Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.     

Glucagon effect on rat liver protein synthesis in vivo

The in vivo effect of glucagon administration on hepatic polyribosomal profiles has been studied. Glucagon did not change significantly total, free or bound polyribosomal fractions 30–45 minutes after its administration. The combined administration of glucagon plus antiinsulin serum failed to show any significant effect of glucagon over the antiinsulin serum treated control. Glucagon increased valine production in the perfused isolated liver. These results suggest that the well known amino acid catabolic action of glucagon may be preferentially mediated through an increased proteolysis. Since it is known that glucagon increases considerably in vivo the liver cyclic AMP levels then its lack of effect on polyribosomal profiles might indicate that the postulated role for the cyclic nucleotide on liver protein synthesis must be taken cautiously.     

Effects of glucagon and insulin on fatty acid synthesis and glycogen degradation in the perfused liver of normal and genetically obese (ob/ob) mice.

1. Rapid effects of hormones on glycogen metabolism and fatty acid synthesis in the perfused liver of the mouse were studied. 2. In perfusions lasting 2h, of livers from normal mice, glucagon in successive doses, each producing concentrations of 10(-10) or 10(-9)M, inhibited fatty acid and cholesterol synthesis. In perfusions lasting 40--50 min, in which medium was not recycled, inhibition of fatty acid synthesis was only observed with glucagon at concentrations greater than 10(-9)M. This concentration was about two orders of magnitude higher than that required for the stimulation of glycogen breakdown. Glucagon did not inhibit the activity of acetyl-CoA carboxylase, assayed 10 or 20 min after addition of glucagon (10(-9) or 10(-10)M). It is proposed that the action of glucagon on hepatic fatty acid biosynthesis could be secondary in time to depletion of glycogen. Insulin prevented the effect of glucagon (10(-10)M) on glycogenolysis, but not that of vasopressin. 3. Livers of genetically obese (ob/ob) mice did not show significant inhibition of lipid biosynthesis in response to glucagon, although there was normal acceleration of glycogen breakdown. This resistance to glucagon action was not reversed by food deprivation. Livers of obese mice exhibited resistance to the counteraction by insulin of glucagon-stimulated glycogenolysis, which was reversible by partial food deprivation.     

Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-translational processing

Glucagon is a pancreatic hormone of 29 amino acids that regulates carbohydrate metabolism and glicentin is an intestinal peptide of 69 amino acids that contains the sequence of glucagon flanked by peptide extensions at the amino and carboxy termini. The glucagon gene encodes a precursor containing glucagon and two additional, structurally related, glucagon-like peptides separated by an intervening peptide. These peptides are encoded in separate exons. To determine whether the pancreatic and intestinal forms of glucagon arise by alternative RNA and/or protein processing, we used antisera to synthetic glucagon-like peptides and exon-specific, complementary oligonucleotides for analyses of proteins and mRNAs in pancreatic and intestinal extracts. Preproglucagon mRNAs are identical, but different and highly specific peptides are liberated in the two tissues. Immunocytochemistry shows colocalization of glucagon and the two glucagon-like peptides in identical cells. We conclude that diversification of preproglucagon gene expression occurs at the level of cell-specific post-translational processing.     

Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria.

Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.     

Glucagon induces translocation of glucokinase from the cytoplasm to the nucleus of hepatocytes by transfer between 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase-2 and the glucokinase regulatory protein

Glucokinase activity is a major determinant of hepatic glucose metabolism and blood glucose homeostasis. Liver glucokinase activity is regulated acutely by adaptive translocation between the nucleus and the cytoplasm through binding and dissociation from its regulatory protein (GKRP) in the nucleus. Whilst the effect of glucose on this mechanism is well established, the role of hormones in regulating glucokinase location and its interaction with binding proteins remains unsettled. Here we show that treatment of rat hepatocytes with 25 mM glucose caused decreased binding of glucokinase to GKRP, translocation from the nucleus and increased binding to 6-phosphofructo 2-kinase/fructose 2,6 bisphosphatase-2 (PFK2/FBPase2) in the cytoplasm. Glucagon caused dissociation of glucokinase from PFK2/FBPase2, concomitant with phosphorylation of PFK2/FBPase2 on Ser-32, uptake of glucokinase into the nucleus and increased interaction with GKRP. Two novel glucagon receptor antagonists attenuated the action of glucagon. This establishes an unequivocal role for hormonal control of glucokinase translocation. Given that glucagon excess contributes to the pathogenesis of diabetes, glucagon may play a role in the defect in glucokinase translocation and activity evident in animal models and human diabetes.     

