REVIEW

Qs next: the diverse functions of glutamine in metabolism, cell biologyand cancer

RJ DeBerardinis1,2 and T Cheng1

1Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA and 2McDermott Centerfor Human Growth and Development, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA

Several decades of research have sought to characterizetumor cell metabolism in the hope that tumor-specicactivities can be exploited to treat cancer. Havingoriginated from Warburgs seminal observation of aerobicglycolysis in tumor cells, most of this attention has focusedon glucose metabolism. However, since the 1950s cancerbiologists have also recognized the importance of gluta-mine (Q) as a tumor nutrient. Glutamine contributes toessentially every core metabolic task of proliferatingtumor cells: it participates in bioenergetics, supports celldefenses against oxidative stress and complements glucosemetabolism in the production of macromolecules. Theinterest in glutamine metabolism has been heightenedfurther by the recent ndings that c-myc controlsglutamine uptake and degradation, and that glutamineitself exerts inuence over a number of signaling pathwaysthat contribute to tumor growth. These observations arestimulating a renewed effort to understand the regulationof glutamine metabolism in tumors and to developstrategies to target glutamine metabolism in cancer. Inthis study we review the protean roles of glutamine incancer, both in the direct support of tumor growth and inmediating some of the complex effects on whole-bodymetabolism that are characteristic of tumor progression.Oncogene (2010) 29, 313324; doi:10.1038/onc.2009.358;published online 2 November 2009

Keywords: glutamine; metabolism; cancer; Warburgeffect; cachexia

Introduction

Tumors are metabolic entities. They draw nutrientsfrom the bloodstream, consume them through bio-chemical pathways and secrete waste products that thenbecome substrates for metabolism elsewhere in thebody. The metabolism of tumors has fascinated cancerbiologists since Warburgs experiments in the 1920s,which showed that ascites tumor cells from the mouse

were capable of unexpectedly high rates of glucoseconsumption and lactate secretion in the presence ofoxygen (the Warburg effect). Because this behavior wasso different from the differentiated tissues he used incomparison, those early observations stimulated thehope that the metabolic idiosyncrasies of tumors couldbe exploited to benet cancer patients (Warburg, 1925,1956). In terms of tumor imaging, this has turned out tobe true; the utility of 2-[18F]-uoro-2-deoxy-D-glucosepositron emission tomography and 1H magnetic reso-nance spectroscopy in human cancer depend preciselyon the ability of these modalities to detect glucoseuptake and lactate production. Interest in the Warburgeffect as an Achilles heel to be exploited in cancertreatment has been further stimulated by showing thatenhanced glucose metabolism is a common consequenceof many of the mutations responsible for human cancer,and therefore may be a central process essential fortumor growth (Flier et al., 1987; Shim et al., 1997;Osthus et al., 2000; Elstrom et al., 2004; Matoba et al.,2006; Kroemer and Pouyssegur, 2008).In terms of developing strategies to treat cancer,

however, tumor metabolism has so far proven to be moreof a Holy Grail than an Achilles heel. Part of thedifculty lies in the exibility of metabolic systems andthe panoply of nutrients to which tumors have access.Thus, a complete picture of the metabolism of any tumormust consider the contribution of multiple nutrientssimultaneously. Chief among the other nutrients avail-able to tumors is glutamine, the most abundant aminoacid in the plasma and the major carrier of nitrogenbetween organs. Glutamine is also second only to glucosein terms of persistent interest in its role in tumor cellmetabolism, which now dates back more than 50 years(Eagle, 1955; Kvamme and Svenneby, 1960). In culture,tumor cells are avid glutamine consumers, metabolizingit at rates far in excess of any other amino acid (Eagle,1955). The same is true in tumors implanted ontovascular pedicles in rats to allow precise measurements ofthe rates of amino acid extraction from the blood (Saueret al., 1982; Sauer and Dauchy, 1983). These observa-tions led to the notion that glutamine metabolism stoodwith the Warburg effect as a major component of thegeneral metabolic phenotype of proliferating tumor cells(Kovacevic and McGivan, 1983).Glutamines importance in tumor cell metabolism

derives from the characteristics that it shares withReceived 3 July 2009; revised 14 September 2009; accepted 23 September2009; published online 2 November 2009

Correspondence: Dr RJ DeBerardinis, Department of Pediatrics,University of Texas Southwestern Medical Center, 5323 Harry HinesBoulevard, Room K4.216, Dallas, TX 75390-9063, USA.E-mail: ralph.deberardinis@utsouthwestern.edu

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glucose. Both nutrients help to satisfy two impor-tant needs for proliferating tumor cells: bioenergetics(adenosine triphosphate (ATP) production) and theprovision of intermediates for macromolecular synthesis(DeBerardinis et al., 2008b). A number of excellentdiscussions on the use of glutamine in cancer havepreviously been presented (Medina et al., 1992; Souba,1993). Surprisingly, however, only recently it has beenreported that oncogenes inuence glutamine metabolismas they do for glucose, and that tumor genetics candictate cellular dependence on glutamine for survival.Furthermore, other studies have been uncoveringdiverse and unexpected roles for glutamine and its by-products in cell signaling, linking glutamine metabolismto cell survival and growth in ways beyond its roles inintermediary metabolism. In this study we reviewglutamines metabolic and non-metabolic functions intumor cells, the integration of glucose and glutaminemetabolism in tumor growth and aspects of whole-bodyglutamine metabolism that may inuence the morbidityand mortality of cancer patients.

