NADPH composite index analysis quantifies the relationship between compartmentalized NADPH dynamics and growth rates in cancer cells.

NADPH, a highly compartmentalized electron donor in mammalian cells, plays essential roles in cell metabolism. However, little is known about how cytosolic and mitochondrial NADPH dynamics relate to cancer cell growth rates in response to varying nutrient conditions. To address this issue, we present NADPH composite index analysis, which quantifies the relationship between compartmentalized NADPH dynamics and growth rates using genetically encoded NADPH sensors, automated image analysis pipeline, and correlation analysis. Through this analysis, we demonstrated that compartmentalized NADPH dynamics patterns were cancer cell-type dependent. Specifically, cytosolic and mitochondrial NADPH dynamics of MDA-MB-231 decreased in response to serine deprivation, while those of HCT-116 increased in response to serine or glutamine deprivation. Furthermore, by introducing a fractional contribution parameter, we correlated cytosolic and mitochondrial NADPH dynamics to growth rates. Using this parameter, we identified cancer cell lines whose growth rates were selectively inhibited by targeting cytosolic or mitochondrial NADPH metabolism. Mechanistically, we identified citrate transporter as a key mitochondrial transporter that maintains compartmentalized NADPH dynamics and growth rates. Altogether, our results present a significant advance in quantifying the relationship between compartmentalized NADPH dynamics and cancer cell growth rates, highlighting a potential of targeting compartmentalized NADPH metabolism for selective cancer cell growth inhibitions.

Differential substrate use in EGF- and oncogenic KRAS-stimulated human mammary epithelial cells.

Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate and provide a model to distinguish the metabolic influences of oncogenic mutations and nononcogenic growth.

Metabolic requirements for cancer cell proliferation.

BACKGROUND: The study of cancer metabolism has been largely dedicated to exploring the hypothesis that oncogenic transformation rewires cellular metabolism to sustain elevated rates of growth and division. Intense examination of tumors and cancer cell lines has confirmed that many cancer-associated metabolic phenotypes allow robust growth and survival; however, little attention has been given to explicitly identifying the biochemical requirements for cell proliferation in a rigorous manner in the context of cancer metabolism. RESULTS: Using a well-studied hybridoma line as a model, we comprehensively and quantitatively enumerate the metabolic requirements for generating new biomass in mammalian cells; this indicated a large biosynthetic requirement for ATP, NADPH, NAD(+), acetyl-CoA, and amino acids. Extension of this approach to serine/glycine and glutamine metabolic pathways suggested lower limits on serine and glycine catabolism to supply one-carbon unit synthesis and significant availability of glutamine-derived carbon for biosynthesis resulting from nitrogen demands alone, respectively. We integrated our biomass composition results into a flux balance analysis model, placing upper bounds on mitochondrial NADH oxidation to simulate metformin treatment; these simulations reproduced several empirically observed metabolic phenotypes, including increased reductive isocitrate dehydrogenase flux. CONCLUSIONS: Our analysis clarifies the differential needs for central carbon metabolism precursors, glutamine-derived nitrogen, and cofactors such as ATP, NADPH, and NAD(+), while also providing justification for various extracellular nutrient uptake behaviors observed in tumors. Collectively, these results demonstrate how stoichiometric considerations alone can successfully predict empirically observed phenotypes and provide insight into biochemical dynamics that underlie responses to metabolic perturbations.

Comprehensive metabolic modeling of multiple 13C-isotopomer data sets to study metabolism in perfused working hearts.

