Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 micromol/l). ATPgammaS (10 micromol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 micromol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPgammaS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Effects of glucagon, 3′,5′-AMP and 3′,5′-GMP on ion fluxes and transmembrane potential in perfused livers of normal and adrenalectomized rats

The effects of glucagon, 3′,5′-AMP, 3′,5′-GMP and dexamethasone on ion fluxes and transmembrane-potential changes were compared in perfused livers from normal and adrenalectomized rats. Glucagon and cyclic nucleotide administration resulted in a similar redistribution of Na⁺ and K⁺ and membrane hyperpolarization in both groups. Dexamethasone at a dose which restores the gluconeogenic response after adrenalectomy, had no effect on either the ion movements or membrane potential and did not alter the responses to cyclic nucleotides or glucagon in either normal or adrenalectomized rat livers. These results suggest that the permissive effect of glucacorticoids on gluconeogenesis might be related to an event following ion movement.     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K⁺ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba²⁺ (BaCl₂, 5 mmol/l) and the blocker of the Na⁺/K⁺ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B₄ receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 μmol/l). ATPγS (10 μmol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 μmol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPγS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule.

The effect of glucagon on gluconeogenesis was measured in periportal and pericentral regions of the liver lobule by monitoring changes in rates of O2 uptake on the surface of the perfused liver with miniature O2 electrodes after infusion of lactate. When lactate (2 mM) was infused into livers from starved rats perfused in the anterograde direction, O2 uptake was increased 2.5-fold more in periportal than in pericentral regions, reflecting increased energy demands for glucose synthesis. Under these conditions, glucagon infusion in the presence of lactate increased O2 uptake exclusively in periportal regions of the liver lobule. Thus, when perfusion is in the physiological anterograde direction, the metabolic actions of glucagon predominate in periportal regions of the liver lobule under gluconeogenic conditions in the starved state. When livers were perfused in the retrograde direction, however, glucagon stimulated O2 uptake exclusively in pericentral regions. Thus glucagon only stimulates gluconeogenesis in 'upstream' regions of the liver lobule irrespective of the direction of flow.     

Substrate-induced membrane potential changes in the perfused rat liver

In the hemoglobin-free perfused liver, administration of pyruvate, lactate fructose, alanine and palmitate elicited a sustained hyperpolarization of the cell membrane. In contrast, glucose, galactose, lysine, acetate or α-aminoisobutyric acid had no effect on the membrane potential. The pattern of the substrate induced hyperpolarization was different from glucagon- or cyclic AMP-induced hyperpolarization in the onset and duration of the response and ouabain sensitivity. The effect of cyclic AMP (5 · 10^-4 M) on membrane potential was additive to the effect of the hyperpolarizing substrates and seems to involve a mechanism different from the substrate-induced potential changes.     

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.     

Hypophysectomy does not alter the acinar zonation of gluconeogenesis or the mitochondrial redox state in rat liver.

The biochemical and functional heterogeneity of hepatocytes in different zones of the liver acinus may be related to the concentrations of hormones within the liver acinus. We examined the effects of hypophysectomy, which causes marked changes in plasma hormone levels and in activities of hepatic enzymes that are normally heterogeneously distributed, on the degree of metabolic zonation within the liver acinus. In hypophysectomized rats the activity of alanine aminotransferase was increased, but its normal zonation (predominance in the periportal zone) was preserved. The activity in cultured periportal and perivenous hepatocytes was increased by dexamethasone, but not by glucagon. Periportal hepatocytes from hypophysectomized rats expressed higher rates of gluconeogenesis in culture than did perivenous hepatocytes, irrespective of the absence or presence of dexamethasone, glucagon or insulin. Similar differences in rates of ketogenesis and in the mitochondrial redox state in response to glucagon were observed between periportal and perivenous hepatocytes from hypophysectomized rats as between cell populations from normal rats. Although hypophysectomy causes marked changes in hepatic enzyme activities, it does not alter the degree of zonation of alanine aminotransferase, gluconeogenesis or the mitochondrial redox state within the liver acinus.     