Glutamines roles in intermediary metabolism: functionsand consequences

Glutamine has traditionally been viewed as a nones-sential amino acid whose primary functions are to storenitrogen in the muscle and to trafc it between organs.Although it contributes only 4% of the amino acid inmuscle protein, glutamine accounts for more than 20%of the free amino acid pool in plasma and more than40% in muscle (Bergstrom et al., 1974; Kuhn et al.,1999). Mammals can synthesize glutamine in mosttissues, but during periods of rapid growth or illness,the cellular demand for glutamine outstrips its supplyand glutamine becomes essential (hence its designationas a conditionally essential amino acid). Proliferatingcells show an intense appetite for glutamine, reectingits incredible versatility as a nutrient and mediator ofother processes (Figure 1).The metabolic fates of glutamine can roughly be

divided into reactions that use glutamine for itsg-nitrogen (nucleotide synthesis and hexosamine synth-esis) and those that use either the a-nitrogen or thecarbon skeleton. The reactions in the second categoryuse glutamate, not glutamine, as the substrate. Althoughtumor cells tend to have large intracellular pools ofglutamate, maintaining these pools rests on the abilityto convert glutamine into glutamate, because glutamineis an abundant extracellular nutrient and glutamate isnot. This process is largely because of the activity ofphosphate-dependent glutaminase (GLS), a mitochon-drial enzyme that is highly expressed in tumors andtumor cell lines. Classical experiments have shown thatGLS activity correlates with tumor growth rates in vivo(Knox et al., 1969; Linder-Horowitz et al., 1969), andexperimental models to limit GLS activity resulted indecreased growth rates of tumor cells and xenografts(Lobo et al., 2000; Gao et al., 2009). Thus, although notall the metabolic fates of glutamine require GLS

activity, this enzyme is essential for the metabolicphenotype of many tumors.The following are the major aspects of glutamine-

based intermediary metabolism and their relevance totumor cell growth. They are summarized in Figure 1.

Nucleotide biosynthesisGlutamine is a required nitrogen donor for the de novosynthesis of both purines and pyrimidines and is thereforeessential for the net production of nucleotides during cellproliferation. The g (amido) nitrogens of two glutaminemolecules are added to the growing purine ring, and athird is used in the conversion of xanthine monopho-sphate to guanosine monophosphate. Other nitrogens aresupplied by glycine and aspartate, but many of these arealso initially derived from glutamine (the a-nitrogen).Pyrimidine rings contain one nitrogen from glutaminesamido group and one from aspartate, and an additionalamido nitrogen is added to uridine triphosphate to formcytidine triphosphate. The importance of glutaminenitrogen in nucleotide biosynthesis probably explainswhy some transformed cells show delayed transit throughS phase in low-glutamine conditions (Gaglio et al., 2009).However, the glutamine utilization rate exceeds nucleicacid synthesis by more than an order of magnitude inproliferating cells, and thus nitrogen donation to nucleo-tides accounts for only a small fraction of total glutamineconsumption (Ardawi et al., 1989).

Hexosamine biosynthesis and glycolsylation reactionsThe rate-limiting step in the formation of hexosamineis catalysed by glutamine:fructose-6-phosphate amido-transferase, which transfers glutamines amido group tofructose-6-phosphate to form glucosamine-6-phosphate,a precursor for N-linked and O-linked glycolsyl-ation reactions. These reactions are necessary to modifyproteins and lipids for their participation in signaltransduction, trafcking/secretion and other processes.Impairment of glucosamine-6-phosphate production isthus predicted to reduce cell growth and to interferewith cell signaling. Surprisingly, glutamine:fructose-6-phosphate amidotransferase activity could be sup-pressed by expressing an antisense GLS complementaryDNA in breast cancer cells. This resulted in disturbancesof O-linked glycolsylation pathways, altering theglycosylation status of the transcription factor Sp-1and increasing its transcriptional activity (Donadioet al., 2008). The mechanism by which loss of GLSactivity inuences glycolsylation is unclear, but thendings suggest that glutamine:fructose-6-phosphateamidotransferase and perhaps other components of theglycosylation machinery are responsive to intracellularglutamine availability, as determined by GLS.

Nonessential amino acidsBecause of the high expression of GLS, tumor cells arepoised to produce glutamate rapidly from glutamine,and a sizable fraction of their glutamate pool carriesglutamines a (amino) nitrogen. This nitrogen is thendispersed into various pools of nonessential amino

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acids through the activity of transaminases, particularlyalanine aminotransferase and aspartate aminotrans-ferase. These enzymes catalyse the reversible transferof amino groups between glutamate and alanine oraspartate, respectively. Alanine is used in proteinsynthesis, but is also avidly secreted by tumor cells,carrying some of the excess carbon from glycolysis.Aspartate, in contrast, remains inside the cell and contri-butes to the synthesis of proteins and nucleotides and toelectron transfer reactions through the malate-aspartate

shuttle. In this shuttle, aspartate exits the mitochondriaand is converted to oxaloacetate by aspartate amino-transferase. Oxaloacetate is then reduced to malate,using electrons donated by NADH generated inglycolysis. The malate then enters the mitochondriaand donates the electrons to complex I of the electrontransport chain. Thus, the shuttle facilitates ongoingATP production in both the mitochondria (throughoxidative phosphorylation) and the cytosol (by re-supplying NAD for glycolysis).

