Triidothyronine and epinephrine rapidly modify myocardial substrate selection: a (13)C isotopomer analysis

Triiodothyronine (T(3)) exerts direct action on myocardial oxygen consumption (MVO(2)), although its immediate effects on substrate metabolism have not been elucidated. The hypothesis, that T(3) regulates substrate selection and flux, was tested in isovolumic rat hearts under four conditions: control, T(3) (10 nM), epinephrine (Epi), and T(3) and Epi (TE). Hearts were perfused with [1,3-(13)C]acetoacetic acid (AA, 0.17 mM), L-[3-(13)C]lactic acid (LAC, 1.2 mM), U-(13)C-labeled long-chain free fatty acids (FFA, 0.35 mM), and unlabeled D-glucose (5.5 mM) for 30 min. Fractional acetyl-CoA contribution to the tricarboxylic acid cycle (Fc) per substrate was determined using (13)C NMR and isotopomer analysis. Oxidative fluxes were calculated using Fc, the respiratory quotient, and MVO(2). T(3) increased (P < 0.05) Fc(FFA), decreased Fc(LAC), and increased absolute FFA oxidation from 0.58 +/- 0.03 to 0.68 +/- 0.03 micromol. min(-1). g dry wt(-1) (P < 0.05). Epi decreased Fc(FFA) and Fc(AA), although FFA flux increased from 0.58 +/- 0.03 to 0.75 +/- 0.09 micromol. min(-1). g dry wt(-1). T(3) moderated the change in Fc(FFA) induced by Epi. In summary, T(3) exerts direct action on substrate pathways and enhances FFA selection and oxidation, although the Epi effect dominates at a high work state.     

Cardioselective dominant-negative thyroid hormone receptor (Delta337T) modulates myocardial metabolism and contractile efficiency

Dominant-negative thyroid hormone receptors (TRs) show elevated expression relative to ligand-binding TRs during cardiac hypertrophy. We tested the hypothesis that overexpression of a dominant-negative TR alters cardiac metabolism and contractile efficiency (CE). We used mice expressing the cardioselective dominant-negative TRbeta(1) mutation Delta337T. Isolated working Delta337T hearts and nontransgenic control (Con) hearts were perfused with (13)C-labeled free fatty acids (FFA), acetoacetate (ACAC), lactate, and glucose at physiological concentrations for 30 min. (13)C NMR spectroscopy and isotopomer analyses were used to determine substrate flux and fractional contributions (Fc) of acetyl-CoA to the citric acid cycle (CAC). Delta337T hearts exhibited rate depression but higher developed pressure and CE, defined as work per oxygen consumption (MVo(2)). Unlabeled substrate Fc from endogenous sources was higher in Delta337T, but ACAC Fc was lower. Fluxes through CAC, lactate, ACAC, and FFA were reduced in Delta337T. CE and Fc differences were reversed by pacing Delta337T to Con rates, accompanied by an increase in FFA Fc. Delta337T hearts lacked the ability to increase MVo(2). Decreases in protein expression for glucose transporter-4 and hexokinase-2 and increases in pyruvate dehydrogenase kinase-2 and -4 suggest that these hearts are unable to increase carbohydrate oxidation in response to stress. These data show that Delta337T alters the metabolic phenotype in murine heart by reducing substrate flux for multiple pathways. Some of these changes are heart rate dependent, indicating that the substrate shift may represent an accommodation to altered contractile protein kinetics, which can be disrupted by pacing stress.     

Triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass

Triiodothyronine (T3) supplementation improves clinical outcomes in infants after cardiac surgery using cardiopulmonary bypass by unknown mechanisms. We utilized a translational model of infant cardiopulmonary bypass to test the hypothesis that T3 modulates pyruvate entry into the citric acid cycle (CAC), thereby providing the energy support for improved cardiac function after ischemia-reperfusion (I/R). Neonatal piglets received intracoronary [2-(13)Carbon((13)C)]pyruvate for 40 min (8 mM) during control aerobic conditions (control) or immediately after reperfusion (I/R) from global hypothermic ischemia. A third group (I/R-Tr) received T3 (1.2 μg/kg) during reperfusion. We assessed absolute CAC intermediate levels and flux parameters into the CAC through oxidative pyruvate decarboxylation (PDC) and anaplerotic carboxylation (PC) using [2-(13)C]pyruvate and isotopomer analysis by gas and liquid chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectroscopy. When compared with I/R, T3 (group I/R-Tr) increased cardiac power and oxygen consumption after I/R while elevating flux of both PDC and PC (～4-fold). Although neither I/R nor I/R-Tr modified absolute CAC levels, T3 inhibited I/R-induced reductions in their molar percent enrichment. Furthermore, (13)C-labeling of CAC intermediates suggests that T3 may decrease entry of unlabeled carbons at the level of oxaloacetate through anaplerosis or exchange reaction with asparate. T3 markedly enhances PC and PDC fluxes, thereby providing potential substrate for elevated cardiac function after reperfusion. This T3-induced increase in pyruvate fluxes occurs with preservation of the CAC intermediate pool. Our labeling data raise the possibility that T3 reduces reliance on amino acids for anaplerosis after reperfusion.     

