Metformin Decreases Glucose Oxidation and Increases the ... · Metformin Decreases Glucose...

Therapeutics, Targets, and Chemical Biology

Metformin Decreases Glucose Oxidation and Increases theDependency of Prostate Cancer Cells on ReductiveGlutamine Metabolism

Sarah-Maria Fendt1, Eric L. Bell2, Mark A. Keibler1, Shawn M. Davidson2,3, Gregory J. Wirth4, Brian Fiske2,3,Jared R. Mayers2,3, Matthias Schwab11,12, Gary Bellinger6, Alfredo Csibi8, Akash Patnaik5,6,Marie Jose Blouin10, Lewis C. Cantley6,7, Leonard Guarente2, John Blenis8, Michael N. Pollak10,Aria F. Olumi4, Matthew G. Vander Heiden2,3,9, and Gregory Stephanopoulos1

AbstractMetformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased

survival in patients with cancer takingmetformin; however, themechanism bywhichmetformin improves canceroutcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed howmetformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreasedglucose oxidation and increased dependency on reductive glutaminemetabolism in both cancer cell lines and in amouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin furtherattenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced bymetformin. These data suggest that interfering with glutamine may synergize with metformin to improveoutcomes in patients with prostate cancer. Cancer Res; 73(14); 4429–38. �2013 AACR.

IntroductionAltered cellular metabolism is a hallmark of cancer cells

(1–3). Tumor cells adapt their metabolism to provide theneeded quantities of ATP, redox equivalents, and biosyn-thetic precursors necessary for cell growth and uncontrolledproliferation (4–6). Individual cancers appear to have dis-tinct metabolic dependencies to sustain growth and prolif-

eration. Addiction to glutamine (7–9), glucose (1, 10), andother amino acids (11) have been described, but how to besttarget these unique dependencies for therapeutic benefitremains unknown (12).

One potential approach has focused on targeting specificmetabolic enzymes including pyruvate kinase (11, 13), lactatedehydrogenase (14), and glutaminase (15, 16), but the feasibilityof targeting these enzymes in patients with cancer remainsunknown. Metabolism-altering drugs in general, however, havebeen used in the clinic and are well-tolerated by humans. Forinstance, dichloroacetate, a compound used to treat lacticacidosis (17), can alter mitochondrial metabolism in humantumors (18) and can induce death of cancer cells in culture thatfail to adapt to increased pyruvate oxidation (19). The antidi-abetic drug metformin is another widely prescribed compoundthat can directly impact cell metabolism. Metformin improvesglycemic control in diabetes, at least in part, by modulatingsignaling pathways that regulate metabolism in the liver, aprocess thought to be mediated though activation of AMPK(20). How exactly metformin acts as an anti-diabetic agentremains controversial, but one target of metformin in cells ismitochondrial complex I (20). Inhibition of this complex bymetformin can induceenergy stress as onemechanismofAMPKactivation, leading to reduced rates of hepatic gluconeogenesis.As the drug is effective and very well-tolerated by patients, it isamong the most widely prescribed anti-diabetic drugs.

Some reports provide evidence that diabetics treated withmetformin have lower-than-expected cancer burden relative todiabetics taking other agents despite similar glucose control(21–24), but how metformin accomplishes this is not known(25). In vitro antineoplastic activity of about 5 mmol/L met-formin has been confirmed bymany laboratories in a variety of

Authors' Affiliations: Departments of 1Chemical Engineering and 2Bio-logy, Massachusetts Institute of Technology; 3Koch Institute for CancerResearch, Massachusetts Institute of Technology, Cambridge; 4Depart-ment of Urology, Massachusetts General Hospital; 5Division of Hemato-logy/Oncology, 6Division of Signal Transduction, Department of Medicine,Beth Israel Deaconess Cancer Center, Beth Israel Deaconess MedicalCenter; Departments of 7Systems Biology and 8Cell Biology, HarvardMedical School; 9Dana-Farber Cancer Institute, Boston, Massachusetts;10Department of Oncology, McGill University and Jewish General Hospital,Montreal, Quebec, Canada; and 11Doctor Margarete Fischer-Bosch-Insti-tute of Clinical Pharmacology, Stuttgart; and 12Department of ClinicalPharmacology, Institute of Experimental and Clinical Pharmacology andToxicology, University Hospital, Tubingen, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

B. Fiske, J.R. Mayers, M. Schwab, and G. Bellinger contributed equally tothis work.

Current address for Sarah-Maria Fendt: Vesalius Research Center, VIB,Leuven, Belgium

Corresponding Authors: Gregory Stephanopoulos, Massachusetts Insti-tute of Technology, 77MassAve., Cambridge,MA02139. Phone: 617-253-4583; Fax: 617-258-6876; E-mail: [email protected] and Matthew G.Vander Heiden, Koch Institute of Integrated Cancer Research, MIT, 77Massachusetts Ave., Cambridge, MA 02139. Phone: 617-715-4471; Fax:617-253-3189; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-0080

�2013 American Association for Cancer Research.