Interrelation between gluconeogenesis and hepatic protein sysnthesis

Acute administration of glucagon to the rat in vivo inhibits hepatic polypeptide chain elongation by about 30% This effect was not observed in adrenalectomized rats, despite the significant increases in the hepatic content of cyclic AMP. Fatty acid administration mimics the glucagon action on protein systhesis; however, in adrenalectomized animals they were ineffective. Whether glucagon or fatty acids were administered, there was a significant increase in the state of reduction of the NAD system in normal as well as in adrenalectomized rats. This observation rules out the change in the cellular state of reduction as the mediator of their action on protein synthesis. A correlation was observed between the ability of glucagon or fatty acids to inhibits proteinp synthesis and to stimulate gluconeogenesis. An increased phosphorylation state of as reflected by an increased gluconeogenesis flux is accompanied by a decreased phosphorylation state of adenine nucleotides that might be responsible for the inhibitory effect on protein sysnthesis. In adrenalectomized animals in which neither glucagon nor fatty acids stimulate gluconeogenesis, no effects on phosphorylation state or on the rate of protein synthesis were detected.     

Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate.

1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogeneous precursors, 10 mM-propionate and 10mM-galactose, were linear for 1 h and were both stimulated by 1 muM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37 degrees, 22 degrees and * degrees C. Even under the best of the three conditions of storage that were tested (i.e. at 22 degrees C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceeded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 muM) and 2mM-butyrate accelerated the rate of glucose formation of liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 muM-glucagon. For lactate+pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1muM-glucagon and for both lactate+pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-cell gluconeogenesis by different mechanisms.     

Physiological effect of circulating glucagon on the hepatic membrane potential

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane under various conditions. Here we investigated the physiological relevance of this effect by testing the influence of infusions of glucagon antiserum on the liver cell membrane potential in vivo. Intracellular microelectrode recordings of liver cells (up to 60/rat over 2 h) were done in anesthetized male rats. Livers were fixed in place, and recordings were done 10-30 min after intraperitoneal injections of glucagon or hepatic portal vein infusions of glucagon or specific polyclonal glucagon antibodies raised in rabbits. The isotonic lactose vehicle was used as a control for glucagon, and equal amounts of nonimmunized rabbit IgG were used as a control for glucagon antibodies. Intraperitoneal glucagon (400 microg/kg) hyperpolarized the liver cell membrane up to 12 mV, and intraportal glucagon (10 or 60 microg/kg) dose dependently hyperpolarized the liver cell membrane by 3-7 mV. Intraportal infusion of glucagon antiserum (in vitro binding capacity of 4 ng glucagon/rat) significantly depolarized the liver cell membrane by approximately 2.5 mV. The effects of both glucagon and glucagon antiserum reversed after 60-90 min. We conclude that glucagon is a physiologically important modulator of the liver cell membrane potential.     

Membrane potential measurements during rat liver regeneration

Membrane potential was measured in perfused rat liver and was shown to increase from ?33 ± 1.0 mV in livers from normal rats to ?50 ± 1.1 mV in livers from rats 12 hr after partial hepatectomy. The hyperpolarization of the membrane in regenerating liver was no longer evident after perfusion with 1 mM ouabain for 5 min. Ouabain had a small (4 mV) depolarizing effect on membrane potential in normal liver. The potential measured in normal and regenerating liver decreased as a function of the external potassium concentration above 5 mM; however, the potential was more electronegative in regenerating liver compared to normal liver at all values of external potassium concentration, and the differences in potential between the two kinds of cells did not decrease at higher concentrations of external potassium. Thus, a plot of membrane potential vs external potassium concentration resulted in approximately parallel curves for the two different cell types. We conclude that hyperpolarization of the liver cell membrane is an early event during rat liver regeneration and results from an electrogenic Na-K pump.     

