Compartmentation between glycolysis and gluconeogenesis in rat liver

1. The specific radioactivity-time relationships of glucose, glucose 6-phosphate, glycerol 1-phosphate and UDP-glucose were determined in rat liver after the intravenous injection of [U-(14)C]fructose, and a kinetic analysis was carried out. The glucose 6-phosphate pool was found to be compartmented into gluconeogenic and glycolytic components, and evidence was obtained that the triose phosphates were similarly compartmented. The glycolytic pathway was fed by glycogenolysis and glucose phosphorylation. There was no direct evidence that glycogenolysis fed only the glycolytic pathway, but this interpretation would make the liver resemble other organs in this respect. 2. UDP-glucose was not formed solely from gluconeogenic glucose 6-phosphate, as there was some dilution of label in the intervening glucose 1-phosphate pool, probably from glycogenolysis, though other pathways cannot be excluded. 3. The data cannot be explained by isotopic exchange.     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014     

A reassessment of glycolysis and gluconeogenesis in higher plants

Sung, S.-J. S., Xu, D.-P., Galloway, C. M. and Black, C. C., Jr. 1988. A reassessment of glycolysis and gluconeogenesis in higher plants. - Physiol. Plant. 72: 650–654.
Sucrose is the starting point of glycolysis and end point of gluconeogenesis in higher plants. During both glycolysis and gluconeogenesis alternative enzymes are present at various steps to carry out parallel pathways; alternatives are available for utilizing nucleotide triphosphates and pyrophosphate; fructose 2,6-bisphosphate serves as a strong internal regulator; and plants use these cytoplasmic alternatives as they develop and as their environments change.     

Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures.

The effects of the glucocorticoid dexamethasone on fatty acid and pyruvate metabolism were studied in rat hepatocyte cultures. Parenchymal hepatocytes were cultured for 24 h with nanomolar concentrations of dexamethasone in either the absence or the presence of insulin (10 nM) or dibutyryl cyclic AMP (1 microM BcAMP). Dexamethasone (1-100 nM) increased the rate of formation of ketone bodies from 0.5 mM-palmitate in both the absence and the presence of BcAMP, but inhibited ketogenesis in the presence of insulin. Dexamethasone increased the proportion of the palmitate metabolized that was partitioned towards oxidation to ketone bodies, and decreased the cellular [glycerol 3-phosphate]. The latter suggests that the increased partitioning of palmitate to ketone bodies may be associated with decreased esterification to glycerolipid. The Vmax. of carnitine palmitoyltransferase (CPT) and the affinity of CPT for palmitoyl-CoA were not affected by dexamethasone, indicating that the increased ketogenesis was not due to an increase in enzymic capacity for long-chain acylcarnitine formation. Dexamethasone and BcAMP, separately and in combination, increased gluconeogenesis. In the presence of insulin, however, dexamethasone inhibited gluconeogenesis. Changes in gluconeogenesis thus paralleled changes in ketogenesis. Dexamethasone decreased the [3-hydroxybutyrate]/[acetoacetate] ratio, despite increasing the rate of ketogenesis and presumably the mitochondrial production of reducing equivalents. The more oxidized mitochondrial NADH/NAD+ redox couple with dexamethasone is probably due either to an increased rate of electron transport or to increased transfer of mitochondrial reducing equivalents to the cytoplasm.     

Influence of tamoxifen on gluconeogenesis and glycolysis in the perfused rat liver

