A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts

Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by beta-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal transmission between mitochondria and cytosol in the normoxic heart.     

Direct antagonism of ethanol''s effects on GABA(A) receptors by increased atmospheric pressure

We investigated hepatic blood flow, O2 exchange and metabolism in porcine endotoxic shock (Control, n = 8; Endotoxin, n = 10) with administration of hydroxyethylstarch to maintain arterial pressure (MAP)>60 mmHg. Before and 12, 18 and 24 h after starting continuous i.v. endotoxin we measured portal venous and hepatic arterial blood flow, intracapillary haemoglobin O2 saturation (Hb-O2%) of the liver surface and arterial, portal and hepatic venous lactate, pyruvate, glycerol and alanine concentrations. Glucose production rate was derived from the plasma isotope enrichment during infusion of [6,6-2H2]-glucose. Despite a sustained 50% increase in cardiac output endotoxin caused a progressive, significant fall in MAP. Liver blood flow significantly increased, but endotoxin affected neither hepatic O2 delivery and uptake nor mean intracapillary Hb-O2% and Hb-O2% frequency distributions. Endotoxin nearly doubled endogenous glucose production rate while hepatic lactate, alanine and glycerol uptake rates progressively decreased significantly. The lactate uptake rate even became negative (P<0.05 vs Control). Endotoxin caused portal and hepatic venous pH to fall significantly concomitant with significantly increased arterial, portal and hepatic venous lactate/pyruvate ratios. During endotoxic shock increased cardiac output achieved by colloid infusion maintained elevated liver blood flow and thereby macro- and microcirculatory O2 supply. Glucose production rate nearly doubled with complete dissociation of hepatic uptake of glucogenic precursors and glucose release. Despite well-preserved capillary oxygenation increased lactate/pyruvate ratios reflecting impaired cytosolic redox state suggested deranged liver energy balance, possibly due to the O2 requirements of gluconeogenesis.     

Comparative effects of englitazone and glyburide on gluconeogenesis and glycolysis in the isolated perfused rat liver

Englitazone (CP 68,722, Pfizer) is a member of a family of drugs known as thiazolidinediones. One member of this family, troglitazone (Rezulin), is currently utilized in the treatment of Type 2 diabetes. Previous studies have focused on the ability of englitazone to increase insulin sensitivity in various tissues. However, little information is available regarding the direct effect of englitazone on hepatic glucose metabolism in the absence of insulin. Therefore, the following studies were conducted to comparatively evaluate the effect of englitazone and glyburide (a representative sulfonylurea) on gluconeogenesis and glycolysis from various substrates in the isolated perfused rat liver (IPRL). In isolated perfused rat livers of 24-hr fasted rats infused with lactate (2 mM), englitazone (6.25 to 50 microM) produced a concentration-dependent decrease (32-93%) in hepatic gluconeogenesis. When dihydroxyacetone (1 mM) and fructose (1 mM) were used as metabolic substrates, englitazone inhibited gluconeogenesis by 31 and 15%, respectively, while increasing glycolysis by 42 and 50%. Similar effects on gluconeogenesis and glycolysis were observed with glyburide, even though the effects with glyburide were more acutely evident, reversible, and of a greater magnitude. Such data suggest alterations in hepatic glucose production may contribute to the decrease in plasma glucose concentrations observed in individuals treated with englitazone and glyburide. These alterations may include effects on several regulatory enzymes (e.g. fructose-1,6-bisphosphatase, pyruvate kinase, and phosphoenolpyruvate carboxykinase), which warrant further investigation.     

The role of cytokines in bacterial pneumonia: an inflammatory balancing act

The effect of 2 mM ethanol, a concentration indicative of daily alcohol consumption, was investigated on trichloroethylene (TRI) metabolism in perfused Wistar rat liver. The study consisted of two parts: The first part studied TRI administration with or without ethanol. In the second study chloral hydrate (CH), an intermediate in TRI metabolism, was administered in the absence or presence of ethanol to phenobarbital (PB) treated or non-PB-treated rats. The concentrations of the metabolites, total trichloroethanol (TCE), and trichloroacetic acid (TCA) were measured by gas chromatography and intracellular reduced pyridine nucleotides by surface fluorometry. In the first study, ethanol infusion significantly increased the TCE/TCA ratio, TCE production rate, and percentage of reduced pyridine nucleotides, and decreased TCA production rate without an associated change in the sum of TCE and TCA formation rates. In the second study, ethanol infusion in the absence or presence of PB produced similar significant increases in the TCE/TCA ratio, TCE production rate, and percentage of reduced pyridine nucleotides, accompanied by a decrease in TCA formation. The observed shift in TRI metabolism in the presence of ethanol, from oxidation to TCA to reduction to TCE, suggests that alcohol exerts alterations in hepatic intracellular oxidation-reduction (redox) states.     

