Interrelations between ureogenesis and gluconeogenesis in isolated hepatocytes. The role of antion transport and the competition for energy.

1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.     

Accumulation of amino acids by the perfused rat liver in the presence of ethanol

1. The rate of gluconeogenesis from alanine in the perfused rat liver is affected by the presence of other metabolizable substances, especially fatty acids, ornithine and ethanol. Gluconeogenesis is accelerated by oleate and by ornithine. When both oleate and ornithine were present the acceleration was greater than expected on the basis of mere additive effects. 2. Much NH(3) and some urea were formed from alanine when no ornithine was added. With ornithine almost all the nitrogen released from alanine appeared as urea. 3. Lactate was a major product of alanine metabolism. Addition of oleate, and especially of oleate plus ornithine, decreased lactate formation. 4. Ethanol had no major effect on gluconeogenesis from alanine when this was the sole added precursor. Gluconeogenesis was strongly inhibited (87%) when oleate was also added, but ethanol greatly accelerated gluconeogenesis when ornithine was added together with alanine. 5. In the absence of ethanol the alanine carbon and alanine nitrogen removed were essentially recovered in the form of glucose, lactate, pyruvate, NH(3) and urea. 6. In the presence of ethanol the balance of both alanine carbon and alanine nitrogen showed substantial deficits. These deficits were largely accounted for by the formation of aspartate and glutamine, the formation of which was increased two- to three-fold. 7. When alanine was replaced by lactate plus NH(4)Cl, ethanol also caused a major accumulation of amino acids, especially of aspartate and alanine. 8. Earlier apparently discrepant results on the effects of ethanol on gluconeogenesis from alanine are explained by the fact that under well defined conditions ethanol can inhibit, or accelerate, or be without major effect on the rate of gluconeogenesis. 9. It is pointed out that in the synthesis of urea through the ornithine cycle half of the nitrogen must be supplied in the form of asparate and half in the form of carbamoyl phosphate. The accumulation of aspartate and other amino acids suggests that ethanol interferes with the control mechanisms which regulate the stoicheiometric formation of aspartate and carbamoyl phosphate.     

Proline metabolism in isolated rat liver cells

The metabolism of proline was studied in liver cells isolated from starved rats. The following observations were made. 1. Consumption of proline could be largely accounted for by production of glucose, urea, glutamate and glutamine. 2. At least 50% of the total consumption of oxygen was used for proline catabolism. 3. Ureogenesis and gluconeogenesis from proline could be stimulated by partial uncoupling of oxidative phosphorylation. 4. Addition of ethanol had little effect on either proline uptake or oxygen consumption, but strongly inhibited the production of both urea and glucose and caused further accumulation of glutamate and lactate. Accumulation of glutamine was not affected by ethanol. 5. The effects of ethanol could be overcome by partial uncoupling of oxidative phosphorylation. 6. The apparent K_m values of argininosuccinate synthetase (EC 6.3.4.5) for aspartate and citrulline in the intact hepatocyte are higher than those reported for the isolated enzyme. 7. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.32), greatly enhanced cytosolic aspartate accumulation during proline metabolism, but inhibited urea synthesis. 8. It is concluded that when proline is provided as a source of nitrogen to liver cells, production of ammonia by oxidative deamination of glutamate is inhibited by the highly reduced state of the nicotinamide nucleotides within the mitochondria. 9. Conversion of proline into glucose and urea is a net-energy-yielding process, and the high state of reduction of the nicotinamide nucleotides is presumably maintained by a high phosphorylation potential. Thus when proline is present as sole substrate, the further oxidation of glutamate by glutamate dehydrogenase (EC 1.4.1.3) is limited by the rate of energy expenditure of the cell.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.     

