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 α-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in U-13Cglutamine or 5-13Cglutamine 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 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-13Cglutamine or 5-13Cglutamine 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. Glutamine is utilized at a high rate by rapidly growing cells, including almost all cultured cell lines, where it is required at super-physiological concentrations of 2–4 mm for optimal growth (1Newsholme E.A. Crabtree B. Ardawi M.S. Biosci. Rep. 1985; 5: 393-400Crossref PubMed Scopus (296) Google Scholar). Recently, we evaluated the role of glutamine as a substrate for lipogenesis in a transformed wild type (WT) 5The abbreviations used are: WT, wild type; ISA, isotopomer spectral analysis; CAC, citric acid cycle; IDH, isocitrate dehydrogenase; MID, mass isotopomer distribution; IDPm, mitochondrial NADP-dependent isocitrate dehydrogenase; DMEM, Dulbecco's modified Eagle's medium. and IRS-1 knock-out brown adipose cell lines developed by Kahn and co-workers (2Fasshauer M. Klein J. Kriauciunas K.M. Ueki K. Benito M. Kahn C.R. Mol. Cell. Biol. 2001; 21: 319-329Crossref PubMed Scopus (151) Google Scholar). Using isotopomer spectral analysis (ISA) we found that WT cells utilized glutamine for over 40% of their lipogenic acetyl-CoA (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Glutamine was the largest precursor for lipogenic carbon, supplying more acetyl-CoA units than glucose or any other single source. This unexpected result led us to investigate glutamine metabolism in brown fat cell lines in more detail. The pathway for glutamine utilization in rapidly dividing cell is generally described as “glutaminolysis” where glutamine enters the citric acid cycle (CAC) as α-ketoglutarate traversing the cycle to oxaloacetate and exits as pyruvate or aspartate (1Newsholme E.A. Crabtree B. Ardawi M.S. Biosci. Rep. 1985; 5: 393-400Crossref PubMed Scopus (296) Google Scholar, 4Mazurek S. Eigenbrodt E. Anticancer Res. 2003; 23: 1149-1154PubMed Google Scholar). Pyruvate then could be converted to acetyl-CoA and citrate in the lipogenic pathway. A major argument for glutaminolysis is the need for a large supply of anaplerotic substrates for rapidly growing cells, which explains the high rate of glutamine utilization (5Board M. Humm S. Newsholme E.A. Biochem. J. 1990; 265: 503-509Crossref PubMed Scopus (189) Google Scholar). An alternative to glutaminolysis is the reductive carboxylation pathway where glutamine enters the CAC as α-ketoglutarate and is converted to citrate via isocitrate dehydrogenase (IDH) operating in reverse of the CAC direction. Evidence for reductive carboxylation in transformed cells is the finding that 5-14Cglutamine labels acetyl-CoA-derived carbons of lipids (6Holleran A.L. Briscoe D.A. Fiskum G. Kelleher J.K. Mol. Cell. Biochem. 1995; 152: 95-101Crossref PubMed Scopus (47) Google Scholar, 7D'Adamo A.F. Haft D.E. J. Biol. Chem. 1989; 240: 613-617Abstract Full Text PDF Google Scholar). Additionally, reversal of IDH has been detected in perfused liver and heart as evidenced by the mass isotopomer distribution (MID) of 13Ccitrate following perfusion with 13Cglutamine (8Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Comte B. Vincent G. Bouchard B. Benderdour M. Rosiers Des. C. Am. J. Physiol. 2002; 283: H1505-H1514Crossref PubMed Scopus (58) Google Scholar). Although previous studies have detected label in citrate and lipids via the reductive carboxylation pathway, fluxes through the metabolic network connecting glutamine metabolism to lipids have not been quantified. A complication in the analysis of the lipogenic pathway from glutamine is the role of the three mammalian IDH enzymes. The high mitochondrial NADH/NAD ratio will favor flux through the mitochondrial NAD-dependent isocitrate dehydrogenase toward α-ketoglutarate. Thus, the CAC enzyme is unlikely to be involved in lipogenesis from glutamine. Likewise, in rapidly growing cells the need for cytosolic NADPH for biosynthesis will favor flux of the cytosolic NADP-dependent enzyme toward α-ketoglutarate as shown recently in 3T3-L1 cells (10Koh H.J. Lee S.M. Son B.G. Lee S.H. Ryoo Z.Y. Chang K.T. Park J.W. Park D.C. Song B.J. Veech R.L. Song H. Huh T.L. J. Biol. Chem. 2004; 279: 39968-39974Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). This study also found that overexpression of cytosolic NADP-dependent enzyme in mice leads to increased lipogenesis in liver and fat, consistent with a reaction producing cytosolic NADPH. Thus, the candidate for flux in reverse of the CAC is the mitochondrial NADP-dependent enzyme (IDPm). Early studies established the feasibility of reductive carboxylation flux through this enzyme in hepatic models (11Dalziel K. Londesborough J.C. Biochem. J. 1968; 110: 223-230Crossref PubMed Scopus (67) Google Scholar, 12Wanders R.J. van Doorn H.E. Tager J.M. Eur. J. Biochem. 