Stimulation of gluconeogenesis with 1,3-butanediol in the perfused rat liver

An intensified synthesis of glucose is observed in gluconeogenesis from endogenous precursor only for the first 30 min of perfusion. Pyruvate introduction into the medium raises phosphoenolpyruvate carboxykinase and fructose-1,6-diphosphatase activities in the liver and determines maintenance of the glucose formation high rate for 90 min of perfusion. 1,3-butanediol is found to have a stimulating effect on gluconeogenesis from pyruvate. Introduction of 1,3 bytanediol into perfusate decreases the redox state of free NAD-pairs, increases the content of phosphoenolpyruvate, malate. ATP and the phosphoenolpyruvate carboxykinase and fructose-1.6-diphosphatase activity in the perfused liver.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.     

The effect of therapeutic doses of paracetamol on liver function in the rat perfused liver

The isolated liver perfusion technique was used to study the effect of therapeutic doses of paracetamol on hepatic gluconeogenesis and bromosulphthalein clearance from the perfusate and biliary excretion of the dye in the rat. Six groups of rats were studied; those in the three experimental groups were given 0.02 g kg-1 paracetamol daily for ninety days. The livers of animals in the control group and in one of the experimental groups were perfused with a medium containing pyruvate. The animals in the second experimental and control group were perfused with a medium containing bromosulphthalein (10 mg/100 mL). The livers of the third experimental and control group were subjected to histological examination. The rate of glucose formation and glucose concentrations were decreased, while, lactate levels and lactate: pyruvate ratios were increased in paracetamol-treated rats. The mean concentration of bromosulphthalein in the perfusate and biliary excretion of the dye were decreased. Macro and micro vesicular fatty change was present in the livers of paracetamol-treated rats. This study demonstrates that chronic administration of therapeutic doses of paracetamol to rats adversely affects liver function, as evidenced by impaired gluconeogenesis and bromosulphthalein clearance from the perfusate, and excretion of the dye into the bile, and provides histological evidence of hepatic damage in rats.     

Glucose production and gluconeogenesis in postabsorptive and starved normal and streptozotocin-diabetic rats

Using a 3-hour primed-continuous infusion of [3-3H]glucose and [2-13C]glycerol, we measured glucose production, gluconeogenesis from glycerol, and total gluconeogenesis (using mass isotopomer distribution analysis [MIDA] of glucose) in postabsorptive and starved normal and streptozotocin-diabetic rats. In normal rats, 48 hours of starvation increased (P < .01) the percent contribution of both gluconeogenesis from glycerol (from 14.4% +/- 1.8% to 25.5% +/- 4.0%) and total gluconeogenesis (from 52.2% +/- 3.9% to 89.8% +/- 1.3%) to glucose production, but the absolute gluconeogenic fluxes were not modified, since glucose production decreased. Diabetic rats showed increased glucose production in the postabsorptive state; this decreased with starvation and was comparable to the of controls after 48 hours of starvation. Gluconeogenesis was increased in postabsorptive diabetic rats (69.0% +/- 1.3%, P < .05 v controls). Surprisingly, this contribution of gluconeogenesis to glucose production was not found to be increased in 24-hour starved diabetic rats (64.4% +/- 2.4%). These rats had significant liver glycogen stores, but gluconeogenesis was also low (42.8% +/- 2.1%) in 48-hour starved diabetic rats deprived of glycogen stores. Moreover, in 24-hour starved diabetic rats infused with [3-13C]lactate, gluconeogenesis was 100% when determined by comparing circulating glucose and liver pyruvate enrichment, but only 47% +/- 3% when calculated from the MIDA of glucose. Therefore, MIDA is not a valid method to measure gluconeogenesis in starved diabetic rats. This was not explained by differences in the labeling of liver and kidney triose phosphates: functional nephrectomy of starved diabetic rats decreased glucose production, but gluconeogenesis calculated by the MIDA method was only 48% +/- 3.3%. We conclude that (1) diabetic rats have increased glucose production and gluconeogenesis in the postabsorptive state; (2) starvation decreases glucose production and increases the contribution of gluconeogenesis, but MIDA is not an appropriate method in this situation; and (3) the kidneys contribute to glucose production in starved diabetic rats.     