Effects of Y. pestis mouse toxin on carbohydrate metabolism in rats

Effects of intravenous Y. pestis mouse toxin (LD50) injection on glucose, lactate glucagon, insulin blood levels and cAMP liver content in dynamics of intoxication development were studied. Hypoglycemia, observed 2 hours after toxin administration seems not to be due to the enhanced glucose utilization in peripheral tissues because insulin blood level during this period was decreased and lactate concentration has not been changed. Glucagon content by 2-5 hour of shock was strong elevated. Proposal is made that Y. pestis mouse toxin might induce carbohydrate metabolism alterations via direct liver glucose synthesising enzymes inhibition rather than cAMP-dependent glycogenolysis and gluconeogenesis regulation disturbances in this organ.     

Effect of glucagon on phenylalanine metabolism and phenylalanine-degrading enzymes in the rat

Glucagon administered subcutaneously to rats for 10 days had no significant effect on liver phenylalanine hydroxylase activity, but induced liver dihydropteridine reductase more than twofold. In rats administered a phenylalanine load orally, glucagon treatment stimulated oxidation and depressed urinary phenylalanine excretion. These responses could not be related to an effect of glucagon on hepatic tyrosine-alpha-oxoglutarate aminotransferase activity. Even in rats with phenylalanine hydroxylase activity depressed to 50% of control values by p-chlorophenylalanine administration, glucagon treatment increased the phenylalanine-oxidation rate substantially. Although hepatic phenylalanine-pyruvate aminotransferase was increased tenfold in glucagon-treated rats, glucagon treatment did not increase urinary excretion of phenylalanine transamination products by rats given a phenylalanine load. Glucagon treatment did not affect phenylalanine uptake by the gut or liver, or the liver content of phenylalanine hydroxylase cofactor. It is suggested that dihydropteridine reductase is the rate-limiting enzyme in phenylalanine degradation in the rat, and that glucagon may regulate the rate of oxidative phenylalanine metabolism in vivo by promoting indirectly the maintenance of the phenylalanine hydroxylase cofactor in its active, reduced state.     

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.     

Glucagon regulation of amino acid transport in hepatocytes: Effect of cell enucleation

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na⁺-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.     

The role of calcium ion as a mediator of the effects of angiotensin II, catecholamines, and vasopressin on the phosphorylation and activity of enzymes in isolated hepatocytes.

Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.     

Effects of glucagon on gluconeogenesis from lactate and propionate in the perfused rat liver.

Quinolinic acid (Q.A.) which inhibits gluconeogenesis at the site of phosphoenolpyruvate (PEP) synthesis, reduced the content of PEP while elevating that of aspartate and malate in rat livers perfused with a medium containing 10 mM L-lactate. Glucagon at 10(-9) M did not affect Q.A. inhibition of lactate gluconeogenesis nor the depression of PEP level, but further elevated malate and aspartate accumulation. Exogenous butyrate had the same effect as glucagon on these parameters. Butylmalonate (BM), an inhibitor of mitochondrial malate transport, inhibited lactate and propionate gluconeogenesis to similar extents. The addition of 10(-9) M glucagon had no effect on BM inhibition of lactate gluconeogenesis, but almost completely reversed BM inhibition of propionate gluconeogenesis. These results suggest that glucagon may act on at least two sites, resulting in elevated hepatic gluconeogenesis. First, it may stimulate dicarboxylic acid synthesis (malate and oxaloacetate, specifically) through activation of pyruvate carboxylation. Secondly, it may stimulate synthesis of other dicarboxylic acids (fumarate, for example) by activating certain steps of the tricarboxylic acid cycle. The stimulatory effect of glucagon on gluconeogenesis in the perfused rat liver is well documented (1, 2). Exton et al., who earlier located the site of stimulation between pyruvate and PEP synthesis (3), proposed that glucagon stimulated PEP synthesis in the perfused rat liver (4), while reports from Williamson et al. (5) suggested the pyruvate-carboxylase reaction as the site of glucagon action. Stimulation at sites above PEP formation and of portions of the tricarboxylic acid cycle (4) by glucagon have also been suggested (6). In the present experiments, we have used substrates entering at different parts of the gluconeogenic pathway, and specific inhibitors to further resolve the action of glucagon.     

