cordycepin or cycloheximide. The effects of the hormone and the cyclic nucleotide were not additive. The induction of the enzyme by glucagon suggests a physiological role for the hormone in the expression of TAT activity during chick embryonic development.     

Effects of glucagon and 3,5,3'-triiodo-L-thyronine on oxidative phosphorylation of thyroidectomized rat liver mitochondria

In normal or thyroidectomized rat liver mitochondria, glucagon produced fast but transient stimulation of respiration rates in state 3 and state 4 whatever the substrates. Stimulation reached its maximum 20 to 30 minutes after glucagon injection. However, the effects of glucagon are less marked after removal of the thyroid gland, since the increases observed in the oxygen consumption and basal metabolic rates were only half those shown in normal rats. The activating effects of triiodothyronine and glucagon on the ADP phosphorylation rates were found to be additive. Pretreatment with cycloheximide blocked the activation induced by glucagon but not that induced by triiodothyronine. Both hormones therefore stimulate oxidative phosphorylation but by different mechanisms. Thyroidectomy did not alter the early rise in glycaemia observed in response to glucagon. It may therefore be assumed that the hypothyroid rat's sensitivity to glucagon is not directly connected with the change in cAMP metabolism.     

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.     

Increased glucagon receptors in chronically hypersomatotrophic and hyperglucagonemic rats

The effect of increased levels of growth hormone on glucagon binding by isolated hepatocytes and on the cellular cyclic AMP response to glucagon was evaluated in rats bearing growth hormone-secreting tumor (Mt-T-W15) and in rats treated with rat growth hormone. An increased binding, due to an increased number of receptors, was observed in both groups of animals. Glucagon binding did not correlate with plasma glucagon levels, suggesting a failure of down regulation, possibly due to an effect of growth hormone and insulin on the number of receptors. Tumor-bearing and growth hormone-treated rats had larger hepatocytes so that, when hormone binding was expressed in terms of square micrometer of membrane surface, it appeared decreased. When the tumor was removed the increase in the number of glucagon receptors per cell persisted, even though the average cell size returned toward normal. It is suggested that this retention of the receptors may have been the result of continuing hyperinsulinism. Basal cAMP levels were elevated in hepatocytes of tumor-bearing and growth hormone-treated animals, possibly due to cell hypertrophy. On the other hand, the maximum cAMP response to glucagon was not altered by the experimental procedures. A negative effect of insulin on cAMP accumulation may explain this apparent paradox. Indeed, hepatocytes isolated from rats following tumor removal, but with continuing hyperinsulinemia, had a lower maximum cAMP response, even though the glucagon binding per cell or per unit of cell surface was increased.     

Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes

Although many effects of leptin are mediated through the central nervous system, leptin can regulate metabolism through a direct action on peripheral tissues, such as fat and liver. We show here that leptin, at physiological concentrations, acts through an intracellular signaling pathway similar to that activated by insulin in isolated primary rat hepatocytes. This pathway involves stimulation of phosphatidylinositol 3-kinase (PI3K) binding to insulin receptor substrate-1 and insulin receptor substrate-2, activation of PI3K and protein kinase B (AKT), and PI3K-dependent activation of cyclic nucleotide phosphodiesterase 3B, a cAMP-degrading enzyme. One important function of this signaling pathway is to reduce levels of cAMP, because leptin-mediated activation of both protein kinase B and phosphodiesterase 3B is most marked following elevation of cAMP by glucagon, and because leptin suppresses glucagon-induced cAMP elevation in a PI3K-dependent manner. There is little or no expression of the long form leptin receptor in primary rat hepatocytes, and these signaling events are probably mediated through the short forms of the leptin receptor. Thus, leptin, like insulin, induces an intracellular signaling pathway in hepatocytes that culminates in cAMP degradation and an antagonism of the actions of glucagon.     

Studies on the alpha-andrenergic activation of hepatic glucose output. II. Investigation of the roles of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in the actions of phenylephrine in isolated hepatocytes.