Gln

Gln

Gln

EAA

EAA mTORC1

Gln

Glu

-KG

NH4+ NH4+ NH4+?

mito

chon

drion

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}

}

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Lac Lac

GROWTH,PROLIFERATION

SURVIVAL

SURVIVAL,GROWTH,

PROLIFERATION

SURVIVAL,GROWTH,

PROLIFERATION

GROWTH

Glu

GluNucleotidesGlucosamine

NEAA Protein

GluCys-CysCys

GSH

Cys-Cys

ATP

SLC7A5SLC1A5

Xc-

MCT

GLS

ME

mGluRERK signalingPI3K signaling

Cit

Ac-CoA OAA

Figure 1 Glutamine supports cell survival, growth and proliferation through metabolic and non-metabolic mechanisms. After itsimport through surface transporters such as SLC1A5, glutamine (Gln) is either exported in exchange with the import of essentialamino acids (EAA) or consumed in various pathways that together support the basic metabolic functions needed for cell survival,growth and proliferation. In cancer cells, the mitochondrial enzyme glutaminase (GLS) seems to account for the largest fraction of netglutamine consumption. This enzyme produces NH4

, which is exported, perhaps through carrier-mediated mechanisms. Ac-CoA,acetyl-CoA; Cit, citrate; Cys, cysteine; Cys-Cys, cystine; ERK, extracellular signal-regulated protein kinase; GLS, glutaminase; Glu,glutamate; GSH, glutathione; Lac, lactate; Mal, malate; ME, malic enzyme; mGluR, metabotropic glutamate receptor; mTORC1,mammalian target of rapamycin complex 1; NEAA, nonessential amino acids; OAA, oxaloacetate; PI3K, phosphatidylinositol30-kinase; Pyr, pyruvate; SLC1A5, solute carrier family 1 (neutral amino acid transporter), member 5; SLC7A5, solute carrier family 7(cationic amino acid transporter, y+ system), member 5; TCA, tricarboxylic acid; a-KG, a-ketoglutarate.

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Glutathione (GSH)At a concentration of approximately 5mM, GSH is themajor thiol-containing endogenous antioxidant andserves as a redox buffer against various sources ofoxidative stress. In tumors, maintaining a supply ofGSH is critical for cell survival because it allows cells toresist the oxidative stress associated with rapid metabo-lism, DNA-damaging agents, inammation and othersources (Estrela et al., 2006). GSH is a tripeptide ofglutamate, cysteine and glycine and its formation ishighly dependent on glutamine. Not only does gluta-mine metabolism produce glutamate, but the glutamatepool is also necessary for cells to acquire cysteine, thelimiting reagent for GSH production. This occurs byvirtue of the Xc

antiporter, which exports glutamate andimports cystine, as shown in Figure 1. Cystine can thenbe converted to cysteine inside the cell and used in GSHsynthesis. Because of the key role of the Xc

antiporter inmaintaining GSH levels, it has been suggested as atarget for cancer therapy (Lo et al., 2008).

Respiratory substrateOxidation of glutamines carbon backbone in the mito-chondria is a major metabolic fate of glutamine and aprimary source of energy for proliferating cells, includinglymphocytes, enterocytes, broblasts and some cancercell lines (Reitzer et al., 1979; Miller, 1999; DeBerardiniset al., 2008b). This requires conversion of glutamine toa-ketoglutarate, typically through GLS activity followed byconversion of glutamate to a-ketoglutarate by eithertransaminases or glutamate dehydrogenase. However, otherprocesses, such as the donation of glutamines amidonitrogen to nucleotides or hexosamines, could contribute afraction of the glutamate pool as well. Overall, thiscontribution is small, but it might become more prominentduring stages of the cell cycle characterized by transientincreases in nucleotide biosynthesis or other activities. Thus,proliferating cells, which show high uxes through anumber of biosynthetic pathways, are well-positioned tomake use of glutamines carbon skeleton as a respiratorysubstrate. Complete oxidation of glutamine carbon involvesexit from the tricarboxylic acid (TCA) cycle as malate,conversion to pyruvate and then acetyl-CoA, and nally re-entry into the cycle (Figure 1). It should be noted, however,that careful studies of tumor cell glutamine metabolismreveal that neither its nitrogen nor its carbon are used tocompletion in vitro. Rather, a high fraction of both ofglutamines nitrogens are secreted from glioblastoma cellsas they proliferate (as ammonia, alanine and glutamate),and at least half of its carbon is secreted as lactate(DeBerardinis et al., 2007). This form of rapid glutamineutilization and secretion of glutamine by-products, similarto the Warburg effect in its apparent inefciency, has beenproposed to be an additional hallmark of tumor cellmetabolism (Mazurek et al., 2005).

Reducing equivalentsOne of the benets of converting glutamine to pyruvateis the reduction of NADP to NADPH by malicenzyme (Figure 1). NADPH is a required electron donor

for reductive steps in lipid synthesis, in nucleotidemetabolism and in maintaining GSH in its reducedstate. Therefore, proliferating cells must produce a largesupply of it. Although cells contain numerous potentialsources of NADPH, in glioblastoma cells the malicenzyme ux was estimated to be high enough to supplyall of the reductive power needed for lipid synthesis(DeBerardinis et al., 2007).

AmmoniagenesisGlutaminase activity generates free ammonia, a poten-tially toxic metabolite. In some cell lines, the rate atwhich ammonia is secreted into the extracellular space isabout 75% of the rate of glutamine disappearance fromthe medium (our unpublished observations), consistentwith a high fraction of glutamine being metabolized inthe mitochondria by GLS. Without a mechanism todispose of ammonia rapidly, intracellular ammoniaconcentrations would reach several hundred mmol/lwithin a few hours. It is not known how tumor cellsdispose of ammonia during rapid glutamine catabolism.The traditional view held that passive diffusion of thegaseous form (NH3) across the lipid bilayer accounted foressentially all ammonia transport. This simple model nolonger holds for some tissues with a high demand forammonia transport. In the kidney, in which ammoniametabolism is a key mediator of acid-base homeostasis, anumber of protein transporters exist to trafc ammoniaas NH3 and/or NH4

. These systems include ion channels,aquaporins and Rh glycoproteins (Weiner and Hamm,2007), some of which are overexpressed in tumors.Although the exact mechanism of tumor cell ammoniasecretion has not been established, the process carriestherapeutic potential. Blocking ammonia secretion wouldpresumably either suppress net glutamine consumptionor cause toxic intracellular accumulation of ammonia,both of which might impair cell survival and growth.