In many forms of cardiomyopathy, alterations in energy substrate metabolism play a key role in disease pathogenesis. Stable isotope tracing in rodent heart perfusion systems can be used to determine cardiac metabolic fluxes, namely those relative fluxes that contribute to pyruvate, the acetyl-CoA pool, and pyruvate anaplerosis, which are critical to cardiac homeostasis. Methods have previously been developed to interrogate these relative fluxes using isotopomer enrichments of measured metabolites and algebraic equations to determine a predefined metabolic flux model. However, this approach is exquisitely sensitive to measurement error, thus precluding accurate relative flux parameter determination. In this study, we applied a novel mathematical approach to determine relative cardiac metabolic fluxes using 13C-metabolic flux analysis (13C-MFA) aided by multiple tracer experiments and integrated data analysis. Using 13C-MFA, we validated a metabolic network model to explain myocardial energy substrate metabolism. Four different 13C-labeled substrates were queried (i.e., glucose, lactate, pyruvate, and oleate) based on a previously published study. We integrated the analysis of the complete set of isotopomer data gathered from these mouse heart perfusion experiments into a single comprehensive network model that delineates substrate contributions to both pyruvate and acetyl-CoA pools at a greater resolution than that offered by traditional methods using algebraic equations. To our knowledge, this is the first rigorous application of 13C-MFA to interrogate data from multiple tracer experiments in the perfused heart. We anticipate that this approach can be used widely to study energy substrate metabolism in this and other similar biological systems.

Neuregulin-activated ERBB4 induces the SREBP-2 cholesterol biosynthetic pathway and increases low-density lipoprotein uptake.

Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2-regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2-regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.

Isotopomer Spectral Analysis: Utilizing Nonlinear Models in Isotopic Flux Studies.

We present the principles underlying the isotopomer spectral analysis (ISA) method for evaluating biosynthesis using stable isotopes. ISA addresses a classic conundrum encountered in the use of radioisotopes to estimate biosynthesis rates whereby the information available is insufficient to estimate biosynthesis. ISA overcomes this difficulty capitalizing on the additional information available from the mass isotopomer labeling profile of a polymer. ISA utilizes nonlinear regression to estimate the two unknown parameters of the model. A key parameter estimated by ISA represents the fractional contribution of the tracer to the precursor pool for the biosynthesis, D. By estimating D in cells synthesizing lipids, ISA quantifies the relative importance of two distinct pathways for flux of glutamine to lipid, reductive carboxylation, and glutaminolysis. ISA can also evaluate the competition between different metabolites, such as glucose and acetoacetate, as precursors for lipogenesis and thereby reveal regulatory properties of the biosynthesis pathway. The model is flexible and may be expanded to quantify sterol biosynthesis allowing tracer to enter the pathway at three different positions, acetyl CoA, acetoacetyl CoA, and mevalonate. The nonlinear properties of ISA provide a method of testing for the presence of gradients of precursor enrichment illustrated by in vivo sterol synthesis. A second ISA parameter provides the fraction of the polymer that is newly synthesized over the time course of the experiment. In summary, ISA is a flexible framework for developing models of polymerization biosynthesis providing insight into pools and pathway that are not easily quantified by other techniques.

Compartmentation of Metabolism of the C12-, C9-, and C5-n-dicarboxylates in Rat Liver, Investigated by Mass Isotopomer Analysis: ANAPLEROSIS FROM DODECANEDIOATE.

We investigated the compartmentation of the catabolism of dodecanedioate (DODA), azelate, and glutarate in perfused rat livers, using a combination of metabolomics and mass isotopomer analyses. Livers were perfused with recirculating or nonrecirculating buffer containing one fully (13)C-labeled dicarboxylate. Information on the peroxisomal versus mitochondrial catabolism was gathered from the labeling patterns of acetyl-CoA proxies, i.e. total acetyl-CoA, the acetyl moiety of citrate, C-1 + 2 of ß-hydroxybutyrate, malonyl-CoA, and acetylcarnitine. Additional information was obtained from the labeling patterns of citric acid cycle intermediates and related compounds. The data characterize the partial oxidation of DODA and azelate in peroxisomes, with terminal oxidation in mitochondria. We did not find evidence of peroxisomal oxidation of glutarate. Unexpectedly, DODA contributes a substantial fraction to anaplerosis of the citric acid cycle. This opens the possibility to use water-soluble DODA in nutritional or pharmacological anaplerotic therapy when other anaplerotic substrates are impractical or contraindicated, e.g. in propionic acidemia and methylmalonic acidemia.