A (13)C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca(2+)](o)

Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility. Hearts were frozen at various times after exposure to these substrates, were extracted, and were then analyzed by (13)C NMR spectroscopy. The time-dependent multiplets observed in the (13)C NMR resonances of glutamate in all hearts and in malate and aspartate in hearts perfused with high-pyruvate/low-lactate concentrations were analyzed using a kinetic model of the tricarboxylic acid (TCA) cycle. The analysis showed that TCA cycle flux (V(TCA)) and exchange flux (V(X)) that involved cycle intermediates were both sensitive to cell redox and altered Ca(2+) concentration, and the ratio of these fluxes (V(X)/V(TCA)) varied >10-fold.     

A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts

Ammonia assimilation in chloroplasts occurs via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. To determine the extent to which these enzymes contribute to the control of ammonia assimilation, a metabolic control analysis was performed on isolated barley (Hordeum vulgare L.) leaf chloroplasts. Pathway flux was measured polarographically as ammonium-plus-2-oxoglutarate-plus-glutamine-dependent O2 evolution in illuminated chloroplasts. Enzyme activity was modulated by titration with specific, irreversible inhibitors of GS (phosphinothricin) and GOGAT (azaserine). Flux control coefficients (CJ0E0) were determined (a) by differentiation of best-fit hyperbolic curves of the data sets (flux versus enzyme activity), and (b) from estimates of the deviation indices (D/[prime]E0). Both analyses gave similar values for the coefficients. The control coefficient for GS was relatively high and the value did not change significantly with changes in 2-oxoglutarate concentration (C/0E0 = 0.58 at 5 mM 2-oxoglutarate and 0.40 at 20 mM 2-oxoglutarate). The control coefficient for GOGAT decreased with decreasing glutamine concentrations, from 0.76 at 20 mM glutamine to 0.19 at 10 mM glutamine. Thus, at high concentrations of glutamine, GOGAT exerts a major control over flux with a significant contribution also from GS. At lower concentrations of glutamine, however, GOGAT exerts far less control over pathway flux.     

Glutamine cycling in isolated working rat heart

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.     

5-3H]glucose overestimates glycolytic flux in isolated working rat heart: role of the pentose phosphate pathway

We set out to study the pentose phosphate pathway (PPP) in isolated rat hearts perfused with [5-3H]glucose and [1-14C]glucose or [6-14C]glucose (crossover study with 1- then 6- or 6- then 1-14C-labeled glucose). To model a physiological state, hearts were perfused under working conditions with Krebs-Henseleit buffer containing 5 mM glucose, 40 microU/ml insulin, 0.5 mM lactate, 0.05 mM pyruvate, and 0.4 mM oleate/3% albumin. The steady-state C1/C6 ratio (i.e., the ratio from [1-14C]glucose to [6-14C]glucose) of metabolites released by the heart, an index of oxidative PPP, was not different from 1 (1.06 +/- 0.19 for 14CO2, and 1.00 +/- 0.01 for [14C]lactate + [14C]pyruvate, mean +/- SE, n = 8). Hearts exhibited contractile, metabolic, and 14C-isotopic steady state for glucose oxidation (14CO2 production). Net glycolytic flux (net release of lactate + pyruvate) and efflux of [14C]lactate + [14C]pyruvate were the same and also exhibited steady state. In contrast, flux based on 3H2O production from [5-3H]glucose increased progressively, reaching 260% of the other measures of glycolysis after 30 min. The 3H/14C ratio of glycogen (relative to extracellular glucose) and sugar phosphates (representing the glycogen precursor pool of hexose phosphates) was not different from each other and was <1 (0.36 +/- 0.01 and 0.43 +/- 0.05 respectively, n = 8, P < 0.05 vs. 1). We conclude that both transaldolase and the L-type PPP permit hexose detritiation in the absence of net glycolytic flux by allowing interconversion of glycolytic hexose and triose phosphates. Thus apparent glycolytic flux obtained by 3H2O production from [5-3H]glucose overestimates the true glycolytic flux in rat heart.     

