Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

Substrate-dependent effect of 1-34 human parathyroid hormone fragment, dibutyryl cAMP and cAMP on gluconeogenesis in rabbit renal tubules

In the presence of 0.5 mM extracellular Ca2+ concentration both 1-34 human parathyroid hormone fragment (0.5 micrograms/ml) as well as 0.1 mM dibutyryl cAMP stimulated gluconeogenesis from lactate in renal tubules isolated from fed rabbits. However, these two compounds did not affect glucose synthesis from pyruvate as substrate. When 2.5 mM Ca2+ was present the stimulatory effect of the hormone fragment on gluconeogenesis from lactate was not detected but dibutyryl cAMP increased markedly the rate of glucose formation from lactate, dihydroxyacetone and glutamate, and inhibited this process from pyruvate and malate. Moreover, dibutyryl cAMP was ineffective in the presence of either 2-oxoglutarate or fructose as substrate. Similar changes in glucose formation were caused by 0.1 mM cAMP. As concluded from the 'crossover' plot the stimulatory effect of dibutyryl cAMP on glucose formation from lactate may result from an acceleration of pyruvate carboxylation due to an increase of intramitochondrial acetyl-CoA, while an inhibition by this compound of gluconeogenesis from pyruvate is likely due to an elevation of mitochondrial NADH/NAD+ ratio, resulting in a decrease of generation of oxaloacetate, the substrate of phosphoenolpyruvate carboxykinase. Dibutyryl cAMP decreased the conversion of fracture 1,6-bisphosphate to fructose 6-phosphate in the presence of both substrates which may be secondary to an inhibition of fructose 1,6-bisphosphatase.     

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.     

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

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

Substrate-dependent effect of 1–34 human parathyroid hormone fragment,dibutyryl cAMP and cAMP on gluconeogenesis in rabbit renal tubules

In the presence of 0.5 mM extracellular Ca²⁺ concentration both 1–34 human parathyroid hormone fragment (0.5 μg/ml) as well as 0.1 mM dibutyryl cAMP stimulated gluconeogenesis from lactate in renal tubules isolated from fed rabbits. However, these two compounds did not affect glucose synthesis from pyruvate as substrate. When 2.5 mM Ca²⁺ was present the stimulatory effect of the hormone fragment on gluconeogenesis from lactate was not detected but dibutyryl cAMP increased markedly the rate of glucose formation from lactate, dihydroxyacetone and glutamate, and inhibited this process from pyruvate and malate. Moreover, dibutyryl cAMP was ineffective in the presence of either 2-oxoglutarate or fructose as substrate. Similar changes in glucose formation were caused by 0.1 mM cAMP. As concluded from the ‘crossover’ plot the stimulatory effect of dibutyryl cAMP on glucose formation from lactate may result from an acceleration of pyruvate carboxylation due to an increase of intramitochondrial acetyl-CoA, while an inhibition by this compound of gluconeogenesis from pyruvate is likely due to an elevation of mitochondrial NADH/NAD⁺ ratio, resulting in a decrease of generation of oxaloacetate, the substrate of phosphoenolpyruvate carboxykinase. Dibutyryl cAMP decreased the conversion of fracture 1,6-bisphosphate to fructose 6-phosphate in the presence of both substrates which may be secondary to an inhibition of fructose 1,6-bisphosphatase.     

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [14C]pyruvate and [14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the 'cross-over' plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD+ ratio resulting in a decline of flux through lactate dehydrogenase.     

Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.     

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.     

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.     

Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.     

Interaction of oxamate with the gluconeogenic pathway in rat liver

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.     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

Control of gluconeogenesis in rat liver cells. I. Kinetics of the individual enzymes and the effect of glucagon

Control of gluconeogenesis from lactate was studied by titrating rat liver cells with lactate and pyruvate in a ratio of 10:1 in a perifusion system. At different steady states of glucose formation, the concentration of key gluconeogenic intermediates was measured and plotted against gluconeogenic flux (J glucose). Complete saturation was observed only in the plot relating J glucose to the extracellular pyruvate concentration. Measurement of pyruvate distribution in the cell showed that the mitochondrial pyruvate translocator operates close to equilibrium at high lactate and pyruvate concentrations. It can therefore be concluded that pyruvate carboxylase limits maximal gluconeogenic flux. Addition of glucagon did not cause a shift in the plots relating J glucose to glucose 6-phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, and phosphoenolpyruvate. It can thus be concluded that glucagon does not affect the kinetic parameters of the enzymes involved in the conversion of phosphoenolpyruvate to glucose. Addition of glucagon led to a shift in the curves relating J glucose to the concentration of cytosolic oxalacetate and extracellular pyruvate. The shift in the curve relating J glucose to oxalacetate is due to glucagon-induced inhibition of pyruvate kinase. The stimulation of gluconeogenesis by glucagon can be accounted for almost completely by inhibition of pyruvate kinase. There was almost no stimulation by glucagon of pyruvate carboxylation. In the absence of glucagon, control on gluconeogenesis from lactate is distributed among different steps including pyruvate carboxylase and pyruvate kinase. Assuming that in the presence of glucagon all pyruvate kinase flux is inhibited, the control of gluconeogenesis in the presence of the hormone is confined exclusively to pyruvate carboxylase.     