Short-term stimulation of Na+-dependent amino acid transport by dibutyryl cyclic AMP in hepatocytes. Characteristics and partial mechanism.

The short-term protein-synthesis-independent stimulation of alanine transport in hepatocytes was further investigated. Cyclic AMP increased the Vmax. of alanine transport. Amino acid transport via systems A, ASC and N was stimulated. A good correlation was found between the initial rate of transport and the cell membrane potential as calculated from the distribution of Cl-. Cyclic AMP increased the rate of alanine transport, stimulated Na+/K+ ATPase (Na+/K+-transporting ATPase) activity and caused membrane hyperpolarization. The time courses and cyclic AMP dose-dependencies of all three effects were similar. Ouabain abolished the effect of cyclic AMP on Cl- distribution and on transport of alanine. The effect of cyclic AMP on alanine transport and Cl- distribution was mimicked by the antibiotic nigericin; the effect of nigericin was also abolished by ouabain. It is concluded that the effect of cyclic AMP on transport is mediated via membrane hyperpolarization. It is suggested that the primary action of cyclic AMP is to increase the activity of an electroneutral Na+/K+-exchange system in the liver cell plasma membrane, thus hyperpolarizing the membrane by stimulating the electrogenic Na+/K+ ATPase.     

Improved response of isolated liver cells to glucagon in the presence of rat serum.

The stimulation by glucagon of gluconeogenesis from 10 mM lactate and urea formation in intact isolated rat liver cells is enhanced from about 33 % to 61 % and 92 %, respectively, by the addition of rat serum (30 % v/v) to the incubation medium. This effect is exerted by (a) dialysable, heat-labile serum component(s), different from known stimulators of gluconeogenesis, such as fatty acids, lysine, ammonium chloride or calcium, which do not improve the response of hepatocytes to glucagon. The factor(s) could provide a useful tool for the study of glucagon action(s) on liver metabolism.     

Hormonal modulation of hepatic plasma membrane lactate transport in cultured rat hepatocytes

Hormonal modulation of hepatic plasma membrane lactate transport was studied in primary cultures of isolated hepatocytes from fed rats to examine the mechanism for the known enhancement of lactate transport in starvation and diabetes. Total cellular lactate entry was increased by 14% in the presence of dexamethasone; this was accounted for by an approximately 40% increase in the carrier-mediated component of entry with no effect on diffusion. A trend of similar magnitude was evident with glucagon. The effects of dexamethasone and glucagon on lactate transport constitute an additional potential mechanism for enhancement of gluconeogenesis by these hormones.     

Effects of hormones and of ethanol on the fructose 6-P-fructose 1,6-P2 futile cycle during gluconeogenesis in the liver

Carbon-14 was incorporated into C-6 of glucose from [1-¹⁴C]galactose during gluconeogenesis from dihydroxyacetone in liver cells from fasted rats, proving the existence of a futile cycle between fructose-6-P and fructose-1,6-P₂ under the conditions used. Using a steady-state model and assumed values for the rates of aldolase and glucose-6-P isomerase, the rates of phosphofructokinase were estimated, ranging from about 15% to nearly 40% of the net rate of gluconeogenesis. Glucagon depressed the rate of phosphofructokinase by as much as 85% and increased the rate of gluconeogenesis by up to 45%. l-epinephrine in the range from 10 to 100 μm also depressed phosphofructokinase, being nearly as effective as glucagon only at high concentrations. The effect of epinephrine was only partially reversed by 10 μm dl-propranolol. Ethanol (10 mm) depressed phosphofructokinase flux nearly as well as glucagon, but had no significant effect on the rate of gluconeogenesis from dihydroxyacetone.     