NTFD--a stand-alone application for the non-targeted detection of stable isotope-labeled compounds in GC/MS data.

SUMMARY: Most current stable isotope-based methodologies are targeted and focus only on the well-described aspects of metabolic networks. Here, we present NTFD (non-targeted tracer fate detection), a software for the non-targeted analysis of all detectable compounds derived from a stable isotope-labeled tracer present in a GC/MS dataset. In contrast to traditional metabolic flux analysis approaches, NTFD does not depend on any a priori knowledge or library information. To obtain dynamic information on metabolic pathway activity, NTFD determines mass isotopomer distributions for all detected and labeled compounds. These data provide information on relative fluxes in a metabolic network. The graphical user interface allows users to import GC/MS data in netCDF format and export all information into a tab-separated format. AVAILABILITY: NTFD is C++- and Qt4-based, and it is freely available under an open-source license. Pre-compiled packages for the installation on Debian- and Redhat-based Linux distributions, as well as Windows operating systems, along with example data, are provided for download at http://ntfd.mit.edu/.

Cofactor balance by nicotinamide nucleotide transhydrogenase (NNT) coordinates reductive carboxylation and glucose catabolism in the tricarboxylic acid (TCA) cycle.

Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)(+) cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)(+) ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle.

Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia.

Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.

Measuring deuterium enrichment of glucose hydrogen atoms by gas chromatography/mass spectrometry.

We developed a simple and accurate method for determining deuterium enrichment of glucose hydrogen atoms by electron impact gas chromatography mass spectrometry (GC/MS). First, we prepared 18 derivatives of glucose and screened over 200 glucose fragments to evaluate the accuracy and precision of mass isotopomer data for each fragment. We identified three glucose derivatives that gave six analytically useful ions: (1) glucose aldonitrile pentapropionate (m/z 173 derived from C4-C5 bond cleavage; m/z 259 from C3-C4 cleavage; m/z 284 from C4-C5 cleavage; and m/z 370 from C5-C6 cleavage); (2) glucose 1,2,5,6-di-isopropylidene propionate (m/z 301, no cleavage of glucose carbon atoms); and (3) glucose methyloxime pentapropionate (m/z 145 from C2-C3 cleavage). Deuterium enrichment at each carbon position of glucose was determined by least-squares regression of mass isotopomer distributions. The validity of the approach was tested using labeled glucose standards and carefully prepared mixtures of standards. Our method determines deuterium enrichment of glucose hydrogen atoms with an accuracy of 0.3 mol %, or better, without the use of any calibration curves or correction factors. The analysis requires only 20 µL of plasma, which makes the method applicable for studying gluconeogenesis using deuterated water in cell culture and animal experiments.

Tracking cellular metabolomics in lipoapoptosis- and steatosis-developing liver cells.

Palmitate (PA) is known to induce reactive oxygen species (ROS) formation and apoptosis in liver cells, whereas concurrent treatment of oleate (OA) with PA predominately induces steatosis without ROS in liver cells. We previously reported that PA treatment induces the decoupling of glycolysis and tricarboxylic acid cycle (TCA cycle) fluxes, but OA co-treatment restored most metabolic fluxes to their control levels. However, the mechanisms by which metabolites are linked to metabolic fluxes and subsequent lipoapoptotic or steatotic phenotypes remain unclear. To determine the link, we used GC-MS-based polar and non-polar metabolic profiling in lipoapoptosis- or steatosis-developing H4IIEC3 hepatoma cells, to examine the metabolome at different time points after treatment with either PA alone (PA cells) or both PA and OA (PA/OA cells). Metabolic profiles revealed various changes in metabolite levels for TCA cycle intermediates, pentose phosphate pathway (PPP) intermediates, and energy storage metabolites between PA and PA/OA cells. For example, adenosine was markedly increased only in PA cells, whereas gluconate was increased in PA/OA cells. To assess the interaction among these metabolites, the metabolite-to-metabolite correlations were calculated and correlation networks were visualized. These correlation networks demonstrate that a dissociation among PPP metabolites was introduced in PA-treated cells, and this dissociation was restored in PA/OA-treated cells. Thus, our data suggest that abnormal PPP fluxes, in addition to increased adenosine levels, might be related to the decoupling of glycolysis and the resulting lipoapoptotic phenotype.

