Metabolic phenotyping of the diseased rat heart using 13C-substrates and ex vivo perfusion in the working mode

The objective of the present study was to compare energy substrate fluxes through metabolic pathways leading to mitochondrial citrate synthesis and release in normal and diseased rat hearts using ¹³C-substrates and mass isotopomer analysis by gas chromatography-mass spectrometry (GCMS). This study was prompted by our previous finding of a modulated citrate release by perfused rat hearts and by the possibility that a dysregulated myocardial citrate release represents a specific chronic alteration of energy metabolism in cardiac patients. The 15-week-old spontaneously hypertensive rat (SHR) was chosen as our animal model of disease and the Wistar-Kyoto (WKY) rat as its matched control. Ex vivo work-performing hearts were perfused with a semi-recirculating buffer containing physiological concentrations of unlabeled (glucose) and ¹³C-labeled ([U-¹³C₃](lactate + pyruvate) and/or [1-¹³C]oleate) substrates. In parallel to the continuous monitoring of indices of the heart's functional and physiological status, the following metabolic parameters were documented: (i) citrate release rates and citric acid cycle intermediate tissue levels, (ii) the contribution of fatty acids as well as pyruvate decarboxylation and carboxylation to citrate synthesis, and (iii) lactate and pyruvate uptake and efflux rates. Working hearts from both rat species showed a similar percent contribution of carbohydrates for citrate synthesis through decarboxylation (70%) and carboxylation (10%). SHR hearts showed the following metabolic alterations: a higher citrate release rate, which was associated with a parallel increase in its tissue level, a lower contribution of oleate -oxidation to citrate synthesis, and an accelerated efflux rate of unlabeled lactate from glycolysis. These metabolic changes were not explained by differences in myocardial oxygen consumption, cardiac performance or efficiency, nor correlated with indices of tissue necrosis or ischemia. This study demonstrates how the alliance between ex vivo semi-recirculating working perfused rat hearts with ¹³C-substrates and mass isotopomer analysis by GCMS, can provide an unprecedented insight into the metabolic phenotype of normal and diseased rat hearts. The clinical relevance of metabolic alterations herein documented in the SHR heart is suggested by its resemblance to those reported in cardiac patients. Taken altogether, our results raise the possibility that the increased citrate release of diseased hearts results from an imbalance between citrate synthesis and utilization rates, which becomes more apparent under conditions of substrate abundance.     

Quantitative assessment of anaplerosis from propionate in pig heart in vivo

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-(13)C(3)]propionate (0.25 mM) was infused in a coronary artery branch for 1 h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half (P < 0.05). Propionate extraction and uptake were 57.4 +/- 3.3% and 0.078 +/- 0.009 micromol x min(-1) x g(-1). Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 +/- 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 +/- 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart.     

Profiling substrate fluxes in the isolated working mouse heart using 13C-labeled substrates: focusing on the origin and fate of pyruvate and citrate carbons

The availability of genetically modified mice requires the development of methods to assess heart function and metabolism in the intact beating organ. With the use of radioactive substrates and ex vivo perfusion of the mouse heart in the working mode, previous studies have documented glucose and fatty acid oxidation pathways. This study was aimed at characterizing the metabolism of other potentially important exogenous carbohydrate sources, namely, lactate and pyruvate. This was achieved by using (13)C-labeling methods. The mouse heart perfusion setup and buffer composition were optimized to reproduce conditions close to the in vivo milieu in terms of workload, cardiac functions, and substrate-hormone supply to the heart (11 mM glucose, 0.8 nM insulin, 50 microM carnitine, 1.5 mM lactate, 0.2 mM pyruvate, 5 nM epinephrine, 0.7 mM oleate, and 3% albumin). The use of three differentially (13)C-labeled carbohydrates and a (13)C-labeled long-chain fatty acid allowed the quantitative assessment of the metabolic origin and fate of tissue pyruvate as well as the relative contribution of substrates feeding acetyl-CoA (pyruvate and fatty acids) and oxaloacetate (pyruvate) for mitochondrial citrate synthesis. Beyond concurring with the notion that the mouse heart preferentially uses fatty acids for energy production (63.5 +/- 3.9%) and regulates its fuel selection according to the Randle cycle, our study reports for the first time in the mouse heart the following findings. First, exogenous lactate is the major carbohydrate contributing to pyruvate formation (42.0 +/- 2.3%). Second, lactate and pyruvate are constantly being taken up and released by the heart, supporting the concept of compartmentation of lactate and glucose metabolism. Finally, mitochondrial anaplerotic pyruvate carboxylation and citrate efflux represent 4.9 +/- 1.8 and 0.8 +/- 0.1%, respectively, of the citric acid cycle flux and are modulated by substrate supply. The described (13)C-labeling strategy combined with an experimental setup that enables continuous monitoring of physiological parameters offers a unique model to clarify the link between metabolic alterations, cardiac dysfunction, and disease development.     

Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy

A previous 13C NMR technique (Malloy, C. R., Sherry, A.D., and Jeffrey, F.M.H. (1987) FEBS Lett. 212, 58-62) for measuring the relative flux of molecules through the oxidative versus anaplerotic pathways involving the citric acid cycle of the rat heart has been extended to include a complete analysis of the entire glutamate 13C spectrum. Although still simple in practice, this more sophisticated model allows an evaluation of 13C fractional enrichment of molecules entering both the oxidative and anaplerotic pathways under steady-state conditions. The method was used to analyze 13C NMR spectra of intact hearts or their acid extracts during utilization of 13C-enriched pyruvate, propionate, acetate, or various combinations thereof. [2-13C]Pyruvate was used to prove that steady-state flux of pyruvate through pyruvate carboxylase is significant during co-perfusion of pyruvate and acetate, and we demonstrate for the first time that a nine-line 13C multiplet may be detected in an intact, beating heart. Acetate or pyruvate alone provided about 86% of the acetyl-CoA; in combination, about 65% of the acetyl-CoA was derived from acetate, about 30% was derived from pyruvate, and the remainder from endogenous sources. Propionate reduced the contribution of exogenous acetate to acetyl-CoA to 77% and also reduced the oxidation of endogenous substrates. Equations are presented which allow this same analysis on multiply labeled substrates, making this technique extremely powerful for the evaluation of substrate selection and relative metabolic flux through anaplerotic and oxidative pathways in the intact heart.     

A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism

NMR spectroscopy and gas chromatography-mass spectrometry (GCMS) have both been used to study cardiac metabolism using substrates labeled with the stable isotope carbon-13. ¹³C-NMR studies of substrate oxidation are based on the assumption that the ¹³C-enrichment of glutamate reflects that of 2-ketoglutarate (2-KG). This assumption appears reasonable; however, it has not been thoroughly validated. The higher sensitivity of GCMS enables the direct determination of ¹³C-enrichment of 2-KG and other tricarboxylic acid (TCA) cycle intermediates. Therefore, using extracts from normal and diabetic hearts perfused with physiological concentrations of unlabeled glucose and ¹³C-labeled substrates, [3-¹³C](lactate + pyruvate) and [U-¹³C]palmitate, we compared the mass isotopomer distribution (MID) of citrate, 2-KG, succinate and malate measured directly by GCMS with that extrapolated from ¹³C-NMR glutamate isotopomer analysis. A significant correlation between the absolute molar percent enrichments (MPE) of the various mass isotopomers of glutamate determined by ¹³C-NMR and 2-KG determined by GCMS was observed for all sixteen-heart samples. This correlation was improved if the contribution from unlabeled 2-KG was removed (i.e. relative MPE) indicating that ¹³C-NMR under estimated the unlabeled fraction. We attribute this discrepancy in the measurement of unlabeled 2-KG to the fact that GCMS measures M0 directly, while the NMR analysis calculates it by difference, since unlabeled glutamate is not detected by ¹³C-NMR spectroscopy. Despite the differences between the two methods, ¹³C-MID of glutamate determined by NMR provides a simple and reliable indicator of fluxes of ¹³C-enriched substrates through the TCA cycle. It is also clear that MID analysis of TCA cycle intermediates by GCMS is a sensitive and direct approach to assess substrate selection for citrate synthesis as well as a potential indicator of sites and extent of anaplerosis and/or compartmentation. This study demonstrates that the alliance of NMR and GCMS represents a powerful approach for investigating the control and regulation of cardiac carbon metabolism.     

Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

A new mathematical model, based on the observation of ¹³C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-¹³C]Glc or [1,2-¹³C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the ¹³C-enrichment of acetyl-CoA can be deduced from ¹³C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-¹³C]glucose in the absence of insulin, was investigated by ¹³C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate ↔ 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G_o) or aspartate (A_O) were approximately the same for all hearts and remained constant during perfusion: G₀ = (0.74 ±0.03) μmol/g; A₀ = (1.49±0.05) μmol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min⁻¹ and 0.72 min⁻¹, respectively; the flux of this cycle is about (1.07±0.02) μmol min^-1 g^-1. Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.     

