Glucagon-stimulated Cyclic AMP Accumulation Research Articles

Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

Diadenosine polyphosphates and the control of cyclic AMP concentrations in isolated rat liver cells

Extracellular diadenosine polyphosphates (Ap n A), through their interactions with appropriate P 2 receptors, influence a diverse range of intracellular activities. In particular, Ap 4A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap 4A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap 4A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides.

Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.

Analysis of the adenylate cyclase signalling system, and alterations induced by culture with insulin, in a novel SV40-DNA-immortalized hepatocyte cell line (P9 cells)

An immortalized cell line, called P9, was derived from hepatocytes by transfection with SV40 DNA. These cells expressed enzyme activities characteristic of hepatocytes, namely glucose-6-phosphatase, glycogen phosphorylase, bilirubin glucuronyltransferase and both glucagon- and prostaglandin E1 (PGE1)-stimulated adenylate cyclase activities, albeit at decreased levels compared with native hepatocytes. Levels of the G-protein subunits alpha-Gi-2, alpha-Gi-3, G beta and the 'long' form of alpha-G2 (45 kDa) were approximately 4-fold higher relative to native hepatocytes, whereas those of the 'short' form of alpha-G2 (42 kDa) were lower by approximately 40%. Associated with this were marked alterations in the guanine nucleotide regulation of adenylate cyclase. Receptor-mediated stimulation, achieved by either PGE1 or glucagon, was apparent in P9 cells, although the latter was only evident upon amplification with forskolin. Glucagon-stimulated cyclic AMP accumulation in P9 cells did not exhibit desensitization, as in hepatocytes, nor was the phosphorylation of alpha-Gi-2 evident. Culture of P9 cells with insulin led to a dose-dependent decrease (EC50 0.2 +/- 0.1 nM) in the ability of PGE1 to stimulate adenylate cyclase activity, with the maximum effect attained after approximately 6 h. A comparable attenuation of stimulation was seen for glucagon- and guanine-nucleotide-stimulated adenylate cyclase activities. In cells cultured with insulin, lower levels of GTP were required to stimulate adenylate cyclase, ADP-ribosylation of the 45 kDa form of alpha-Gs with cholera toxin was attenuated, and the expression of both alpha Gi-2 and alpha-Gi-3 was increased. It is suggested that the expression of alpha-Gi-2 and alpha-Gi-3 may be directly regulated by the action of insulin in hepatocytes and P9 cells.

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 dibutyril cyclic AMP (But 2-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 [Ca 2+] induced by glucagon. THG or But 2-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 alter 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 rat seems to be related to the latter action.

Decreased glucagon-stimulated cyclic AMP production by isolated liver cells of rats with type 2 diabetes

This study was undertaken to investigate the effect of experimental type 2 diabetes in the rat on the insulin and glucagon receptors and on the early steps of glucagon action. The binding of insulin and glucagon and the glucagon-stimulated cyclic AMP accumulation in the presence of a phosphodiesterase inhibitor (IBMX, 0.1 mmoles/l) were studied in liver cells isolated from 7–9-month-old rats with chronic type 2 diabetes and from control rats. No significant change was observed in [ 125I]insulin binding and [ 125I]glucagon binding of diabetic liver cells as compared to controls. Scatchard analysis of the competition experiments indicated that affinity and number of insulin and glucagon receptors were not significantly changed in the liver cells of diabetic rats. The basal cyclic AMP level was significantly lower in the diabetic hepatocytes (2.3 ± 0.9 pmoles/10 6 cells) than in the controls (4.0 ± 0.6 pmoles/10 6 cells). Cyclic AMP response to physiological concentrations of glucagon (0.1–1 nmoles/l) was about 2 times lower in the diabetic hepatocytes than in the controls. Furthermore, the basal liver membrane adenylate cyclase activity and the fluoride-activatable adenylate cyclase activity were about 2 times lower in the diabetics as compared to control rats, while the liver cyclic AMP and cyclic GMP phosphodiesterase activities were unchanged. The ability of glucagon to stimulate liver membrane adenylate cyclase over a 10 −12-10 −6 M concentration range was decreased in diabetic rats. Taken together, these data are consistent with the thesis that the impairment of the liver cyclic AMP response to glucagon in rats with type 2 diabetes is caused by a decrease in the amount of adenylate cyclase in the liver plasma membranes.

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production.

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.

The effect of streptozotocin-induced diabetes on the early steps of glucagon action in isolated rat liver cells.

