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SummaryThe use of isotopic tracers and metabolic flux analysis (MFA) has unveiled a number of metabolic pathways differentially activated in cancer cells. To support efforts to design effective metabolic therapies for cancer, we sought to distinguish metabolic behavior in cancer versus normal cells growing at the same rate. To this end, we performed13C-isotope tracing and MFA in human mammary epithelial cells (HMECs) harboring different combinations of oncogenes. By introducing a new quantity termed metabolic flux intensity, defined as pathway flux divided by specific growth rate, we showed that metabolism is dually controlled by proliferation and oncogenotypes.13C-MFA further revealed that oxidative pentose phosphate pathway (oxPPP), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) were most enhanced in cancerous HMECs. Drug targeting of these pathways selectively reduced growth in the tumorigenic HMEC line. Our study provides direct evidence that metabolism of cancer cells is different than that of normal proliferating cells.

Isotopic tracers have been widely used to probe specific pathways within metabolic networks. Tracers are metabolized through various metabolic routes, generating differential labeling patterns. Cor...

Many metabolic phenotypes in cancer cells are also characteristic of proliferating non-transformed 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 non-oncogenic growth.

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 using13C-metabolic flux analysis (13C-MFA) aided by multiple tracer experiments and integrated data analysis. Using13C-MFA, we validated a metabolic network model to explain myocardial energy substrate metabolism. Four different13C-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 of13C-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.

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 13C-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 β-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.

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.

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., National Institutes of Health (U.S.) (Grant 1R01DK075850-01), National Institutes of Health (U.S.) (Grant 1R01CA160458-01A1)

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.

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.

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.

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 α-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-13C]glutamine or [5-13C]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 α-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.

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.

Metabolic fluxes, estimated from stable isotope studies, provide a key to quantifying physiology in fields ranging from metabolic engineering to the analysis of human metabolic diseases. A serious drawback of the flux estimation method in current use is that it does not produce confidence limits for the estimated fluxes. Without this information it is difficult to interpret flux results and expand the physiological significance of flux studies. To address this shortcoming we derived analytical expressions of flux sensitivities with respect to isotope measurements and measurement errors. These tools allow the determination of local statistical properties of fluxes and relative importance of measurements. Furthermore, we developed an efficient algorithm to determine accurate flux confidence intervals and demonstrated that confidence intervals obtained with this method closely approximate true flux uncertainty. In contrast, confidence intervals approximated from local estimates of standard deviations are inappropriate due to inherent system nonlinearities. We applied these methods to analyze the statistical significance and confidence of estimated gluconeogenesis fluxes from human studies with [U–13C]glucose as tracer and found true limits for flux estimation in specific human isotopic protocols.

We developed gas chromatography-mass spectrometry assays for the relative concentration and for the mass isotopomer distribution of gluconeogenic and citric acid cycle intermediates in tissues. The assay involves (i) spiking the sample with one or more internal standards, (ii) chloroform–methanol extraction at −25 °C, (iii) Folch wash of the extract, (iv) treatment of the water-methanol phase with methoxylamine, (v) evaporation and trimethylsilyl derivatization, and (vi) ammonia positive chemical ionization gas chromatography-mass spectrometry. For metabolomic computations, indices of concentrations for all compounds assayed are calculated as (Area of analyte)/(Area of reference compound). The assay was applied to a study of the effect of mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, on the profile of gluconeogenic intermediates in rat livers perfused with pyruvate. Crossover analysis of concentrations indices, compared to a control group, yielded very similar profiles as previous enzymatic assays, and correctly identified the site of action of mercaptopicolinate. Principal component analysis distinguished between control and drug treated samples. A loadings plot was used to identify the site of action of the drug in the metabolic pathway. Since metabolite concentrations do not address the flux through a pathway, perfusions with [1,4-13C2] succinate dimethylester were conducted to assess fluxes around PEPCK. This allowed a dynamic metabolomics analysis which indicated that considerable flux through the pathway remained in the presence of mercaptopicolinate. This study illustrates the power of dynamic metabolomics to complement concentration based metabolomic studies.