1981; 116: 609-614Crossref PubMed Scopus (11) Google Scholar). Flux of NADP-dependent enzyme toward α-ketoglutarate in vivo is supported by the affinity constant of the enzyme for CO2 (Km ∼ 1.6 mm), which is very close to physiological CO2 concentration (1.5 mm) and the affinity of the enzyme for NADPH, which is 100-fold higher than for NADP+ (13Reynolds C.H. Kuchel P.W. Dalziel K. Biochem. J. 1978; 171: 733-742Crossref PubMed Scopus (22) Google Scholar). Additionally, reductive carboxylation flux through IDPm has been hypothesized to play a key role in metabolic regulation of CAC cycle flux via a substrate cycle involving NAD/NADP transhydrogenase located at the inner mitochondrial membrane (14Sazanov L.A. Jackson J.B. FEBS Lett. 1994; 344: 109-116Crossref PubMed Scopus (180) Google Scholar). However, there may be other roles for IDPm. Recent studies have provided evidence for flux through IDPm toward α-ketoglutarate generating NADPH in cellular defense against oxidative stress (15Kim H.J. Kang B.S. Park J.W. Free Radic. Res. 2005; 39: 441-448Crossref PubMed Scopus (37) Google Scholar, 16Jo S.H. Son M.K. Koh H.J. Lee S.M. Song I.H. Kim Y.O. Lee Y.S. Jeong K.S. Kim W.B. Park J.W. Song B.J. Huh T.L. J. Biol. Chem. 2001; 276: 16168-16176Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). This role for IDPm may explain the high levels of the enzyme in non-lipogenic tissues, including heart and skeletal muscle. Although it is reasonable that the flux through the enzyme may differ in lipogenic and non-lipogenic tissues, it has been difficult to study such complicated pathways systematically. The major difficulty may be the absence of a comprehensive method for the analysis of metabolic fluxes through all of the relevant pathways. Here, we have tested the hypothesis that flux of glutamine to lipogenic acetyl-CoA involves a reorganization of CAC metabolism including reductive carboxylation flux through IDPm. Studies were conducted with stable isotopes and mass spectrometry. The data analysis included our previously described ISA approach (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, we enhanced the analysis by employing a novel comprehensive modeling approach to quantify many additional fluxes from isotopic data. This powerful method provided quantitative flux estimates of the network describing glutamine metabolism in brown adipocytes. The details of this approach and its implementation with software (Metran) will be provided elsewhere. Cell Culture and Adipocyte Differentiation—Brown adipocyte cells were cultured with the same procedure as in Yoo et al. (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Briefly, WT brown preadipocytes were grown in 6-well plates to confluence in differentiation medium, DMEM containing 25 mm glucose, 4 mm glutamine, 20 nm insulin, 1 nm triiodothyronine, and 10% fetal bovine serum as well as 44 mm NaHCO3 (day 0). The medium was then replaced with fresh induction medium, which was differentiation medium plus 0.125 mm indomethacin, 0.25 mm isobutylmethylxanthine, and 5 μm dexamethasone. On days 2 and 4, the medium was replaced with fresh differentiation medium. To estimate metabolic fluxes, 13C-labeled glutamine and glucose were substituted for the unlabeled metabolites in the medium as described for specific experiments. To evaluate the effect of inhibitors of IDP, the differentiation medium was supplemented with oxalomalate or 2-methylisocitrate as indicated and incubated at 37 °C for 10 min, followed by addition of 13C-labeled substrates. After a 6-h incubation at 37 °C, the medium was removed and cells were extracted for organic/amino acids or lipids. Stock solution of 2-methylisocitrate was prepared from 2-methylisocitrate lactone M. C. A. Eur. J. Biochem. 