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.     

Stimulation of glycogenolysis by epinephrine and glucagon and its inhibition by insulin in isolated rat liver hepatocytes

Rat liver hepatocytes were isolated by collagenase $\text{in vitro}$ perfusion technique and the effect of epinephrine, glucagon and insulin on glycogenolysis was studied. Both glucagon and epinephrine at the concentration of 10^?6M, stimulated gluconeogenesis by 80–100%. Addition of insulin (33 μUnits/ml) completely abolished the epinephrine-stimulated glycogenolysis whereas only 50% inhibition was observed with insulin in glucagon stimulated glycogenolysis. This stimulation was observed within 2–5 min after the addition of the hormones. These results suggest that hepatocytes isolated with low concentrations of collagenase retain glucagon, epinephrine and insulin receptor sites.     

Studies of glucagon resistance in large rat adipocytes: 125I-labeled glucagon binding and lipolytic capacity

This study is concerned with potential modifications of large fat cells from adult rats (400-450 g) that make them resistant to stimulation by glucagon. The lipolytic capacity and (125)I-labeled glucagon-binding capability of these cells were compared with these properties of small glucagon-sensitive cells from young rats (130-160 g). As determined by maximal stimulation with theophylline, dibutyryl cAMP, or epinephrine, the lipolytic capacity of large cells was not markedly different from small cells, which suggests that an alteration contributing to glucagon insensitivity is not present in the enzymes involved with hormone-mediated lipolysis. Glucagon-binding studies did indicate a difference between the two cell types. Both large cells and particulate fractions from large cells bound less (125)I-labeled glucagon than small cells or small-cell particles. That diminished binding is not a consequence of glucagon degradation is indicated by the similar amounts of (125)I-labeled glucagon degraded by both cell types. The decrease in (125)I-labeled glucagon binding was not as marked as the decrease in lipolytic response to glucagon stimulation. This lack of correlation and the relationship between elevated phosphodiesterase levels and glucagon insensitivity described in the accompanying report suggest that diminished binding explains only in part the marked resistance to glucagon found in large cells.     

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.     

Studies of the interaction between glucagon and alpha-adrenergic agonists in the control of hepatic glucose output

alpha-Adrenergic stimulation of hepatocytes prevented, in a dose-dependent manner, the stimulation of [U-14C]lactate conversion to [14C]glucose by glucagon and exogenously added cAMP and Bt2cAMP. The inhibition was referable to an interaction with adrenergic receptors which resulted in a small decrease in hepatic cAMP levels. Low concentrations of epinephrine (10 nM) were able to inhibit phosphorylase activation and glucose output elicited by low doses of glucagon (5 X 10(-11) M to 2 X 10(-10) M). The ability of epinephrine (acting via alpha 1-adrenergic receptors), vasopressin, and angiotensin II to elicit calcium efflux was inhibited by glucagon, suggesting that intracellular redistributions of Ca2+ are importantly involved in the gluconeogenic process. It is proposed that vasopressin, angiotensin II, and catecholamines, acting primarily via alpha 1-adrenergic receptors, are responsible for inhibition of glucagon mediated stimulation of gluconeogenesis by altering subcellular calcium redistribution and decreasing cAMP levels.     

Gluconeogenesis in rabbit liver. III. The influences of glucagon, epinephrine, alpha- and beta-adrenergic agents on gluconeogenesis in isolated hepatocytes