Ketogenic essential amino acids modulate lipid synthetic pathways and prevent hepatic steatosis in mice.

BACKGROUND: Although dietary ketogenic essential amino acid (KAA) content modifies accumulation of hepatic lipids, the molecular interactions between KAAs and lipid metabolism are yet to be fully elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We designed a diet with a high ratio (E/N) of essential amino acids (EAAs) to non-EAAs by partially replacing dietary protein with 5 major free KAAs (Leu, Ile, Val, Lys and Thr) without altering carbohydrate and fat content. This high-KAA diet was assessed for its preventive effects on diet-induced hepatic steatosis and whole-animal insulin resistance. C57B6 mice were fed with a high-fat diet, and hyperinsulinemic ob/ob mice were fed with a high-fat or high-sucrose diet. The high-KAA diet improved hepatic steatosis with decreased de novo lipogenesis (DNL) fluxes as well as reduced expressions of lipogenic genes. In C57B6 mice, the high-KAA diet lowered postprandial insulin secretion and improved glucose tolerance, in association with restored expression of muscle insulin signaling proteins repressed by the high-fat diet. Lipotoxic metabolites and their synthetic fluxes were also evaluated with reference to insulin resistance. The high-KAA diet lowered muscle and liver ceramides, both by reducing dietary lipid incorporation into muscular ceramides and preventing incorporation of DNL-derived fatty acids into hepatic ceramides. CONCLUSION: Our results indicate that dietary KAA intake improves hepatic steatosis and insulin resistance by modulating lipid synthetic pathways.

Nontargeted elucidation of metabolic pathways using stable-isotope tracers and mass spectrometry.

Systems level tools for the quantitative analysis of metabolic networks are required to engineer metabolism for biomedical and industrial applications. While current metabolomics techniques enable high-throughput quantification of metabolites, these methods provide minimal information on the rates and connectivity of metabolic pathways. Here we present a new method, nontargeted tracer fate detection (NTFD), that expands upon the concept of metabolomics to solve the above problems. Through the combined use of stable isotope tracers and chromatography coupled to mass spectrometry, our computational analysis enables the quantitative detection of all measurable metabolites derived from a specific labeled compound. Without a priori knowledge of a reaction network or compound library, NTFD provides information about relative flux magnitudes into each metabolite pool by determining the mass isotopomer distribution for all labeled compounds. This novel method adds a new dimension to the metabolomics tool box and provides a framework for global analysis of metabolic fluxes.

Effect of anaplerotic fluxes and amino acid availability on hepatic lipoapoptosis.