CancerResearch

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Published OnlineFirst May 17, 2013; DOI: 10.1158/0008-5472.CAN-13-0080

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in model systems. While some activity of biguanides in vivomay be attributable to systemic effects such as reduction ofinsulin levels, there is considerable interest in possible "direct"effects of these compounds under conditions where sufficientintracellular drug concentrations are achieved (26). Indeed, theability of cancer cells to take upmetformin has been challenged(26). Nevertheless, numerous studies have shown antiproli-ferative effects of metformin in various cancer cell lines, and inprostate cancer cells, these metformin effects appear to beindependent of AMPK. Instead metformin appears to activatep53, leading to subsequent-REDD1mediatedmTOR and cyclinD1 inhibition (27–29). Yet, in line with other tumor types, itremains possible that metformin induces antineoplastic activ-ity via direct effects on metabolism. Whether metformin has adirect effect on prostate cancer cell metabolism is unexploredand consequently how best to use the antiproliferative effect ofmetformin for cancer therapy might be missed.

To understand how to best use metformin in patients withprostate cancer, we sought to understand whether and howmetformin affects the metabolism of prostate cancer cells. Wefound that both glucose and glutamine metabolism are affect-ed by metformin via complex I inhibition. Prostate cancer cellswith higher glucose oxidation appear to be the most sensitiveto metformin. Moreover, we identified reductive glutaminemetabolism as essential for maintaining modest proliferationin the presence ofmetformin, thus suggesting that interruptionof glutamine metabolism may have a synergistic effect withmetformin. In agreement with this hypothesis, we found thatmetformin also increased reductive glutamine metabolism intumors arising in an SV40-driven mouse model of prostatecancer.

Materials and MethodsCell lines and cell culture conditions

All cell lines were obtained from American Type CultureCollection. All experiments were carried out in RPMI (Med-iatech), with the exception of Huh7 cells which were culturedin Dulbecco's Modified Eagle's Media, each supplementedwith 10% FBS (Invitrogen) and 1% penicillin/streptomycin(Mediatech). For labeling experiments, FBS was replacedwith dialyzed FBS (Invitrogen), and glucose or glutamine wasreplaced with U-13C glucose (CLM-1396 Cambridge Isotopes),U-13C glutamine (605166 Sigma), or 5–13C glutamine (CLM-1166 Cambridge Isotopes). Cell lines were treated with met-formin or any other small molecule for 3 days in all experi-ments.

PhysiologyGrowth rates were determined using a hemocytometer or an

automated cell counter (Nexcelom). To determine uptake andsecretion rates, glucose and lactate concentrations weredetected by a Waters Alliance 2695 HPLC (Waters) with aWaters 410 Differential Refractometer and a Bio-Rad HPX-87Hcolumn (Bio-Rad). The column was eluted at 50�C with 14mmol/L of sulfuric acid at aflow rate of 0.7mL/min. Glutamineconsumption and glutamate secretion were measured with anYSI 7100MBS (YSI Life Sciences) according to the manufac-turer's protocol.

Carbon contribution to tricarboxylic acid cycle andpalmitate

Labeled tissue cultures were washed with saline andmetab-olism was quenched with�20�C cold 70%methanol. After cellscraping in 70%methanol (containing internal standard norva-line),�20�C cold chloroform was added and the samples werevortexed at 4�C to extract metabolites. Phase separation wasachieved by centrifugation at 4�C. Methanol phase (polarmetabolites) and chloroform phase (fatty acids) were separat-ed and dried by applying constant air flow. Dried metabolitesamples were stored at �80�C.

Polar metabolite samples were derivatized with methoxya-mine (TS-45950 Thermo Scientific) for 90 minutes at 40�Cand subsequently with N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamid, with 1% tert-butyldimethylchlorosilane(375934 Sigma) for 60 minutes at 60�C. Fatty acids wereesterified with 2% sulfuric acid in methanol for 120 minutesat 60�C and subsequently extracted with hexane. Isotopomerdistributions of polar metabolites and fatty acids were mea-sured with a 6890N GC system (Agilent Technologies) com-bined with a 5975B Inert XLMS system (Agilent Technologies).