Glutamines roles in cell signaling and gene expression

Beyond its roles in intermediary metabolism, glutamineexerts other effects that support cell survival and growth.Reecting the importance of glutamine to anabolicmetabolism, cells have developed glutamine-dependentmechanisms to control growth, including the modulationof signal transduction pathways. A recent study revealedthe necessity of glutamine for the well-known stimula-tory effect of essential amino acids on the mammaliantarget of rapamycin (mTOR) pathway (Nicklin et al.,2009). In the study, stimulation of mTOR complex-1(mTORC1) in HeLa cells required bidirectional trans-port of glutamine: the cells imported glutamine throughthe Na-dependent transporter, solute carrier family 1(neutral amino acid transporter), member 5, and thenexported it through the Na -independent transporter,solute carrier family 7 (cationic amino acid transporter,y system), member 5 (Figure 1). The latter step wasaccompanied by import of essential amino acids andsubsequent activation of mTORC1, stimulating cell growth

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while suppressing autophagy. It was the sequential importand export of glutamine itself and not the by-products ofglutamine metabolism that facilitated the uptake of essentialamino acids, because addition of glutamate or a-ketoglu-tarate failed to rescue mTORC1 activation in the absence ofglutamine. Thus, this mechanism of control relies onglutamine abundance exceeding the metabolic needs ofthe cells, suggesting that glutamine excess is a signal topromote cell growth and suppress catabolism.Other data have identied a role for glutamine in

extracellular signal-regulated protein kinase (ERK)signaling pathways. This has been best characterized inintestinal epithelial cells, which consume glutamine astheir major bioenergetic substrate and require glutaminefor both proliferation and survival. Addition ofglutamine was sufcient to stimulate ERK signalingwithin a few minutes in porcine intestinal epithelial cells,and it enhanced 3H-thymidine incorporation (Rhoadset al., 1997). In rat intestinal epithelial cells, glutaminewas comparable to serum in preventing apoptosis, and itstimulated a sustained activation of ERK signaling(Larson et al., 2007). In those cells, inhibitors of theERK pathway eliminated the protective effect ofglutamine supplementation. It was not clear from thesestudies whether glutamine import alone was required forthe effects, or whether the cells needed to metabolizeglutamine to activate ERK signaling.Glutamate also inuences signaling and tumor growth

if it is secreted by tumor cells. In the central nervoussystem, the rate of glutamate secretion by glioma cellscorrelated with the ability of those cells to elicit regionalneuronal cell death and to form tumors in the ratstriatum. Both these effects were due to glutamatesignaling through ionotropic receptors, because theywere blocked by N-methyl D-aspartate receptor antago-nists (Takano et al., 2001). Glutamate also has non-ionotropic signaling properties through its effects onmetabotropic glutamate receptors (mGluRs) (Figure 1).These G-protein-coupled receptors are widely expressedin both neuronal and non-neuronal tissues, implying thatthey have signaling duties beyond their traditionalfunction in synaptogenesis (Skerry and Genever, 2001;Nicoletti et al., 2007). Activation of mGluR isoformsleads to stimulation of ERK and phosphatidylinositol30-kinase signaling, supporting cell survival, growth andproliferation. This may translate into a role for glutamatesignaling in cancer. The isoform mGluR1 is expressed inhuman melanoma cells but not in melanocytes or benignnevi, and overexpression of mGluR1 in mouse melano-cytes caused hyperproliferation and occasional transfor-mation into melanoma (Pollock et al., 2003). In mice withdoxycycline-repressible mGluR1 expression in melano-cytes, the growth of existing melanomas could beimpaired by administering doxycycline, and this wascorrelated with a reduction in activated ERK in thetumors (Ohtani et al., 2008). Human glioblastomas alsoexpress mGluRs, and a small molecule inhibitor againstmGluR2 and 3 inactivated ERK and phosphatidylinosi-tol 30-kinase signaling in glioma cell lines, inhibited cellproliferation and suppressed the growth of tumors inboth the brain and subcutaneous tissue (Arcella et al.,

2005). Thus, these receptors might be useful targets incancer therapy. Brain tumors may have access tosufcient extracellular glutamate to signal throughthese receptors without requiring glutamate secretionby the tumor itself. However, the glutamate concentra-tion in the plasma is quite low (B50mM). For melanomasand other tumors outside the central nervous system,mGluR signaling may require that the tumor secreteglutamate produced from glutamine metabolism. Thiscould result in autocrine or paracrine effects on cellgrowth.Consistent with glutamines effects on cell signaling, a