This study was undertaken to investigate the effect of experimental diabetes on the early steps of glucagon action. The binding of glucagon and glucagon-stimulated cyclic AMP accumulation in the presence of a potent phosphodiesterase inhibitor (IBMX, 0.1 mmol/l) were studied in liver cells isolated from control and streptozotocin-induced (65 mg/kg) diabetic rats. Comparative studies of insulin binding indicated that hepatocytes of diabetic rats bound twice as much 125I-insulin (10.8 +/- 2.0%) as those of control rats (5.7 +/- 1.3%). Scatchard analysis and the competition plots of the data suggested that this was due to an increased number of receptors rather a change in their affinity. No significant change was observed in 125I-glucagon binding of diabetic liver cells (5.8 +/- 0.5%( as compared to controls (6.8 +/- 0.4%). The number of molecules of glucagon bound to high and low affinity binding sites of control liver cells was (51 +/- 2) X 10(3) and (1300 +/- 134) X 10(3) sites/cell, respectively. The corresponding numbers in streptozotocin-treated rats were (45 +/- 5) X 10(3) and (1000 +/- 167( X 10(3) sites/cell, respectively. Cyclic AMP response to concentration of glucagon below 1 nmol/l was significantly lower in diabetics than in normals: for 0.3 nmol/l and 0.6 nmol/l of glucagon, cyclic AMP production was 48 +/- 7 pmol/10(6) cells and 78 +/- 8 pmol/10(6) cells in diabetics, as compared to 72 +/- 9 and 110 +/- 9 pmol/10(6) cells in normals. At concentrations of glucagon that are maximally efficient (greater than or equal to 7 nmol/l) cyclic AMP production was higher in diabetic (202 +/- 20 pmol/10(6) cells) than in normal rats (156 +/- 7 pmol/10(6) cells). Thus, diabetes seems to increase the quantity of adenylate cyclase and decrease its affinity for glucagon. Those changes are not related to a modification of the glucagon binding sites and are associated to an increase of insulin receptors.

Biological evaluation of the third pancreatic hormone (APP): Hepatocyte and adipocyte effects

The effects of a third pancreatic hormone, avian pancreatic polypeptide (APP), on metabolism in isolated chicken adipocytes and hepatocytes were studied. APP inhibits glucagon-stimulated lipolysis within the chicken adipocyte by as much as 45%. Additionally, APP causes a marked reduction in glucagon-stimulated cyclic AMP accumulation, suggesting that APP in some way interferes with the glucagon-sensitive adenylate cyclase system. APP binds specifically to the chicken adipocyte; two orders of receptor sites were demonstrated: a high affinity ( K d = 5.6 × 10 −9), low capacity (6.75 × 10 −20 mol/cell) site and a low affinity ( K d = 8.3 × 10 −7), high capacity (3.18 × 10 −18 mol/cell) site. APP does not alter the rate of glycogenolysis or triglyceride synthesis at the chicken hepatocyte; neither does it stimulate glucose utilization by the chicken adipocyte.

Somatostatin Inhibition of Glucagon-Stimulated Adenosine 3'-5'-Monophosphate Accumulation in Isolated Hepatocytes

SummaryThe cyclic form of somatotropin release inhibitor factor over a concentration range from 0.50 to 8.0 μg/ml was found to inhibit glucagon-stimulated cyclic AMP accumulation in isolated rat hepato-cytes.The authors are grateful to Nancy Bridgforth, Ann Miller, Sue Wills, and Lucinda Carr for skilled technical assistance and to Deborah Barr and Carol Grimme for typing of the manuscript. The authors are also very grateful to Dr. R. Deghenghi of Ayerst Laboratories, Montreal, Quebec, Canada, for the supply of cyclic form somatostatin, Lot No. Ay-24,910.

Effect of Feeding and Fasting on the Early Steps of Glucagon Action in Isolated Rat Liver Cells

The effect of the nutritional state (fasted or fed) on the binding of glucagon and on the glucagon-stimulated cyclic AMP accumulation in the presence of theophylline was studied in isolated rat liver cells. The binding of glucagon was higher in cells from fed than in those from fasted rats at each concentration of glucagon tested between 0.1 and 36 nM. The specific binding of the hormone was about 2-fold higher in cells from fed than from fasted rats. At concentrations of glucagon between 0.1 and 2.2 nM, the accumulation of cyclic AMP in the presence of 1 mM theophylline was higher in the cells from fed rats. Furthermore, 4 times as much glucagon was required to elicit half-maximal cyclic AMP accumulation in the cells of fasted (1.47 nM) than in the cells of fed (0.35 nM) rats. These data suggest that, in isolated rat liver cells, both glucagon binding to receptor sites and glucagon-stimulated cyclic AMP levels in the presence of theophylline can be affected by the nutritional status of the animal.