To identify metabolic pathways involved in hepatic lipoapoptosis, metabolic flux analysis using [U-(13)C(5)]glutamine as an isotopic tracer was applied to quantify phenotypic changes in H4IIEC3 hepatoma cells treated with either palmitate alone (PA-cells) or both palmitate and oleate in combination (PA/OA-cells). Our results indicate that palmitate inhibited glycolysis and lactate dehydrogenase fluxes while activating citric acid cycle (CAC) flux and glutamine uptake. This decoupling of glycolysis and CAC fluxes occurred during the period following palmitate exposure but preceding the onset of apoptosis. Oleate co-treatment restored most fluxes to their control levels, resulting in steatotic lipid accumulation while preventing apoptosis. In addition, palmitate strongly increased the cytosolic NAD(+)/NADH ratio, whereas oleate co-treatment had the opposite effect on cellular redox. We next examined the influence of amino acids on these free fatty acid-induced phenotypic changes. Increased medium amino acids enhanced reactive oxygen species (ROS) generation and apoptosis in PA-cells but not in PA/OA-cells. Overloading the medium with non-essential amino acids induced apoptosis, but essential amino acid overloading partially ameliorated apoptosis. Glutamate was the most effective single amino acid in promoting ROS. Amino acid overloading also increased cellular palmitoyl-ceramide; however, ceramide synthesis inhibitors had no effect on measurable indicators of apoptosis. Our results indicate that free fatty acid-induced ROS generation and apoptosis are accompanied by the decoupling of glycolysis and CAC fluxes leading to abnormal cytosolic redox states. Amino acids play a modulatory role in these processes via a mechanism that does not involve ceramide accumulation.

Dynamics of glutathione and ophthalmate traced with 2H-enriched body water in rats and humans.

We developed a LC-MS-MS assay of the (2)H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from (2)H-enriched body water. In rats whose body water was 2.5% (2)H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [(13)C(2),(15)N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 micromol.min(-1).kg(-1), and the half-life of plasma GSH/GSSR was 6-8 min. In healthy humans whose body fluids were 0.5% (2)H enriched, the (2)H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.

Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. I. Interrelation between gluconeogenesis and cataplerosis; formation of methoxamates from aminooxyacetate and ketoacids.

We conducted a study coupling metabolomics and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle. Rat livers were perfused with lactate or pyruvate +/- aminooxyacetate or mercaptopicolinate in the presence of 40% enriched NaH(13)CO(3). Other livers were perfused with dimethyl [1,4-(13)C(2)]succinate +/- mercaptopicolinate. In this first of two companion articles, we show that a substantial fraction of gluconeogenic carbon leaves the liver as citric acid cycle intermediates, mostly alpha-ketoglutarate. The efflux of gluconeogenic carbon ranges from 10 to 200% of the rate of liver gluconeogenesis. This cataplerotic efflux of gluconeogenic carbon may contribute to renal gluconeogenesis in vivo. Multiple crossover analyses of concentrations of gluconeogenic intermediates and redox measurements expand previous reports on the regulation of gluconeogenesis and the effects of inhibitors. We also demonstrate the formation of adducts from the condensation, in the liver, of (i) aminooxyacetate with pyruvate, alpha-ketoglutarate, and oxaloacetate and (ii) mercaptopicolinate and pyruvate. These adducts may exert metabolic effects unrelated to their effect on gluconeogenesis.

Metabolomic and mass isotopomer analysis of liver gluconeogenesis and citric acid cycle: II. Heterogeneity of metabolite labeling pattern.

In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and citric acid cycle labeled from NaH(13)CO(3) or dimethyl [1,4-(13)C(2)]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase + aconitase reactions, even in the absence of a source of alpha-ketoglutarate. In addition, in many cases, a number of labeling incompatibilities were found as follows: (i) glucose versus triose phosphates and phosphoenolpyruvate; (ii) differences in the labeling ratios C-4/C-3 of glucose versus (glyceraldehyde 3-phosphate)/(dihydroxyacetone phosphate); and (iii) labeling of citric acid cycle intermediates in tissue versus effluent perfusate. Overall, our data show that gluconeogenic and citric acid cycle intermediates cannot be considered as sets of homogeneously labeled pools. This probably results from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial versus extramitochondrial metabolites. Our data have implications for the use of labeling patterns for the calculation of metabolic rates or fractional syntheses in liver, as well as for modeling liver intermediary metabolism.

Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis.

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of (2)H from orally supplied (2)H(2)O into stable C-H bonds on the tripeptide. The incorporation of (2)H(2)O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with (2)H(2)O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N=6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with (2)H(2)O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of (2)H(2)O and a statistical basis for estimating N.

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.