Hepatic gluconeogenesis in chickens

Summary Gluconeogenesis by isolated hepatocytes resulted in glucose release but insignificant rates of glycogen synthesis. The effectiveness of precursors was similar for hepatocytes from fed and starved chickens except for impaired gluconeogenesis from pyruvate when compared to lactate in lactate in starved chicken hepatocytes. The impairment was caused by limitations in cytosolic NADH production as a result of the mitochondrial location of phosphoenolpyruvate carboxykinase in chicken liver. The order of effectiveness of precursors on hepatic gluconeogenesis was generally similar to the effects of precursors on increasing the plasma glucose concentration in vivo. The exceptions were caused by interactions with other precursors in vivo.The alteration of the NADH/NAD⁺ ratio by ethanol and ATP/ADP ratio by adenosine could play significant roles in the control of precursor conversion to glucose. Physiological glucagon concentrations stimulated gluconeogenesis from precursors entering the pathway both above and below the level of triose phosphates, and its effect were mimicked by dibutyryl cyclic AMP.Previous results on the effects of precursor and glucagon injection on the plasma glucose concentration of chickens in vivo can largely be explained by effects at the hepatic level.Isolated chicken and rat hepatocytes share many common features. Qualitatively the ordering of gluconeogenic effectiveness was similar but quantitive differences existed as a result of differing activities and cellular locations of enzymes. Neither preparation readily synthesised glycogen and the sensitivity to glucagon was similar.     

Effect of epidermal growth factor (EGF) on gluconeogenesis in isolated rat hepatocytes. Dependency on the red-ox state of the substrate

It was found that EGF decreased both the basal- and the glucagon-stimulated gluconeogenesis from lactate alone or from a high lactate/pyruvate ratio and that it enhanced both the basal- and the glucagon-inhibited glucose synthesis from pyruvate alone or from a low lactate/pyruvate ratio. These findings demonstrate that the effect of both EGF and glucagon on glucose production by isolated hepatocytes depends on the red-ox state of the substrate.     

Effect of ornithine and lactate on urea synthesis in isolated hepatocytes.

1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline.     

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.     

Gluconeogenesis by isolated guinea-pig liver parenchymal cells

1. Guinea-pig hepatocytes were prepared by collagenase digestion of the perfused liver. 2. The highest rates of gluconeogenesis were obtained from fructose, followed by pyruvate, xylitol and lactate, glycerol and propionate in that order. Maximum rates of gluconeogenesis were attained at 6-10mm substrate. 3. An initial 15-min lag period occurred during gluconeogenesis from lactate. This lag was abolished by preincubating the cells or by preincubation plus the addition of NH(4)Cl or lysine. 4. The lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were increased during the lag and adjusted to values favouring rapid gluconeogenesis from lactate after 15min. 5. The data suggest that the low glucose synthesis during the lag resulted from a limitation of the glutamate-aspartate shuttle and from the unusual redox state of the NAD(+) couple prevailing during this period. 6. At 0.1mm, amino-oxyacetate, a transaminase inhibitor, decreased gluconeogenesis from lactate by 80%, but had a negligible effect on glucose production from pyruvate. Gluconeogenesis from lactate was also inhibited (20%) by 10mm-dl-3-hydroxybutyrate.     

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Summary Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.Incubating eel hepatocytes with 10 mM substrates, the following results were obtained: glycerol, alanine and lactate, in that order, were effective gluconeogenic substrates; these three substrates reduced glucose release from glycogen stores, while aspartate had no such effect; lactate, alanine and aspartate led to high rates of glycerol production, with subsequent incorporation into lipid; incorporation into glycogen was low from all substrates; and, alanine oxidation was seven times higher than that observed with other substrates.When eel hepatocytes were incubated with low or physiological substrate concentrations gluconeogenic rates from lactate were twice those from alanine; rates from aspartate were very low. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis, and had no significant effect on glycogenolysis. Cortisol increased gluconeogenic rates from 1 mM lactate.Thus, in the presence of adequate substrate, eel liver gluconeogenesis is preferentially stimulated relative to glycogenolysis to produce plasma glucose. These data support three important roles for gluconeogenesis: the recycling of muscle lactate, the synthesis of glucose from dietary amino acids to supplement glucose levels, and the production of glycerol for lipogenesis.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

Induction of rat kidney gluconeogenesis during acute liver intoxication by carbon tetrachloride.