Fatty acid oxidation and its impact on response of spontaneously hypertensive rat hearts to an adrenergic stress: benefits of a medium-chain fatty acid

The spontaneously hypertensive rat (SHR) is a model of cardiomyopathy characterized by a restricted use of exogenous long-chain fatty acid (LCFA) for energy production. The aims of the present study were to document the functional and metabolic response of the SHR heart under conditions of increased energy demand and the effects of a medium-chain fatty acid (MCFA; octanoate) supplementation in this situation. Hearts were perfused ex vivo in a working mode with physiological concentrations of substrates and hormones and subjected to an adrenergic stimulation (epinephrine, 10 microM). (13)C-labeled substrates were used to assess substrate selection for energy production. Compared with control Wistar rat hearts, SHR hearts showed an impaired response to the adrenergic stimulation as reflected by 1) a smaller increase in contractility and developed pressure, 2) a faster decline in the aortic flow, and 3) greater cardiac tissue damage (lactate dehydrogenase release: 1,577 +/- 118 vs. 825 +/- 44 mU/min, P < 0.01). At the metabolic level, SHR hearts presented 1) a reduced exogenous LCFA contribution to the citric acid cycle flux (16 +/- 1 vs. 44 +/- 4%, P < 0.001) and an enhanced contribution of endogenous substrates (20 +/- 4 vs. 1 +/- 4%, P < 0.01); and 2) an increased lactate production from glycolysis, with a greater lactate-to-pyruvate production ratio. Addition of 0.2 mM octanoate reduced lactate dehydrogenase release (1,145 +/- 155 vs. 1,890 +/- 89 mU/min, P < 0.001) and increased exogenous fatty acid contribution to energy metabolism (23.7 +/- 1.3 vs. 15.8 +/- 0.8%, P < 0.01), which was accompanied by an equivalent decrease in unlabeled endogenous substrate contribution, possibly triglycerides (11.6 +/- 1.5 vs. 19.0 +/- 1.2%, P < 0.01). Taken altogether, these results demonstrate that the SHR heart shows an impaired capacity to withstand an acute adrenergic stress, which can be improved by increasing the contribution of exogenous fatty acid oxidation to energy production by MCFA supplementation.     

Determination of full 13C isotopomer distributions for metabolic flux analysis using heteronuclear spin echo difference NMR spectroscopy

13C-isotopomer labeling experiments play an increasingly important role in the analysis of intracellular metabolic fluxes for genetic engineering purposes. 13C NMR spectroscopy is a key technique in the experimental determination of isotopomer distributions. However, only subsets of isotopomers can be quantitated using this technique due to redundancies in the scalar coupling patterns and due to invisibility of the 12C isotope in NMR. Therefore, we developed and describe in this paper a 1H NMR spectroscopy method that allows to determine the complete isotopomer distribution in metabolites having a backbone consisting of up to at least four carbons. The proposed pulse sequences employ up to three alternately applied frequency-selective inversion pulses in the 13C channel. In a first application study, the complete isotopomer distribution of aspartate isolated from [1-13C]ethanol-grown Ashbya gossypii was determined. A tentative model of the central metabolism of this organism was constructed and used for metabolic flux analysis. The aspartate isotopomer NMR data played a key role in the successful determination of the flux through the peroxisomal glyoxylate pathway. The new NMR method can be highly instrumental in generating the data upon which isotopomer labeling experiments for flux analysis, that are becoming increasingly important, are based.     

Isolated cardiomyocytes in conjunction with NMR spectroscopy techniques to study metabolism and ion flux.

To distinguish cellular from vascular responses to physiological and pathophysiological stimuli, we developed methods to perform NMR spectroscopy on isolated ventricular cardiomyocytes. Isolated adult rat cardiomyocytes, placed in agarose beads and superfused with phosphate-free buffer (Media 199 (GIBCO 400-1100) gassed with 95% O2, 5% CO2), were used to evaluate a variety of cellular processes during different pharmacological and physiological interventions. Bioenergetic function was monitored with 31P NMR. Intermediary metabolism, gluconeogenesis, and glycolysis were monitored with 13C NMR. Sodium flux was monitored with 23Na NMR. Calcium flux was monitored with 19F NMR in conjunction with an intracellular calcium-chelating agent, 5F-1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid. Creatine kinase kinetics (forward rate constant (Kf) and flux of phosphocreatine to ATP) were estimated with 31P NMR saturation transfer data. Various combinations of NMR parameters were monitored simultaneously so that the interaction of metabolism and ion flux could be evaluated. We have demonstrated that it is possible to simultaneously monitor a variety of cellular processes in intact heart cells in real time, without the confounding influences of perfusion, contractile function, and extrinsic blood-borne neurohumoral agents. This model will be useful for longitudinal studies of myocyte metabolism and ion flux.     

Analysis of tumor metabolism reveals mitochondrial glucose oxidation in genetically diverse human glioblastomas in the mouse brain in vivo

Dysregulated metabolism is a hallmark of cancer cell lines, but little is known about the fate of glucose and other nutrients in tumors growing in their native microenvironment. To study tumor metabolism in vivo, we used an orthotopic mouse model of primary human glioblastoma (GBM). We infused (13)C-labeled nutrients into mice bearing three independent GBM lines, each with a distinct set of mutations. All three lines displayed glycolysis, as expected for aggressive tumors. They also displayed unexpected metabolic complexity, oxidizing glucose via pyruvate dehydrogenase and the citric acid cycle, and using glucose to supply anaplerosis and other biosynthetic activities. Comparing the tumors to surrounding brain revealed obvious metabolic differences, notably the accumulation of a large glutamine pool within the tumors. Many of these same activities were conserved in cells cultured ex vivo from the tumors. Thus GBM cells utilize mitochondrial glucose oxidation during aggressive tumor growth in vivo.     