The actions of tamoxifen, a selective estrogen receptor modulator used in chemotherapy and chemo-prevention of breast cancer, on glycolysis and gluconeogenesis were investigated in the isolated perfused rat liver. Tamoxifen inhibited gluconeogenesis from both lactate and fructose at very low concentrations (e.g., 5 μM). The opposite, i.e., stimulation, was found for glycolysis from both endogenous glycogen and fructose. Oxygen uptake was unaffected, inhibited or stimulated, depending on the conditions. Stimulation occurred in both microsomes and mitochondria. Tamoxifen did not affect the most important key-enzymes of gluconeogenesis, namely, phosphoenolpyruvate carboxykinase, pyruvate carboxylase, fructose 1,6-bisphosphatase and glucose 6-phosphatase. Confirming previous observations, however, tamoxifen inhibited very strongly NADH- and succinate-oxidase of freeze–thawing disrupted mitochondria. Tamoxifen promoted the release of both lactate dehydrogenase (mainly cytosolic) and fumarase (mainly mitochondrial) into the perfusate. Tamoxifen (200 μM) clearly diminished the ATP content and increased the ADP content of livers in the presence of lactate with a diminution of the ATP/ADP ratio from 1.67 to 0.79. The main causes for gluconeogenesis inhibition are probably: (a) inhibition of energy metabolism; (b) deviation of intermediates (malate and glucose 6-phosphate) for the production of NADPH required in hydroxylation and demethylation reactions; (c) deviation of glucosyl units toward glucuronidation reactions; (d) secondary inhibitory action of nitric oxide, whose production is stimulated by tamoxifen; (e) impairment of the cellular structure, especially the membrane structure. Stimulation of glycolysis is probably a compensatory phenomenon for the diminished mitochondrial ATP production. The multiple actions of tamoxifen at relatively low concentrations can represent a continuous burden to the overall hepatic functions during long treatment periods.     

Control of glycolysis and gluconeogenesis in rat kidney cortex slices

1. Glucose uptake or glucose formation has been studied in kidney cortex slices to investigate metabolic control of phosphofructokinase and fructose-diphosphatase activities. 2. Glucose uptake is increased and glucose formation is decreased by anoxia, cyanide or an uncoupling agent. Under these conditions the intracellular concentrations of glucose 6-phosphate and ATP decreased whereas that of fructose diphosphate either increased or remained constant, and the concentrations of AMP and ADP increased. 3. Glucose uptake was decreased, and glucose formation from glycerol or dihydroxyacetone was increased, by the presence of ketone bodies or fatty acids, or after starvation of the donor animal. Under these conditions, the concentrations of glucose 6-phosphate and citrate were increased, whereas those of fructose diphosphate and the adenine nucleotides were unchanged (see also Newsholme & Underwood, 1966). 4. It is concluded that anoxia and cell poisons increase glucose uptake and decrease gluconeogenesis by stimulating phosphofructokinase and inhibiting fructose diphosphatase, whereas ketone bodies, fatty acids or starvation increase gluconeogenesis and decrease glucose uptake through the citrate inhibition of phosphofructokinase.     

The control of glycolysis and gluconeogenesis by protein phosphorylation

Fructose 2,6-bisphosphate has been discovered as a potent stimulator of liver phosphofructokinase. It is also an inhibitor of fructose 1,6-biphosphatase and a stimulator of PPi: fructose 6-phosphate phosphotransferase from higher plants. It is formed from fructose 6-phosphate and ATP by a 6-phosphofructo 2-kinase and hydrolysed by a fructose 2,6-bisphosphatase. These two enzymes have very similar physicochemical properties and could not be separated from each other. They are substrates for cyclic-AMP-dependent protein kinase, which inactivates the first enzyme and activates the second.     

Adenosine phosphates and the control of glycolysis and gluconeogenesis in yeast

1. Changes in dry weight, protein, RNA and DNA were measured in yeast during adaptation to glycolytic metabolism. 2. Only RNA increased significantly during the lag phase, but during the exponential phase all these cellular components increased in parallel. 3. The concentrations of ATP, ADP, AMP and glucose 6-phosphate were measured in respiring yeast and during the transition to glycolytic metabolism. 4. In respiring cells the concentration of AMP was at its highest and that of ATP was at its lowest; this relationship was reversed in glycolysing cells. 5. ADP concentration was similar in respiring and glycolysing cells, but glucose 6-phosphate concentration was much higher in the glycolysing cells. 6. A possible reason for mitochondrial repression is suggested. 7. It is concluded that adenosine phosphates do not control the direction of glycolytic flux in yeast and an alternative control of glycolysis and gluconeogenesis by enzyme activation and inactivation is suggested.     