2002; PubMed Scopus Google to as of 2-methylisocitrate lactone of and in 1 solution was incubated at °C for 20 and then to with were from and were from were from and of and of organic/amino the procedure described in et al. J. L. Chem. PubMed Scopus Google was modified as of and 25 of were to well of the 6-well after of the medium. After of incubation at the was with of and of in a was followed by at for The was and the was at for The was then to a and The was in of in and After of incubation at 37 °C, organic/amino acids were for analysis with of at °C for of lipids and of were as described previously (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). with 5-13Cglutamine and U-13Cglutamine were analyzed for isotopomer distribution as previously reported (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). of was from of at the analysis of organic/amino acid were a with a mm 0.25 mm 0.25 to an operating by at were in the of mass units at The of the was °C, for 5 min, increased to °C at 10 and for 5 of the carbon was from the mass isotopomer of the following pyruvate aspartate glutamine citrate were for and mass isotopomer were by Kelleher J.K. Stephanopoulos G. Chem. PubMed Scopus Google Scholar). isotopomer data as for the of all mass of 13C-labeled carbon to fatty acid and new synthesis of fatty acids were from the mass isotopomer distribution of the of ISA as reported previously (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). fluxes and their were by of fluxes and mass isotopomer of glutamine, and citrate to a metabolic network of using the software estimates fluxes by the between the and using an procedure and isotopomer Kelleher J.K. Stephanopoulos G. PubMed Scopus Google Scholar). The of this is to evaluate a of fluxes that for the and flux After metabolic fluxes were analysis was to and of fluxes by the of the with to fluxes as described by et al. Kelleher J.K. Stephanopoulos G. PubMed Scopus Google Scholar). Flux was by a for the the for To a global flux was at with were with and To fluxes in to we for the synthesis ISA analysis we the new synthesis of synthesis was then as synthesis flux The was from where of glucose and glutamine were as reported previously (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). the WT brown adipocyte cells were incubated from 2 to of differentiation in medium containing 25 mm unlabeled glucose and 4 mm the of was to ISA of indicated that glutamine provided of the acetyl-CoA for synthesis and that of was de novo over the The was the flux through specific pathways from glutamine to lipogenic novo fatty acid using glutamine as carbon may by either of two which and of glutamine to and reductive which and of glutamine to To evaluate the fluxes from glutamine to via reductive carboxylation and glutaminolysis we the of 5-13Cglutamine and U-13Cglutamine to lipogenic acetyl-CoA using ISA that U-13Cglutamine of the lipogenic The ISA for U-13Cglutamine the of fluxes through the two pathways. Thus, we estimate that of the glutamine flux to lipogenic acetyl-CoA was via reductive carboxylation in this cell These results that one of the IDH is NADPH or to citrate used for The of to by of of the large flux to via the reductive carboxylation pathway, we the effect of two reported inhibitors of NADP-dependent IDH, oxalomalate and were incubated in DMEM in the of 4 mm U-13Cglutamine and either oxalomalate or 10 mm) or 2-methylisocitrate or 4 The effect of oxalomalate the of that oxalomalate flux of glutamine to This is from the in the higher mass with A result was with 2-methylisocitrate ISA analysis indicated that the major effect of these was the of glutamine to lipogenic acetyl-CoA than the synthesis A and in at higher concentration of oxalomalate or 2-methylisocitrate was whereas from with three concentrations of oxalomalate not studies with in DMEM that these compounds not the flux of glucose to lipogenic acetyl-CoA Thus, the inhibitor studies supported a role for IDPm cytosolic NADP-dependent enzyme in the reductive carboxylation of concentration of oxalomalate and 2-methylisocitrate and of synthesis from U-13Cglutamine and from with the inhibitor The effect of oxalomalate and 2-methylisocitrate the of glutamine to fatty acid synthesis was investigated by the of other metabolites in the pathways of glutamine The effect of oxalomalate was in isotopomer of and which in the of glutamine to fatty acid synthesis The mass isotopomer of increased in with higher concentration of