The mechanism of the hormonal activation of respiration in isolated hepatocytes and its importance in the regulation of gluconeogenesis.

The effects of hormones on the cytochrome spectra of isolated hepatocytes were recorded under conditions of active gluconeogenesis from L-lactate. Glucagon, phenylephrine, vasopressin and valinomycin, at concentrations that caused stimulation of gluconeogenesis, increased the reduction of the components of the cytochrome bc1 complex, just as has been observed in liver mitochondria isolated from glucagon-treated rats [Halestrap (1982) Biochem. J. 204, 37-47]. The effects of glucagon and phenylephrine were additive. The time courses of the increased reduction of cytochrome c/c1 and NAD(P)H/NAD(P)+ caused by hormones, valinomycin, A23187 and ethanol were measured by dual-beam spectrophotometry and fluorescence respectively. Ethanol (14 mM) produced a substantial rise in NAD(P)H fluorescence, beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, no change in cytochrome c/c1 reduction, a 10% decrease in O2 consumption and a 60% decrease in gluconeogenesis. Glucagon, phenylephrine and vasopressin caused a substantial and transient rise in NAD(P)H fluorescence, but a sustained increase in cytochrome c/c1 reduction and the rates of O2 consumption and gluconeogenesis. The transience of the fluorescence response was greater in the absence of Ca2+, when the cytochrome c/c1 response also became transient. The fluorescence response was smaller and less transient, but the cytochrome c/c1 response was greater, in the presence of fatty acids. Both responses were greatly decreased by the presence of 1 mM-pent-4-enoate. Valinomycin (2.5 nM) caused a decrease in NAD(P)H fluorescence coincident with an increase in cytochrome c/c1 reduction and the rate of gluconeogenesis and O2 consumption. A23187 (7.5 mM) caused increases in both NAD(P)H fluorescence and cytochrome c/c1 reduction. The effects of hormones and valinomycin on the time courses of NAD(P)H fluorescence, cytochrome c/c1 reduction and light-scattering by hepatocytes were compared with those of 0.5 microM-Ca2+ or 1 nM-valinomycin on the same parameters of isolated liver mitochondria. It is concluded that hormones increase respiration by hepatocytes in a biphasic manner. An initial Ca2+-dependent activation of mitochondrial dehydrogenases rapidly increases the mitochondrial [NADH], which is followed by a volume-mediated stimulation of fatty acid oxidation and electron flow between NADH and cytochrome c. 10. Amytal (0.5 mM) was able to reverse the effects of hormones on the reduction of cytochromes c/c1 and the rates of gluconeogenesis and O2 consumption without significantly lowering tissue [ATP].(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat liver mitochondria prepared in mannitol media demonstrate increased mitochondrial volumes compared with mitochondria prepared in sucrose media. Relationship to the effect of glucagon on mitochondrial function.

Liver mitochondria isolated from glucagon-treated rats by using both mannitol- and sucrose-based media showed enhanced uncoupled succinate oxidation, pyruvate metabolism and citrulline synthesis. Mitochondria prepared in mannitol medium showed some stimulation of these parameters compared with those prepared in sucrose medium. This was accompanied by an increase in matrix volume of about 20%. Some [14C]mannitol became permanently associated with mitochondria during preparation. It is suggested that mannitol may enter mitochondria during their preparation and cause swelling. The presence of 4mM-phosphate in the sucrose isolation medium stimulated the same parameters as did glucagon treatment, and also caused an increase in matrix volume of about 20%. These results confirm the conclusion that the mitochondrial volume may be important in the regulation of mitochondrial metabolism. They contradict the conclusion of others [Siess (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 279-290, 835-838] that mannitol rather than sucrose should be used when studying hormonal effects on mitochondrial metabolism. Reasons for the discrepancies in the results between groups studying the effects of hormones on mitochondrial metabolism are discussed.     