Competition between sodium reabsorption and gluconeogenesis in kidneys of steroid-treated rats

Kidneys of rats treated with methylprednisolone show altered substrate requirements for sodium reabsorption when perfused in vitro. Such kidneys synthesize glucose from lactate at twice the rate of control. Optimum sodium reabsorption is not seen with glucose, which is normally the preferred substrate. Sodium reabsorption is restored toward normal by the combination of glucose and butyrate, by pyruvate, or by 3-mercaptopicolinate. All of these results point to a metabolic adaptation in the kidney; butyrate may improve sodium reabsorption by sparing glucose, pyruvate is a gluconeogenic precursor and an effective fuel of respiration, and 3-mercaptopicolinate is an inhibitor of gluconeogenesis. In kidneys from rats treated with methylprednisolone there is an increased requirement for metabolic energy because of the increased rate of gluconeogenesis. It is suggested that the availability of energy from glucose oxidation is limited in part by the diversion of pyruvate back to glucose. Under these special circumstances, gluconeogenesis competes with sodium reabsorption in the intact kidney.     

Effects of rho-chloroamphetamine on locomotor activity and brain 5-hydroxyindoles

The conversion of 14C-maltose into glucose, lactate and 14 CO2 was studied in perfused livers from fed and fasted rats and in isolated hepatocytes. Maximal glucose production was 30 mM x g-1 x h-1; half-maximal rates were found with 3 mM maltose. About 0.01 % of the radioactivity infused was recovered as 14CO2. The addition of maltose had no effect on rates of oxygen consumption, lactate production or ketogenesis. The data suggest that maltose did not serve as a major substrate for biosynthetic or energy producing processes under the conditions of the perfused rat liver.     

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells

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.     

Performance and cost-effectiveness of selective screening criteria for Chlamydia trachomatis infection in women. Implications for a national Chlamydia control strategy

At 9 mM glucose, experimental results show that mitochondrial phosphate depletion (induced by glucose phosphorylation, catalyzed by mitochondrial hexokinase) reduces the activities of the respiratory chain, oxidative phosphorylation, and glutaminase. Consequently, the 14C-lactate oxidation to 14CO2 is lowered in the presence of glucose. The fall of ATP level triggers a high aerobic glycolysis by deinhibiting fructose-6-P kinase. NADH, generated by enhanced glyceraldehyde-3-P dehydrogenase activity, increases the reducing power. Moreover, the lactate dehydrogenase (LDH) system is shifted toward lactate formation, while NAD+ is regenerated and the oligomycin-inhibited ATP production is replaced by the iodoacetate-inhibited ATP production. From 14CO2 production and lactate accumulation it is calculated that about 60% of 14C-glucose which disappears is channelled into extraglycolytic reactions. On the contrary, 82% of glucose below l mM is metabolized through non-glycolytic reactions. The pyruvate kinase-M2 (PK-M2) inhibition does not limit the glycolytic flow from 9 mM glucose, but it may cause sustained gluconeogenesis.     

The applicability of the partial C4-complement fixation reaction to the serologic diagnosis of syphilis and brucellosis

The absolute rate of fatty acid synthesis was measured in slices of mammary gland from lactating rats by incubation in [3H]2O-labeled medium containing 10 mM D-3-hydroxybutyrate or acetoacetate alone and paired in combination with 10 mM glucose, lactate, or pyruvate. When compared with our previous studies, the ketone bodies alone supported significant fatty acid synthesis; the rate of synthesis from either ketone body and lactate was higher than that from pyruvate and lactate, and that from pyruvate and glucose; the rate of synthesis from D-3-hydroxybutyrate and lactate was the highest we have observed in the absence of an exogenous substrate for the hexose monophosphate pathway. This study confirms our previous contention that, in rat mammary gland, substrates formed in the mitochondria can be utilized in the cytosol to provide some of the NADPH necessary for fatty acid synthesis.     

Malate-aspartate shuttle, cytoplasmic NADH redox potential, and energetics in vascular smooth muscle

The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0. 4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2 consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism.     

