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This work aimed to determine the role played by the adrenal gland in the fatty acid control of gluconeogenesis in isolated perfused rat livers. The gluconeogenic substrate concentration responses were not altered in adrenalectomized (ADX) rats. This observation indicates that glucocorticoids are not essential to maintain normal basal gluconeogenic rates. In contrast, fatty acid failed to stimulate gluconeogenesis from lactate and elicited attenuated stimulation with pyruvate as substrate in livers from ADX rats. Fatty acid-induced stimulation of respiration and ketone body production were similar in control and ADX rats. Thus the diminished responsiveness of the gluconeogenic pathway to fatty acid cannot be the result of different rates of energy production and/or generation of reducing power. Fatty acids did not inhibit pyruvate decarboxylation in livers from ADX rats. Even though mitochondria isolated from livers of ADX rats showed normal basal rates of pyruvate metabolism, fatty acids failed to inhibit pyruvate decarboxylation and the activity of the pyruvate dehydrogenase complex. This novel observation of the glucocorticoid effect in controlling the pyruvate dehydrogenase complex responsiveness indicates that the mitochondrial partitioning of pyruvate between carboxylation and decarboxylation reactions may be altered in livers from ADX rats. We propose that the diminished effect of fatty acid in stimulating gluconeogenesis in livers from ADX rats is the result of a limited pyruvate availability for the carboxylase reaction due to a lack of inhibition of flux through the pyruvate dehydrogenase complex.

Fatty acids produced a stimulation of gluconeogenesis and either inhibition or no effect on ureagenesis in livers perfused with gluconeogenic substrates and having NH4Cl plus ornithine as the nitrogen source. This finding indicates that stimulation of flux through pyruvate carboxylase is not sufficient to enhance urea production from ammonia. The metabolic action of fatty acids showed the following characteristics: (1) it was concentration-dependent, showing saturation-type kinetics similar to those described for fatty acid oxidation; (2) the stimulatory action on gluconeogenesis was constant and independent of NH4Cl concentration, whereas the inhibition of ureagenesis was variable and dependent on NH4Cl concentration and the degree of reduction of the gluconeogenic substrate; and (3) fatty acids produced apparent reciprocal changes in the state of reduction of the cytosolic and mitochondrial NAD systems. Fatty acid oxidation exerted its effect mainly, if not exclusively, by preventing the gluconeogenic substrate-induced stimulation of ureagenesis. Fatty acids also inhibited ureagenesis without stimulating gluconeogenesis (lactate < 1 mmol/L), ruling out a limiting energy availability as the cause of the inhibition. One or both of the following two mechanisms seem to account for the fatty acid-induced inhibition of ureagenesis from NH4Cl. First, a decreased uptake of ornithine, and second, decreased flux through pyruvate dehydrogenase and probably other NAD(P)-linked mitochondrial dehydrogenases. The correlation found between the ability of fatty acids to inhibit ureagenesis and the state of activation of pyruvate dehydrogenase supports the latter point.

Stimulation of ureogenesis by ornithine and/or NH4Cl inhibited gluconeogenesis from lactate but not from equimolar concentrations of pyruvate in perfused rat liver. Neither a shortage of energy nor a decrease in alpha-ketoglutarate availability seems to be responsible for this inhibition. With lactate as substrate the extracellular concentration of pyruvate attained was approximately equal to 0.15 mM that assuming reflects its cytosolic concentration it would be limiting for its mitochondrial transport. Stimulation of ureogenesis from NH4Cl enhances flux through pyruvate dehydrogenase. Furthermore, activation of pyruvate dehydrogenase by dichloroacetate led to stimulation of ureogenesis and inhibition of glucose production. Conversely, inhibition of pyruvate dehydrogenase flux by fatty acid enhanced glucose production and inhibited ureogenesis. Thus, ornithine and/or NH4Cl seem to inhibit lactate to glucose flux by shifting the mitochondrial partitioning of pyruvate from carboxylation towards decarboxylation with the result of a decreased oxaloacetate formation. Gluconeogenic substrates enhanced the hepatic uptake of ornithine. However, no correlation seems to exist between the uptake of ornithine, ornithine-induced stimulation of ureogenesis and total rates of urea production. Ornithine produced a concentration-dependent acidification of the hepatic outflow perfusate, suggesting that it may be transported in exchange for H+.