1. Gluconeogenesis from various substrates has been demonstrated in hepatocytes from 48 h fasted rabbits. Maximum rates of gluconeogenesis (expressed as mumol glucose formed/30 min per 10(8) cells) are: D-fructose, 9.86; dihydroxyacetone, 5.28; L-lactate, 5.26; L-lactate/pyruvate, 3.83; pyruvate, 3.32; glycerol, 2.92; L-alanine, 2.24. 2. Gluconeogenesis from L-lactate is enhanced 1.3--1.5-fold over control values by glucagon, L-epinephrine, L-norepinephrine, dibutyryl cyclic AMP, L-phenylephrine and L-isoproterenol. Glucogenesis from both dihydroxyacetone and D-fructose is stimulated 1.7--2.0-fold of control values by glucagon, epinephrine and dibutyryl cyclic AMP. 3. Gluconeogenesis from lactate is enhanced by both alpha- and beta-adrenergic stimulations based on findings with alpha- and beta-agonists and antagonists. 4. Enhancement of gluconeogenesis by epinephrine and norepinephrine is apparently due to both alpha- and beta-adrenergic effects, as either propranolol or phentolamine partially inhibits such enhancement. The consistently more pronounced inhibition produced by propranolol implies that stimulation of glucose formation by catecholamines is more strongly beta-adrenergic related. Epinephrine-induced glycogenolysis in rabbit hepatocytes is severely inhibited by propranolol but insensitive to phentolamine, suggesting that glycogen breakdown is solely beta-adrenergic related. These observations contrast with those of others that stimulation of both gluconeogenesis and glycogenolysis by catecholamines while sensitive to both alpha- and beta-adrenergic stimulation in rats, at least young rats, is primarily alpha-adrenergic mediated, especially in adult rats.     

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.     

Adrenergic regulation of pyruvate kinase and gluconeogenesis in hepatocytes from phosphorylase kinase-deficient (gsdgsd) rats

Hepatocytes were prepared from a strain of rats deficient in hepatic phosphorylase $\text{b}$ kinase and were used to assess the role of this enzyme in the adrenergic regulation of pyruvate kinase and gluconeogenesis. Epinephrine (10 μM) stimulated glucose output and gluconeogenesis from 1.8 mM lactate but did not significantly affect the concentration of hepatocyte glycogen. In addition epinephrine treatment led to an inhibition of pyruvate kinase. The stimulation of gluconeogenesis and the inhibition of pyruvate kinase by epinephrine were blocked by both α- and β-antagonists: similar effects with epinephrine were observed in cells from control animals. It is concluded that mechanisms for the adrenergic regulation of pyruvate kinase and gluconeogenesis are similar in hepatocytes from both phosphorylase kinase-deficient and normal rats.     

"Hormone-like" effect of adenosine and inosine on gluconeogenesis from lactate in isolated hepatocytes

In isolated rat hepatocytes adenosine and inosine showed a dose-dependent increase in the rate of glucose synthesis from lactate with a Ka of 7.5 x 10(-8) and 9 x 10(-8) M, respectively. Absence of this action was recorded with: IMP, xanthosine, adenine, hypoxanthine, and uric acid. A reciprocal inhibition of individual gluconeogenic stimulation was found in cells incubated with glucagon or epinephrine and adenosine, but not with inosine. 5'-(N-ethyl) carboxamido adenosine was more potent than adenosine, whereas N6-(L-2-phenylisopropyl)-adenosine antagonized the stimulation of gluconeogenesis by adenosine. Neither of the analogs used modified the stimulatory role of inosine on the studied pathway. Adenosine and inosine may be involved in the short term regulation of gluconeogenesis.     

Selective loss of adipose cell responsiveness to glucagon with growth in the rat

In isolated fat cells, the same maximal rate of glycerol production can be induced by epinephrine or ACTH, alone or in combination with each other or with glucagon. With fat cells from rats weighing 150-175 g, the maximal rate of lipolysis attained with glucagon was 75-80% of that produced by epinephrine or ACTH, and with increasing size of the donor rat, the magnitude of the effect of glucagon relative to that of the other hormones declined markedly. In particulate preparations from fat cells of rats weighing 100-125 g, the maximal effect of glucagon on adenyl cyclase activity was about 60% of that of epinephrine, and was significantly less (30%) in preparations from 350-400 g rats. These data are consistent with the hypothesis that with growth of the rat there is a selective decline in the number of glucagon receptors relative to those for epinephrine or ACTH in the fat cell membrane.     