A study of regulation of gluconeogenesis and the supply of cytosolic reducing equivalents for lactate formation in rat kidney-cortical-tubule fragments incubated with pyruvate.

1. Tubule fragments were isolated after treatment of rat kidney cortex with collagenase. The formation of glucose and lactate on incubation with 5mM-pyruvate was then measured under various conditions. 2. When tubule fragments were isolated from fed rats in the absence of Ca2+ and then incubated with various Ca2+ concentrations, an incubation period of 15--30 min was necessary to establish a metabolic steady state. Under these conditions glucose formation was increased by Ca2+, adrenaline or 3':5'-cyclic AMP to a greater extent than was lactate formation. Data show that appreciable lactate formation could not have resulted from glycolytic metabolism of glucose formed by gluconeogenesis during incubation. 3. When tubule fragments were isolated from fed rats in the presence of 1.27 mM-Ca2+ and adjustments made to the Ca2+ concentration at the commencement of incubation, metabolic steady state was rapidly established. Under these conditions lactate formation was almost insensitive to Ca2+ concentration (0.16--4.5 mM), whereas glucose formation varied with Ca2+ concentration in a sigmoidal manner. 3':5'-Cyclic AMP decreased this sigmoidicity. 4. Ca2+ depletion of the tissue before incubation appeared to change permanently the relationship between extracellular Ca2+ concentration and the measured rates of metabolic processes. 5. Under conditions of metabolic steady state, glucose formation by tubule fragments from fed rats was less sensitive than lactate formation to inhibition by 3-mercaptopicolinate or 2-n-butylmalonate. Lactate formation by tubule fragments prepared from 48 h-starved rats was more sensitive to these inhibitors. 6. Estimates were made of the rate of futile cycling of C3 species through pyruvate kinase. This was greater in the starved than in the fed state, was decreased by 3':5'-cyclic AMP in both the fed and the starved state, but was unaffected by Ca2+. 7. These results suggested that formation of lactate and glucose is less tightly linked in kidney cortex than in liver. A considerable amount of the supply of reducing equivalents for lactate formation did not appear to be associated with an energy-dependent translocation from mitochondria to cytosol involving a pyruvate leads to oxaloacetate leads to phosphoenolpyruvate leads to pyruvate cycle.     

Effects of oleate,palmitate, and octanoate on gluconeogenesis in isolated rabbit liver cells

The effect of oleate, palmitate, and octanoate on glucose formation was studied with lactate or pyruvate as substrate. Octanoate was much more quickly oxidized and utilized for ketone body production than were oleate and palmitate. Among fatty acids studied, only octanoate resulted in a marked increase of the 3-hydroxybutyrate/acetoacetate (3-$\text{OHB}\text{AcAc}$) ratio. Each of the fatty acids studied stimulated glucose synthesis from pyruvate. The enhancement of gluconeogenesis by long-chain fatty acids was abolished after the addition of ammonia. As concluded from the “crossover” plot, the stimulatory effect of fatty acids was due to: (i) a stimulation of pyruvate carboxylation, (ii) a provision of reducing equivalents for glyceraldehyde phosphate dehydrogenase, and (iii) an acceleration of flux through hexose diphosphatase. Moreover, palmitate and oleate resulted in an increased generation of mitochondrial phosphpenolpyruvate, while in the presence of octanoate, the activity of mitochondrial phosphoenolpyruvate carboxykinase was diminished. When lactate was used as the glucose precursor, palmitate and oleate increased glucose production by about 50% but did not affect the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis. In contrast, in spite of the stimulation of both pyruvate carboxylase and hexose diphosphatase, as judged from the crossover plot, the addition of octanoate resulted in a marked inhibition of both glucose formation and mitochondrial generation of phosphoenolpyruvate. The inhibitory effect of octanoate was reversed by ammonia. Results indicate that fatty acids and ammonia are potent regulatory factors of both the rate of glucose formation and the contribution of mitochondrial phosphoenolpyruvate carboxykinase to gluconeogenesis in hepatocytes of the fasted rabbit.     

