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Suramin is the drug of choice for the treatment of African trypanosomiasis and onchocerciasis. It is also tested for its potential use as an anticancer agent and chemosensitizer. As suramin has been reported to induce hyperglycaemia, its effect on glucose formation has been studied in isolated rabbit hepatocytes and kidney-cortex tubules. In contrast to hepatocytes, in kidney-cortex tubules suramin augments glucose production and decreases lactate formation. Suramin-induced changes in intracellular gluconeogenic/glycolytic intermediates indicate a decrease in flux through pyruvate-phosphoenolpyruvate step. Moreover, this compound diminishes pyruvate kinase activity in kidney-cortex cytosolic fraction, while fructose-1,6-bisphosphate ameliorates its inhibitory action. As (i) kidneys are important contributors to the whole body glucose homeostasis and (ii) suramin is known to accumulate in kidney, suramin-induced stimulation of glucose formation in renal tubules might be responsible for hyperglycaemia observed in patients undergoing suramin treatment.

Effects of various cAMP analogues on gluconeogenesis in isolated rabbit kidney tubules have been investigated. In contrast to N(6),2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) and cAMP, which accelerate renal gluconeogenesis, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) and 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) inhibit glucose production. Stimulatory action of cAMP and db-cAMP may be evoked by butyrate and purinergic agonists generated during their extracellular and intracellular metabolism resulting in an increase in flux through fructose-1,6-bisphosphatase and in consequence acceleration of the rate of glucose formation. On the contrary, Br-cAMP is poorly metabolized in renal tubules and induces a fall of flux through glyceraldehyde-3-phosphate dehydrogenase. The contribution of putative extracellular cAMP receptors to the inhibitory Br-cAMP action is doubtful in view of a decline of glucose formation in renal tubules grown in the primary culture supplemented with forskolin. The presented data indicate that in contrast to hepatocytes, in kidney-cortex tubules an increased intracellular cAMP level results in an inhibition of glucose production.

The intracellular glutathione redox state and the rate of glucose formation were studied in rabbit kidney-cortex tubules. In the presence of substrates effectively utilized for glucose formation, ie, aspartate + glycerol + octanoate, alanine + glycerol + octanoate, malate, or pyruvate, the intracellular reduced glutathione/oxidized glutathione (GSH/GSSG) ratios were significantly higher than those under conditions of negligible glucose production. Changes in the intracellular GSH/GSSG ratio corresponded to those in glucose-6-phosphate content and reduced nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP(+)) ratio obtained from malate/pyruvate measurements. Gluconeogenesis stimulation by extracellular adenosine triphosphate (ATP) or inosine caused an elevation of the intracellular GSH/GSSG and NADPH/NADP(+) ratios, as well as glucose-6-phosphate level. Surprisingly, in the presence of 5 mmol/L glucose, both the intracellular GSH/GSSG and NADPH/NADP(+) ratios and glucose-6-phosphate content were almost as low as under conditions of negligible glucose synthesis. L-buthionine sulfoximine (BSO)-induced decline in both the intracellular glutathione level and redox state resulted in inhibition of gluconeogenesis accompanied by accumulation of phosphotrioses and a decrease in fructose-1,6-bisphosphate content, while cysteine precursors altered neither GSH redox state nor the rate of glucose formation. In view of the data, it seems likely that: (1) intensive gluconeogenesis rather than extracellular glucose is responsible for maintaining a high intracellular GSH/GSSG ratio due to effective glucose-6-phosphate delivery for NADPH generation via the pentose phosphate pathway; (2) a decline in the intracellular glutathione level and/or redox state causes a decrease in glucose synthesis resulting from a diminished flux through aldolase; (3) induced by cysteine precursors, elevation of the intracellular GSH level does not affect the rate of glucose formation, probably due to no changes in the intracellular GSH/GSSG ratio.