The effects of the alpha-adrenergic agonist phenylephrine on the levels of adenosine 3':5'-monophosphate (cAMP) and the activity of the cAMP-dependent protein kinase in isolated rat liver parenchymal cells were studied. Cyclic AMP was very slightly (5 to 13%) increased in cells incubated with phenylephrine at a concentration (10(-5) M) which was maximally effective on glycogenolysis and gluconeogenesis. However, the increase was significant only at 5 min. Cyclic AMP levels with 10(-5) M phenylephrine measured at this time were reduced by the beta-adrenergic antagonist propranolol, but were unaffected by the alpha-blocker phenoxybenzamine, indicating that the elevation was due to weak beta activity of the agonist. When doses of glucagon, epinephrine, and phenylephrine which produced the same stimulation of glycogenolysis or gluconeogenesis were added to the same batches of cells, there were marked rises in cAMP with glucagon, minimal increases with epinephrine, and little or no changes with phenylephrine, indicating that the two catecholamine stimulated these processes largely by mechanisms not involving cAMP accumulation. DEAE-cellulose chromatography of homogenates of liver cells revealed two major peaks of cAMP-dependent protein kinase activity. These eluted at similar salt concentrations as the type I and II isozymes from rat heart. Optimal conditions for preservation of hormone effects on the activity of the enzyme in the cells were determined. High concentrations of phenylephrine (10(-5) M and 10(-4) M) produced a small increase (10 tp 16%) in the activity ratio (-cAMP/+cAMP) of the enzyme. This was abolished by propranolol, but not by phenoxybenzamine, indicating that it was due to weak beta activity of the agonist. The increase in the activity ratio of the kinase with 10(-5) M phenylephrine was much smaller than that produced by a glycogenolytically equivalent dose of glucagon. The changes in protein kinase induced by phenylephrine and the blockers and by glucagon were thus consistent with those in cAMP. Theophylline and 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, potentiated the effects of phenylephrine on glycogenolysis and gluconeogenesis. The potentiations were blocked by phenoxybenzamine, but not by propranolol. Methylisobutylxanthine increased the levels of cAMP and enhanced the activation of protein kinase in cells incubated with phenylephrine. These effects were diminished or abolished by propanolol, but were unaffected by phenoxybenzamine. It is concluded from these data that alpha-adrenergic activation of glycogenolysis and gluconeogenesis in isolated rat liver parenchymal cells occurs by mechanisms not involving an increase in total cellular cAMP or activation of the cAMP-dependent protein kinase. The results also show that phosphodiesterase inhibitors potentiate alpha-adrenergic actions in hepatocytes mainly by a mechanism(s) not involving a rise in cAMP.     

Regulation of hepatic glycogenolysis by glucagon in male and female rats. Role of cAMP and Ca2+ and interactions between epinephrine and glucagon

The effects of 10(-10) to 10(-7) M glucagon on cAMP, phosphorylase a, cell calcium, and glucose production, and glucagon interactions with epinephrine were studied in isolated hepatocytes from adult male and female rats. At physiological concentrations (10(-10) - 10(-9) M), glucagon activated phosphorylase by increasing cAMP and not by raising the cytosolic free calcium. At supra-physiologic concentrations (and in the male only), glucagon slightly increased the cytosolic free calcium, the fractional efflux of calcium, and, after 2 h, decreased the cell calcium content. Exposure of hepatocytes to the simultaneous administration of 10(-9) M glucagon and 10(-7) M epinephrine resulted in a prolongation of the activation of phosphorylase a and a greater release of glucose from glycogen stores than exposure to either agonist alone. In the male, the effects of low concentrations of the two hormones on phosphorylase a activity were additive. Cytosolic free calcium was increased by 10(-6) M epinephrine from 280 to 500 nM while physiological concentrations of glucagon did not change it. In these intact cells, there was no evidence of an alpha 2-adrenergic inhibition of adenyl cyclase and no indication that cAMP depresses the rise in cell calcium induced by alpha-adrenergic stimuli.     

Regulation of hepatic glycine catabolism by glucagon

Glucagon stimulates 14CO2 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%) of decarboxylation of glycine by hepatocytes was achieved when the concentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured. Inclusion of beta-hydroxybutyrate (10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activity of intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14CO2 production from [1-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondria isolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.     