number of reports have shown that it also inuencesgene expression (Brasse-Lagnel et al., 2009). In cell lines,addition of glutamine increases expression of the pro-proliferation factors c-jun and c-myc within a fewminutes (Kandil et al., 1995; Rhoads et al., 1997) andpromotes cell survival through negative effects ongrowth-inhibitory and pro-apoptotic factors, such asCHOP, GADD45, Fas and ATF5 (Abcouwer et al.,1999; Huang et al., 1999; Yeo et al., 2006; Watataniet al., 2007). In Ehrich ascites tumor cells, GLSknockdown led to enhanced phosphorylation, DNAbinding and transcriptional activity of Sp1 (Seguraet al., 2005). In HepG2 hepatoma cells, glutamine wasrequired for the induction of manganese superoxidedismutase expression that accompanied the depletion ofessential amino acids (Aiken et al., 2008). Glutaminesinvolvement in this expression was blocked by inhibitingthe TCA cycle, ERK1/2 or mTOR, suggesting that anintegration between mitochondrial glutamine metabo-lism and signal transduction facilitates the effect.Abundant evidence suggests that glutamine also modu-

lates immune responses, although it is unclear exactly howthese changes are achieved. Conceivably, glutamine couldexert its effects through redox homeostasis, bioenergetics,nitrogen balance or other functions (Eliasen et al., 2006;Roth, 2007). During radiation-induced oxidative stress inthe rat abdomen, pre-treatment of the animals withglutamine signicantly decreased tissue inammation andexpression of nuclear factor-kB, suggesting that in this caseglutamine was used to buffer the redox capacity (Erbilet al., 2005). Nuclear factor-kB seems to be a key mediatorthat links glutamine availability with stress responses, asother reports also show an inverse correlation betweenglutamine abundance and nuclear factor-kB-mediatedgene expression (Bobrovnikova-Marjon et al., 2004;Hubert-Buron et al., 2006). The role of glutamine as animmunomodulator in cancer has not been explored at themolecular level, but this is an area worth further study. Ifthe avid consumption of glutamine by tumors reduces itsavailability for neighboring cells, this could modulate localnuclear factor-kB signaling and expression of inamma-tory mediators in the stroma.

Integrating glutamine and glucose metabolism in tumorcell growth

Because tumor cells are exposed to many nutrientssimultaneously, achieving a comprehensive view of

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tumor metabolism requires an understanding of howcells integrate these pathways into an over-archingmetabolic phenotype. Consequently, discussions aboutglutamine cannot ignore the rapid glucose utilizationthat also accompanies cell proliferation. In fact, therates of glucose and glutamine consumption far outpacethe utilization of other nutrients available to the cell.Presumably, this type of metabolism supports bothbioenergetics and the production of precursor poolswhile sparing other energy-rich substrates, such as fattyacids and essential amino acids, for direct incorporationinto macromolecules (DeBerardinis et al., 2006).Warburgs work and many studies since then have

focused on the production of lactate from glucose, onthe low relative rate of glucose oxidation and on thehigh apparent contribution of glycolysis to overallenergetics in tumor cells. These have led to the general-ization that tumors do not or cannot engage in oxidativemetabolism, and that aerobic glycolysis (that is,conversion of glucose to lactate in the presence of ampleoxygen) is a sine qua non for the tumor metabolicphenotype. A few points should be made about theseassumptions. First, metabolism of glucose to lactate isnot limited to tumor cells, but is a common feature ofrapid cell proliferation (Wang et al., 1976; Brand, 1985).The potential advantages of this form of metabolism forproliferating cells have been reviewed (DeBerardiniset al., 2008a). Second, although many tumor cell lines doshow high glycolytic rates, the contribution of glycolysisto total cellular ATP content varies widely, from over50%, as Warburg found, to less than 5% in other cells(Zu and Guppy, 2004). Thus, oxidative phosphorylationis not universally impaired in tumor cells. Third,mitochondrial metabolism directly contributes to cellgrowth because many macromolecular precursors areproduced in the TCA cycle (DeBerardinis et al., 2008b).Even when the glycolytic rate is high enough to supportmost of the need of the cells for ATP synthesis, growthrequires that the cells produce lipids, proteins andnucleic acids, and building blocks for these moleculescome from the TCA cycle. Therefore, the aerobicglycolysis discovered by Warburg is only one piece ofthe puzzle of anabolic metabolism in tumor cells.Our growing understanding of glutamine metabolism

promises to help ll in this picture, as the concurrentconsumption of glucose and glutamine has obvioustheoretical advantages (Figure 2). The simultaneousmetabolism of these two nutrients would supportbioenergetics in cells showing the Warburg effect bydelivering glutamine-derived a-ketoglutarate to theTCA cycle for oxidation. Moreover, the entry of a-ketoglutarate offsets the export of intermediates used inbiosynthetic pathways. The process of replenishing TCAcycle intermediates during cell growth (anaplerosis) is akeystone of biomass production and is much less activein quiescent cells. In rapidly proliferating culturedglioblastoma cells, for example, most of the acetyl-CoA pool comes from glucose, whereas essentially all ofthe anaplerotic carbon (that is, the oxaloacetate) comesfrom glutamine (DeBerardinis et al., 2007). This resultsin citrate molecules that contain carbon from both

glucose and glutamine (purple arrows in Figure 2). Afterthis citrate leaves the TCA cycle, the two glucose-derived carbons are released as acetyl-CoA and used inlipid synthesis. Protein and nucleic acid synthesis alsorequire precursor molecules derived from glucose andglutamine metabolism. Thus, during cell proliferation,glucose and glutamine are the two major nutrientinputs, and the primary outputs are biomass (nucleicacids, proteins and lipids) and the by-products secretedas a result of this type of metabolism: lactate, alanineand ammonia (Figure 2).The importance of glucose and glutamine metabo-

lism to tumor cell biology is underscored by the factthat mutations in tumor suppressors and oncogenesallow cells to by-pass the normal, growth factor-dependent handling of these two nutrients. This has

Pyr

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{

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{ {Outputs:LactateAlanineNH4+

