Effects of the specific cAMP antagonist, (Rp)-adenosine cyclic 3',5'-phosphorothioate, on the cAMP-dependent protein kinase-induced activity of hepatic glycogen phosphorylase and glycogen synthase

The cAMP-dependent protein kinase-induced effects on phosphorylase and glycogen synthase activities and glucose production were studied in hepatocytes isolated from fed rats in the presence of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS. Incubation of hepatocytes with (Sp)-cAMPS or glucagon, both of which lead to cAMP-dependent protein kinase activation, resulted in a concentration-dependent increase in glycogen phosphorylase activity and a decrease in glycogen synthase activity. Incubation of hepatocytes with the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, in the absence of an agonist, had no significant effect on phosphorylase or glycogen synthase activities. Incubation of hepatocytes with a half-maximally inhibitory concentration of (Rp)-cAMPS shifted the agonist-induced activation curves for phosphorylase and the agonist-induced inhibition curves for glycogen synthase to 5-fold higher concentrations for both (Sp)-cAMPS and glucagon. Phosphorylase activity was very sensitive to the rapid, concentration-dependent inhibition by (Rp)-cAMPS of agonist-induced activation of cAMP-dependent protein kinase. The effects on phosphorylase activity were observable in 30 s and were concentration-dependent with half-maximal inhibition at 10 microM, similar to that observed for cAMP-dependent protein kinase. In contrast, glycogen synthase activity was less sensitive to (Rp)-cAMPS inhibition of agonist-induced activation of cAMP-dependent protein kinase. The effects on glycogen synthase activity lagged behind those on phosphorylase activity and the concentration dependence did not parallel the cAMP-dependent protein kinase effect, but was shifted to higher concentrations of (Rp)-cAMPS with half-maximal inhibition at 60 microM. Glucose (10 to 40 mM) increased the sensitivity of glycogen synthase to (Rp)-cAMPS inhibition of cAMP-dependent protein kinase over a narrow range of agonist concentration, but had no significant effect throughout most of the agonist-induced activation range. Thus, the diastereomers, (Sp)- and (Rp)-cAMPS, influence glycogen metabolism and the glycogenolytic enzymes through their modulation of cAMP-dependent protein kinase levels.     

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.     

A study of the mechanism of glucagon-induced protein phosphorylation in isolated rat hepatocytes using (Sp)-cAMPS and (Rp)-cAMPS, the stimulatory and inhibitory diastereomers of adenosine cyclic 3',5'-phosphorothioate

Maximal doses of glucagon increase the phosphorylation state of 12 cytosolic proteins in isolated hepatocytes from fasted rats (Garrison, J. C., and Wagner, J. D. (1982) J. Biol. Chem. 257, 13135-13143). Incubation of hepatocytes with lower concentrations of glucagon indicates that a hierarchy of substrates exists with the concentration of glucagon required for half-maximal increases in phosphorylation varying 5-15-fold. The proteins whose phosphorylation state is most sensitive to low concentrations of glucagon are pyruvate kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, both of which play key roles in the regulation of gluconeogenesis. Treatment of hepatocytes with (Sp)-cAMPS, the stimulatory diastereomer of adenosine cyclic 3',5'-phosphorothioate, mimics the response seen with glucagon. When hepatocytes are pretreated with the cAMP antagonist, (Rp)-cAMPS, the phosphorylation response is abolished at low concentrations of glucagon, and the dose of glucagon required for half-maximal stimulation of phosphorylation is increased 5-10-fold. The (Sp)-cAMPS-stimulated increases in phosphorylation state are also blunted by (Rp)-cAMPS. These results provide direct pharmacological evidence for the activation of the cAMP-dependent protein kinase in response to glucagon in the intact cell. Although low doses of glucagon appear to stimulate protein phosphorylation via the cAMP-dependent protein kinase, high doses of glucagon also cause a small increase in the concentration of free intracellular Ca2+ in hepatocytes. The glucagon-stimulated increases in the level of Ca2+ can be mimicked by (Sp)-cAMPS and inhibited by pretreatment with (Rp)-cAMPS. These results suggest that glucagon can elevate intracellular Ca2+ via cAMP and the cAMP-dependent protein kinase.     

Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

Regulation of glycogen synthesis in rat-hepatocyte cultures by glucose, insulin and glucocorticoids.