Gluconeogenic substrates, lactate or pyruvate, or ornithine produced 100% increase of urea synthesis from NH4Cl. The combined administration of ornithine and lactate (or pyruvate) produced more than additive effects, indicating that they acted at different steps in a potentiating manner. The uptake of ornithine was enhanced by gluconeogenic substrates. This finding may explain, at least in part, the stimulating effect of these substrates on ureagenesis from NH4Cl and ornithine. The gluconeogenic substrate-induced stimulation of ureagenesis from NH4Cl was still observed under conditions of reduced flux through pyruvate carboxylase, ruling out that their action was exclusively mediated by the anaplerotic effect of this enzyme. Pyruvate was a more potent stimulator of ureagenesis than lactate and its effect less sensitive to pyruvate carboxylase inhibition. These observations indicate that a correlation exists between stimulation of ureagenesis by gluconeogenic substrates and flux through pyruvate dehydrogenase. It is concluded that gluconeogenic substrates may stimulate ureagenesis from NH4Cl by 1) increasing intracellular ornithine availability and/or 2) enhancing flux through pyruvate dehydrogenase and consequently the tricarboxylic acid cycle activity.

Inhibition of endogenous long chain fatty acids oxidation by tetradecylglycidate (TDGA) impeded gluconeogenesis from lactate or from low concentrations of pyruvate (less than 0.5 mM). The inhibitory effect of TDGA was overcome by medium and short chain fatty acid or by concentrations of pyruvate about 0.5 mM, but not by 10-fold higher concentrations of lactate. Despite decreased energy demand when gluconeogenesis was inhibited by TDGA, the pyruvate-induced increase in hepatic oxygen consumption was similar to the control, indicating that pyruvate transport across the mitochondrial membrane and/or its decarboxylation was not altered, and therefore can not be responsible for the inhibition of gluconeogenesis. Neither does a deficiency of acetyl-CoA explain the decrease in the gluconeogenic flux since high pyruvate loads (greater than 0.5 mM), beta-hydroxybutyrate or even ethanol was capable of overcoming the inhibitory effect of TDGA in the absence of significant changes in the hepatic content of acetyl-CoA. At low (less than 0.3 mM), presumably physiological, pyruvate concentrations, its rate of mitochondrial utilization is limited by the activity of the monocarboxylate transporter. Agents that reduced the mitochondrial NAD system, and therefore reduced flux through pyruvate dehydrogenase, like beta-hydroxybutyrate or ethanol, stimulated gluconeogenesis when fatty acid oxidation was inhibited. The latter observations indicate that the primary role of endogenous fatty acid, when substrate availability is limiting, is to spare mitochondrial pyruvate by decreasing its oxidation, and therefore shifting the partitioning between the carboxylation and decarboxylation reactions toward the former.

Inhibition of hepatic long chain fatty acid oxidation by 2-5-4 chlorophenylpentyloxirane-2-carboxylate (POCA) leads to decreased gluconeogenic rates from lactate or from low concentrations of pyruvate. The inhibitory effect is fully overcome by concentrations of pyruvate above 0.8 mM or by the simultaneous administration of a medium chain fatty acid. At low pyruvate availability the energy cost of gluconeogenesis is mainly supported by fatty acid oxidation and POCA-induced inhibition of glucose production is secondary to a decreased energy availability. This is supported by the following observations: (i) POCA decreases hepatic respiration and phosphorylation potential: (ii) the rate of pyruvate-induced respiration was the same regardless of whether gluconeogenesis was inhibited or not by POCA: and (iii) concentrations of pyruvate above 0.8 mM, at which gluconeogenesis is not inhibited, prevented the POCA-induced decrease in the phosphorylation potential. It is concluded that inhibition of long chain fatty acid oxidation by POCA leads to a switch of energy fuel, and results in the oxidation of more pyruvate to meet the cellular energy demands. When pyruvate availability is low and thus, presumably, its mitochondrial transport restricted, pyruvate carboxylation most probably becomes limiting as a result of the increased flux through pyruvate dehydrogenase, in the presence of POCA.

Calcium depletion induced by perfusing livers with calcium-free buffer did not alter the rates of basal glucose production from pyruvate or from increasing concentrations of lactate. However, calcium deficiency selectively prevented the fatty acid-induced stimulation of gluconeogenesis from lactate. This effect is not related to the higher NAD redox potential consistently observed in Ca2(+)-deficient livers. On the other hand, octanoate was capable of inducing dose-dependent changes in the [pyruvate]0.5 in calcium-depleted livers perfused with lactate, ruling out that low cellular calcium content could perturb the mitochondrial transport of pyruvate. The observation that the effect of calcium deficiency can be overcome by supraphysiological concentrations of pyruvate supports the proposal that stimulation of the maximal capacity of the gluconeogenic pathway by fatty acid relies largely on the tricarboxylic acid cycle activity, restricted in calcium deficiency conditions.