Measurement of fractional lipogenesis by condensation polymerization methods assumes constant enrichment of lipogenic acetyl-CoA in all hepatocytes. mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) represent such methods and are based on the combinatorial analyses of mass isotopomer distributions (MIDs) of fatty acids and sterols. We previously showed that the concentration and enrichment of [13C]acetate decrease markedly across the dog liver because of the simultaneous uptake and production of acetate. To test for zonation of the enrichment of lipogenic acetyl-CoA, conscious dogs, prefitted with transhepatic catheters, were infused with glucose and [1,2-13C2]acetate in a branch of the portal vein. Analyses of MIDs of fatty acids and sterols isolated from liver, bile, and plasma very low density lipoprotein by a variant of ISA designed to detect gradients in precursor enrichment revealed marked zonation of enrichment of lipogenic acetyl-CoA. As control experiments where no zonation of acetyl-CoA enrichment would be expected, isolated rat livers were perfused with 10 mm [1,2-13C2]acetate. The ISA analyses of MIDs of fatty acids and sterols from liver and bile still revealed a zonation of acetyl-CoA enrichment. We conclude that zonation of hepatic acetyl-CoA enrichment occurs under a variety of animal models and physiological conditions. Failure to consider gradients of precursor enrichment can lead to underestimations of fractional lipogenesis calculated from the mass isotopomer distributions. The degree of such underestimation was modeled in vitro, and the data are reported in the companion paper (Bederman, I. R., Kasumov, T., Reszko, A. E., David, F., Brunengraber, H., and Kelleher, J. K. (2004) J. Biol. Chem. 279, 43217-43226).

This study was undertaken to explore the metabolic fate of acetyl-L-carnitine in rat brain. To measure the flux of carbon atoms into anabolic processes occurring at regional levels, we have injected [1-(14)C]acetyl-L-carnitine into the lateral brain ventricle of conscious rats. After injection of [1-(14)C]acetyl-L-carnitine, the majority of radioactivity was recovered as 14CO2 expired (60% of that injected). The percentage of radioactivity recovered in brain was 1.95, 1.60, 1.30, and 0.93% at 1, 3, 6, and 22 h, respectively. Radioactivity distribution in various lipid components indicated that the fatty acid moiety of phospholipid contained the majority of radioactivity. The radioactive profile of these fatty acids showed that the acetyl moiety of acetyl-L-carnitine was incorporated into saturated (60%), monounsaturated (15%), and polyunsaturated (25%) fatty acids [mainly present in 20:4 (5.2%) and 22:6 (7.8%)]. Injection in the brain ventricle of radioactive glucose, the major source of acetyl-CoA in the CNS, revealed that glucose was a precursor of saturated (85%) and monounsaturated (15%) but not of polyunsaturated fatty acids. Thus, this study demonstrated distinct fates of glucose and acetyl-L-carnitine following intracerebroventricular injection. In summary, these data implicate acetyl-L-carnitine as an important member of a complex acetate trafficking system in brain lipid metabolism.

Phospholipids and sterols are known to have multiple functions in reproductive tissue of mammals. High concentrations of the cholesterol precursor desmosterol have been described in testis, epididymis, and spermatozoa of various species. These findings and the recent discovery of some cholesterol precursors as meiosis-activating sterols suggest important functions of cholesterol precursors in fertility. Many sterol intermediates appear from the 19-step conversion of lanosterol, the first sterol synthesized in the cascade of cholesterol synthesis, to cholesterol. The bio- chemical basis of the genetically inherited Smith-Lemli- Opitz syndrome has been described as a defective conver- sion of 7-dehydrocholesterol to cholesterol. Since this dis- covery, interest has focused on this special cholesterol pre- cursor. Here, we report high concentrations of 7- and 8-dehydrocholesterol in caput epididymidis and spermato- zoa derived from caput epididymidis of Sprague-Dawley and Wistar rats, which comprised up to 30% of total sterols. In contrast to caput epididymidis, 7- and 8-dehydrocholes- terol were barely detected in cauda epididymidis or testis. Desmosterol increased several times from caput to cauda epididymidis. This is the first report of the natural ap- pearance of high concentrations of dehydrocholesterols in mammalian tissue, and it underlines the putative impor- tance of cholesterol precursors in reproductive tissue. — Lindenthal, B., T. A. Aldaghlas, J. K. Kelleher, S. M. Henkel, R. Tolba, G. Haidl, and K. von Bergmann. Neutral sterols of rat epididymis: high concentrations of dehydrocholesterols in rat caput epididymidis. J. Lipid Res. 2001. 42: 1089-1095.

