Pyruvate cycling involving possible oxaloacetate decarboxylase activity

With high concentrations of pyruvate as substrate for hepatocytes from fasted rats, high rates of cycling between pyruvate and the dicarboxylic acids occur, as shown isotopically. This rate of cycling is adequate to account for the hydrogen translocation from the mitochondria to the cytosol to furnish NADH for lactate formation. Addition of sufficiently high concentrations of mercaptopicolinate to block almost completely glucose formation from pyruvate, depresses isotopic cycling and lactate formation by only about 50–75%. Under some conditions, when the normal phosphoenolpyruvate carboxykinase activity is inhibited, cytosolic oxaloacetate may be decarboxylated directly to pyruvate, possibly via the decarboxylase activity of phosphoenolpyruvate carboxykinase.     

3-Aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes and increases release of gluconeogenic precursors from peripheral tissues

3- Aminopicolinate , a hyperglycemic agent that activates purified phosphoenolpyruvate carboxykinase in the presence of Fe2+, inhibits glucose synthesis from lactate, pyruvate, asparagine, monomethyl succinate, or glutamine but does not affect that from fructose, dihydroxyacetone, sorbitol, or glycerol in hepatocytes isolated from rats fasted for 24 h. Lactate production from monomethyl succinate by hepatocytes is also inhibited by 3- aminopicolinate . This compound elevates the concentrations of pyruvate, malate, and aspartate but decreases that of phosphoenolpyruvate in hepatocytes incubated with lactate plus pyruvate. In rats, the ability of 3- aminopicolinate to elevate blood glucose concentration is unimpaired by renalectomy . The drug does not significantly affect glycemia in functionally hepatectomized rats but accelerates blood lactate and pyruvate accumulation to higher maximum concentrations even when kidney function is also ablated. It is concluded that 3- aminopicolinate inhibits phosphoenolpyruvate carboxykinase in hepatocytes, that the reported stimulation of renal glutaminase and glutamine gluconeogenesis by this compound does not contribute significantly to its hyperglycemic property, and that the drug increases gluconeogenic substrate supply from peripheral tissues.     

Bcl-2 inhibits tumor necrosis factor-alpha-mediated increase of glycolytic enzyme activities and enhances pyruvate carboxylase activity

To understand the effects of bcl-2 on glucose metabolism and tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity, the activities of glycolytic enzymes (hexokinase, 6-phosphofructo-1-kinase, and pyruvate kinase), lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were examined with or without TNF-alpha treatment in TNF-alpha sensitive L929 cells and TNF-alpha resistant bcl-2 transfected L929 cells. In TNF-alpha-treated L929 cells, the activities of the glycolytic enzymes and lactate dehydrogenase greatly increased, but there was no detectable change in phosphoenolpyruvate carboxykinase. Pyruvate carboxylase activity decreased by about 25% between 6 and 12 h after TNF-alpha treatment. The activities of the glycolytic enzymes and lactate dehydrogenase in bcl-2 transfected L929 cells were lower than in L929 cells upon TNF-alpha treatment. On the other hand, the activity of pyruvate carboxylase was 20-100% greater after 6 h of TNF-alpha treatment than in the L929 cells. The activity of phosphoenolpyruvate carboxykinase of bcl-2 trasfected L929 cells was lower by up to 25% than in L929 cells after 12 h. The increase of pyruvate carboxylase activity and decrease of phosphoenolpyruvate carboxykinase activity in bcl-2 transfected L929 cells may contribute to the protective effects of bcl-2 against TNF-alpha mediated cytotoxicity.     

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.     

Regulation of glucose formation from lactate and pyruvate in isolated tubules of chicken kidney.

1. Isolated kidney tubules from chicken have been used to study the actions of ethanol, ouabain and aminooxyacetate on glucose formation from lactate and pyruvate. 2. In kidney tubules from well-fed chickens the rate of glucose production from lactate was higher than from pyruvate. Ethanol (10 mM) and ouabain (0.1 mM) were found to increase glucose formation from pyruvate but not from lactate. 3. It is concluded that in the presence of ethanol the fluxes of pyruvate through pyruvate dehydrogenase are in favour of the pyruvate carboxylase reaction restricted. 4. Glucose formation from lactate is decreased by aminooxyacetate (0.1 mM) and ouabain (0.1 mM). 5. Aminooxyacetate inhibited glucose formation from lactate, although chicken phosphoenolpyruvate carboxykinase is located intramitochondrially. 6. The results indicate that the effect of aminooxyacetate like that of ouabain is caused by the restricted formation of pyruvate.     

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.     

Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver.