Inhibition of hepatocyte proteolysis and lactate gluconeogenesis by chloroquine

Chloroquine (50 μm) is rapidly taken up by isolated hepatocytes in a temperature-dependent manner. It inhibits glucose synthesis from lactate, but not from pyruvate or dihydroxyacetone. The inhibition is reversed by lysine or ammonia but not by oleate or carnitine. Ammonia inhibits chloroquine uptake by the hepatocytes but lysine does not. Chloroquine also inhibits urea synthesis, the release of ninhydrin-reacting substances, the accumulation of amino acids, and the lactate-dependent accumulation of glutamate. Ethanol oxidation in the presence of lactate is also inhibited, and this too is reversed by lysine. Chloroquine increases the redox state of the cytosolic compartment, as evidenced by lactate-to-pyruvate ratios, of hepatocytes prepared from both 48-h fasted and meal-fed rats. The above findings are consistent with chloroquine entering the lysosomes of the hepatocytes and inhibiting proteolysis by raising the lysosomal pH. Isolated hepatocytes are deficient in amino acids and, chloroquine inhibition of proteolysis prevents replenishment of the amino acid pools. Thus, chloroquine prevents reconstitution of the malate-aspartate shuttle required for the movement of reducing equivalents into the mitochondrion during lactate gluconeogenesis, ethanol oxidation, and glycolysis. The metabolic competency of freshly isolated hepatocytes, therefore, depends on the replenishment of amino acid pools by lysosomal breakdown of endogenous protein. Furthermore, chloroquine uptake may be an index of lysosomal function with isolated hepatocytes.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

The effects of varying oxygen tensions on organ cultures

Summary 1. Human embryonic organ slices survived better in organ culture when the oxygen tension was relatively low. Necrosis, presumably due to oxygen poisoning was seen at an oxygen pressure of 2.0 atmospheres.2. Slices of organs from adult laboratory animals varied in their reponse to high oxygen pressuresin vitro.3. In some organs, e. g. liver and kidney, considerably thicker slices survived in culture at pressures of 2.0 and 3.0 atmospheres than at 1.0 atmosphere.4. Some tissues, zona fasciculata of the adrenal cortex and brown fat, which do not survive at 1.0 atmosphere were found to do well at raised pressures of oxygen.5. Other tissues, notably the adrenal medulla, survived well at 1.0 atmosphere, but were destroyed by high oxygen pressure.6. Rat liver slices, cultivated under high oxygen pressure maintained a reasonably normal histological appearance for 10–14 days, but rapidly lost their glycogen content. Their oxygen consumption remained essentially the same for at least 6 days. Attempts to prolong the survival of liver slices by the use of feeder cultures and by extracts of liver were unsuccessful.7. Glucuronide synthesis rapidly fell off in slices of mouse liver maintained at 3.0 atmospheres. A similar result was obtained with guinea-pig kidney, but in this case, the addition of extra glucose to the medium had a marked effect in delaying the fall.

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Die Wirkungen verschiedener Sauerstoffspannungen auf Organkulturen