NH4+

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GLS

Acetyl-CoAOAA

Figure 2 Cooperativity between glucose and glutamine metabo-lism in growing tumors. The major nutrients consumed by tumorsare glutamine and glucose, which provide precursors for nucleicacids, proteins and lipids, the three classes of macromoleculesneeded to produce daughter cells. The metabolism of glutamine(blue arrows) and glucose (red arrows) are complementary,converging on the production of citrate (purple arrows). Glutaminemetabolism produces oxaloacetate (OAA) and NADPH, both ofwhich are required to convert glucose carbon into macromolecules.Glutamine metabolism also supplements the pyruvate pool, whichis predominantly formed from glucose. As a consequence of therapid metabolism of these two nutrients, lactate, alanine and NH4

are secreted by the tumor. Fum, fumarate; GLS, glutaminase; Glu,glutamate; Lac, lactate; Mal, malate; NADPH, nicotinamideadenine dinucleotide phosphate; Pyr, pyruvate; Succ, succinate;TCA, tricarboxylic acid; a-KG, a-ketoglutarate.

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been extensively studied for glucose metabolism. Asearly as the 1980s, it was showed that overexpression ofRas or Myc was sufcient to drive glucose uptake inbroblasts (Flier et al., 1987). Subsequently, mutationsin many other tumor suppressors and oncogenes havebeen implicated as drivers of the Warburg effect intumor cells (reviewed in DeBerardinis, 2008). Althoughmuch less is known about the regulation of glutaminemetabolism, several reports have recently implicatedc-Myc as a major player. Enhanced c-Myc activity wassufcient to drive glutamine metabolism and to impaircell survival in low-glutamine conditions (Yuneva et al.,2007; Wise et al., 2008). c-Myc regulates glutaminemetabolism in part by stimulating the expression ofsurface transporters (Wise et al., 2008). Interestingly,c-Myc also indirectly regulates the protein expression ofGLS through effects on the microRNAs, miR23a andmiR23b. Normally, these microRNAs bind to the GLS30-untranslated region and prevent translation of themessage. However, c-Myc suppresses miR-23a/b expres-sion, and thus enhanced c-Myc activity de-repressedGLS translation and facilitated glutamine oxidation inthe mitochondria (Gao et al., 2009).The simplest mechanism to explain the enhanced

use of both glutamine and glucose by tumor cells isthat metabolism of the two nutrients is co-regulated.However, recent ndings suggest that they can beregulated by independent signaling pathways withinthe same cells. In a glioblastoma cell line with genomicc-myc amplication, inhibition of Akt signaling ledto a decrease in glycolysis but had no effect onglutamine metabolism, which was only inhibited whenc-Myc was suppressed to normal levels (Wise et al.,2008). This raises the possibility that the complexmetabolic phenotype observed in tumor cells is theresult of multiple different signaling inputs, presumablythrough multiple mutations. This notion is consistentwith the observation that serial transduction of humanbroblasts leads to step-wise changes in the metabolicprole and dependence of the cells on particularpathways to sustain ATP supply (Ramanathan et al.,2005).Tumors often show regional heterogeneity in oxygen

availability, and this can signicantly inuence inter-mediary metabolism independently of tumor genetics.Stabilization of the transcription factor, hypoxia-indu-cible factor-1a, in hypoxic cells enhances the expressionof glucose transporters, glycolytic enzymes, and inhibi-tory kinases for the pyruvate dehydrogenase complex,all of which serve to increase the production of lactatefrom glucose (Semenza, 2003; Kim et al., 2006;Papandreou et al., 2006). Little is known about theeffects of hypoxia on glutamine metabolism. Presum-ably, to survive, cells would at least need to maintainsome of the proximal steps of glutamine metabolism tosatisfy homeostatic requirements for amino acid andnucleotide synthesis. One study showed that hypoxiacould stimulate the import of glutamine in neuroblas-toma cells, although downstream metabolism was notexamined (Soh et al., 2007). On the other hand, ratpheochromocytoma cells showed increased rates of

endogenous glutamine synthesis under hypoxia, perhapsreecting a need for glutamine that exceeded transportcapacity (Kobayashi and Millhorn, 2001). Further workis needed to determine how hypoxia inuences gluta-mine metabolism and whether glutamine inuences cellsurvival during hypoxic stress.Glucose and glutamine metabolism also have over-

lapping functions in redox homeostasis, as both canproduce NADPH, and glutamine has the additional roleof supporting GSH biosynthesis. This is an importantconsideration during cell proliferation, becauseNADPH is required for biosynthetic reactions andbecause some production of reactive oxygen species isinevitable during rapid nutrient metabolism. Glutaminewithdrawal led to decreased GSH pools in broblastswith enhanced c-Myc activity, and to frank oxidativedamage in hybridoma cells (Guerin et al., 2006; Yunevaet al., 2007). In neither case, however, was the redoxstress sufcient to explain the loss of cell viability. Onthe other hand, in P-493 B-lymphoma cells and PC3prostate cancer cells the loss of cell proliferation andviability triggered by glutamine deprivation or GLSknockdown could be partially reversed with anti-oxidants (Gao et al., 2009). Thus, although impairedglutamine metabolism limits the availability of GSH,this effect is not always responsible for the death ofglutamine-dependent cells. It is likely that tumor cellsdiffer in their use of glucose and glutamine to maintainredox balance, and exploiting these differences may betherapeutically useful, especially during therapies thatinduce oxidative stress.

Tumor growth and whole-body metabolism: is the tumorthe tail that wags the dog?