Effect of epinephrine and other lipolytic agents on intracellular lipolysis and lipoprotein lipase activity in 3T3-L1 adipocytes

3T3-L1 adipocytes were used to test the hypothesis that hormone-sensitive lipolysis and lipoprotein lipase activity might be regulated in a reciprocal manner. Intracellular lipolysis was stimulated by catecholamine, dibutyryl cAMP, and ACTH, but not by glucagon. The effects of epinephrine on lipolysis were blocked by the beta-antagonist propanolol but not by the alpha-antagonist phentolamine. Hormone-stimulated lipolysis was not changed by acute (45 min) or chronic (2 days) treatment of the cells with insulin whereas the latter treatment augmented lipoprotein lipase activity about fivefold. Epinephrine did not affect the lipoprotein lipase activity of insulin-stimulated cells. Withdrawal of glucose from the medium decreased lipoprotein lipase activity and the effect of epinephrine on lipolysis. Effects of lipolytic agents on activity of lipoprotein lipase were variable and concentration-dependent. Lipoprotein lipase activity was decreased only by concentrations of epinephrine greater than those inducing maximal intracellular lipolysis, and the decrease in activity occurred about 30 min after the increase in glycerol release. There seems to be no relationship between the level of activity of lipoprotein lipase and the maximal rate of hormone-stimulated lipolysis in 3T3-L1 cells. Unlike in adipose tissue and adipocytes of rats, hormone-stimulated lipolysis and lipoprotein lipase activity in murine 3T3-L1 adipocytes appear to be regulated independently.     

Hormonal regulation of key gluconeogenic enzymes and glucose release in cultured hepatocytes: effects of dexamethasone and gastrointestinal hormones on glucagon action

Hormonal regulation of key gluconeogenic enzymes and glucose release by glucagon, dexamethasone, secretin and somatostatin was evaluated in maintenance cultured rat hepatocytes. (i) Phosphoenolpyruvate (PEP)-carboxykinase activity declined rapidly during the first 24 h in serum- and hormone-free culture with a further slight decay during the following 2 days. Dexamethasone and glucagon independently increased PEP-carboxykinase and acted synergistically when added in combination. Glucose-6-phosphatase activity declining linearly during hormone-free culture was stimulated by glucagon. Dexamethasone itself was without significant effects but completely abolished glucagon action. Fructose-1,6-diphosphatase was maintained at its initial level during the first day under control conditions and declined thereafter. Neither glucagon nor dexamethasone affected total activity or substrate (fructose-1,6-diphosphate) affinity of this enzyme. In short-term experiments on cells cultured under control conditions, protein synthesis-dependent stimulation of PEP-carboxykinase by glucagon and the permissive action of dexamethasone was demonstrated. Glucose-6-phosphatase and fructose-1,6-diphosphatase were not altered by hormones within this period. (ii) Stimulation by glucagon of gluconeogenesis was independent of its action on PEP-carboxykinase. Dexamethasone inhibited glycogenolysis but maintained glucose release at control levels probably by stimulation of gluconeogenesis. When added in combination, the glycogen-preserving action of dexamethasone acutely reduced the glucose release in response to glucagon. Glucagon sensitivity remained unchanged. (iii) The gastrointestinal hormones secretin and somatostatin were ineffective in modulating basal or glucagon-stimulated glucose release and gluconeogenic key enzymes. They are therefore unlikely to play a physiological role in hepatic glucose metabolism.     

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.     

Gluconeogenesis in rat liver parenchymal cells in primary culture: Permissive effect of the glucocorticoids on glucagon stimulation of gluconeogenesis

Primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion procedure and maintained as a monolayer in a serum-free culture medium were used to study glucoeogenesis and the role that the glucocorticoids play in the control of this pathway. These cells carried out gluconeogenesis from three-carbon precursors (alanine and lactate) in response to glucagon and dexamethasone added alone or in combination. Maximum glucose production was observed with cells pretreated for several hours with dexamethasone and glucagon prior to addition of substrate and glucagon (8- to 12-fold increase over basal glucose production). Half-maximum stimulation of gluconeogenesis was seen with 3.6 × 10^?10 M glucagon and 3.6 × 10^?8 M dexamethasone. Maximum stimulation was oberved with 10^?7 M glucagon and 10^?6 M dexamethasone. The length of time of dexamethasone pretreatment was found to be important in demonstrating the effect of glucocorticoids on glucagon-stimulated gluconeogenesis. Treeatment of cells with dexamethasone for 2 hours did not result in an increase in glucose production over identical experimental conditions in the absence of dexamethasone, wherease pretreatment for 5 hours (1.2-fold increase) or 15 hours (1.7-fold increase) did result in an increase in glucose production. The results establish that the adult rat liver parenchymal cells in primary culture are a valid model system to study hepatic gluconeogenesis. In addition, we have established directly that the glucocorticoids amplify the glucagon stimulation of gluconeogenesis.     