oxalomalate The of isotopomer of which from isotopomer of α-ketoglutarate via reductive in oxalomalate concentration from to 5 and 10 mm The mass isotopomer of and from the same as in the of or 10 mm oxalomalate in These data that the inhibitor has the of The is to these data in to estimates of fluxes in the metabolic To reductive carboxylation of α-ketoglutarate to citrate is a cytosolic or the of an inhibitor of the mitochondrial acid were investigated S. Cell Res. 1989; PubMed Scopus Google Scholar). were incubated with mm) in DMEM containing U-13Cglutamine for Flux of glutamine to lipogenic acetyl-CoA was from the of with We found that the of glutamine to lipogenic acetyl-CoA from to Additionally, the new from to This to an in flux of glutamine to lipid. Thus, these findings the mitochondrial synthesis of citrate and reductive carboxylation flux through IDPm in de novo more metabolic studies of brown fluxes required that the pathway was in isotopic after of An in flux from data is that in metabolic and isotopic that the of metabolites not in has that substrate utilization was over consistent with metabolic (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, these studies synthesis and were our for to To the modeling study we evaluated CAC and acids of incubation in 4 mm The isotopomer distribution of metabolites was at 4, and The of key including citrate and was constant at the metabolic was found to be at isotopic and metabolic the metabolic flux of glutamine carbon to fatty acid synthesis was in with a metabolic network from metabolic of fatty acid pyruvate and the The included between α-ketoglutarate and and and and glutamine, and α-ketoglutarate. The was with a software the described Kelleher J.K. Stephanopoulos G. Chem. PubMed Scopus Google Scholar, Kelleher J.K. Stephanopoulos G. PubMed Scopus Google Scholar). Flux and of metabolic network of metabolic fluxes was for WT The network was analyzed with data 4 of differentiation in DMEM with 4 mm U-13Cglutamine and 25 mm unlabeled glucose in medium supplemented with 10 mm oxalomalate or 4 mm fluxes for the were by mass isotopomer there were for all the of was than the of for at that all flux models were data of the fluxes in the of either 10 mm oxalomalate or 4 mm 2-methylisocitrate in of oxalomalate and 2-methylisocitrate the flux in WT brown flux of reductive carboxylation a flux in the from citrate to CO2 to CO2 and citrate equilibrium equilibrium of is as equilibrium equilibrium of is as from ISA new synthesis from ISA flux of reductive carboxylation a flux in the from citrate to equilibrium of is as in a new of of glutamine to α-ketoglutarate was glutamine WT cells the this flux of was then flux of to and a flux of to citrate via reductive carboxylation. addition to the fluxes, mass isotopomer data also of the of carbon between α-ketoglutarate and citrate. We almost equilibrium of the two We also a large anaplerotic flux of glutamine CAC at α-ketoglutarate. We as the ratio of this flux to the of citrate and glutamine to α-ketoglutarate. Using data for we at of CAC This is with a estimate of glutamine from Thus, we have the ISA results with the more the of 5 and 10 mm the flux of reductive carboxylation was to and flux to flux from citrate to consistent with specific of The flux of α-ketoglutarate to not while glutamine to and for 5 and 10 mm the flux from to pyruvate by enzyme was from to 4 and concentration of The inhibitors not citrate of WT cells with or 4 mm 2-methylisocitrate to for the fluxes of α-ketoglutarate to citrate as in the with The in glutamine flux was to and in a in the flux from α-ketoglutarate to from to 1.6 and with the addition of 2-methylisocitrate effect pyruvate cycling fluxes, enzyme and pyruvate carboxylation. Taken together, the results shown in 1 demonstrate flux from α-ketoglutarate to citrate with the detected an pyruvate cycle between and of Pyruvate additional for the pyruvate a with 25 mm and 4 mm unlabeled glutamine was The of and aspartate of consistent with large flux and high of pyruvate via either pyruvate or An alternative of the pyruvate cycle involving was there was of from Thus, the the pyruvate carboxylation flux by the of U-13Cglutamine metabolism The provided the fluxes of the The large of pyruvate indicated that glucose from the medium was not the source of consistent with rapid pyruvate The of and mass in pyruvate indicated that flux