Insulin and glucagon regulate the activation of two distinct membrane-bound cyclic AMP phosphodiesterases in hepatocytes.

Glucagon (10 nM) caused a transient elevation of intracellular cyclic AMP concentrations, which reached a peak in around 5 min, and slowly returned to basal values in around 30 min. When 1 mM-3-isobutyl-1-methylxanthine (IBMX) was present, this process yielded a Ka of 1 nM for glucagon. The addition of insulin (10 nM) after 5 min exposure to glucagon (10 nM) caused intracellular cyclic AMP concentrations to fall dramatically, attaining basal values within 10 min. The regulation of this process was dose-dependent, exhibiting a Ka of 0.4 nM for insulin. If insulin and glucagon were added together to hepatocytes, then insulin decreased the magnitude of the cyclic AMP response to glucagon. IBMX (1 mM) prevented insulin antagonizing the action of glucagon in both of these instances. A gentle homogenization procedure followed by a rapid subcellular fractionation of hepatocytes on a Percoll gradient was developed. This was used to resolve subcellular membrane fractions and to identify cyclic AMP phosphodiesterase activity in both membrane and cytosol fractions. Glucagon and insulin only affected the activity of two distinct membrane-bound species, a plasma-membrane enzyme and a 'dense vesicle' enzyme. Glucagon (10 nM), insulin (10 nM), IBMX (1 mM), dibutyryl cyclic AMP (10 microM) and cholera toxin (1 microgram/ml) all elicited the activation of the 'dense vesicle' enzyme. The plasma-membrane enzyme was not activated by glucagon, IBMX or dibutyryl cyclic AMP, although insulin and cholera toxin both led to its activation. The degree of activation of the plasma-membrane enzyme produced by insulin was increased in the presence of IBMX or dibutyryl cyclic AMP. Glucagon pretreatment (5 min) of hepatocytes blocked the ability of insulin to activate the plasma-membrane enzyme. The activity state of these phosphodiesterases is discussed in relation to the observed changes in intracellular cyclic AMP concentrations. It is suggested that insulin exerts its action on the plasma-membrane phosphodiesterase through a mechanism involving a guanine nucleotide-regulatory protein.     

A reassessment of structure-function relationships in glucagon. Glucagon1-21 is a full agonist.

Glucagon1-21 has been prepared by treating native glucagon with carboxypeptidase A. Purified glucagon1-21 did not contain detectable methionine (less than 0.001 residue/mol) and the activity of the compound did not change after treatment with cyanogen bromide as has been shown with native glucagon. Glucagon1-21 stimulates hepatic adenylate cyclase activity to the same extent as native glucagon but with 0.1% the potency. Glucagon1-21 also displayed 0.1% the binding affinity of native glucagon to the glucagon receptor in hepatic membranes. Glucagon22-29 alone or in combination with glucagon1-21 did not activate adenylate cyclase or displase 125I-glucagon from its receptor. The finding that glucagon1-21 is a full agonist on adenylate cyclase is discussed in relation to the structure-function relationships required for the biological action of glucagon.     