Kurzfassung Die Sauerstoffbedürfnisse embryonaler und adulter Organe sind recht unterschiedlich. Erstere werden durch hohe Sauerstoffspannungen getötet, letztere überleben vielfach nicht bei niedrigen Spannungen. Im günstigsten Falle können nur dünne Scheiben adulter Organe lebend erhalten werden, selbst wenn die Luft im Kulturgefäß durch Sauerstoff ersetzt wird. Organkulturen wurden daher in einer Druckkammer durchgeführt unter Sauerstoffpartialdrucken zwischen 0,2 und 4,0 Atmosphären (absolut). Die höheren Drücke begünstigten das Überleben einiger Organe von adulter Ratte und Maus, z. B. Nebennierenrinde, Leber und braunes Fett, welche bis zu Drücken von 1,0 Atmosphären nicht gut gedeihen. Andererseits überlebten embryonale Organe und bestimmte adulte Organe — wie etwa Nebennierenmark — besser bei relativ niedrigen Sauerstoffdrücken und wurden durch höhere Drücke stark geschädigt. Im Falle der adulten Leber wurden QO₂-Bestimmungen und Studien der bei der Glucuronid-Synthese beteiligten Enzyme benutzt, um die herkömmlichen histologischen Kriterien für das Überlebensvermögen nach mehreren Kulturtagen zu ergänzen. Es wird die Schlußfolgerung gezogen, daß die Anwendung hoher Sauerstoffdrücke von beträchtlichem Wert sein könnte für Arbeiten mit Organkulturen.

     

Regulation of hepatic glycolysis and gluconeogenesis by atrial natriuretic peptide.

Recently we reported the presence of both the guanylyl cyclase-linked (116 kDa) and the ANF-C (66 kDa) atrial natriuretic peptide receptors in the rat liver. Since ANF 103-125 (atriopeptin II) stimulates cGMP production in livers and because cGMP has previously been shown to mimic the actions of cAMP in regulating hepatic carbohydrate metabolism, studies were performed to investigate the effects of atriopeptin II on hepatic glycolysis and gluconeogenesis. Additionally, employing analogs of atrial natriuretic hormone [des-(Q116, S117, G118, L119, G120) ANF 102-121 (C-ANF) and des-(C105,121) ANF 104-126 (analog I)] which bind only the ANF-C receptors, the role of the ANF-C receptors in the hepatic actions of atriopeptin II was evaluated. In perfused livers of fed rats atriopeptin II, but not C-ANF and analog I, inhibited hepatic glycolysis and stimulated glucose production. Moreover, analog I did not alter the ability of atriopeptin II to inhibit hepatic glycolysis. Atriopeptin II, but not C-ANF and analog I, also stimulated cGMP production in perfused rat livers. Furthermore, while atriopeptin II inhibited the activity ratio of pyruvate kinase by 30%, C-ANF did not alter hepatic pyruvate kinase activity. Finally, in rat hepatocytes, atriopeptin II stimulated the synthesis of [14C]glucose from [2-14C]pyruvate by 50% and this effect of atriopeptin II was mimicked by the exogenously supplied cGMP analog, 8-bromo cGMP. Thus atriopeptin II increases hepatic gluconeogenesis and inhibits glycolysis, in part by inhibiting pyruvate kinase activity, and the effects of atriopeptin II are mediated via activation of guanylyl cyclase-linked ANF receptors which elevate cGMP production.     

Effects of training, exercise and diet on muscle glycolysis and liver gluconeogenesis.

Trained and untrained rats were fed either a control, high-fat, or high-carbohydrate diet and then sacrificed in either a rested or exhausted state. The $\text{in vitro}$ activity of several muscle glycolytic and liver gluconeogenic enzymes was measured. Muscle hexokinase, phosphorylase, and phosphofructokinase activities were increased after training. Hexokinase was decreased in exhausted rats. Phosphorylase and phosphofructokinase were increased in untrained-exhausted rats but were unchanged in trained-exhausted rats. Liver pyruvate carboxylase and phosphoenolpyruvate carboxykinase activities were increased in trained-rested rats fed a high-fat diet. In trained-exhausted rats phosphoenolpyruvate carboxykinase activity was increased regardless of diet fed. Blood glucose was decreased in trained-exhausted rats, but it was increased in untrained-exhausted rats. Plasma glucocorticoid level was increased in exhausted rats. This study showed that training was associated with an increased muscle glycolytic capacity. Training was also related to the ability of liver to increase phosphoenolpyruvate carboxykinase activity during exercise, thereby increasing gluconeogenic capacity.