through the oxidative of the pathway was to the high rate of these transformed previous that the of glutamine to lipogenic acetyl-CoA was than that of glucose for brown (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This result was unexpected glutamine is not generally as a lipogenic Glutamine metabolism in transformed cells has been described as an oxidative pathway for in the CAC and for generating NADPH through of enzyme Cell Biol. Rep. PubMed Scopus Google Scholar). The pyruvate by glutaminolysis could be either converted to and or converted to acetyl-CoA for utilization in fatty acid brown of glutamine to lipids is supported by findings of high of in J.M. Newsholme E.A. Biochem. J. PubMed Scopus Google and in vivo A.F. Newsholme Newsholme E.A. J. Biochem. 1989; 21: PubMed Scopus Google Scholar). in vivo of NADP-dependent IDH was shown to be higher in brown adipose than in adipose G. J. Biochem. PubMed Scopus Google Scholar). The studies glutaminolysis not isotopes to estimate fluxes. Thus, the studies a more quantitative analysis of glutamine metabolism in brown adipocytes. We found that of the glutamine carbon entering the CAC follows the oxidative as by However, one third of the glutamine to citrate via reductive carboxylation through IDH in reverse of the CAC direction. This reductive carboxylation flux is not isotopic of the units for de novo Thus, we found flux via an alternative to the glutaminolysis pathway in which glutamine lipogenic carbon via reductive carboxylation. These results were at mm glutamine, which is than physiological glutamine is that the glutamine concentration the of reductive carboxylation. Thus, our findings relevant for the metabolic of cells high levels of glutamine for optimal The metabolic fluxes in this study used and ISA was used to quantify lipogenesis from the two glutamine over and was used for fluxes of CAC and pathways. ISA and models to fit the data and found for reductive carboxylation flux of glutamine to lipogenic However, there The ISA used the of a single fatty such as as whereas models the of all of the metabolic detected in the relevant pathways. The ISA is to two and to models and estimate a large of a the more a metabolic network from an isotopic study by data additional compounds in the cell or This as to and data with network metabolic flux data. A major finding in this study is the role of reductive carboxylation flux in This flux was by near between cellular α-ketoglutarate and citrate Thus, the reductive carboxylation reaction a high flux to the CAC This near equilibrium of citrate and α-ketoglutarate has for the of flux in this pathway. Studies with inhibitors supported a role for IDPm in reductive carboxylation of α-ketoglutarate to To IDPm to as a source of mitochondrial NADPH. The rapid cycling between α-ketoglutarate and citrate is consistent with of a substrate cycle involving IDPm and the NAD/NADP transhydrogenase located at the inner mitochondrial membrane (14Sazanov L.A. Jackson J.B. FEBS Lett. 1994; 344: 109-116Crossref PubMed Scopus (180) Google Scholar). The cycle at the inner mitochondrial membrane where the of from to NADPH. NADPH be by reductive carboxylation where IDPm isocitrate and is then via the near equilibrium from the of this reductive carboxylation and citrate has three (1Newsholme E.A. Crabtree B. Ardawi M.S. Biosci. Rep. 1985; 5: 393-400Crossref PubMed Scopus (296) Google Scholar). may be oxidized via mitochondrial NAD-dependent isocitrate dehydrogenase (2Fasshauer M. Klein J. Kriauciunas K.M. Ueki K. Benito M. Kahn C.R. Mol. Cell. Biol. 2001; 21: 319-329Crossref PubMed Scopus (151) Google Scholar). may the the citrate and be converted to α-ketoglutarate via cytosolic NADP-dependent enzyme with of cytosolic NADPH (3Yoo H. Stephanopoulos G. Kelleher J.K. J. Lipid Res. 2004; 45: 1324-1332Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). may the citrate in the and lipogenic acetyl-CoA plus 1 and 2 to the near between cellular α-ketoglutarate and citrate the large of glutamine carbon to lipogenic acetyl-CoA This cycle is by to in transformed cells, high for biosynthesis and for mitochondrial These brown NADPH for de novo lipogenesis and the transformed cells of their from cytosolic the conducted a novel of metabolic fluxes in transformed brown that a role of glutamine as a precursor for The brown cell was provided by C. Kahn lactone was a from with