A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors and gluconeogenic intermediates at near-physiological concentrations was shown to form hexose 6-phosphates linearly from lactate + pyruvate + glutamate at a rate of 0.82 +/- 0.05 mumol/min per g of liver (mean +/- S.E.M., n = 8, 37 degrees C). The indicated rates were measured between 20 min and 60 min incubation time, when the system was near steady state. Experiments with either [1-14C]lactate or [U-14C]glutamate revealed that the incorporation of radioactive label into hexose 6-phosphates was proportional to the utilization of lactate + pyruvate and of glutamate during incubation and that both served as gluconeogenic substrates at a ratio of about 2:1. When the [ATP]/[ADP] ratio was lowered from 60 to 19 by addition of ATPase, the rate of hexose 6-phosphate formation fell to one-third. This decrease in gluconeogenic flux was mainly due to a decreased flow through the phosphoglycerate kinase step. Hexose 6-phosphate formation could also be decreased by increasing the ratio [NADH]/[NAD+], either by addition of ethanol or by increasing the initial concentration of lactate + pyruvate at a fixed ratio of 10:1. The observed inhibition was linked to a limitation in the availability of oxaloacetate for the phosphoenolpyruvate carboxykinase reaction and to an increased formation of sn-glycerol 3-phosphate. Finally, the rates of hexose 6-phosphate formation in incubations with cytosols from fed rats were only 50% of those observed with cytosols from animals starved for 48 h. One of the limiting steps was found to be the flow through the phosphoenolpyruvate carboxykinase step.     

Induction in primary culture of 'gluconeogenic' and 'glycolytic' hepatocytes resembling periportal and perivenous cells

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1. Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity 'periportal'. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply 'perivenous'. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2. Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3. Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 muM dexamethasone) gluconeogenesis was 1.5-fold faster in the 'periportal' cells and glycolysis was 2.4-fold faster in the 'perivenous' cells. 4. Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in 'periportal' cells containing high phosphoenolpyruvate carboxykinase activity, not in the 'perivenous' cells with a low level of this enzyme. 5. A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in 'periportal' cells and glycolysis was eightfold faster in 'perivenous' cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.     

Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus)

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.     

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

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

Intracellular distribution of some enzymes of the glutamine utilisation pathway in rat lymphocytes

In lymphocytes of the rat, pyruvate kinase, phosphoenolpyruvate carboxykinase and NADP+-linked malate dehydrogenase (decarboxylating) are distributed almost exclusively in the cytosol whereas pyruvate carboxylase is distributed almost entirely in the mitochondria. For NAD+-linked malate dehydrogenase and aspartate aminotransferase approximately 80% and 40%, respectively, are in the cytosolic compartment. Since glutaminase is present in the mitochondria, glutamine is converted to malate within the mitochondria but further metabolism of the malate is likely to occur in the cytosol. Hence pyruvate produced from this malate, via oxaloacetate and phosphoenolpyruvate carboxykinase, may be rapidly converted to lactate, so restricting the entry of pyruvate into the mitochondria and explaining why very little glutamine is completely oxidised in these cells despite a high capacity of the Krebs cycle.     

Estimation of the relative contributions of enhanced production of oxalacetate and inhibition of pyruvate kinase to acute hormonal stimulation of gluconeogenesis in rat hepatocytes. An analysis of the effects of glucagon, angiotensin II, and dexamethasone on gluconeogenic flux from lactate/pyruvate

Hepatocytes, isolated from fasted rats, were incubated with graded concentrations of lactate and pyruvate, at a mean constant ratio of 10-13:1, to alter systematically the concentrations of gluconeogenic intermediate metabolites and rates of glucose production. By analyzing glucose production rates as a function of corresponding concentrations of extracellular pyruvate, cytosolic oxalacetate, and cellular 3-phosphoglycerate in the presence and absence of hormones and assuming no primary activation of phosphoenolpyruvate carboxykinase, estimates were made of the relative contributions of stimulation of formation of cytosolic oxalacetate and inhibition of pyruvate kinase to hormonal stimulations of gluconeogenesis. Addition of dexamethasone, glucagon, or angiotensin II did not cause a shift in the relationship between cellular 3-phosphoglycerate concentrations and rates of glucose production, indicating that there was no effect of these agents on the reactions involved in conversion of phosphoenolpyruvate to glucose. All three agents shifted the relationships between rates of glucose production and both cytosolic oxalacetate and extracellular pyruvate. The following conclusions were drawn from computer analyses of these results. At low concentrations of pyruvate, stimulation of oxalacetate production and pyruvate kinase inhibition were approximately equally contributory to the overall stimulations of gluconeogenesis by angiotensin II and dexamethasone. At higher pyruvate concentrations, pyruvate kinase inhibition by angiotensin II played a greater role, accounting for 90% of the overall stimulation. For dexamethasone, as the pyruvate concentration was increased, stimulation of gluconeogenesis resulting from enhanced formation of oxalacetate diminished as did overall stimulation of gluconeogenesis. Glucagon addition resulted in an inhibition of pyruvate kinase flux that accounted for 75% of the hormone's overall effect at low pyruvate concentrations; this increased to 95% at high pyruvate concentrations.     