Metabolic physiologists and metabolic engineers share the need to estimate flux. However, the physiologist often works with systems that do not maintain steady state for long. Many sites cannot be sampled, and calculating mass and isotopic balance for the entire system may not be feasible. To deal with these constraints, metabolic physiologists have developed specialized isotopic techniques that may be unfamiliar to metabolic engineers. A selection of these techniques is presented here, not because it is anticipated that they would be used by engineers exactly as in the physiologist's setting, but because they illustrate novel applications of tracer methodology. Creative engineers may find new adaptations of these tools in metabolic engineering and opportunities to increase redundancy. Physiologists, entering into a dialog with engineers, may see more clearly the potential of comprehensive models and revisit the impediments to a more complete analysis of human metabolic systems.

Objective—To determine effects of dietary lipid and protein on plasma lipoprotein and free fatty acid concentrations and hepatic fatty acid synthesis during weight gain and rapid weight loss in cats. Animals—24 ovariohysterectomized cats. Procedure—Cats were fed a high energy diet until they gained 30% of their ideal body weight and then randomly assigned to receive 1 of 4 weight reduction diets (6 cats/diet) at 25% of maintenance energy requirements. Diets contained a low or high quality protein source and a lipid source deficient or sufficient in long chain essential fatty acids. Plasma samples and liver biopsy specimens were obtained before and after weight gain and during and after weight loss for determination of free fatty acid, triglyceride, and lipoprotein concentrations. Synthesis of these substances was measured by use of isotope enrichment. Results—Plasma total cholesterol concentration and concentration of lipoprotein fractions increased after weight gain, compared with baseline values. Weight loss resulted in a significant decrease in concentrations of all lipoprotein fractions except high density lipoprotein. High density lipoprotein concentration was significantly greater in cats fed diets containing an oil blend, compared with cats fed diets containing corn oil. Fatty acid synthesis after weight loss was below the detection limit of the measurement technique. Conclusions and Clinical Relevance—In cats undergoing rapid weight loss there is neither increased triglyceride synthesis nor decreased transport of very low density lipoproteins from the liver, suggesting that their involvement in the development of hepatic lipidosis may be minimal. (Am J Vet Res 2000;61:566–572)

Recently three equations for estimating gluconeogenesis in vivo have been proposed, two by J. A. Tayek and J. Katz [ Am. J. Physiol. 270 ( Endocrinol. Metab. 33): E709–E717, 1996, and Am. J. Physiol. 272 ( Endocrinol. Metab. 35): E476–E484, 1997] and one by B. R. Landau, J. Wahren, K. Ekberg, S. F. Previs, D. Yang, and H. Brunengraber [ Am. J. Physiol.274 ( Endocrinol. Metab. 37): E954–E961, 1998]. Both groups estimate gluconeogenesis from cycling of [U-13C]glucose to lactate and back to glucose, detected by mass spectrometry. Landau’s approach is based on analysis of labeled molecules, whereas Tayek and Katz’s is based on labeling of carbon atoms by use of the concept of “molar enrichment,” which weights each mass isotopomer by the number of labeled carbons. We derived an equation very similar to Landau’s using binomial probability. Our analysis demonstrates that the molecular-based approach is correct. Additionally, equations appropriate for 14C studies are not appropriate for 13C studies, because the method used to detect14C, decay of atoms, differs from13C mass isotopomers detected as labeled molecules. We conclude that the molar enrichment carbon-based approach is not useful in the derivation of equations for the polymerization of molecules detected by mass spectrometry of molecules, and we confirm the findings of Landau et al.