The interaction between the cytosolic pyridine nucleotide redox potential and gluconeogenesis from lactate/pyruvate in isolated rat hepatocytes. Implications for investigations of hormone action

By using very low concentrations of cells to minimize alterations in substrate concentrations, we demonstrated that the lactate/pyruvate ratio of the incubation medium, which determines the cytosolic NADH/NAD+ ratio, affects gluconeogenic flux in suspensions of isolated hepatocytes from fasted rats. At a fixed extracellular pyruvate concentration of 1 mM and with the lactate/pyruvate ratio varied from 0.6 to 10 and to 50, glucose production rates increased from 2.5 to 5.5 and then decreased to 1.8 nmol/mg of cell protein/min. This finding paralleled the observation of Sugano et al. (Sugano, T., Shiota, M., Tanaka, T., Miyamae, Y., Shimada, M., and Oshino, N. (1980) J. Biochem. (Tokyo) 87, 153-166) who noted a similar biphasic response in the perfused liver system when lactate was held constant and pyruvate varied. The biphasic relationship can be explained by the influence of the NADH/NAD+ ratio on the near-equilibrium reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase in the hepatocyte cytosol. By shifting the equilibrium of the glyceraldehyde-3-phosphate dehydrogenase reaction, a rise in the NADH/NAD+ ratio decreases the concentration of 3-phosphoglycerate which, because of the linkage of 3-phosphoglycerate to phosphoenolpyruvate through two near-equilibrium reactions, reduces the concentration of phosphoenolpyruvate and therefore causes a decline in flux through pyruvate kinase. This decrease in pyruvate kinase flux results in an enhanced gluconeogenic flux. At higher NADH/NAD+ ratios, however, the oxalacetate concentration drops to such an extent that the consequent decreased flux through phosphoenolpyruvate carboxykinase exceeds the decline in flux through pyruvate kinase, producing a decrease in gluconeogenic flux. The lactate/pyruvate ratio was found to influence the actions of three hormones thought to stimulate gluconeogenesis by different mechanisms. Except for an inhibition by glucagon seen at the lowest lactate/pyruvate ratio tested, the stimulations by this hormone were relatively insensitive to lactate/pyruvate ratios, while angiotensin II produced greater stimulations of gluconeogenesis as the lactate/pyruvate ratio was increased. Dexamethasone, added in vitro, stimulated gluconeogenesis significantly only at very low and very high lactate/pyruvate ratios.     

Inhibitory effect of gentamicin on gluconeogenesis from pyruvate, propionate, and lactate in isolated rabbit kidney-cortex tubules

The effect of gentamicin on glucose production in isolated rabbit renal tubules was studied with lactate, propionate, malate, 2-oxoglutarate, and succinate as substrates. This antibiotic at 5 mM concentration inhibited gluconeogenesis from lactate by about 60% and that from either pyruvate or propionate by about 30%. In contrast, it did not alter the rate of glucose formation from other substrates studied. The rate of gluconeogenesis was higher at 1 mM propionate than at increasing concentrations of this substrate and was stimulated in the presence of 1 mM carnitine. However, the addition of carnitine did not affect the degree of inhibition of glucose formation by gentamicin. Since the mitochondrial free coenzyme A level was significantly lower in the presence of 10 than 1 mM propionate and increased on the addition of carnitine to the reaction medium, the inhibitory effect of propionate concentrations above 1 mM on gluconeogenesis in rabbit renal tubules may be due to a depletion of the free mitochondrial coenzyme A level, resulting in an inhibition of the mitochondrial coenzyme A-dependent reactions. In intact rabbit kidney cortex mitochondria incubated in State 4 as well as in Triton X-100-treated mitochondria, 5 mM gentamicin inhibited by about 30-40% the incorporation of 14CO2 into both pyruvate and propionate. The results indicate that the inhibitory effect of gentamicin on glucose formation in isolated kidney tubules incubated with lactate, pyruvate, or propionate is likely due to a decrease of the rate of carboxylation reactions.     

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

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

Hepatic redox state and gluconeogenesis from lactate in vivo in the rat

1. To examine the role of the hepatic redox state on the rate of gluconeogenesis the effects of sodium crotonate injection (6mmol/kg body wt.) on rat liver metabolite concentrations and gluconeogenesis from lactate were studied in vivo. 2. Crotonate caused a marked oxidation of cytoplasmic and mitochondrial redox couples; decreases were observed in the ratios of [lactate]/[pyruvate], [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [hydroxybutyrate]/[acetoacetate] and measured [NAD(+)]/[NADH]. 3. Increases occurred in the liver concentrations of all gluconeogenic intermediates from pyruvate through to glucose 6-phosphate, but there was no change in lactate concentration. 4. To determine whether gluconeogenesis from lactate was altered by the more-oxidized hepatic redox state l-[2-(14)C]lactic acid was infused into the inferior vena cava (50mumol/min per kg body wt.) and the incorporation of radioactivity into blood glucose was measured. 5. Administration of crotonate transiently decreased the rate of lactate incorporation into glucose but within a few minutes the rate of incorporation returned to that of the controls. 6. The results indicate that in these experiments alteration of the NAD(+)-NADH systems of cytoplasm and mitochondria to a more-oxidized state did not change the rate of gluconeogenesis.     

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.