Control of gluconeogenesis and of enzymes of glycogen metabolism in isolated rat hepatocytes. A parallel study of the effect of phenylephrine and of glucagon

Hepatocytes isolated from the livers of fed rats were used for a comparative study of the effects of phenylephrine, vasopressin and glucagon on gluconeogenesis and on enzymes of glycogen metabolism. When hepatocytes were incubated in the presence of Ca²⁺, phenylephrine stimulated gluconeogenesis from pyruvate less than did glucagon, but, in contrast with this hormone, it did not affect the activities of protein kinase and pyruvate kinase, nor the concentration of phosphoenolpyruvate, and it did not decrease the release of ³H₂O from [6-³H]glucose. The effects of vasopressin were similar to those of phenylephrine. Gluconeogenesis from fructose was also stimulated by phenylephrine and, more markedly, by glucagon at the expense of the conversion of fructose into lactate. Insulin was able to antagonize the stimulatory effect of phenylephrine on gluconeogenesis from pyruvate. When Ca²⁺ was removed from the incubation medium, phenylephrine still stimulated gluconeogenesis from pyruvate, but it also caused an activation of protein kinase and an inactivation of pyruvate kinase; accordingly, the concentration of phosphoenolpyruvate was increased, and, in contrast, vasopressin had no effect on all these parameters. The property of phenylephrine to cause the activation of glycogen phosphorylase was decreased by glucose or by the absence of Ca²⁺; it was abolished when these two conditions were combined. Glycogen synthase was inactivated by phenylephrine in the presence or the absence of Ca²⁺, although presumably by different mechanisms.     

The intramitochondrial volume measured using sucrose as an extramitochondrial marker overestimates the true matrix volume determined with mannitol.

The matrix volume of isolated liver and heart mitochondria has been estimated at various osmolarities and in various osmotic supports using 36Cl- and [14C]sucrose, D-mannitol, D-3-methoxyglucose and choline as extramitochondrial markers. The use of 3-methoxyglucose was only possible at 0 degree C since it entered mitochondria at physiological temperatures. All extramitochondrial markers used gave linear plots of apparent matrix volume against the reciprocal of the osmolarity, but the slope of this plot was greater when sucrose was used than with the other extramitochondrial markers. When extrapolated to infinite osmolarity the mean matrix volume was zero when mannitol was used, but about 0.6 microliter/mg of protein for sucrose and Cl- and -0.4 microliter/mg of protein when choline was used. At physiological osmolarity (about 330 m-osmol) the mean matrix volume of de-energized liver mitochondria in KCl medium estimated using mannitol was 0.46 microliter/mg of protein, whereas that obtained using sucrose was 1.68 microliters/mg of protein. Values in mannitol, choline and sucrose media were similar when mannitol but not sucrose was used as extramitochondrial marker. It is argued that the 3H2O/[14C]mannitol space more accurately reflects the true mitochondrial matrix volume than does the 3H2O/[14C]sucrose space. The consequences of this for measurements of the protonmotive force and the intramitochondrial concentration of metabolites are discussed.     

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.     

Evidence that the flux control coefficient of the respiratory chain is high during gluconeogenesis from lactate in hepatocytes from starved rats. Implications for the hormonal control of gluconeogenesis and action of hypoglycaemic agents.

1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.     

The glucagon-induced activation of pyruvate dehydrogenase in hepatocytes is diminished by 4 beta-phorbol 12-myristate 13-acetate. A role for cytoplasmic Ca2+ in dehydrogenase regulation.

