Transcriptional activation of the rat glucagon gene by the cyclic AMP-responsive element in pancreatic islet cells.

The 5'-flanking region of the rat glucagon gene contains, from nucleotides -291 to -298, a sequence (TGA CGTCA) which mediates cyclic AMP (cAMP) responsiveness in several genes (cAMP-responsive element [CRE]). However, because of nonpermissive bases surrounding the CRE octamer, the glucagon CRE does not confer cAMP responsiveness to an inert heterologous promoter in placental JEG cells that do not express the glucagon gene. This report describes transient transfection experiments with glucagon-reporter fusion genes that show that glucagon gene expression is activated by cAMP-dependent protein kinase A in a glucagon-expressing pancreatic islet cell line. This activation is mediated through the glucagon CRE.     

Inhibition of the glycogenolytic effects of alpha-adrenergic stimulation and glucagon by cobalt ions in perfused rat liver

Cobalt ions (2 mM) inhibited the glycogenolysis induced by phenylephrine and glucagon in perfused rat liver. Cobalt ions also inhibited 45Ca++ efflux from prelabelled livers induced by phenylephrine and glucagon. In addition, they inhibited the rise in tissue levels of cyclic AMP caused by glucagon, but did not inhibit the stimulation of 45Ca++ efflux or glycogenolysis by cyclic AMP or dibutyryl cyclic AMP. The specific binding of glucagon and alpha-agonist to hepatocytes was not inhibited by cobalt ions. These data suggest that cobalt ions, presumably through their high affinity for calcium binding sites on membranes inhibit the stimulation of glycogenolysis by phenylephrine and glucagon in distinct ways; one by inhibiting calcium mobilization and the other by inhibiting cyclic AMP production. Therefore, it is conceivable that membrane-bound calcium plays an important role in stimulating Ca++ mobilization by phenylephrine, and cyclic AMP production by glucagon.     

Nalpha-trinitrophenyl glucagon: an inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis.

Nalpha-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AM-independent process.     

Regulation of coenzyme A biosynthesis by glucagon and glucocorticoid in adult rat liver parenchymal cells

We studied the effects of glucagon, dibutyryl cyclic AMP and dexamethasone on the rate of [(14)C]pantothenate conversion to CoA in adult rat liver parenchymal cells in primary culture. The presence of 30nm-glucagon increased the rate by about 1.5-fold relative to control cultures (range 1.4-2.3) and 2.4-fold relative to cultures containing 1-3m-i.u. of insulin/ml. The half-maximal effect was obtained at 3nm-glucagon. Dibutyryl cyclic AMP plus theophylline also enhanced the rate by about 1.5-fold. Dexamethasone acted synergistically with glucagon; glucagon at 0.3nm had no effect when added alone, but resulted in a 1.7-fold enhancement when added in the presence of dexamethasone (maximum effect at 50nm). The 1.4-fold enhancement caused by the addition of saturating glucagon concentrations was increased to a 3-fold overall enhancement by the addition of dexamethasone. However, dexamethasone added alone over the range 5nm to 5mum had no effect on the rate of [(14)C]pantothenate conversion to CoA. The stimulatory effect of dibutyryl cyclic AMP plus theophylline was also enhanced by the addition of dexamethasone. Changes in intracellular pantothenate concentration or radioactivity could not account for the stimulatory effects of glucagon, dibutyryl cyclic AMP or dexamethasone. Addition of 18mum-cycloheximide, an inhibitor of protein synthesis, decreased the rate of incorporation of [(14)C]pantothenate into CoA and the enhancement of this rate by glucagon and dibutyryl cyclic AMP plus theophylline in a reversible manner. These results demonstrate an influence of glucagon, dibutyryl cyclic AMP and glucocorticoids on the intracellular mechanism regulating total CoA concentrations in the liver.     

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.     

Nα-Trinitrophenyl glucagon An inhibitor of glucagon-stimulated cyclic AMP production and its effects on glycogenolysis

N^α-Trinitrophenyl glucagon was prepared by reaction with trinitrobenzene sulfonic acid and purified by ion-exchange chromatography. This derivative has essentially no ability to activate adenylate cyclase from rat liver nor to increase the levels of cyclic AMP in isolated hepatocytes nor to stimulate protein kinase activity. This derivative also can act as a glucagon antagonist with regard to cyclic AMP production and can decrease the degree of stimulation of adenylate cyclase caused by glucagon, as well as lowering the glucagon-stimulated elevation of cyclic AMP levels in intact hepatocytes. Nevertheless, this derivative is capable of activating glycogenolysis.in isolated hepatocytes and in augmenting the effect of glucagon on glycogenolysis. This metabolic effect of the glucagon derivative thus appears to occur independent of changes in cyclic AMP levels. These results suggest that glucagon can also activate glycogenolysis by a cyclic AMP-independent process.     