Acetyl-L-carnitine is known as a reservoir of activated acetyl units and as a modulator of metabolic function. The objective of this study was to quantify the fate of the acetyl moiety of acetyl-L-carnitine in lipogenic pathways. Lipogenesis was studied in an adipocyte model, differentiated 3T3-L1 cells, and a hepatoma cell, HepG2 cells. Lipogenesis and ketogenesis were examined in rat hepatocytes. Both de novo synthesis and elongation of fatty acids were investigated using gas chromatography/mass spectrometry and [1,2-(13)C]acetyl-L-carnitine. Comparisons were performed with [13C]glucose and [13C]acetate. Isotopomer Spectral Analysis, a stable isotope method for differentiating between the enrichment of the precursor and the amount of synthesis was used to analyze the data. Acetyl-L-carnitine was generally less effective than acetate as a precursor for de novo lipogenesis. The effects of acetyl-L-carnitine were not identical to those of acetate plus carnitine as expected if acetyl-L-carnitine flux to acetyl CoA is controlled by carnitine acetyl transferase. Acetyl-L-carnitine (2 mM) contributed approximately 10% of the lipogenic acetyl-CoA used for synthesis and elongation as well as 6% of the ketogenic acetyl-CoA. No differences were found between the precursor enrichment for de novo lipogenesis and for elongation of saturated fatty acids. Flux of acetyl-L-carnitine to lipid was increased, not decreased, by the ATP citrate lyase inhibitor, -hydroxycitrate. In contrast, flux of glucose to lipid was dramatically decreased by this inhibitor. These results indicate that flux of acetyl-L-carnitine to lipid can bypass citrate and utilize cytosolic acetyl-CoA synthesis.

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/., Belgian National Foundation for Scientific Research (ATTRACT A10/03), Belgian National Foundation for Scientific Research (AFR 1328318), German Science Foundation (HI1400/I-1)

Cholesterol synthesis from 13C-labeled precursors produces a discrete spectrum of mass isotopomers detectable using gas chromatography-mass spectrometry. The isotopomer spectral analysis (ISA) method matches the observed spectrum of cholesterol isotopomers with a mathematical model to obtain the best fit of model spectrum to data spectrum. The model was based on multinomial probability expressions that simulate cholesterol synthesis as a condensation of mevalonate fragments. As many as four unknown parameters, representing fluxes between compartments, were included in the model. Models were developed to assess cholesterol synthesis from 13C-enriched precursors including mevalonate, acetate, acetoacetate or octanoate. Models were tested in the human hepatoma cell line, Hep G2, which readily incorporated the 13C substrates into cholesterol. The ISA approach was used to estimate the fractional amount of the cholesterol precursors derived from the 13C substrate and the fraction of total cellular cholesterol synthesized in the presence of the 13C substrate. The study demonstrated the feasibility of the ISA approach for a condensation biosynthesis that is not a simple polymerization and for models with more than two unknown parameters.

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetyl CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells. The major findings were: 1. Acetoacetate was utilized by AS-30D cells, with 14C-lipid and 14CO2 as major products of [3-14C] acetoacetate. 2. Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium. 3. Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate. 4. Succinyl CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria. 5. Addition of acetoacetate, but not beta-hydroxybutyrate decreased conversion of [1-14C] acetate to 14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer. 6. In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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., National Institutes of Health (U.S.) (Grant R01 DK075850-01), American Cancer Society (Postdoctoral Fellowship), National Institutes of Health (U.S.), Burroughs Wellcome Fund, Smith Family Foundation, Damon Runyon Cancer Research Foundation, National Cancer Institute (U.S.)

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 lipogensis (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., National Institutes of Health (U.S) (Bioengineering Research Partnership Grant DK58533), National Institutes of Health (U.S) (NIH Metabolomics Roadmap Initiative DK070291), National Institutes of Health (U.S) (DK072856)

We developed a LC-MS-MS assay of the2H 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 from2H-enriched body water. In rats whose body water was 2.5%2H 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 [13C2,15N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 μmol·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%2H enriched, the2H 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.

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.

Metabolic flux analysis based on stable-isotope labeling experiments and analysis of mass isotopomer distributions (MID) of cellular metabolites is a tool of great significance for metabolic engineering and study of human disease. This method relies on accurate and precise measurements of mass isotopomers by gas chromatography/mass spectrometry. To improve flux estimates, we assessed potential errors in determining MID of tert-butyldimethylsilyl-derivatized amino acids, which were attributed to (i) the choice of integration algorithm, (ii) concentration effects, and (iii) overlapping fragments. We report 29 amino acid fragments that are useful for flux analysis and another 18 fragments that should be rejected, most importantly Val-302, Leu-200, Leu-302, Ile-302, Ser-302, and Asp-316. In addition, we provide a protocol to minimize errors for determining MID to less than 0.4 mol % for accepted fragments.