1. Glucose production from L-lactate was completely inhibited 24h after carbon tetrachloride treatment in liver from 48h-starved rats. The activities of phosphoenolpyruvate carboxykinase, fructose diphosphatase and glucose 6-phosphatase were decreased by this treatment in fed and starved rats, whereas lactate dehydrogenase activity was only decreased in fed animals. 2. The production of glucose by renal cortical slices from fed rats previously treated with carbon tetrachloride was enhanced when L-lactate, pyruvate and glutamine but not fructose were used as glucose precursors. Renal phosphoenolpyruvate carboxykinase activity was increased in this condition. 3. This increase was counteracted by cycloheximide or actinomycin D, suggesting that the effect was due to the synthesis de novo of the enzyme. 4. The pattern of hepatic gluconeogenic metabolites in treated animals was characterized by an increase in lactate, pyruvate, malate and citrate as well as a decrease in glucose 6-phosphate, suggesting an impairment of liver gluconeogenesis in vivo. 5. In contrast, the profile of renal metabolites suggested that gluconeogenesis was operative in the treated rats, as indicated by the marked increase in the content of phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate and glucose 6-phosphate. 6. It is postulated that renal gluconeogenesis could contribute to the maintenance of glycaemia in carbon tetrachloride-treated rats.     

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.     

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.     

Stimulation of gluconeogenesis by somatostatin in rat kidney cortex slices.

The effect of somatostatin on gluconeogenesis was studied in kidney cortex slices. Addition of somatostatin (2 μg) stimulated gluconeogenesis from lactate, pyruvate and glutamine by 42%, 50% and 68% respectively. Stimulation of glucose synthesis from lactate by somatostatin was found to be linear with time and dose dependent between 0.1 and 20 μg. Somatostatin-stimulated gluconeogenesis was inhibited by phentolamine (10 μM) but not by propranolol (10 μM) suggesting that somatostatin action is mediated by α-adrenergic stimuli.     

Effect of alloxan-diabetes on gluconeogenesis and ureogenesis in isolated rabbit liver cells.

1. Neither alloxan-diabetes nor starvation affected the rate of glucose production in hepatocytes incubated with lactate, pyruvate, propionate or fructose as substrates. In contrast, glucose synthesis with either alanine or glutamine was increased nearly 3- and 12-fold respectively, in comparison with that in fed rabbits. 2. The addition of amino-oxyacetate resulted in about a 50% decrease in glucose formation from lactate in hepatocytes isolated from fed, alloxan-diabetic and starved rats, suggesting that both mitochondrial and cytosolic forms of rabbit phosphoenolpyruvate carboxykinase function actively during gluconeogenesis. 3. Alloxan-diabetes resulted in about 2-3-fold stimulation of urea production from either amino acid studied or NH4Cl as NH3 donor, whereas starvation caused a significant increase in the rate of ureogenesis only in the presence of alanine as the source of NH3. 4. As concluded from changes in the [3-hydroxybutyrate]/[acetoacetate] ratio, in hepatocytes from diabetic animals the mitochondrial redox state was shifted toward oxidation in comparison with that observed in liver cells isolated from fed rabbits.     

Gluconeogenesis in the perfused rat liver

1. A modification of the methods of Miller and of Schimassek for the perfusion of the isolated rat liver, suitable for the study of gluconeogenesis, is described. 2. The main modifications concern the operative technique (reducing the period of anoxia during the operation to 3min.) and the use of aged (non-glycolysing) red cells in the semi-synthetic perfusion medium. 3. The performance of the perfused liver was tested by measuring the rate of gluconeogenesis, of urea synthesis and the stability of adenine nucleotides. Higher rates of gluconeogenesis (1mumole/min./g.) from excess of lactate and of urea synthesis from excess of ammonia (4mumoles/min./g. in the presence of ornithine) were observed than are likely to occur in vivo where rates are limited by the rate of supply of precursor. The concentrations of the three adenine nucleotides in the liver tissue were maintained within 15% over a perfusion period of 135min. 4. Ca(2+), Na(+), K(+), Mg(2+) and phosphate were found to be required at physiological concentrations for optimum gluconeogenesis but bicarbonate and carbon dioxide could be largely replaced by phosphate buffer without affecting the rate of gluconeogenesis. 5. Maximal gluconeogenesis did not decrease maximal urea synthesis in the presence of ornithine and ammonia and vice versa. This indicates that the energy requirements were not limiting the rates of gluconeogenesis or of urea synthesis. 6. Addition of lactate, and especially ammonium salts, increased the uptake of oxygen more than expected on the basis of the ATP requirements of the gluconeogenesis and urea synthesis.     