Modulation of glucagon actions by phorbol myristate acetate in isolated hepatocytes. Effect of hypothyroidism

Phorbol myristate acetate (PMA) inhibits glucagon-stimulated cyclic AMP accumulation and shifts to the right the dose-response curve to glucagon for ureagenesis. In cells from hypothyroid rats the effect of PMA on glucagon-stimulated ureagenesis was much more pronounced, but its effect on cyclic AMP accumulation was similar to that observed in the control cells. The stimulations of ureagenesis by the glucagon analogue THG and dibutyryl cyclic AMP (But2-cAMP) were also diminished by PMA, to a greater extent in cells from hypothyroid rats than in those from euthyroid rats. PMA inhibited the increases in cytoplasmic [Ca2+] induced by glucagon. THG or But2-cAMP; the effect of PMA was much more marked in cells from hypothyroid rats than in the controls. Treatment of the cells with glucagon or THG increased the production of citrulline by subsequently isolated mitochondria, whereas PMA diminished their effects. The results suggest that PMA alters glucagon actions at least at two levels; (i) cyclic AMP production and (ii) elevation of cytosol calcium. The increased sensitivity to PMA of some glucagon effects in hypothyroid rats seems to be related to the latter action.     

Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.     

Absence of insulinotropic glucagon-like peptide-I(7-37) receptors on isolated rat liver hepatocytes.

The effects of glucagon and the glucagon-like peptide GLP-1(7-37) were compared in rat liver hepatocytes. Glucagon elevated cAMP, elevated intracellular free calcium ([Ca2+]i), activated phosphorylase and stimulated gluconeogenesis, whereas GLP-1(7-37) was without effect on any of these parameters. GLP-1(7-37) did not block any of the actions of glucagon. The glucagon analog, des His1[Glu9] glucagon amide, was a partial agonist in liver, but also was an effective antagonist of glucagon actions in liver but not those of GLP-1(7-37) in islet B cells. It was concluded that in the rat, GLP-1(7-37) is a potent insulin secretagogue [1] but is without effect on liver.     

Effects of [1-Nα-trinitrophenylhistidine, 12-homoarginine]glucagon on cyclic AMP levels and free fatty acid release in isolated rat adipocytes

[1-N^α-Trinitrophenylhistidine, 12-homoarginine]glucagon (THG) stimulated, in a concentration-dependent fashion, lipolysis (2-fold) and cyclic AMP accumulation (50% over basal) in isolated rat adipocytes, but was much less effective than glucagon, which stimulated lipolysis 4-fold and cyclic AMP accumulation 10–15-fold. THG displaced to the right the concentration-response curves for glucagon and diminished in a concentration-dependent fashion the effects of a fixed concentration of glucagon. The data indicate that THG is a mixed agonist-antagonist (partial agonist) in isolated rat fat cells.     

Dominant role of glucagon in the initial induction of phosphoenolpyruvate carboxykinase mRNA in cultured hepatocytes from fetal rats.

The injection of streptozotocin to 18-day-old rat fetuses induced, 2 days later, a 50% fall in plasma insulin and a twofold increase in plasma glucagon concentrations and liver cAMP levels. Phosphoenolpyruvate carboxykinase mRNA that were undetectable in the fetal rat liver, accumulated 48 h after streptozotocin injection, their concentration being 30% of that found in the liver of 1-day-old newborn rats in whom liver phosphoenolpyruvate carboxykinase gene expression is maximal. Physiological concentrations of glucagon (0.7 +/- 0.2 nM) induced, within 2 h, phosphoenolpyruvate carboxykinase mRNA accumulation in cultured hepatocytes from 20-day-old fetuses. The addition of insulin (0.01-100 nM) inhibits, by no more than 30%, the glucagon-induced phosphoenolpyruvate carboxykinase mRNA accumulation. Exposure of fetal hepatocytes to insulin for 24 h did not change the glucagon dose/response curve and did not lead to a more efficient inhibition of the glucagon-induced phosphoenolpyruvate carboxykinase mRNA accumulation, despite a clear stimulatory effect on the rate of lipogenesis. In contrast, when hepatocytes were cultured in the presence of dexamethasone, the glucagon-induced phosphoenolpyruvate carboxykinase mRNA accumulation can be totally inhibited by pharmacological concentrations of insulin (10 nM). From these in-vivo and in-vitro studies, it is concluded that, under physiological conditions, the postnatal rise in plasma glucagon concentration is more important than the fall in the plasma insulin concentration for the primary induction of liver phosphoenolpyruvate carboxykinase gene expression.