Effects of Potassium and Glutamine on Metabolism of Glucose in Astrocytes

The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-¹³C]glucose and [2-¹³C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the ¹³C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-¹³C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.     

Isotopomer analysis of myocardial substrate metabolism: a systems biology approach

The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network.     

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts.

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.     

Reverse flux through cardiac NADP(+)-isocitrate dehydrogenase under normoxia and ischemia

Little is known about the role of mitochondrial NADP(+)-isocitrate dehydrogenase (NADP(+)-ICDH) in the heart, where this enzyme shows its highest expression and activity. We tested the hypothesis that in the heart, NADP(+)-ICDH operates in the reverse direction of the citric acid cycle (CAC) and thereby may contribute to the fine regulation of CAC activity (Sazanov and Jackson, FEBS Lett 344: 109-116, 1994). We documented a reverse flux through this enzyme in rat hearts perfused with the medium-chain fatty acid octanoate using [U-(13)C(5)]glutamate and mass isotopomer analysis of tissue citrate (Comte et al., J Biol Chem 272: 26117-26124, 1997). In this study, we assessed the significance of our previous finding by perfusing hearts with long-chain fatty acids and tested the effects of changes in O(2) supply. We showed that under all of these conditions citrate was enriched in an isotopomer containing five (13)C atoms. This isotopomer can only be explained by substrate flux through reversal of the NADP(+)-ICDH reaction, which is evaluated at 3-7% of flux through citrate synthase. Small variations in reversal fluxes induced by low-flow ischemia that mimicked hibernation occurred despite major changes in contractile function and O(2) consumption of the heart as well as citrate and succinate release rates and tissue levels. Our data show a reverse flux through NADP(+)-ICDH and support its hypothesized role in the fine regulation of CAC activity in the normoxic and O(2)-deprived heart.     

Metabolic heterogeneity of carbon substrate utilization in mammalian heart: NMR determinations of mitochondrial versus cytosolic compartmentation.

Carbon-13 (13C) nuclear magnetic resonance (NMR) spectroscopy can be used to target specific pathways of intermediary metabolism within intact tissues and was employed in this study to evaluate the compartmentation of pyruvate metabolism between the cytosol and mitochondrial matrix. The distribution of 13C into the tissue alanine, lactate, and glutamate pools was evaluated during metabolism of [3-13C]-pyruvate in intact, isolated perfused rabbit hearts with and without activation of pyruvate dehydrogenase activity by dichloroacetate (5 mM). Equilibrium between the intracellular alanine and pyruvate pools was in evidence from the rapid evolution of the steady-state 13C signal arising from the 3-carbon of alanine in intact hearts perfused with 2.5 mM 99.4% [3-13C]pyruvate. Augmented pyruvate oxidation, in response to perfusion with dichloroacetate, was evident within 13C NMR spectra of intact hearts as a relative increase in signal intensity of 53-62% (p less than 0.05) from the 4-carbon resonance of 13C-enriched glutamate when compared to the unaffected alanine signal. The increased bulk flow of [3-13C]pyruvate into the tricarboxylic acid cycle in response to dichloroacetate resulted in elevated fractional enrichment of glutamate from 68% in controls to 83% in the treated group (p less than 0.04), via interconversion with alpha-ketoglutarate, without changes in the actual tissue content of glutamate. Evidence of metabolic heterogeneity of cytosolic and mitochondrial pyruvate pools was also obtained from analysis of tissue extracts with in vitro NMR spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

C Isotopomer Analysis of Glutamate by Tandem Mass Spectrometry

Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with (13)C NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-(13)C]acetate, 5 mM [1-(13)C]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-(13)C]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 +/- 2, 53 +/- 2, and 84 +/- 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 +/- 3, 25 +/- 8, and 16 +/- 8%. Similar results were obtained with (13)C NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using (13)C-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR.     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