The changes in glycogen content and in its rate of synthesis in two-day-old primary cultures of rat hepatocytes were assessed under various conditions. Hepatocytes cultivated in serum-free and hormone-free medium switch from glycogen degradation to glycogen deposition at 10.3 mM glucose. After pretreatment of the cells with glucocorticoids this threshold was reduced, in the absence or presence of insulin, to 5.4 or 1.2 mM glucose, respectively. The rate of glycogen synthesis in the presence of 10 mM glucose was amplified from 5 nmol x h-1 x mg protein-1 to 20 nmol glucose x h-1 x mg protein-1 after pretreatment with triamcinolone. Glucagon pretreatment also significantly increased the subsequent glycogen synthesis rate. Insulin addition accelerated glycogen synthesis about twofold regardless of the pretreatment. The dose-response relationship between insulin concentration and glycogen synthesis rate showed half-maximal effect at 0.62 +/- 0.22 nM (mean +/- S.D.) insulin. Pretreatment of hepatocytes with glucocorticoids, glucagon, insulin or combinations of these hormones did not significantly change the concentration which gives the half-maximal effect.     

Short-term stimulation of net glycogen production by insulin in rat hepatocytes

Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.     

Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon. 1. The mRNA of PEPCK was induced maximally 2-3 h after addition of 10 nM glucagon, as detected by Northern-blot analysis after hybridization with a biotinylated antisense RNA of PEPCK. 2. The synthesis rate of PEPCK increased maximally 2-3 h after application of glucagon as revealed by pansorbin-linked immunoprecipitation of [35S]methionine-labelled PEPCK. 3. The enzyme amount and activity was maximally induced 4 h after glucagon application. 4. The mRNA of PEPCK was half-maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half-maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin. The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon-dependent increase in mRNA and enzyme-synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1-1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range, with glucagon being the dominant hormone.     

Inhibition of hepatic gluconeogenesis by the Rp-diastereomer of adenosine cyclic 3'',5''-phosphorothioate.

The specific intracellular cyclic AMP-dependent protein kinase antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), inhibited both basal and cyclic AMP-agonist-induced rates of gluconeogenesis in hepatocytes isolated from fasted rats. Incubation of the cells in the presence of pyruvate and lactate and either the Sp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS) or glucagon produced a concentration-dependent increase in the rate of gluconeogenic glucose production which was shifted to higher concentrations of Sp-cAMPS or glucagon in the presence of Rp-cAMPS. Incubation of the cells with Rp-cAMPS in the absence of agonist produced no increase in the rate of glucose production and, in most cases, 100 microM-Rp-cAMPS resulted in 14-20% decrease in the substrate-stimulated rate of glucose production. Sp-cAMPS-induced gluconeogenesis was inhibited half-maximally at 1 microM-Rp-cAMPS and glucagon-induced gluconeogenesis was inhibited half-maximally at 12 microM-Rp-cAMPS. Approx. 10-15% of the inhibition of gluconeogenesis observed in the presence of Rp-cAMPS was due to conversion of glucose 6-phosphate to liver glycogen, consistent with Rp-cAMPS-induced reactivation of glycogen synthase. The remaining 85-90% inhibition of gluconeogenic glucose production resulted from the action of Rp-cAMPS on the cyclic AMP-sensitive enzymes controlling the rate of gluconeogenesis.     

Role of cortisol on the glycogenolytic effect of glucagon and on the glycogenic response to insulin in fetal hepatocyte culture.

The effects of insulin and glucagon on glycogen metabolism were studied in cultured fetal hepatocytes transplanted from 15-day-old fetuses. The effects of these hormones were examined just after transplantation, when the cells contained only minute amounts of glycogen, and during the 3 to 4 day culture period, when the hepatocytes were exposed to 10 muM cortisol and actively accumulated glycogen. At all stages of the culture, glucagon addition (10 nM) was followed by a rapid depletion of labeled glycogen, previously synthesized during a pulse labeling with [14C]glucose: this effect was mimicked by N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) (0.3 to 1 nM). Such a glycogenolytic effect of glucagon was observed even 6 hours after transplantation, i.e. at a time when cortisol was not present. In addition, glucagon clearly induced cyclic adenosine 3':5'-monosphosphate (cyclic AMP) accumulation in cells grown for 18 hours in the absence of cortisol. With cells grown for 3 days in the presence of cortisol, glucagon-dependent glycogenolysis was also obtained when cortisol was removed from the medium 20 hours before hormone addition. Thus the presence of cortisol is not necessary either to maintain a response to glucagon or for the onset of the glycogenolytic effect of glucagon. Insulin addition (10 nM) stimulated [14C]glucose incorporation into glycogen at all stages of the culture when grown in the presence of cortisol; no glycogenic response to insulin was observed 6 hours after transplantation where cortisol was not previously introduced. In addition, if the hepatocytes were grown in the presence of insulin alone (i.e. in the absence of cortisol) no significant storage of glycogen occurred. Maximal storage (or labeling) of glycogen was observed when hepatocytes were grown in the presence of both cortisol and insulin. The presence of cortisol was therefore necessary for the expression of the glycogenic effect of insulin. These data show that marked difference exist between the onset of developmental responses towards glucagon and insulin. The glucagon-dependent regulatory pathway should be present very early in fetal development and should not depend on cortisol. On the contrary, the onset of the insulin-dependent regulatory pathway seems to be induced during culture, and it is likely that this is caused by cortisol.     