Ion fluxes changes during early stages of Schistosoma mansoni. Evaluation of complement effect

The relationship between the average membrane potential (delta psi av) and sensitivity to complement action of the Schistosoma mansoni parasite was explored. The average membrane potential was estimated by measuring the uptake of [3H]tetraphenyl phosphonium ([3H]Ph4P+). The parasites take up Ph4P+ indicating the existence of a negative internal plasma potential which is in part dependent on the transmembrane K+ gradient, maintained by an active Na+/K+-ATPase. Values for Ph4P+ uptake could be corrected for mitochondrial accumulation by employing the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses the mitochondrial potential. The plasma membrane potential derived by this technique was in the range of -60 mV. Transformation of this parasite, from its early cercaria stage to the adult worm, was associated with changes in the average membrane potential. The apparent hyperpolarization, which accompanies transformation, may be related to changes in ionic permeability and morphology which occur concomitantly. Complement acting through both the classical and alternative pathways was found to affect the potential of the parasite in its early development stages. The correlation between effects on delta psi av and sensitivity to complement action, indicates that the complement-induced changes in delta psi av are indeed tightly associated with its mode of action. Treatment of the parasite with complement resulted in net hyperpolarization of the membrane indicating that hyperpolarization rather than depolarization of the membrane is linked to the primary non-lethal action of complement.     

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.     

Carbohydrate metabolism of the perfused rat liver

1. The rates of gluconeogenesis from most substrates tested in the perfused livers of well-fed rats were about half of those obtained in the livers of starved rats. There was no difference for glycerol. 2. A diet low in carbohydrate increased the rates of gluconeogenesis from some substrates but not from all. In general the effects of a low-carbohydrate diet on rat liver are less marked than those on rat kidney cortex. 3. Glycogen was deposited in the livers of starved rats when the perfusion medium contained about 10mm-glucose. The shedding of glucose from the glycogen stores by the well-fed liver was greatly diminished by 10mm-glucose and stopped by 13.3mm-glucose. Livers of well-fed rats that were depleted of their glycogen stores by treatment with phlorrhizin and glucagon synthesized glycogen from glucose. 4. When two gluconeogenic substrates were added to the perfusion medium additive effects occurred only when glycerol was one of the substrates. Lactate and glycerol gave more than additive effects owing to an increased rate of glucose formation from glycerol. 5. Pyruvate also accelerated the conversion of glycerol into glucose, and the accelerating effect of lactate can be attributed to a rapid formation of pyruvate from lactate. 6. Butyrate and oleate at 2mm, which alone are not gluconeogenic, increased the rate of gluconeogenesis from lactate. 7. The acceleration of gluconeogenesis from lactate by glucagon was also found when gluconeogenesis from lactate was stimulated by butyrate and oleate. This finding is not compatible with the view that the primary action of glucagon in promoting gluconeogenesis is an acceleration of lipolysis. 8. The rate of gluconeogenesis from pyruvate at 10mm was only 70% of that at 5mm. This ;inhibition' was abolished by oleate or glucagon.     

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.     

Distinct effects of glucagon and vasopressin on proline metabolism in isolated hepatocytes. The role of oxoglutarate dehydrogenase.

The hormonal regulation of gluconeogenesis and ureogenesis in isolated rat hepatocytes with 5 mM-proline as precursor was studied, with the following results. (1) The formation of glucose and urea in a 30 min interval were stimulated more by vasopressin than by glucagon, and the effects of the two hormones in combination were additive. (2) The rates of gluconeogenesis during the 30 min were constant under control, glucagon-stimulated and glucagon-plus-vasopressin-stimulated conditions. The stimulated rate in the presence of vasopressin diminished with time; glucagon in combination with vasopressin prevented this diminution, resulting in an additive effect. (3) Coincident with these changes in gluconeogenesis, vasopressin caused a decrease in cell oxoglutarate concentration, which, in contrast with the decrease caused by glucagon, was greater, but not sustained unless glucagon was also present. Changes in cell glutamate concentration similar to those observed for oxoglutarate occurred. (4) The data suggest that activation of oxoglutarate dehydrogenase (EC 1.2.4.2) by glucagon and vasopressin by different mechanisms may explain the relative effects of the hormones alone and in combination on gluconeogenesis from proline.