Phenylephrine, vasopressin and glucagon each increased the amount of active (dephospho) pyruvate dehydrogenase (PDHa) in isolated rat hepatocytes. Treatment with 4 beta-phorbol 12-myristate 13-acetate (PMA) opposed the increase in PDHa caused by both phenylephrine and glucagon, but had no effect on the response to vasopressin: PMA alone had no effect on PDHa. As PMA is known to prevent the phenylephrine-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and to diminish the increase [Ca2+]c caused by glucagon, while having no effect on the ability of vasopressin to increase [Ca2+]c, these data are consistent with the notion that in intact cells an increase in [Ca2+]c results in an increase in the mitochondrial free Ca2+ concentration, which in turn leads to the activation of PDH. In the presence of 2.5 mM-Ca2+, glucagon caused an increase in NAD(P)H fluorescence in hepatocytes. This increase is taken to reflect an enhanced activity of mitochondrial dehydrogenases. PMA alone had no effect on NAD(P)H fluorescence; it did, however, compromise the increase produced by glucagon. When the extracellular free [Ca2+] was decreased to 0.2 microM, glucagon could still increase NAD(P)H fluorescence. Vasopressin also increased fluorescence under these conditions; however, if vasopressin was added after glucagon, no further increase in fluorescence was observed. Treatment of the cells with PMA resulted in a smaller increase in NAD(P)H fluorescence on addition of glucagon: the subsequent addition of vasopressin now caused a further increase in fluorescence. Changes in [Ca2+]c corresponding to the changes in NAD(P)H fluorescence were observed, again supporting the idea that [Ca2+]c indirectly regulates intramitochondrial dehydrogenase activity in intact cells. PMA alone had no effect on pyruvate kinase activity, and the phorbol ester did not prevent the inactivation caused by glucagon. The latter emphasizes the different mechanisms by which the hormone influences mitochondrial and cytoplasmic metabolism.     

The r?le of mitochondrial pyruvate transport in the stimulation by glucagon and phenylephrine of gluconeogenesis from L-lactate in isolated rat hepatocytes.

The sensitivity of glucose production from L-lactate by isolated liver cells from starved rats to inhibition by alpha-cyano-4-hydroxycinnamate was studied. A small percentage of the maximal rate of gluconeogenesis was insensitive to inhibition by alpha-cyano-4-hydroxycinnamate, and evidence is presented to show that this is due to pyruvate entry into the mitochondria as alanine. After subtraction of this rate, Dixon plots of the reciprocal of the rate of gluconeogenesis against inhibitor concentration were linear both in the absence and presence of glucagon, phenylephrine or valinomycin, each of which stimulated gluconeogenesis by 30-50%. Pyruvate kinase activity was decreased by glucagon, but not by phenylephrine or valinomycin. Inhibition of gluconeogenesis by quinolinate (inhibitor of phosphoenolpyruvate carboxykinase) or monochloroacetate (probably inhibiting pyruvate carboxylation) caused a significant deviation from linearity of the Dixon plot obtained with alpha-cyano-4-hydroxycinnamate. Amytal, however, inhibited gluconeogenesis without affecting the linearity of this plot. These data, coupled with a computer simulation study, suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain. An additional role for pyruvate kinase and pyruvate carboxylase is quite compatible with the data presented.     

Use of 3H and 14C doubly labeled glucose and amino acids in the study of hormonal regulation of gluconeogenesis in rats.

Double isotope procedures (3H and 14C) were used in vivo to investigate a) slow long-term gluconeogenic actions of adrenal glucocorticoids, and b) rapid stimulation of gluconeogenesis by glucagon. [U-14C,6-3H]Glucose was administered to normal and adrenalectomized rats. No effect was observed on the [6-3H]glucose half-life suggesting the dicarboxylic acid shuttle is unaffected by adrenalectomy; the Cori cycle is also not influenced. Loads of [14C]aspartate, [14C]glutamate, or [14C]alanine were given to normal and adrenalectomized rats. Simultaneously, in vivo transaminase activity was studied by measuring the appearance of 3H2O in body water after administration of [2-3H]aspartate, [2-3H]glutamate, or [2-3H]alanine, Adrenalectomy has no influence on the incorporation of glutamate or aspartate into glucose or on their in vivo transaminases. Diminution of incorporation of [14C]alanine into glucose and alanine transaminase activities occurs only when rats are given unphysiological loads. These studies support the contention that glucocorticoid rate-limiting actions occur in extrahepatic tissues to produce an increased flow of glucose precursors to the liver. [U-14C,3-3H]Glucose was used to investigate the effect of glucagon on the hepatic fructose-6-phosphate (F-6-P) cycle. Glucagon administration resulted in a rapid drop in the 3H/14C ratio of circulating glucose, suggesting an increase in F-6-P recycling caused by activation of FDPase with little or no decrease in phosphofructokinase. Such a change would direct substrate flux toward gluconeogenesis.     