Isotopomer distributions were analyzed using theMATLAB-based software Metran (30–33). Total contribution of carbonwas calculated using the following equation (34):

Total contribution of carbon ¼Pn

i¼0 i �mi

n�Pni¼0 mi

where n gives the number of C atoms in the metabolite, i is thedifferent mass isotopomers, and m is the abundance of acertain mass.

Isotopomer distributions of fatty acids were further fitted toan isotopic spectral analysis model, assuming that the mea-sured mass distribution vectors are a function of the rate offatty acid synthesis and contribution of the labeled carbonsource to fatty acids (35, 36).

Net glutamine flux was calculated from glutamine uptakerates and glutamate secretion rates.

Small molecules for in vitro experimentsMetformin (D5035 Sigma) was dissolved in milliQ water and

applied at concentrations of 0.5 to 2.5 mmol/L. Rotenone(R8875 Sigma) was dissolved in dimethyl sulfoxide (DMSO)and applied at concentrations of 10 to 50 ng/mL. BPTES was agift from Agios Pharmaceuticals. BPTES and 968 (AG-690Specs) were dissolved in DMSO and applied at concentrationsof 4 or 8 and 5 or 10 mmol/L, respectively.

TSC2 knockdown and mRNA expression levelsLentivirus with shRNA against TSC2 (TRCN0000040178,

TRCN0000040181; Sigma) was applied. Control cells wereinfected with empty pLKO.1. For quantitative real-time PCR,RNA was isolated using RNeasy Mini Kit (Qiagen), and cDNAwas generated from 1 mg RNA using High Capacity cDNAReverse Transcription Kit (Applied Biosystems) according tomanufacturer's protocols. cDNA was analyzed using the LightCycler 480 II (Roche) with SYBR Green Master Mix from Bio-Rad. Primers sequences were obtained from Primer Bank (37).

Fendt et al.

Cancer Res; 73(14) July 15, 2013 Cancer Research4430




Metformin quantificationTumor tissue samples (20–30 mg) were homogenized in

0.9% NaCl using a mortar and pestle and centrifuged (10minutes, 13,000 rpm). An aliquot of the supernatant was usedfor protein determination; in an additional aliquot, metforminwas quantified. Metforminwas quantified with an Agilent 6460triple quadrupole mass spectrometer (Agilent) coupled to anAgilent 1200 HPLC system. Ionization mode was electrospray(ESI), polarity positive. Electrospray jetstream conditions wereas follows: capillary voltage 3,500 V, nozzle voltage 1,000 V,drying gas flow 11 L/min nitrogen, drying gas temperature350�C, nebulizer pressure 55 psi, sheath gas temperature350�C, and sheath gas flow 11 L/min. The mass spectrometerwas operated in the multiple reaction monitoring (MRM)mode using m/z 130.1 and the product ion m/z 60.1. Dwelltimewas 100ms, the fragmentor was set at 60, and the collisionenergy at 10. High-performance liquid chromatography(HPLC) separation was achieved on a HILIC plus column(50 � 2.1 mm2 I.D., 3.5 mm particle size; Agilent) using 0.1%formic acid in water/acetonitrile 20:80 (v/v) as mobile phase ata flow rate of 0.65 mL/min.

Transgenetic adenocarcinoma of mouse prostateTransgenetic adenocarcinoma of mouse prostate (TRAMP)

mice were injected with 3 consecutive intraperitoneal injec-tions (with a 24-hour interval) of vehicle or metformin (300 or500mg/kg/d). Metformin was dissolved in PBS, and the vehiclewas PBS. Mice were sacrificed 2 hours after the last injection.Prostate tumor tissue was removed and quenched in liquidnitrogen. Pulverized tumor tissue was extracted and analyzedas described above. Metabolite pool sizes were normalized toanalyzed tumor tissue weight. Mass isotopomer distributionsof citrate were normalized to the glutamine enrichment of thetissue.All animal experiments were approved by the Committee on

Animal Care at MIT and/or Harvard.

StatisticsError bars depict SD. R and P values for the depicted

correlations were calculated with the MATLAB integratedfunction corrcoef: [R,P] ¼ corrcoef returns P, a matrix of Pvalues for testing the hypothesis of no correlation. Each P valueis the probability of getting a correlation as large as theobserved value by random chance, when the true correlationis zero. P values were also calculated for bar graphs using theStudent t test (2-tailed, unequal variance) to determine wheth-er 2 samples are likely to have come from the same 2 underlyingpopulations that have the same mean.