The aggressive metabolism of glucose and glutamineby tumors begs the question of how cancer affectsmetabolism in the rest of the body. Two issues areparticularly relevant to this discussion. First, how dotumors acquire the nutrients they need to grow? Andsecond, what is the fate of the waste products secretedby tumor cells? Both these issues relate to the pheno-menon of declining nutrition observed in patients withcancer or other chronic health problems. Cachexia, theprogressive loss of muscle and adipose tissue mass, is awell-known and clinically important dimension ofcancer, affecting more than half of all patients. Itconsists of a combination of complex changes in muscleand liver metabolism, culminating in weight loss.Changes in appetite and taste sensation may accompanycachexia and can be exacerbated by chemotherapy, butthese are not sufcient to explain the phenotype. Weightloss often predates the diagnosis and treatment ofcancer, and normalizing the caloric intake usually doesnot reverse the loss of lean mass. These observationsimply that fundamental changes in whole-body meta-bolism, including an increase in energy expenditure, areat the root of the problem. The clinical importance ofcachexia is emphasized by the nding that it is theprincipal cause of death in one-third or more of cancer

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patients (NCI Website, 2009). Many aspects of thecachectic phenotype are stimulated (directly or indir-ectly) by the tumor, and evidence suggests that thetumor derives benets from altered metabolism in theliver and muscle. Two aspects of cachexia are discussedhere: the Cori cycle and muscle proteolysis, each ofwhich has implications for the metabolism of growingtumors (Figure 3).In the Cori cycle, lactate and alanine released from

glucose-consuming tissues, such as the muscle and brain,are returned to the liver and used as precursors forgluconeogenesis. As early as the 1950s, Hiatt (1957)showed that a high fraction of glucose produced by theliver of tumor-bearing mice had been generated from3-carbon units released by the tumor, suggesting aCori-like cycle between the tumor and the liver. Similarexperiments in humans also documented higher rates ofCori cycle metabolism in individuals with cancer ofvarious types (Reichard GA et al., 1963; Holroyde et al.,1984). The Cori cycle would thus be a way to minimizethe apparent energetic inefciency of the Warburg

effect, as the wasted 3-carbon units could ultimatelybe returned to the tumor as glucose. However, althoughthe cycle produces energy in the tumor (two ATPs perglucose used), this is more than offset by energyexpenditure in the liver (612 ATP equivalents perglucose formed). Thus, the Cori cycle imposes anenergetic burden on whole-body metabolism. Increasedux through this pathway has been postulated to beone of the drivers of energy dissipation in cachecticcancer patients (Tisdale, 2009).Secretion of lactate by hypoxic cells within tumors

can also have local effects on metabolism. If this lactatecan reach better oxygenated cells within the tumor, itcan be taken up and used as a respiratory fuel in themitochondria. Sonveaux et al. (2008) identied thecell surface monocarboxylate transporter type 1 as theenabler of such a metabolic symbiosis in xenografts. Itallowed normoxic cells to take up lactate, presumablysparing glucose for the more hypoxic regions of thetumor. Inhibiting it signicantly reduced tumor growthand increased the effect of radiation therapy.

Inflammation/Cytokines

Glucose

Lactate, Alanine

Glutamine

NH4+

NH4+

NH4+

NH4+

Lactate, Alanine

Lactate, Alanine

Glucose

Gluconeogenesis

TUMOR

LIVER

Urea

Glutamine

CELLGROWTH

Proteins

MUSCLE

Urea

GLUL

CORI

CYC

LE

GLN-NH4+ CYCLE?

ATP ATP

ATP

ATPATP

Glucose

-KG

AAsGlu

Figure 3 Proposed inter-organ metabolic cycles in cachectic cancer patients. As summarized in Figure 2, growth of the tumor involvesconsumption of glucose and glutamine with secretion of lactate, alanine and ammonia. Some of the lactate may be taken up by well-oxygenated regions of the tumor and used as a respiratory fuel. Other lactate and alanine are delivered to the liver and used to produceglucose, which can then return to the tumor (the Cori cycle). Meanwhile, the ammonia can be disposed through the urea cycle, orpossibly delivered to the muscle for incorporation into new glutamine molecules produced during protein catabolism and glucosemetabolism. Both the Cori cycle and the putative glutamine-ammonia cycle deliver energy to the tumor, but cost energy in the otherorgans involved, driving up whole-body energy expenditure as is typically observed in cancer cachexia. AAs, amino acids; Glu,glutamate; GLUL, glutamate-ammonia ligase (that is, glutamine synthetase); a-KG, a-ketoglutarate.

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Cancer also induces a negative nitrogen balance in thehost characterized by loss of protein and amino acidsfrom the muscle, eventually leading to loss of musclemass. This observation predates even Warburgs workby several decades and has been credited to Muller(Parry-Billings et al., 1991), who compared the hostresponse in cancer with that of a prolonged febrileillness. Although the inciting agents that promotemuscle catabolism have not been fully elucidated, manystudies have implicated inammatory mediators, such astransforming growth factor-b, tumor necrosis factor-aand various cytokines, consistent with Mullers hypo-thesis (Tisdale, 2005). Clearly, glutamine metabolism istied to cachexia, as more than 90% of the bodysglutamine stores are in the muscle, and glutamine is themajor amino acid exported from the muscle duringcatabolic stress. Newsholme et al. (1985) suggested thatthe demand for glutamine by rapidly proliferating cells,such as lymphocytes or tumor cells, might be the triggerfor cachexia. Evidence in support of this hypothesisincludes the observation that implantation of tumortissue in rodents rapidly triggers an increase in muscleglutamine output and a drop in muscle glutamine stores(Parry-Billings et al., 1991; Chen et al., 1993). Similarchanges have been suggested to occur with prolongedtumor burden in humans (Souba, 1993).The process of muscle nitrogen loss during cachexia