The effects of extracellular purinergic agonists and their breakdown products on glucose and glutamine synthesis in rabbit kidney-cortex tubules incubated with aspartate + glycerol or alanine + glycerol + octanoate were investigated. A rapid extracellular degradation of ATP was accompanied by an accumulation of AMP, inosine, and hypoxanthine. Extracellular ATP and its breakdown products accelerated glucose synthesis in renal tubules, while ammonium released from adenine-containing compounds enhanced glutamine synthesis and diminished the degree of gluconeogenesis stimulation. In contrast to AMP and inosine, ATP evoked calcium signals, while both ATP and inosine decreased intracellular cAMP content and accelerated the flux through fructose-1,6-bisphosphatase as concluded from changes in gluconeogenic intermediates. Since (i) the activity of partially purified renal fructose-1,6-bisphosphatase was increased upon protein phosphatase-1 treatment and decreased following treatment of previously dephosphorylated enzyme with protein kinase A catalytic subunit and (ii) both 8-bromoadenosine 3',5'-cyclic monophosphate and 8-(4-chlorophenyltio)-cAMP inhibited renal glucose synthesis, it seems likely that in rabbit renal tubules ATP and inosine stimulate gluconeogenesis via cAMP decrease, which favors the appearance of a more active, dephosphorylated form of fructose-1,6-bisphosphatase, a key gluconeogenic enzyme., (Copyright 2002 Elsevier Science (USA).)

Effect of vanadyl acetylacetonate (VAc) and metformin on gluconeogenesis has been studied in isolated hepatocytes and kidney-cortex tubules of rabbit. Glucose formation from alanine+glycerol+octanoate, pyruvate or dihydroxyacetone was inhibited by 50-80% by 100 microM VAc or 500 microM metformin in renal tubules of control and alloxan-diabetic animals, while the inhibitory action of these compounds in hepatocytes was less pronounced (by about 20-30%). In contrast to VAc, metformin increased the rate of lactate formation by about 2-fold in renal tubules incubated with alanine+glycerol+octanoate. In view of VAc-induced changes in intracellular gluconeogenic intermediates and gluconeogenic enzyme activities, it is likely that this compound may decrease fluxes through pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase and glucose-6-phosphatase. In contrast to VAc, metformin-induced decrease in renal gluconeogenesis may result from a decline of cytosolic oxaloacetate level and consequently PEPCK activity. Following 6 days of VAc administration (1.275 mg Vkg(-1) body weight daily) the blood glucose level in alloxan-diabetic rabbits was normalised while blood glucose changes in control animals were not observed. On the contrary, in diabetic animals treated for 6 days with metformin (200 mg kg(-1) body weight day(-1)) a high blood glucose level was maintained. Unfortunately, VAc-treated control and diabetic rabbits exhibited elevated serum urea and creatinine levels. In VAc-treated animals vanadium was accumulated in kidney-cortex up to 7.6+/-0.6 microg Vg(-1) dry weight. In view of a potential vanadium nephrotoxicity a therapeutic application of vanadium compounds needs a critical re-evaluation.

In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-14C]alanine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in 14CO2 fixation and elevation of both cytosolic and mitochondrial NADH/NAD+ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.

The effect of 2-aminobicyclo[2.2.1]heptan-2-carboxylic acid (BCH), an L-leucine nonmetabolizable analogue and an allosteric activator of glutamate dehydrogenase, on glucose and glutamine synthesis was studied in rabbit renal tubules incubated with alanine, aspartate or proline in the presence of glycerol and octanoate, i.e. under conditions of efficient glucose formation. With alanine+glycerol+octanoate the addition of BCH resulted in a stimulation of alanine and glycerol consumption, accompanied by an increased glucose, lactate and glutamine synthesis. In contrast, when alanine was substituted by either aspartate or proline, BCH altered neither glucose formation nor glutamine and glutamate synthesis, while an accelerated glycerol utilization was accompanied by a small increase in lactate production. In view of the BCH-induced changes in intracellular metabolite levels the acceleration of gluconeogenesis by BCH in the presence of alanine+glycerol+octanoate is probably due to (i) increased uptake of alanine via alanine aminotransferase, (ii) stimulation of phosphoenolpyruvate carboxykinase, a key-enzyme of gluconeogenesis, (iii) rise of glucose-6-phosphatase activity, as well as (iv) activation of the malate-aspartate shuttle resulting in an augmented glycerol utilization for lactate and glucose synthesis.