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 synthesis; 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 inhibit protein synthesis and to stimulate gluconeogenesis. An increased biosynthetic activity as reflected by an increased gluconeogenic flux is accompanied by a decreased phosphorylation state of adenine nucleotides that might be responsible for the inhibitory effect on protein synthesis. 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.

Octanoate has been found to activate the gluconeogenic pathway in perfused isolated rat liver. Whether a net increase in the production of glucose is observed is a function of the relative concentrations of the glucose precursor and the fatty acid. The kinetics of octanoate interaction with the gluconeogenic pathway are influenced by the rate changes induced by decreases in pyruvate concentration as a result of the increased NAD redox potential produced by the oxidation of fatty acid. Taking this into account, two distinct effects of octanoate were identified. The first is an increase in the Vmax even at the lowest (25 microM) concentration of the fatty acid tested. The second is a progressive decrease in [pyruvate]0.5 as a function of octanoate concentration. The latter occurs at low (less than 0.1 mM), presumably physiological, pyruvate concentrations, when its mitochondrial transport is limiting, indicating that this process must have been activated. The former is observable even at high (greater than 0.5 mM), supraphysiological, concentrations of pyruvate, when its mitochondrial transport is not limiting, indicating that a distal step, presumably pyruvate carboxylation, is activated. The action of octanoate in increasing gluconeogenesis has been found not to be related to a decreased flux through pyruvate dehydrogenase, neither to changes in the NAD redox potential nor to its ability to increase energy production. Actually, the oxygen uptake induced by octanoate was largely accounted for by the production of ketone body and the latter process was found to be independent of variations in energy demand.

Phenylephrine increases hepatic gluconeogenesis for as long as it is present in the extracellular medium. This effect is accompanied by a parallel increase in oxygen consumption. No apparent stoichiometric relationship exists between the phenylephrine-stimulated respiration and the energy required to meet the demands of gluconeogenesis. In the absence of extracellular calcium, no sustained stimulation of respiration was observed and phenylephrine failed to enhance gluconeogenesis; however, acute and transient effects of the alpha-adrenergic agonist were still observable. The following observations indicate that fatty acids are not involved in the alpha-adrenergic response: (1) the effects of phenylephrine and octanoate on respiration and gluconeogenesis were found to be additive; (2) unlike phenylephrine, octanoate is capable of stimulating gluconeogenesis in calcium-depleted liver; (3) in the absence of calcium, phenylephrine was incapable of further stimulating respiration or gluconeogenesis in the presence of octanoate. It is concluded that the conditions of increased lipid mobilization and/or oxidation are not sufficient to explain the metabolic response to alpha-adrenergic agonists. Fatty acids and alpha-adrenergic stimulation share a common role of stimulating gluconeogenesis in a manner dependent on their ability to stimulate respiration; however, the additive nature of their effects and distinct calcium requirements indicate that they act to trigger different mechanisms.

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.

Oxamate, structural analog of pyruvate, inhibits gluconeogenesis from pyruvate or substrates yielding pyruvate. The inhibitory effect is the result of a decreased mitochondrial pyruvate utilization. Although the inhibition of gluconeogenesis is competitive for pyruvate, in isolated mitochondria oxamate displays a mixed type kinetics inhibitory pattern of pyruvate utilization. Evidence is presented indicating that this mixed type pattern of inhibition is the result of the action of oxamate on two different sites: noncompetitive inhibition of pyruvate carboxylation, and competitive inhibition of pyruvate entry into the mitochondria. At concentrations of pyruvate above 0.4 mM, although pyruvate carboxylation is decreased by 40% by oxamate, no detectable effects on the gluconeogenic flux were observed. This finding strongly indicates that pyruvate carboxylase is not an important rate-limiting step for hepatic gluconeogenesis. Thus, the inhibition of gluconeogenesis at low pyruvate concentrations (less than 0.4 mM) seems to be the result of an interaction of oxamate with the mitochondrial pyruvate translocator, indicating that pyruvate transport across the mitochondrial membrane is the first nonequilibrium step in the gluconeogenic pathway when low physiological concentrations of this substrate are utilized.