Gluconeogenesis in goat hepatocytes is affected by calcium, ammonia and other key metabolites but not primarily through cytosolic redox state

1. Gluconeogenesis from propionate and lactate was studied in caprine hepatocytes. 2. Reducing cytosol with additions of ETOH, ammonium, or lactate decreased [2-14C]propionate conversion to glucose. 3. Calcium oxidized the cytosol and increased gluconeogenesis from propionate by 198% and from lactate by 220%. 4. Cells isolated from lactating does and wethers differed quantitatively in propionate conversion to glucose and response to calcium. 5. Acetoacetate decreased and 3-OH-butyrate slightly increased glucose production from propionate. 6. Neither ketone body had any significant effect on gluconeogenesis from lactate. 7. Results reported herein suggest gluconeogenesis from propionate is not limited by lack of cytosolic reducing equivalents.     

Role of fructose 2,6-bisphosphate in the control by glucagon of gluconeogenesis from various precursors in isolated rat hepatocytes.

Hepatocytes from overnight-starved rats were incubated with 1-20 mM-fructose, -dihydroxyacetone, -glycerol, -alanine or -lactate and -pyruvate with or without 0.1 microM-glucagon. The production of glucose and lactate was measured, as was the content of fructose 2,6-bisphosphate. The concentrations of fructose (below 5 mM) and dihydroxyacetone (above 1 mM) that gave rise to an increase in fructose 2,6-bisphosphate were those at which a glucagon effect on the production of glucose and lactate could be observed. Glycerol had no effect on fructose 2,6-bisphosphate content or on production of lactate, and glucagon did not stimulate the production of glucose from this precursor. With alanine or lactate/pyruvate as substrates, glucagon stimulated glucose production whether the concentration of fructose 2,6-bisphosphate was increased or not. The extent of inactivation of pyruvate kinase by glucagon was not affected by the presence of the various gluconeogenic precursors. The role of fructose 2,6-bisphosphate in the effect of glucagon on gluconeogenesis from precursors entering the pathway at the level of triose phosphates or pyruvate is discussed.     

Role of adenosine monophosphate in regulation of metabolic pathways of perfused rat liver

1. By perfusion of rat livers with 3mm-AMP in the perfusion medium we obtain increased intracellular concentrations of AMP. 2. These high intracellular concentrations of AMP lead to an increased output of glucose and urea into the perfusion medium. 3. The increased output of glucose in livers from fed rats is brought about primarily by an AMP-stimulated breakdown of liver glycogen. In livers from starved rats the increase in glucose output is not as great, reflecting the low contents of glycogen in livers from starved rats. 4. AMP inhibits gluconeogenesis from lactate in perfused livers. In the presence of high concentrations of lactate, however, the counteracting effects of AMP to increase glycogenolysis and to inhibit gluconeogenesis result in little change in the net glucose output. 5. The increased urea output is brought about by increased breakdown of amino acids that are present in the perfusion medium. In livers from starved rats the overall urea production is much higher, indicating increased catabolism of amino acids and other nitrogenous substrates in the absence of carbohydrate substrates. 6. AMP causes an inhibition of incorporation of labelled precursors into protein and nucleic acid. This may result from increased catabolism of precursors of proteins and nucleic acids as reflected by the more rapid breakdown of nitrogenous compounds. In support of this hypothesis, cell-free systems for amino acid incorporation isolated from livers perfused with and without AMP are equally capable of supporting protein synthesis. 7. The labelling pattern of RNA in perfused livers corresponds very closely to those found by pulse-labelling in vivo. AMP in no way alters the qualitative nature of the labelling patterns. 8. We consider these results as supporting evidence for the role of the concentration ratio of AMP to ATP in controlling the metabolic pathways that lead to the formation of ATP.     

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.