The influence of ammonium and calcium lons on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate.Calcium ions (1.3 mm) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca²⁺ and NH₄⁺ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.     

Effect of prostaglandin E1 on hepatic cyclic AMP activity, carbohydrate and lipid metabolism

Prostaglandin E₁ (PGE₁) failed to stimulate rat liver cyclic AMP (cAMP), induce hyperglycemia, glycogenolysis or lipolysis or prevent epinephrine-induced hyperglycemia in isolated perfused rat liver, even though other known glycogenolytic agents (glucagon and epinephrine) activated cAMP in this same system. The data do not support a physiologic role for PGE₁ on hepatic glycogenolysis or lipolysis. Although the effects of PGE₁ on gluconeogenesis, lipogenesis, ureogenesis or amino acid transport in isolated perfused liver were not investigated, if PGE₁ is subsequently found to influence these metabolic parameters, such alterations would probably occur independent of a change in cAMP activity.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes. Heterogeneity of hormone action and of the mitochondrial redox state.

Hepatocytes isolated from the periportal or perivenous zones of livers of fed rats were used to study the long-term (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ketogenesis. Long-term culture with glucagon (100 nM) resulted in a greater increase (P less than 0.01) in gluconeogenesis in periportal than in perivenous cells (93 +/- 16 versus 30 +/- 14 nmol/h per mg of protein; 72% versus 30% increase), but short-term incubation (2 h) with glucagon resulted in similar stimulation in the two cell populations. Rates of ketogenesis (acetoacetate and D-3-hydroxybutyrate production) were not significantly higher in periportal cells cultured without glucagon, compared with perivenous cells. However, after long-term culture with glucagon, the periportal cells had a significantly higher rate of ketogenesis (from either palmitate or octanoate as substrate), but a lower 3-hydroxybutyrate/acetoacetate production ratio, suggesting a more oxidized mitochondrial NADH/NAD+ redox state despite the higher rate of beta-oxidation. Periportal hepatocytes had a higher activity of carnitine palmitoyltransferase but a lower activity of citrate synthase than did perivenous cells. These findings suggest that: (i) glucagon elicits greater long-term stimulation of gluconeogenesis in periportal than in perivenous hepatocytes maintained in culture; (ii) after culture with glucagon, the rates of ketogenesis and the mitochondrial redox state differ in periportal and perivenous hepatocytes.     

Vasopressin and angiotensin II stimulate ureogenesis through increased mitochondrial citrulline production.

Vasopressin, angiotensin II, glucagon and epinephrine (through a cAMP-independent, alpha₁adrenergic mechanism), stimulate ureogenesis in isolated rat hepatocytes. Mitochondria, isolated from hepatocytes which were previously treated with these hormones, displayed an enhanced rate of citrulline synthesis in the presence of NH₄Cl as the nitrogen source. When mitochondria were incubated with glutamine as the nitrogen source, only those mitochondria isolated from hepatocytes previously treated with epinephrine or glucagon displayed an enhanced capacity to synthesize citrulline.When cells were incubated in the absence of extracellular calcium, the effects of vasopressin and angiotensin II on urea synthesis were abolished, whereas those of epinephrine and glucagon were only diminished. Mitochondria isolated from cells incubated under these conditions, showed that the effect of all these hormones on citrulline synthesis could still be observed. However, the effects of glucagon and epinephrine plus propranolol were larger than those of angiotensin II or vasopressin.Phosphatidylinositol labeling was significantly increased by epinephrine, vasopressin and angiotensin II both in the absence or presence of calcium. Cyclic AMP levels were significantly increased by glucagon or epinephrine but not by vasopressin or angiotensin II. The effect of epinephrine on cyclic AMP levels was blocked by propranolol both in the absence or presence of calcium.