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.     

Effects of epidermal growth factor (urogastrone) on gluconeogenesis, glucose oxidation, and glycogen synthesis in isolated rat hepatocytes

Using isolated rat hepatocytes, we studied the effect of epidermal growth factor (urogastrone) (EGF-URO) on the incorporation of [3-14C]pyruvate into glucose and glycogen, on the incorporation of [U-14C]glucose into glycogen, and on the oxidation of [U-14C]glucose to 14CO2. The effects of EGF-URO were compared with those of glucagon and insulin. EGF-URO, with an EC50 of 0.2 nM, enhanced by 34% (maximal stimulation) the conversion of [3-14C]pyruvate into glucose; no effect was observed on the oxidation of glucose to CO2 and on the incorporation of either pyruvate or glucose into glycogen. The effect of EGF-URO on pyruvate conversion to glucose was observed only when hepatocytes were preincubated with EGF-URO for 40 min prior to the addition of substrate. Glucagon (10 nM) increased the incorporation of [3-14C]pyruvate into glucose (44% above control); however, unlike EGF-URO, glucagon stimulated gluconeogenesis better without than with a preincubation period. Neither insulin nor EGF-URO (both 10 nM) affected the incorporation of [U-14C]glucose into glycogen during a 20-min incubation period. However, at longer time periods of incubation with the substrate (60 instead 20 min), insulin (but not EGF-URO) increased the incorporation of [14C]glucose into glycogen; EGF-URO counteracted this stimulatory effect of insulin. In contrast with previous data, our work indicates that EGF-URO can, under certain conditions, counteract the effects of insulin and, like glucagon, promote gluconeogenesis in isolated rat hepatocytes.     

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.     

Epidermal growth factor (urogastrone)-stimulated gluconeogenesis in isolated mouse hepatocytes

In freshly isolated mouse hepatocytes obtained from fasted animals, we have studied the receptors for epidermal growth factor urogastrone (EGF-URO) in terms of the electrophoretic profile, ligand affinity, and numbers of EGF-URO receptors present on the cells, and also in terms of the ability of EGF-URO to stimulate gluconeogenesis, as reflected by the increased incorporation of [3-14C]pyruvate into glucose. The effects of EGF-URO were compared with those of glucagon. Ligand-binding studies revealed that the mouse hepatocytes possess an unusually high number of EGF-URO receptors (about 3 X 10(6) binding sites/cell), with a ligand dissociation constant of 4.4 nM. The binding of EGF-URO by mouse hepatocytes was more than 10-fold higher than the previously measured binding of EGF-URO by rat hepatocytes. Crosslink-labeling studies, coupled with gel electrophoretic analysis, demonstrated the presence of intact EGF-URO receptors, although some receptor processing had occurred during the isolation procedure. EGF-URO was able to stimulate the incorporation of 3-14C-labeled pyruvate into glucose; glucagon was unable to do so. In contrast, in rat hepatocytes isolated and assayed under identical conditions, glucagon (10 nM) caused a marked (250%) stimulation of the incorporation of pyruvate into glucose. Maximally, EGF-URO caused a 34% increase in the incorporation of [3-14C]pyruvate into glucose; a half-maximal effect was observed at a concentration of 2.5 nM EGF-URO. The stimulatory effect of EGF-URO was not dependent on the concentration of pyruvate, lactate, glucose, or calcium in the incubation medium. Although raising the concentration of pyruvate in the incubation medium increased the incorporation of [3-14C]pyruvate into glycogen, EGF-URO did not cause any change in the incorporation of radioactivity into glycogen. Overall, our data point to marked differences between rat and mouse liver preparations, in terms of the hormonal regulation of glucose metabolism, and our work documents a potential role for the remarkably high number of mouse hepatocyte EGF-URO receptors in terms of the modulation of gluconeogenesis in the mouse.     

Epidermal growth factor mimics insulin effects in rat hepatocytes.

Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells

Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.     

Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.     

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.     

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.