Lack of effect of somatostatin on the glucagon-induced alterations of hepatic metabolism of [1-14C]oleate

Livers from normal fed male rats were perfused in a recycling system in vitro. Glucagon was infused in varying quantities to give final concentration in the cell-free perfusate of 4.9 . 10(-10)-4.9 . 10(-7) M after 3 h of perfusion, assuming no degradation of the hormone. Where indicated, cyclic somatostatin was infused simultaneously to give a final concentration of 3.0 . 10(-6) M. In the absence of somatostatin, glucagon at a concentration as low as 4.9 . 10(-10) M increased the release of glucose and increased ketogenesis, but impaired the synthesis and release of perfusate triacylglycerol and very low density lipoprotein lipids. Somatostatin did not affect these actions of glucagon. Somatostatin alone, however, did reduce the output of very low density lipoprotein. It is suggested that the alteration of fatty acid metabolism by somatostatin in vivo results from modulation of pancreatic glucagon secretion, not from interference by somatostatin of the action of glucagon on the liver.     

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.     

Effect of glucagon on the metabolic rate in growing chicken.

The investigations were carried out on 70 growing broiler chickens. The chickens were kept on a higher and the other ones on the lower level of nutrition. As a result of this the rate of growth was different in both groups. Glucagon had a strong calorigenic effect, reaching a peak 30 min after its injection. This effect of glucagon increased progressively with the growth and development of birds reaching a maximum in chickens aged about 7 weeks, and weighing approx. 1200 g. In the birds examined 2 hours after feeding the calorigenic effect of glucagon was most expressed in birds maintained on the low nutrition levels. The fall of RQ after glucagon injection may suggest that this hormone has a strong lipolytic action.     

Effects of glucagon on physiologic and compensatory renal growth in the rat

Glucagon has been implicated as a growth-promoting hormone in the kidneys of diabetic animals, but its role in the nondiabetic state is unknown. We evaluated the effect of subcutaneous glucagon administration on renal growth in intact rats with two kidneys and after 50% reduction in renal mass. The relative kidney weight was increased in intact rats treated with a glucagon infusion for 7 days (p less than 0.01), but decreased in uninephrectomized rats treated with glucagon (p less than 0.05). Absolute kidney weight gain and rates of renal DNA synthesis were also significantly blunted by glucagon infusion in uninephrectomized rats. These data suggest that 'physiologic' and 'compensatory' renal growth are governed by separate processes. Furthermore, the observation that glucagon promotes renal growth in intact nondiabetic animals supports its possible role as a growth factor in the early stages of diabetes.     

Modulation of the sympathetic nerve action on carbohydrate and ketone body metabolism by fatty acids, glucagon und insulin in perfused rat liver

Rat liver was perfused in situ via the portal vein without recirculation: 1) Nerve stimulation (20 Hz, 2 ms, 20 V) increased glucose output and shifted lactate uptake to output; the alterations were diminished by oleate but not octanoate. 2) Glucagon (1nM) stimulated glucose output maximally also in the presence of the fatty acids, so that nerve stimulation could not increase it further. The hormone also enhanced lactate uptake and nerve stimulation counteracted this effect. The counteraction was diminished by oleate but not octanoate. 3) Insulin (100nM) slightly lowered glucose output and had no effect on lactate balance. It antagonized the increase of glucose output by nerve stimulation, but left the shift of lactate uptake to release unaffected. These events were not influenced by the fatty acids. 4) Nerve stimulation decreased ketone body production from oleate and octanoate. 5) Glucagon increased ketogenesis from oleate, but not octanoate. In the presence of glucagon nerve stimulation also lowered ketogenesis. This decrease was diminished in the presence of oleate. 6) Insulin lowered ketogenesis from oleate but not octanoate. In the presence of insulin nerve stimulation decreased ketogenesis; the relative change was independent of the fatty acids. The complex interactions between fatty acids, glucagon and insulin in the modulation of sympathetic nerve actions can be summarized as follows: Oleate, which enters the mitochondria via the carnitine system, but not octanoate, which enters independently from this system, as well as insulin but not glucagon effectively modulated the nerve actions on carbohydrate metabolism. Glucagon but not insulin modulated the nerve effects on ketogenesis from oleate but not octanoate. The regulatory interactions between substrates, hormones and nerves can best be explained on the basis of the model of metabolic zonation.     

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.