Insulin resistance is characterized by high insulin levels and decreased responsiveness of tissues to the clearance of glucose from the bloodstream. This study maintained the diabetes-prone C57BL/6J and obese-resistant A/J mice strains on a high-fat diet for twelve weeks to transcriptionally profile the liver for changes caused by high fat diet. In the eighth week of the experiment, the C57BL/6J mice began exhibiting signs of insulin resistance, while the A/J mice did not show any such indications during the course of the experiment. A regression model of partial least squares between serum insulin measurements and the liver gene expression profile for the C57BL/6J mice on a high-fat diet was constructed in an effort to quantitatively link the physiological measurement with the gene expressions. A series of discriminating genes between high fat and chow fed mice was generated for both the C57BL/6J and A/J strains. These discriminatory genes contain information about the mechanisms responsible for the development of insulin resistance, and the compensation for a high fat diet, respectively. The results identified several genes involved in the development of insulin resistance and serve as a framework for other studies involving other organs affected by this systemic disease.

Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal beta-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.

Metabolic fluxes estimated from stable-isotope studies provide a key to understanding cell physiology and regulation of metabolism. A limitation of the classical method for metabolic flux analysis (MFA) is the requirement for isotopic steady state. To extend the scope of flux determination from stationary to nonstationary systems, we present a novel modeling strategy that combines key ideas from isotopomer spectral analysis (ISA) and stationary MFA. Isotopic transients of the precursor pool and the sampled products are described by two parameters, D and G parameters, respectively, which are incorporated into the flux model. The G value is the fraction of labeled product in the sample, and the D value is the fractional contribution of the feed for the production of labeled products. We illustrate the novel modeling strategy with a nonstationary system that closely resembles industrial production conditions, i.e. fed-batch fermentation of Escherichia coli that produces 1,3-propanediol (PDO). Metabolic fluxes and the D and G parameters were estimated by fitting labeling distributions of biomass amino acids measured by GC/MS to a model of E. coli metabolism. We obtained highly consistent fits from the data with 82 redundant measurements. Metabolic fluxes were estimated for 20 time points during course of the fermentation. As such we established, for the first time, detailed time profiles of in vivo fluxes. We found that intracellular fluxes changed significantly during the fed-batch. The intracellular flux associated with PDO pathway increased by 10%. Concurrently, we observed a decrease in the split ratio between glycolysis and pentose phosphate pathway from 70/30 to 50/50 as a function of time. The TCA cycle flux, on the other hand, remained constant throughout the fermentation. Furthermore, our flux results provided additional insight in support of the assumed genotype of the organism.

Metabolic flux analysis (MFA) has emerged as a tool of great significance for metabolic engineering and mammalian physiology. An important limitation of MFA, as carried out via stable isotope labeling and GC/MS and nuclear magnetic resonance (NMR) measurements, is the large number of isotopomer or cumomer equations that need to be solved, especially when multiple isotopic tracers are used for the labeling of the system. This restriction reduces the ability of MFA to fully utilize the power of multiple isotopic tracers in elucidating the physiology of realistic situations comprising complex bioreaction networks. Here, we present a novel framework for the modeling of isotopic labeling systems that significantly reduces the number of system variables without any loss of information. The elementary metabolite unit (EMU) framework is based on a highly efficient decomposition method that identifies the minimum amount of information needed to simulate isotopic labeling within a reaction network using the knowledge of atomic transitions occurring in the network reactions. The functional units generated by the decomposition algorithm, called EMUs, form the new basis for generating system equations that describe the relationship between fluxes and stable isotope measurements. Isotopomer abundances simulated using the EMU framework are identical to those obtained using the isotopomer and cumomer methods, however, require significantly less computation time. For a typical (13)C-labeling system the total number of equations that needs to be solved is reduced by one order-of-magnitude (100s EMUs vs. 1000s isotopomers). As such, the EMU framework is most efficient for the analysis of labeling by multiple isotopic tracers. For example, analysis of the gluconeogenesis pathway with (2)H, (13)C, and (18)O tracers requires only 354 EMUs, compared to more than two million isotopomers.