Stimulation of mitochondrial pyruvate metabolism and citrulline synthesis by dexamethasone. Effect of isolation and incubation media.

Hepatic mitochondria isolated in 0.3 M-sucrose or 0.3 M-mannitol from rats treated for 3h with dexamethasone displayed stimulated rates of pyruvate carboxylation and decarboxylation and citrulline synthesis when compared with organelles from control animals. Mitochondria isolated in mannitol also displayed elevated rates of pyruvate carboxylation and decarboxylation when compared with those isolated in sucrose, and this stimulation was shown to be independent of the lengthy isolation procedure. Citrulline synthesis proceeded at similar rates in mitochondria isolated in either sugar. The concentration of exchangeable adenine nucleotides was identical in mitochondria isolated in sucrose or mannitol, suggesting that those prepared in the former sugar are not more permeable to metabolites than those prepared in the latter. The matrix volume of mitochondria isolated in mannitol was greater than that of mitochondria isolated in sucrose, and the effect of mannitol on pyruvate metabolism was mimicked by swelling the organelles in hypo-osmotic sucrose. Measurements of the extra-matrix volume by using [14C]sucrose or [14C]mannitol suggest that mannitol can permeate mitochondria to a greater extent than can sucrose. The possibility that mannitol elicits its effect by entering the mitochondrial matrix and so initiating swelling is discussed.     

Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187.

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.     

Effect of phenylephrine on glutamate and glutamine metabolism in isolated perfused rat liver.

Addition of phenylephrine to isolated perfused rat liver is followed by an increased 14CO2 production from [1-14C]glutamate, [1-14C]glutamine, [U-14C]proline and [3-14C]pyruvate, but by a decreased 14CO2 production from [1-14C]pyruvate. Simultaneously, there is a considerable decrease in tissue content of 2-oxoglutarate, glutamate and citrate. Stimulation of 14CO2 production from [1-14C]glutamate is also observed in the presence of amino-oxyacetate, suggesting a stimulation of glutamate dehydrogenase and 2-oxoglutarate dehydrogenase fluxes by phenylephrine. Inhibition of pyruvate dehydrogenase flux by phenylephrine is due to an increased 2-oxoglutarate dehydroxygenase flux. Phenylephrine stimulates glutaminase flux and inhibits glutamine synthetase flux to a similar extent, resulting in an increased hepatic glutamine uptake. Whereas the effects of NH4+ ions and phenylephrine on glutaminase flux were additive, activation of glutaminase by glucagon was considerably diminished in the presence of phenylephrine. The reported effects are largely overcome by prazosin, indicating the involvement of alpha-adrenergic receptors in the action of phenylephrine. It is concluded that stimulation of gluconeogenesis from various amino acids by phenylephrine is due to an increased flux through glutamate dehydrogenase and the citric acid cycle.     

Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin.

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.     

A re-evaluation of the role of mitochondrial pyruvate transport in the hormonal control of rat liver mitochondrial pyruvate metabolism.