The effect of chloroquine and other antimalarial drugs on glutamate dehydrogenase activity was studied in liver and renal mitochondria as well as in kidney-cortex tubules of rabbit. In permeabilized mitochondria, with free access of substrates and drugs to glutamate dehydrogenase, 100 microns chloroquine decreased both glutamate synthesis and glutamate deamination by about 70 and 50%, respectively. Ki value was equal to 49 microns in both liver and renal mitochondria. Other antimalarials (amodiaquine, quinacrine, chinidine and chinine) showed much smaller effect on the enzyme activity. Both ADP and L-leucine, allosteric activators of glutamate dehydrogenase, did not abolish the inhibitory action of chloroquine. Moreover, when added at 200 microns concentrations all drugs besides chinine suppressed glutamate formation in kidney-cortex tubules while chloroquine and quinacrine inhibited also glutamate deamination. Furthermore, chloroquine at 500 microns concentration decreased significantly [14C]glutamate transport into kidney-cortex mitochondria. In view of these observations it seems likely that chloroquine and some other antimalarials may inhibit the rate of glutamate metabolism in both liver and kidney-cortex causing hepatoxicity and nephrotoxicity. A possible action of chloroquine as an inhibitor of glutamate dehydrogenase in Plasmodium falciparum during the clinical treatment of malaria is discussed.

In isolated rabbit renal kidney-cortex tubules 2 mM glycerol, which is a poor gluconeogenic substrate, does not induce glucose formation in the presence of alanine, while it activates gluconeogenesis on substitution of alanine by aspartate, glutamate or proline. The addition of either 5 mM 3-hydroxybutyrate or 5 mM acetoacetate to renal tubules incubated with alanine + glycerol causes a marked induction of glucose production associated with inhibition of glutamine synthesis. In contrast, the rate of the latter process is not altered by ketones in the presence of glycerol and either aspartate, glutamine or proline despite the stimulation of glucose formation. Acceleration of gluconeogenesis by ketone bodies in the presence of amino acids and glycerol is probably due to (i) stimulation of pyruvate carboxylase activity, (ii) activation of malate-aspartate shuttle as concluded from elevated intracellular levels of malate, aspartate and glutamate, as well as (iii) diminished supply of ammonium for glutamine synthesis from alanine resulting from a decrease in glutamate dehydrogenase activity.

The effect of polyamines on glutamate deamination has been studied in both isolated tubules and permeabilized kidney cortex mitochondria of rabbit. Spermine, spermidine and putrescine resulted in a decrease of ammonium release in isolated renal tubules incubated with glutamate in the presence of MSO and AOA, inhibitors of glutamine synthetase and aminotransferases, respectively. This was not due to the inhibition of glutamate transport across renal tubular membranes since transport of [14C]glutamate into brush border membranes vesicles was not decreased by polyamines. In contrast, polyamines stimulated glutamate deamination in permeabilized mitochondria. This effect was additive to the action of ADP, an allosteric activator of glutamate dehydrogenase. Since these compounds decreased both glutamate-induced mitochondrial swelling as well as [14C]glutamate accumulation in mitochondria, the inhibitory effect of polyamines on glutamate deamination in renal tubules might be due to a diminished glutamate transport across the mitochondrial membrane.