The in vivo 13C NMR technique opens up a rich new landscape for metabolic research, providing the ability to detect the flow of label in real time from one metabolite pool to the next in living tissue. However, the abundance of data that is gleaned from these experiments yields up useful results only in via the process of mathematical modeling. The use of tracer kinetics in biological systems has a rich history dating back at least fifty years, and the body of theory expands as each new tracer, detection methodology, or biological system requires a new collection of models tailored to particular strengths and limitations. With 13C NMR, we have been able to monitor multiple intracellular compounds in time, allowing the use of far more detailed and complex models than ever before (Chance et al., 1983). The challenge presented to the investigator now is to use the particular strengths of 13C NMR, those of in situ, time-dependent, and compound-specific detection, to investigate those aspects of biological systems that have constituted the black boxes of assumptions made for other tracer methodologies.

Background Obesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrys and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO)-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions. Results Transcriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/ weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced, hepatic glucose output via phosphoenolpyruvate carboxykinase (PCK1), whose mRNA levels were also elevated. Conclusion These findings demonstrate that hepatocytes respond to DIO and weight reduction by controlling gene transcription in a variety of important molecular pathways. Future studies that characterize the physiological significance of the identified genes in modulating energy homeostasis could provide a better understanding of the mechanisms linking DIO with insulin resistance.

Four patients with cerebrotendinous xanthomatosis (CTX) and 2 healthy controls received a constant proximal intraduodenal infusion of 1- 13 C-acetate as a stable-isotope-labeled marker of sterol synthesis. One patient was treated with pravastatin (20 mg twice daily) and another patient with chenodeoxycholic acid (250 mg tid). Every hour, venous blood and duodenal samples were obtained. Stable-isotope enrichment of neutral and polar sterols in serum and bile was assessed by gas chromatography/mass spectrometry. Isotopomer spectral analysis was performed on cholesterol, lathosterol, Delta-8-cholesterol, methylsterol, and lanosterol. Stable-isotope labeling of cholestanol, bile acids, and bile alcohols was analyzed by assessing the change over time of the ratio of M + 3 to M + 0. Eleven hours after marker infusion, we found up to 50% newly synthesized lathosterol in serum and up to 80% in bile, with similar results for other cholesterol precursors. In cholesterol, stable-isotope labeling could be demonstrated in all study subjects with a more prominent labeling in bile than in serum. No stable-isotope labeling was detected in cholestanol. Only minor stable-isotope incorporation was detectable in polar sterols in some subjects. Therapy with pravastatin did not have any effect on fractional or absolute synthesis rates or on the concentrations of cholestanol or cholesterol precursors compared to untreated patients with CTX. In contrast, therapy with chenodeoxycholic acid markedly lowered the concentrations of cholestanol and cholesterol precursors, led to a disappearance of bile alcohols, and reduced absolute synthesis rates of lathosterol. Isotopomer spectral analysis proved to be a powerful method to assess the endogenous synthesis of cholesterol precursors in patients with CTX. Higher fractional synthesis in bile than in serum may be due to the size of the pools in bile vs serum. Cholestanol exhibits no marker uptake and is therefore probably synthesized from preformed cholesterol. Biliary cholesterol secretion in patients with CTX is decreased compared to healthy controls.

In the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David, F., Wasserman, D. H., Kelleher, J. K., and Brunengraber, H. (2004) J. Biol. Chem. 279, 43207-43216), we demonstrated that, when the hepatic pool of lipogenic acetyl-CoA is labeled from [13C]acetate, the enrichment of this pool decreases across the liver lobule. In addition, estimates of fractional synthesis calculated by isotopomer spectral analysis (ISA), a nonlinear regression method, did not agree with a simpler algebraic two-isotopomer method. To evaluate differences between these methods, we simulated in vitro the synthesis of fatty acids under known gradients of precursor enrichment, and known values of fractional synthesis. First, we synthesized pentadecanoate from [U-13C3]propionyl-CoA and four gradients of [U-13C3]malonyl-CoA enrichment. Second, we pooled the fractions of each gradient. Third, we diluted each pool with pentadecanoate prepared from unlabeled malonyl-CoA to simulate the dilution of the newly synthesized compound by pre-existing fatty acids. This yielded a series of samples of pentadecanoate with known values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data.