Pseudoketogenesis in the perfused rat heart

Ketogenesis is usually measured in vivo by dilution of tracers of (3R)-hydroxybutyrate or acetoacetate. We show that, in perfused working rat hearts, the specific activities of (3R)-hydroxybutyrate and acetoacetate are diluted by isotopic exchanges in the absence of net ketogenesis. We call this process pseudoketogenesis. When hearts are perfused with buffer containing 2.3 mM of [4-3H]- plus [3-14C]acetoacetate, the specific activities of [4-3H] and [3-14C]acetoacetate decrease while C-1 of acetoacetate becomes progressively labeled with 14C. This is explained by the reversibility of reactions catalyzed by mitochondrial 3-oxoacid-CoA transferase and acetoacetyl-CoA thiolase. After activation of labeled acetoacetate, the specific activity of acetoacetyl-CoA is diluted by unlabeled acetoacetyl-CoA derived from endogenous fatty acids or glucose. Acetoacetyl-CoA thiolase partially exchanges 14C between C-1 and C-3 of acetoacetyl-CoA. Finally, 3-oxoacid-CoA transferase liberates weakly labeled acetoacetate which dilutes the specific activity of extracellular acetoacetate. An isotopic exchange in the reverse direction is observed when hearts are perfused with unlabeled acetoacetate plus [1-14C]-, [13-14C]-, or [15-14C]palmitate; here also, acetoacetate becomes labeled on C-1 and C-3. Computations of specific activities of (3R)-hydroxybutyrate, acetoacetate, and acetyl-CoA yield minimal rates of pseudoketogenesis ranging from 19 to 32% of the net uptake of (3R)-hydroxybutyrate plus acetoacetate by the heart.     

Effect of 8-bromo-cAMP and dexamethasone on glutamate metabolism in rat astrocytes

Glutamine synthetase (GS) activity in cultured rat astrocytes was measured in extracts and compared to the intracellular rate of glutamine synthesis by intact control astrocytes or astrocytes exposed to 1 mM 8-bromo-cAMP (8Br-cAMP)+1 M dexamethasone (DEX) for 4 days. GS activity in extracts of astrocytes treated with 8Br-cAMP+DEX was 7.5 times greater than the activity in extracts of control astrocytes. In contrast, the intracellular rate of glutamine synthesis by intact cells increased only 2-fold, suggesting that additional intracellular effectors regulate the expression of GS activity inside the intact cell. The rate of glutamine synthesis by astrocytes was 4.3 times greater in MEM than in HEPES buffered Hank's salts. Synthesis of glutamine by intact astrocytes cultured in MEM was independent of the external glutamine or ammonia concentrations but was increased by higher extracellular glutamate concentrations. In studies with intact astrocytes 80% of the original [U-¹⁴C]glutamate was recovered in the medium as radioactive glutamine, 2–3% as aspartate, and 7% as glutamate after 2 hours for both control and treated astrocytes. The results suggest: (1) astrocytes are highly efficient in the conversion of glutamate to glutamine; (2) induction of GS activity increases the rate of glutamate conversion to glutamine by astrocytes and the rate of glutamine release into the medium; (3) endogenous intracellular regulators of GS activity control the flux of glutamate through this enzymatic reaction; and, (4) the composition of the medium alters the rate of glutamine synthesis from external glutamate.     

Cardiac metabolism in mice: tracer method developments and in vivo application revealing profound metabolic inflexibility in diabetes

Studies of cardiac fuel metabolism in mice have been almost exclusively conducted ex vivo. The major aim of this study was to assess in vivo plasma FFA and glucose utilization by the hearts of healthy control (db/+) and diabetic (db/db) mice, based on cardiac uptake of (R)-2-[9,10-(3)H]bromopalmitate ([3H]R-BrP) and 2-deoxy-D-[U-14C]glucose tracers. To obtain quantitative information about the evaluation of cardiac FFA utilization with [3H]R-BrP, simultaneous comparisons of [3H]R-BrP and [14C]palmitate ([14C]P) uptake were first made in isolated perfused working hearts from db/+ mice. It was found that [3H]R-BrP uptake was closely correlated with [14C]P oxidation (r2 = 0.94, P < 0.001). Then, methods for in vivo application of [3H]R-BrP and [14C]2-DG previously developed for application in the rat were specially adapted for use in the mouse. The method yields indexes of cardiac FFA utilization (R(f)*) and clearance (K(f)*), as well as glucose utilization (R(g)'). Finally, in the main part of the study, the ability of the heart to switch between FFA and glucose fuels (metabolic flexibility) was investigated by studying anesthetized, 8-h-fasted control and db/db mice in either the basal state or during glucose infusion. In control mice, glucose infusion raised plasma levels of glucose and insulin, raised R(g)' (+58%), and lowered plasma FFA level (-48%), K(f)* (-45%), and R(f)* (-70%). This apparent reciprocal regulation of glucose and FFA utilization by control hearts illustrates metabolic flexibility for substrate use. By contrast, in the db/db mice, glucose infusion raised glucose levels with no apparent influence on cardiac FFA or glucose utilization. In conclusion, tracer methodology for assessing in vivo tissue-specific plasma FFA and glucose utilization has been adapted for use in mice and reveals a profound loss of metabolic flexibility in the diabetic db/db heart, suggesting a fixed level of FFA oxidation in fasted and glucose-infused states.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Urea synthesis and CO2/HCO3- compartmentation in isolated perfused rat liver