The inhibitor of mitochondrial pyruvate transport alpha-cyano-beta-(1-phenylindol-3-yl)-acrylate was used to inhibit progressively pyruvate carboxylation by liver mitochondria from control and glucagon-treated rats. The data showed that, contrary to our previous conclusions [Halestrap (1978) Biochem. J. 172, 389-398], pyruvate transport could not regulate metabolism under these conditions. This was confirmed by measuring the intramitochondrial pyruvate concentration, which almost equilibrated with the extramitochondrial pyruvate concentration in control mitochondria, but was significantly decreased in mitochondria from glucagon-treated rats, where rates of pyruvate metabolism were elevated. Computer-simulation studies explain how this is compatible with linear Dixon plots of the inhibition of pyruvate metabolism by alpha-cyano-4-hydroxycinnamate. Parallel measurements of the mitochondrial membrane potential by using [3H]triphenylmethylphosphonium ions showed that it was elevated by about 3 mV after pretreatment of rats with both glucagon and phenylephrine. There was no significant change in the transmembrane pH gradient. It is shown that the increase in pyruvate metabolism can be explained by a stimulation of the respiratory chain, producing an elevation in the protonmotive force and a consequent rise in the intramitochondrial ATP/ADP ratio, which in turn increases pyruvate carboxylase activity. Mild inhibition of the respiratory chain with Amytal reversed the effects of hormone treatment on mitochondrial pyruvate metabolism and ATP concentrations, but not on citrulline synthesis. The significance of these observations for the hormonal regulation of gluconeogenesis from L-lactate in vivo is discussed.     

Hormonal regulation of the tricarboxylic acid cycle in the isolated perfused rat liver

The effect of Ca2+-mobilizing hormones, vasopressin, angiotensin II and the alpha-adrenergic agonist phenylephrine, on the metabolic flux through the tricarboxylic acid cycle was investigated in isolated perfused rat livers. All three Ca2+-mobilizing agonists stimulated 14CO2 production and gluconeogenesis in livers of 24-h-fasted rats perfused with [2-14C]pyruvate. Prazosin blocked the phenylephrine-elicited stimulation of 14CO2 and glucose production from [2-14C]pyruvate whereas the alpha 2-adrenergic agonist, BHT-933, did not affect the rates of 14CO2 and glucose production from [2-14C]pyruvate indicating that the phenylephrine-mediated response involved alpha 1-adrenergic receptors. Phenylephrine, vasopressin and angiotensin II stimulated 14CO2 production from [2-14C]acetate in livers derived from fed rats but not in livers of 24-h-fasted rats. In livers of 24-h-fasted rats, perfused with [2-14C]acetate, exogenously added pyruvate was required for an increase in the rate of 14CO2 production during phenylephrine infusion. This last observation suggests increased pyruvate carboxylation as one of the mechanisms involved in stimulation of tricarboxylic acid cycle activity by the Ca2+-mobilizing agonists, vasopressin, angiotensin II and phenylephrine.     

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.     

Effects of glucagon and alpha- and beta-agonists on glycogenolysis and gluconeogenesis in isolated ovine hepatocytes

(1) The effects of glucagon, dibutyryl cyclic AMP, vasopressin, phenylephrine, and isoproterenol on glycogenolysis and gluconeogenesis were investigated using isolated ovine hepatocytes. (2) Glycogenolysis was stimulated by all effectors except vasopressin. The response to alpha-agonists was greater than that of beta-agonists in older animals. Stimulation by beta-agonists increased after 30 h primary culture. (3) Gluconeogenesis from propionate or L-lactate plus pyruvate was stimulated to a small extent by dibutyryl cyclic AMP, glucagon and isoproterenol but not by vasopressin or phenylephrine. (4) No effects of lactation were observed. (5) Data are compared to results obtained in other species and the physiological significance of the results in relation to the ruminant is discussed.     

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.     

Lack of V1 vasopressin receptors in rabbit hepatocytes.

Vasopressin does not induce glycogenolysis in rabbit hepatocytes; glucagon, angiotensin, phenylephrine and ATP are as potent as with rat hepatocytes, whereas isoprenaline is nearly 10000 times more potent in the rabbit. Binding studies of [3H]vasopressin reveal the complete absence of specific vasopressin receptors on rabbit liver plasma membranes. We verified that vasopressin acts as an antidiuretic and vasopressor agent in the rabbit. We conclude that there is a selective lack of V1 vasopressin receptors in rabbit liver.