In renal tubules isolated from fed rabbits, 1 mM aspartate is mainly utilized for production of glutamine, glutamate, alanine, and serine, while it is not used for glucose synthesis. However, the addition of either 2 mM glycerol or 2 mM lactate, which are poor gluconeogenic substrates in renal tubules, results in acceleration of both glucose formation and incorporation of [14C]aspartate into glucose by several fold, accompanied by about a twofold decrease in glutamine synthesis and marked accumulation of glutamate and alanine. Ammonium release in renal tubules incubated with aspartate in the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, is also decreased on the addition of glycerol and lactate by about two- and threefold, respectively. Since intracellular [glyceraldehyde 3-phosphate]/[3-phosphoglycerate], [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [lactate]/[pyruvate], and intramitochondrial [glutamate]/[2-oxoglutarate] x [NH4+] ratios are increased in comparison with control values determined with aspartate alone, it is likely that the stimulatory effect of lactate and glycerol on glucose formation from aspartate may be due to (i) an increased availability of reducing equivalents in the cytosol resulting in an enhancement of glyceraldehyde-3-phosphate dehydrogenase activity and (ii) elevation of the mitochondrial NADH/NAD- ratio causing a decrease in glutamate dehydrogenase activity resulting in a diminished glutamine synthesis and enhanced provision of carbon skeleton of aspartate for gluconeogenesis. Stimulation of glucose formation in the presence of 1 mM aspartate + glycerol is not related to cell volume changes. However, an increase for about 30% of intracellular water space induced by 10 mM aspartate + glycerol is accompanied by both diminished gluconeogenesis and enhanced glutamine synthesis, compared with values measured with 1 mM aspartate plus glycerol.

The effect of polyamines on glutamate dehydrogenase [L-glutamate: NAD(P) oxidoreductase (deaminating) [EC 1.4.1.3]) activity has been studied in both permeabilized kidney-cortex mitochondria and isolated renal tubules of rabbit. Spermidine was the most potent inhibitor of glutamate synthesis in permeabilized mitochondria resulting in about 80% decrease of the enzyme activity at 5 mM concentration. Putrescine, alpha-monofluoromethylputrescine (MFMP) and (R,R)-delta-methyl-alpha-acetylenic-putrescine (MAP) were more efficient than spermine. The inhibitory action of polyamines was potentiated by an elevated NADH content in the reaction mixture. Increasing concentrations of either NH4Cl, KCl or NaCl in the incubation medium resulted in a decrease of polyamine-induced inhibition of the enzyme activity, indicating that monovalent cations can compete with polyamines for the binding site at glutamate dehydrogenase. The inhibitory action of spermidine on glutamate synthesis was abolished by 2 mM ADP or 10 mM L-leucine, allosteric activators of the enzyme, as well as on the addition of either oxalate or sulphate at 20 mM concentrations. Spermidine did not affect glutamate formation when NADH was substituted by NADPH, suggesting an importance of the NADH binding to the inhibitory site of the enzyme for a decrease of reductive amination of 2-oxoglutarate by polyamine. Although spermidine did not influence glutamate deamination in the presence of NAD+, it stimulated this process by about 70% when NAD+ was substituted by NADP+. In the presence of ADP the stimulatory effect of polyamine was not significant. The data indicate that in permeabilized rabbit kidney-cortex mitochondria the effect of polyamines on both glutamate formation and glutamate deamination via the reaction catalysed by glutamate dehydrogenase is dependent upon the coenzyme utilized by the enzyme. In the presence of NADH their inhibitory effect on the glutamate formation may be alleviated by allosteric activators of the enzyme, and concentrations of potassium, sodium, sulphate and oxalate. In isolated rabbit renal tubules incubated with 5 mM methionine sulfoximine and aminooxyacetate, in order to inhibit glutamine synthetase and aminotransferases, respectively, 5 mM spermidine decreased glutamate formation by about 30%, while putrescine and spermine did not significantly diminish the enzyme activity. In the presence of octanoate glutamate formation was reduced by about 30% by naturally occurring polyamines as well as MFMP and MAP, indicating that under these conditions NADH rather than NADPH is utilized as the coenzyme. In view of these data it is possible to suggest that polyamines may be of importance to control glutamate dehydrogenase activity under physiological conditions.