Fractors affecting the rate of gluconeogenesis from L-cysteine in the perfused rat liver

It has been shown that the net rate of gluconeogenesis from cysteine was only 10% the rate observed from pyruvate. This suggested that the rate limiting step in gluconeogenesis from cysteine was between cysteine and pyruvate. Evidence is presented showing that the cysteine-sulfinate pathway does not play a regulating role in the conversion of cysteine to glucose. Thus, liver cysteine desulfhydrase (CDS) activity and hydrogen sulfide production were evaluated for their potential effects. Liver CDS activity was increased by a 3 day starvation, by feeding a 90% casein diet or a 4% cysteine + 86% casein diet. In all cases the activity of the enzyme was in excess of that required to account for the rate of conversion of cysteine to glucose observed, thus the potential activity of this enzyme was not a rate limiting factor. The possible effect of H2S, an end product of the CDS reaction, on gluconeogenesis from cysteine was evaluated. The addition of NaHS abolished the glucogenic response observed from cysteine, but had very little effect on glucoeogenesis from lactate, suggesting that accumulated H2S may inhibit CDS, marking CDS rate limiting in the conversion of cysteine to pyruvate.     

Abscesses caused by "dropped" stones after laparoscopic cholecystectomy for cholelithiasis: a report of three cases

This presentation gives an overview about the factors involved in the regulation of gluconeogenesis. Then, based on these regulatory principles, the changes seen in impaired liver function are discussed. Gluconeogenesis from lactate and pyruvate is mediated through pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) activity. The PC mediated pathway depends on substrate supply and on the downregulation of the oxidative pathway for pyruvate. Both enzymes need ATP or GTP and, thus, depend on the cellular energy charge. Tissue anoxia can reduce the energy charge and limit the flow through the PEPCK pathway. Thus, one expects a coupling between reduced splanchnic blood flow, limited oxygen supply to the liver, resulting tissue anoxia, and reduced gluconeogenesis. Conditions are shown, where this coupling exists. Since gluconeogenesis is concentrated in the periportal region of the liver, the local oxygen tension is sufficient under many circumstances to maintain a high glucose production level. Also, the enzyme activity of PEPCK can compensate for long term anoxia. Thus, gluconeogenesis is sufficient in most cases, as seen in critically ill patients. However, this could be associated with a reduction in the perivenous oxygen tension, possibly below critical levels. Beta-adrenergic stimulation increases gluconeogenesis. Examples are shown where this stimulation can overlay the dependency on the oxygen tension and substrate supply. Catecholamines are generally used to stabilize the hemodynamic system. This treatment could limit splanchnic bloodflow and, as a consequence, the oxygen supply to the liver with a simultaneous stimulation of gluconeogenesis and can cause severe anoxia in the perivenous region. These negative side effects of catecholamine treatment should be avoided and the ideal treatment should aim at improving splanchnic flow without stimulation of gluconeogenesis.     

Biochemical characterization and localization of transglutaminase in wild-type and cell-death mutants of the nematode Caenorhabditis elegans

Glucose infusion into rats has been shown to sensitize/desensitize insulin secretion in response to glucose. In pancreatic islets from glucose-infused rats (GIR) (48 h, 50%, 2 ml/h) basal insulin release (2.8 mmol/l glucose) was more than fourfold compared with islets from saline-infused controls and the concentration-response curve for glucose was shifted to the left with a maximum at 11.1 mmol/l. The concentration-response curve for 45Ca2+ uptake was also shifted to the left in islets from GIR with a maximum at 11.1 mmol/l glucose. Starting from a high basal level at 2.8 mmol/l glucose KCl produced no insulin release or 45Ca2+ uptake in islets from GIR. Islets from GIR exhibited a higher ATP/ADP ratio in the presence of 2.8 mmol/l glucose and marked inhibition of 86Rb+ efflux occurred even at 3 mmol/l glucose. Moreover, in islets from GIR the redox ratios of pyridine nucleotides were increased. On the other hand insulin content was reduced to about 20%. The data suggest that a 48-h glucose infusion sensitizes glucose-induced insulin release in vitro in concentrations below 11.1 mmol/l. This may, at least in part, be due to enhanced glucose metabolism providing increased availability of critical metabolic factors including ATP which, in turn, decrease the threshold for depolarization and therefore calcium uptake. Calcium uptake may then be further augmented by elevation of the redox state of pyridine nucleotides.     