Metabolic flux analysis in complex isotopolog space. Recycling of glucose in tobacco plants

Tobacco plants grown in vitro were supplied with a mixture of [U-13C6]glucose and unlabelled sucrose via the root system. After 20 days, leaves were harvested and extracted with water. Glucose was isolated from the extract and was analysed by 13C NMR spectroscopy. All 13C signals appeared as complex multiplets due to 13C-13C coupling. The abundance of 21 isotopologous glucose species was determined from the 13C NMR signal integrals by numerical deconvolution using a genetic algorithm. The relative fractions of specific isotopologs in the overall excess of 13C-labelled specimens establish flux contributions via glycolysis/glucogenesis, pentose phosphate pathway, citric acid cycle and Calvin cycle including 13CO2 refixation. The fluxes were modelled and reconstructed in silico by a novel rule-based approach yielding the contributions of circular pathways and the degree of multiple cycling events. The data indicate that the vast majority of the proffered [U-13C6]glucose molecules had been modified by catabolism and subsequent glucogenesis from catabolic fragments, predominantly via passage through the citric acid cycle and the pentose phosphate pathway.     

Partitioning of pyruvate between oxidation and anaplerosis in swine hearts

The goal of this study was to measure flux through pyruvate carboxylation and decarboxylation in the heart in vivo. These rates were measured in the anterior wall of normal anesthetized swine hearts by infusing [U-(13)C(3)]lactate and/or [U-(13)C(3)] pyruvate into the left anterior descending (LAD) coronary artery. After 1 h, the tissue was freeze-clamped and analyzed by gas chromatography-mass spectrometry for the mass isotopomer distribution of citrate and its oxaloacetate moiety. LAD blood pyruvate and lactate enrichments and concentrations were constant after 15 min of infusion. Under near-normal physiological concentrations of lactate and pyruvate, pyruvate carboxylation and decarboxylation accounted for 4.7 +/- 0.3 and 41.5 +/- 2.0% of citrate formation, respectively. Similar relative fluxes were found when arterial pyruvate was raised from 0.2 to 1.1 mM. Addition of 1 mM octanoate to 1 mM pyruvate inhibited pyruvate decarboxylation by 93% without affecting carboxylation. The absence of M1 and M2 pyruvate demonstrated net irreversible pyruvate carboxylation. Under our experimental conditions we found that pyruvate carboxylation in the in vivo heart accounts for at least 3-6% of the citric acid cycle flux despite considerable variation in the flux through pyruvate decarboxylation.     

In vivo 13C NMR studies of compartmentalized cerebral carbohydrate metabolism

Localized 13C nuclear magnetic resonance (NMR) spectroscopy provides a unique window for studying cerebral carbohydrate metabolism through, e.g. the completely non-invasive measurement of cerebral glucose and glycogen metabolism. In addition, label incorporation into amino acid neurotransmitters such as glutamate (Glu), GABA and aspartate can be measured providing information on Krebs cycle flux and oxidative metabolism. Given the compartmentation of key enzymes such as pyruvate carboxylase and glutamine synthetase, the detection of label incorporation into glutamine indicated that neuronal and glial metabolism can be measured in vivo. The purpose of this paper is to provide a critical overview of these recent advances into measuring compartmentation of brain energy metabolism using localized in vivo 13C NMR spectroscopy. The studies reviewed herein showed that anaplerosis is significant in brain, as is oxidative ATP generation in glia and the rate of glial glutamine synthesis attributed to the replenishment of the neuronal Glu pool and that brain glycogen metabolism is slow under resting conditions. This new modality promises to provide a new investigative tool to study aspects of normal and diseased brain hitherto unaccessible, such as the interplay between glutamatergic action, glucose and glycogen metabolism during brain activation, and the derangements thereof in patients with hepatic encephalopathy, neurodegenerative diseases and diabetes.     

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.     

Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and alpha-ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.     

Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat

Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 +/- 2.8, 62.4 +/- 4.5, and 224 +/- 16 mM/s) and exceeded the NMR-determined in vivo reaction velocities by an order of magnitude (4.1 +/- 1.2, 5.1 +/- 1.6, and 18.4 +/- 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues: greater than 99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98% MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p less than 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.     

Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon-13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of (13)C label from the TCA cycle. These results illustrate that (13)NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. (c) 1994 John Wiley & Sons, Inc.