Evidence of cyclic AMP-independent action of glucagon on calcium mobilization in rat hepatocytes

Glucagon increases the cytoplasmic free calcium concentration as measured by aequorin bioluminescence. It has been proposed by Wakelam et al. (Nature 323 (1986) 68-71) that low concentrations of glucagon mobilize calcium from an intracellular pool by causing polyphosphoinositide breakdown. To identify whether cyclic AMP mediates changes in the cytoplasmic free calcium concentration ([Ca2+]c) induced by glucagon, the effects of forskolin and exogenous cyclic AMP on [Ca2+]c were compared with that of glucagon in aequorin-loaded hepatocytes. Although the magnitudes of the [Ca2+]c responses to 250 microM forskolin and 1 mM 8-bromo cyclic AMP were identical to that of 5 nM glucagon, these two agents induced a more prolonged elevation of [Ca2+]c. Glucagon-induced elevation of [Ca2+]c was accompanied by a smaller increase in cyclic AMP than that induced by forskolin. When the cyclic AMP response to glucagon was potentiated by an inhibitor of phosphodiesterase, 3-isobutyl-1-methylxanthine, the glucagon-induced increase in [Ca2+]c was not affected. Conversely, when the cyclic AMP response to glucagon was reduced by pretreatment of the cells with angiotensin II, glucagon-induced changes in [Ca2+]c were rather enhanced. Furthermore, vasopressin potentiated glucagon-induced changes in [Ca2+]c despite the reduction of the cyclic AMP response to glucagon. In the presence of 1 microM extracellular calcium, angiotensin II did not enhance glucagon-induced changes in [Ca2+]c. These results suggest that at least part of the action of 5 nM glucagon on calcium mobilization is independent of cyclic AMP.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Stimulation by glucagon and adenosine-3',5'-cyclic monophosphate of protein degradation in Reuber H35 hepatoma monolayers

Protein degradation in Reuber H35 hepatoma monolayers was measured as release of radioactive trichloroacetic acid-soluble material from intracellular protein labelled with [3H]leucine for 16 hr followed by 3-hr chase period. Proteolysis in this system was stimulated by physiological concentration of glucagon reaching a maximum at 10(-7) M with an increase of 30%. Dibutyryl cyclic AMP also had a stimulatory effect. When both glucagon and dibutyryl cyclic AMP were present at optimal concentrations, their effects were not additive suggesting that glucagon may act via the formation of cyclic AMP. In the presence of protein synthesis inhibitor, cycloheximide or puromycin, proteolysis remained responsive to glucagon. Glucagon counteracted the inhibitory effect of insulin on proteolysis.     

Protein degradation in hepatocyte monolayers. Effects of glucagon, adenosine 3'':5''-cyclic monophosphate and insulin.

1. Hepatocytes were isolated by collagenase perfusion of livers from fed rats and established in stationary monolayer culture. 2. Degradation of intracellular protein was measured in these monolayers after labelling for 16h with [3H]leucine followed by a 3h chase period in medium containing 2mM-leucine. 3. Proteolysis in this system was stimulated by physiological concentrations of glucagon and also by added dibutyryl cyclic AMP. The effects of these two agents were not additive, which is consistent with the view that they act by the same mechanism. 4. A close correlation was found between intracellular cyclic AMP concentrations generated by glucagon and the degree of stimulation of proteolysis elicited by the hormone. 5. Insulin reduced glucagon-stimulated proteolysis, but not glucagon-elevated intracellular cyclic AMP concentrations. 6. The continual presence of either insulin or glucagon was necessary for the full expression of their effects on proteolysis. 7. In the presence of cycloheximide, proteolysis was normally responsive to glucagon but not to insulin. In contrast, proteolysis was not responsive to either hormone in the presence of ammonia, an agent that blocks the final lysosomal step of protein breakdown. 8. We propose that in hepatocyte monolayers glucagon may act via cyclic AMP to increase cellular autophagy and thus increase proteolysis, whereas insulin inhibits these processes independently of cyclic AMP.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E₁ or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue.In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.