Stimulation of weak organic acid uptake in rat renal tubules by cadmium and nystatin

The uphill uptake of a weak organic acid, fluorescein, in superficial proximal tubules of the rat kidney was stimulated by CdCl2 (0.1 mM) or nystatin (20 microM) in the absence of metabolic substrates in the incubation medium. The stimulation could be observed during the initial period of incubation (up to 30 min) only and was prevented completely by ouabain (0.1 mM), fluoroacetate (1 mM), malonate (10 mM), alpha-cyano-4-hydroxycinnamate (0.1 mM), phenylpyruvate (1 mM), D-malate (2 mM) or phenazine methosulfate (20 microM). In the renal cortex fragment suspension, both Cd2+ and nystatin increased the ouabain-sensitive, basal oxygen consumption and inhibited the rate of glucose production from pyruvate, but not from lactate. In the presence of lactate (0.5-5 mM) in the incubation medium, Cd2+ and nystatin rather inhibited fluorescein uptake, while externally added pyruvate did not influence their stimulatory effects. Taken together, these data suggest that both activation of the tricarboxylic acid cycle and export of reducing equivalents from the mitochondria to the cytosol are necessary for the stimulatory effects of Cd2+ and nystatin on the weak organic acid uptake to develop.     

Reductive metabolism of the hypoxia marker pimonidazole is regulated by oxygen tension independent of the pyridine nucleotide redox state

2-Nitroimidazoles, such as pimonidazole, are reduced in cells with low oxygen tension and are, therefore, used as hypoxia markers. However, the effect of the pyridine nucleotide redox state on pimonidazole reduction is not known. Therefore, livers from fed or fasted rats were perfused with oxygen-saturated buffer containing pimonidazole (400 microM) in the presence and absence of an inhibitor of the mitochondrial respiratory chain, potassium cyanide; these treatments were used to modulate the mitochondrial and cytosolic pyridine nucleotide redox states. Pimonidazole-induced increases in oxygen uptake over basal values were as follows: fed, 15.1 +/- 2.4; fasted, 4.2 +/- 0.8; fed + KCN, 32.1 +/- 0.9; fasted + KCN, 0.2 +/- 0.2 micromol x g(-1) x h(-1). However, if NADPH was added in excess, microsomal oxygen uptake due to oxidative metabolism of pimonidazole was independent of treatment. These results indicate that pimonidazole-stimulated O2 uptake, due predominantly to N-oxidation and glucuronidation, is dependent on the NADPH redox state. In contrast, reduced pimonidazole adducts, detected immunochemically, accumulated in pericentral regions in liver. Increasing the NADH redox state by inhibiting the mitochondrial respiratory chain with KCN decreased protein-bound pimonidazole adducts. Concomitantly, the average O2 tension of the liver was increased at least 30%. However, KCN had no effect on total pimonidazole adducts detected by ELISA, although both cytosolic (lactate/pyruvate) and mitochondrial (3-hydroxybutyrate/acetoacetate) NADH redox states were elevated by at least a factor of eight. These results indicate that, unlike oxidative metabolism, the pyridine nucleotide redox state does not determine the rate of reductive metabolism of pimonidazole. Instead, the cellular oxygen tension regulates this process. Therefore, even in cases where the supply of reducing equivalents is increased (e.g., ethanol metabolism), accumulation of the reduced bound product of pimonidazole is oxygen dependent in liver.     

Carbon-14 tracer studies in the metabolism of isolated rat-liver parenchymal cells under conditions of gluconeogenesis from lactate and pyruvate

In rat liver perfusion experiments under conditions of gluconeogenesis from lactate and pyruvate 14C-labeling patterns of metabolites with (1-14C)-labeled and (2-14C)-labeled lactate or pyruvate, [14C]bicarbonate and [1-14C]octanoate as tracers, have been obtained, which do not agree with generally assumed reaction schemes. The experiments have been repeated with incubations of isolated rat-liver parenchymal cells. The results demonstrate that the discrepancies between expected and analysed 14C-labeling patterns of metabolites were still existent. From this observation it may be concluded, that 14C-labeling patterns of metabolites are indicative for the existence of still unknown metabolic relationships in liver parenchymal cells. Furthermore the results of our experiments prove that conclusions based on the exclusive analysis of metabolite levels are of limited value for studying intracellular events, because of uncharacterized compartmentations, which become evident in 14C-tracer studies. It cannot be answered by our studies, whether the apparent existence of differently labeled species of citrate, oxoglutarate or acetyl-CoA, represent intracellular compartmentation or whether it is the result of metabolic heterogeneity of liver parenchymal cells.