The effect of gentamicin on both glutamate synthesis and glutamate deamination was studied in kidney-cortex mitochondria and tubules isolated from both control and gentamicin-treated animals. In kidney-cortex mitochondria which were permeabilized in order to make a free access of substrates and antibiotic to the glutamate dehydrogenase, gentamicin appeared to be a very potent inhibitor of glutamate synthesis, resulting in about 60% decrease of the enzyme activity at 5 mM concentration. Other aminoglycoside antibiotics decreased the enzymatic activity, in the following order: gentamicin > neomycin = tobramycin = kanamycin > biodacyna > amikacin > streptomycin. This, in principle, corresponds to their known nephrotoxic potential observed in vivo. The inhibitory action of antibiotics was abolished by neither ADP nor leucine, allosteric activators of glutamate dehydrogenase. Surprisingly, gentamicin did not decrease the rate of ammonia formation from glutamate when added to both renal tubules and mitochondria isolated from control rabbits. This indicates that the antibiotic exerts its inhibitory effect on glutamate dehydrogenase activity in the direction of glutamate synthesis only. In contrast, the rate of both glutamate deamination and glutamate synthesis was about 40% lower in renal tubules and mitochondria isolated from kidney-cortex of animals which were given antibiotics for 10 days. In view of these results it seems that (i) the depression of ammoniagenesis in gentamicin-treated animals may be due to a decrease of glutamate dehydrogenase content and (ii) under conditions in vitro the aminoglycoside inhibits the enzyme activity in the direction of glutamate synthesis while it does not affect the glutamate deamination.

Rabbits were given gentamicin over a period of 10 days. At 1, 3, 5 and 10 days renal proximal tubules were isolated and glucose synthesis from several substrates was measured. A relationship between the inhibition of renal gluconeogenesis, accompanied by a decline of both pyruvate carboxylase and phosphoenopyruvate carboxykinase (PEPCK) activities, and an increased gentamicin level in kidney-cortex was noticed after 5 days of therapy. Both the rates of glucose formation from various substrates as well as pyruvate carboxylase and the cytosolic PEPCK activity recovered fully within 3 weeks after cessation of antibiotic treatment while an increase of activity of the mitochondrial PEPCK occurred during chronic administration of the drug for 10 days. It is concluded, that gentamicin-induced inhibition of gluconeogenesis is one of the events occurring during complex action of this drug on renal cortex.

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.

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.

In kidney cortex tubules isolated from fed rabbits L-alanine is not utilized as glucose precursor, when added as a sole substrate. However, this amino acid decreases gluconeogenesis from low (up to 1 mM) 2-oxoglutarate concentrations and stimulates this process at higher (2.5-10 mM) ketoacid contents in the suspension medium. Aminooxyacetate, an inhibitor of aminotransferases, abolishes both inhibitory and stimulatory effects of L-alanine on glucose formation. The addition of 2-oxoglutarate increases the incorporation of L-[U-14C]alanine to glucose from 8- to 123-fold, depending upon the ketoacid and alanine concentrations used. In contrast, nonlabelled L-alanine decreases the incorporation of low [U-14C)2-oxoglutarate concentrations into glucose, while it does not affect contribution of 5 mM ketoacid to gluconeogenesis. The data indicate that (i) in the presence of 2-oxoglutarate L-alanine is utilized as glucose precursor in rabbit renal tubules and (ii) this amino acid may decrease the contribution of low extracellular concentrations of the ketoacid to gluconeogenesis.

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.

The effect of octanoate, palmitate and oleate on glucose formation was studied with lactate, pyruvate or malate as substrate in kidney tubules isolated from fasted rabbits. All fatty acids studied inhibited the rate of glucose production by about 30-50% depending on the glucose precursor and fatty acid used, stimulated the oxygen uptake by about 50% and increased the mitochondrial NADH/NAD+ ratio, as manifested by a marked rise of 3-hydroxybutyrate/acetoacetate ratio. Octanoate was twice as quickly utilized for ketone body production than palmitate and oleate were. As concluded from the 'crossover' plot the inhibitory effect of fatty acids on gluconeogenesis in rabbit kidney tubules may be due to: (i) a decrease of mitochondrial generation of phosphoenolpyruvate and (ii) an inhibition of flux through fructose-1,6-bisphosphatase.