involves the transfer of amino acid nitrogen rst toglutamate and then to glutamine, which is secretedinto the bloodstream (Figure 3). Production of gluta-mine from glutamate is catalysed by glutamine synthe-tase (also known as glutamate-ammonia ligase). Tumorburden is associated with increased expression of theglutamate-ammonia ligase mRNA and activity in themuscle of mammals and birds, implicating this enzymeas an evolutionarily conserved mediator of cachexia(Matsuno and Satoh, 1986; Quesada et al., 1988; Chenet al., 1993). Glutamine synthesis consumes energy andthus would tend to increase energy expenditure in themuscle. It also requires ammonia. Interestingly, myo-cytes can take up and metabolize ammonia, and this isone of the mechanisms by which ammonia is clearedduring exercise in humans (Bangsbo et al., 1996). Thesource of muscle ammonia during glutamine secretion incachexia is unknown, but presumably comes from analteration in inter-organ ammonia handling. This couldbe similar to the changes that accompany chronic liverdisease, when the muscle takes up excess ammonia anduses it to produce glutamine as a detoxication strategy(Olde Damink et al., 2009). Thus, it is possible that someof the ammonia secreted by tumors as a result of theiravid glutamine consumption is trafcked to the muscleto re-create glutamine from the intracellular glutamatepool (Figure 3). Although speculative, such a systemwould be analogous to glucose handling in the tumor-liver Cori cycle.The question of whether cancer patients with cachexia

would benet from glutamine supplementation hasgenerated a large amount of interest and debate. Atheoretical concern is that increasing glutamine avail-ability might stimulate tumor growth, but it is not clear

whether well-perfused tumors ever experience glutaminedeprivation in vivo, and one study in sarcoma-bearingrats found no enhancement of tumor growth when thediet of the animals was supplemented with glutamine(Klimberg et al., 1990). A large number of clinicalhuman trials have been performed to assess glutaminesutility in improving muscle mass or in limiting chemo-therapeutic toxicity, alone or in combination with otherdietary agents, but there is still no consensus as towhether glutamine is generally helpful.In principle, one could also attempt to intervene at the

level of glutamine release from the muscle. It is temptingto speculate that if muscle export is the major sourceof glutamine for the tumor, then suppressing eitherproteolysis or glutamine synthesis in the muscle mightsuppress tumor growth by reducing the supply of anessential nutrient. Transcription of the glutamate-ammonia ligase mRNA seems to be the pivotal levelof regulation in this process. In various cell lines,expression of glutamate-ammonia ligase is stimulated byglucocorticoids, which have also been implicated in thedevelopment of cancer cachexia (Gaunitz et al., 2002;Wang and Watford, 2007). Perhaps, targeting thissystem in the muscle could be of some benet.

Conclusions and more questions

A number of landmark reports over the last decade havecemented the intimate connections between oncogenesand glucose metabolism in tumors, and shown that thereliance of transformed cells on the Warburg effectcould be exploited experimentally to suppress tumorgrowth. The next wave of interest in tumor metabolismwill likely focus increased attention on cellular gluta-mine handling, and should re-assess the question ofwhether the appetite for glutamine showed by tumorscan be used against them. The diverse contributions ofglutamine to intermediary metabolism, cell signaling,gene expression and cachexia argue in favor of attemp-ting to develop rational strategies to limit tumorglutamine uptake and to image glutamine metabolismin tumors. Presumably, such maneuvers would beparticularly useful in tumors containing enhancedc-Myc activity.There are also a number of fundamental biological

questions that deserve further study. For example, howdo cells manage to allocate glutamine appropriately intothe numerous pathways that use it? Are there mechan-isms to channel glutamine toward the enzymes andcellular compartments that most need it, and are thesesystems responsive to stresses that change cellularpatterns of glutamine use? The observation that somecells require glutamine export to activate mTORsignaling begs the question of how these cells can sensewhen glutamine is abundant enough to permit itssecretion. It will also be interesting to determine howsignals related to glutamine abundance/deprivation aretransmitted to the nucleus to inuence gene expression.A deeper understanding of these issues should helpexplain why glutamine pervades so many aspects of cell

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biology, and should support future efforts to manipulateglutamine metabolism in cancer and other diseases.

Conict of interest

The authors declare no conicts of interest.

Acknowledgements

We thank Roland Knoblauch and Andrew Mullen forcritically reading this paper. RJD is supported by grants fromthe National Institute of Diabetes and Digestive and KidneyDiseases, National Institutes of Health (DK072565) and theAmerican Cancer Society (ACS-IRG-02-196).

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Qaposs next: the diverse functions of glutamine in metabolism, cell biology and cancerIntroductionGlutamineaposs roles in intermediary metabolism: functions and consequencesNucleotide biosynthesisHexosamine biosynthesis and glycolsylation reactionsNonessential amino acids

Figure 1 Glutamine supports cell survival, growth and proliferation through metabolic and non-metabolic mechanisms.Glutathione (GSH)Respiratory substrateReducing equivalentsAmmoniagenesis

Glutamineaposs roles in cell signaling and gene expressionIntegrating glutamine and glucose metabolism in tumor cell growthFigure 2 Cooperativity between glucose and glutamine metabolism in growing tumors.Tumor growth and whole-body metabolism: is the tumor the tail that wags the dog?Figure 3 Proposed inter-organ metabolic cycles in cachectic cancer patients.Conclusions and more questionsConflict of interestAcknowledgementsReferences