Synthesis of phosphoenolpyruvate from propionate in sheep liver

1. Utilization of propionate by sheep liver mitochondria was stimulated equally by pyruvate or alpha-oxoglutarate, with formation predominantly of malate. Pyruvate increased conversion of propionate carbon into citrate, whereas alpha-oxoglutarate increased formation of phosphoenolpyruvate. The fraction of metabolized propionate converted into phosphoenolpyruvate was about 17% in the presence or absence of alpha-oxoglutarate and about 7% in the presence of pyruvate. Pyruvate consumption was inhibited by 80% by 5mm-propionate. 2. Compared with rat liver, sheep liver was characterized by very high activities of phosphoenolpyruvate carboxykinase and moderately high activities of aconitase in the mitochondria and by low activities of ;malic' enzyme, pyruvate kinase and lactate dehydrogenase in the cytosol. Activities of phosphoenolpyruvate carboxy-kinase were similar in liver cytosol from rats and sheep. Activities of malate dehydrogenase and NADP-linked isocitrate dehydrogenase in sheep liver were about half those in rat liver. 3. The phosphate-dicarboxylate antiport was active in sheep liver mitochondria, but compared with rat liver mitochondria the citrate-malate antiport showed only low activity and mitochondrial aconitase was relatively inaccessible to external citrate. The rate of swelling of mitochondria induced by phosphate in solutions of ammonium malate was inversely related to the concentration of malate. 4. The results are discussed in relation to gluconeogenesis from propionate in sheep liver. It is proposed that propionate is converted into malate by the mitochondria and the malate is converted into phosphoenolpyruvate by enzymes in the cytosol. In this way sufficient NADH would be generated in the cytosol to convert the phosphoenolpyruvate into glucose.     

The effects of adenine nucleotides on carbohydrate metabolism in pigeon-liver homogenates

1. The effects of added AMP on carbohydrate metabolism were investigated in pigeon-liver homogenates, which can degrade glucose and synthesize it from lactate. Suitable experimental conditions were established for studying such effects, including the addition of P(i) (20mm) to stabilize adenine nucleotides and supplementation with NAD(+) (0.5mm). 2. Lactate increased the rate of oxygen consumption and kept the concentration of ATP high and that of AMP relatively low. 3. Added AMP (1.25-5mm) raised the net rate of carbohydrate removal and inhibited the net formation of glucose from lactate, as well as the incorporation of lactate into glucose. These effects were accompanied by a fall in the concentrations of hexose 6-phosphates and a rise in those of fructose diphosphate and triose phosphates. When the activity of glyceraldehyde 3-phosphate dehydrogenase was limited experimentally by a low concentration of NAD(+) or when it was blocked by iodoacetate, the accumulations of fructose diphosphate and triose phosphates were large and accounted for most of the carbohydrate degraded in the presence of AMP. 4. AMP also inhibited the conversion of pyruvate into phosphoenolpyruvate. Data on the concentrations of pyruvate, phosphoenolpyruvate and intermediates of the tricarboxylic acid cycle, as well as on isotope distribution, suggest that the effect was due to inhibition of phosphoenolpyruvate carboxykinase. 5. The results indicate that in the homogenates phosphofructokinase and fructose diphosphatase, controlled in their activity by adenine nucleotides and other cell constituents, are enzymes which regulate the direction of carbohydrate metabolism (degradation or synthesis) in the liver. 6. It is suggested that active transport of adenine nucleotides, citrate, Mg(2+), Ca(2+), P(i) and other cell constituents may play a role in regulating the activity of enzymes which are affected by these substances. 7. A procedure is described for generating alkali in a closed manometer vessel, by mixing mercuric oxide and a solution of sodium iodide, for use in a method for measuring the oxygen consumption at physiological bicarbonate concentrations.     

Gluconeogenesis in developing rat kidney cortex

1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21-22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.     

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.     

Induction and suppression of the key enzymes of glycolysis and gluconeogenesis in isolated perfused rat liver in response to glucose, fructose and lactate

1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.     

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.     

Calcium-dependent effect of gentamicin on glucose formation in isolated rabbit kidney-cortex tubules.

In contrast to the inhibition by gentamicin of glucose production from propionate, pyruvate and lactate in renal tubules incubated at 2.5 mM Ca2+, this antibiotic does not affect gluconeogenesis from propionate and lactate, and significantly stimulates this process from other substrates at 0.5 mM Ca2+. This may be due to the gentamicin-induced increase of the cytosolic manganese content (from 1.7 to 2.7 nmol/mg protein), resulting in a stimulation of cytosolic phosphoenolpyruvate carboxykinase activity. At 2.5 mM Ca2+ the cytosolic Mn content (2.7 nmol/mg protein) seems to be high enough to accomplish activation of the enzyme.