With physiological portal HCO3- and CO2 concentrations of 25mM and 1.2mM in the perfusate, respectively, acetazolamide inhibited urea synthesis from NH4Cl in isolated perfused rat liver by 50-60%, whereas urea synthesis from glutamine was inhibited by only 10-15%. A decreased sensitivity of urea synthesis from glutamine to acetazolamide inhibition was also observed when the extracellular HCO3- and CO2 concentrations were varied from 0-50mM and 0-2.4mM, respectively. Stimulation of intramitochondrial CO2 formation at pyruvate dehydrogenase with high pyruvate concentrations (7mM) was without effect on the acetazolamide sensitivity of urea synthesis from NH4Cl. Urea synthesis was studied under conditions of a limiting HCO3- supply for carbamoyl-phosphate synthesis. In the absence of externally added HCO3- or CO2, when 14CO2 was provided intracellularly by [U-14C]glutamine or [1-14C]-glutamine oxidation, acetazolamide had almost no effect on label incorporation into urea, whereas label incorporation from an added tracer H14CO3- dose was inhibited by about 70%. 14CO2 production from [U-14C]glutamine was about twice as high as from [1-14C]glutamine, indicating that about 50% of the CO2 produced from glutamine is formed at 2-oxoglutarate dehydrogenase. The fractional incorporation of 14CO2 into urea was about 13% with [1-14C]-as well as with [U-14C]glutamine. Addition of small concentrations of HCO3- (1.2mM) to the perfusate increased urea synthesis from glutamine by about 70%. This stimulation of urea synthesis was fully abolished by acetazolamide. The carbonate-dehydratase inhibitor prevented the incorporation of added HCO3- into urea, whereas incorporation of CO2 derived from glutamine degradation was unaffected. Without HCO3- and CO2 in the perfusion medium, when 14CO2 was provided by [1-14C]-pyruvate oxidation, acetazolamide inhibited urea synthesis from NH4Cl as well as 14C incorporation into urea by about 50%. Therefore carbonate-dehydratase activity is required for the utilization of extracellular CO2 or pyruvate-dehydrogenase-derived CO2 for urea synthesis, but not for CO2 derived from glutamine oxidation. This is further evidence for a special role of glutamine as substrate for urea synthesis.     

Effect of phenylephrine on glutamate and glutamine metabolism in isolated perfused rat liver.

Addition of phenylephrine to isolated perfused rat liver is followed by an increased 14CO2 production from [1-14C]glutamate, [1-14C]glutamine, [U-14C]proline and [3-14C]pyruvate, but by a decreased 14CO2 production from [1-14C]pyruvate. Simultaneously, there is a considerable decrease in tissue content of 2-oxoglutarate, glutamate and citrate. Stimulation of 14CO2 production from [1-14C]glutamate is also observed in the presence of amino-oxyacetate, suggesting a stimulation of glutamate dehydrogenase and 2-oxoglutarate dehydrogenase fluxes by phenylephrine. Inhibition of pyruvate dehydrogenase flux by phenylephrine is due to an increased 2-oxoglutarate dehydroxygenase flux. Phenylephrine stimulates glutaminase flux and inhibits glutamine synthetase flux to a similar extent, resulting in an increased hepatic glutamine uptake. Whereas the effects of NH4+ ions and phenylephrine on glutaminase flux were additive, activation of glutaminase by glucagon was considerably diminished in the presence of phenylephrine. The reported effects are largely overcome by prazosin, indicating the involvement of alpha-adrenergic receptors in the action of phenylephrine. It is concluded that stimulation of gluconeogenesis from various amino acids by phenylephrine is due to an increased flux through glutamate dehydrogenase and the citric acid cycle.