ResultsMetformin decreases proliferation and promotesglucose fermentation in prostate cancer cell linesTo begin to characterize the effect of metformin on the

metabolism of 3 different prostate cancer cell lines, wemeasured proliferation, glucose uptake rate, and lactatesecretion rate in the absence or presence of increasing dosesof metformin. For all 3 cell lines, the decrease in proliferationobserved with increasing metformin concentration was

accompanied by an increase in the rate of glucose uptakeand lactate secretion (Fig. 1A). Interestingly, the magnitudeof the decrease in proliferation along with the increase inglucose uptake/lactate secretion was different across all 3cell lines. LNCaP cells exhibited the greatest response, PC3cells showed an intermediate response, and DU145 hardlyresponded to metformin. In the presence of 2.5 mmol/Lmetformin compared with no-treatment, all cell lines dis-played an increase in the amount of lactate secretion relativeto glucose uptake, suggesting that a larger percentage of theglucose is converted to lactate in these cells. However, theincrease in lactate secretion relative to glucose uptake wasdifferent in different cell lines (increase in this ratio forDU145: 13% � 3.9%, PC3: 23% � 2.2%, and LNCaP: 49% �3.0%), suggesting that metformin differently affected carbonmetabolism in these different cell lines. Furthermore, thedegree to which metformin altered glucose metabolismcorrelated with the decrease in proliferation rate when cellswere treated with the drug (Fig. 1B).

The metabolic response to metformin is consistent withmitochondrial complex I inhibition

To understand how metformin influences glucose metab-olism, we first asked whether the known action of metforminas an inhibitor of mitochondrial complex I is also observedin these cells. Complex I is a component of the electrontransport chain that transfers electrons derived from nutri-ent oxidation to oxygen. To determine whether metforminaffected this process in prostate cancer cells, we measuredoxygen consumption in the absence or presence of increas-ing concentrations of metformin. Metformin inhibited oxy-gen consumption in a dose-dependent manner in all 3 celllines (Supplementary Fig. S1), consistent with complex Iinhibition. To determine whether complex I inhibition couldaccount for the differential metformin sensitivity across the3 cell lines, we tested the effect of increasing doses of theestablished complex I inhibitor rotenone on cell prolifera-tion (Fig. 2A). Rotenone inhibited cell proliferation in a dose-dependent manner that varied across cell lines. Consistentwith complex I being involved in metformin toxicity, thesensitivity of all 3 cell lines to rotenone was the same as thatobserved with metformin (i.e., DU145 cells were the leastsensitive, PC3 cell had an intermediate phenotype, andLNCaP cells were the most sensitive). Moreover, additionof metformin did not have any further effect on cells treatedwith the highest dose of rotenone, supporting the hypothesisthat the antiproliferative effect of metformin is mediated bycomplex I inhibition in these cells (Fig. 2A). Furthermore, wetested whether the correlation between decreased prolifer-ation and increased glucose uptake was also observed withrotenone treatment. Consistent with a role for complex Iinhibition in altering glucose metabolism in response tometformin, proliferation, and glucose uptake rates in thepresence of rotenone were highly correlated (Fig. 2B). Final-ly, we tested whether hypoxia affects metformin sensitivity.Hypoxic cells are more dependent on glycolysis as an adap-tation to low oxygen (38), raising the possibility that hypoxiccells could be less sensitive to metformin. Indeed we found

Metformin Increases Dependency on Glutamine Metabolism

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that PC3 cells in hypoxia are unaffected by metformin andthat LNCaP cells were much less affected in hypoxia com-pared with normoxia (Fig. 2C). Together, these data suggestthat complex I inhibition by metformin was responsible forboth altered glucose metabolism and the inhibition of cellproliferation in prostate cancer cell lines and that themagnitude of the effect on glucose metabolism is associatedwith the ability of metformin to suppress cell proliferation.

Metformin decreases entry of glucose carbon into thetricarboxylic acid cycle and increases glutamineanaplerosis

To investigate the metabolic effects of metformin beyond themacroscopic parameters of glucose uptake and lactate secre-tion, we cultured all 3 cell lines in the presence of uniformlylabeled 13C-glucose or uniformly labeled 13C-glutamine andmeasured the incorporation of labeled carbon from either

A LNCaPProstate carcinoma Prostate carcinomaProstate adenocarcinoma

P < 0.001

P < 0.0001

P < 0.001

P < 0.005

P = 0.0017

P = 0.017

P = 0.012

0.0

0

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Figure 1. Metformin decreases proliferation and promotes glucose fermentation in prostate cancer cell lines. A, proliferation, glucose uptake rate, and lactatesecretion rate of cell lines treated with metformin. B, correlation between absolute alteration in proliferation and glucose uptake or lactate secretionrate for different metformin concentrations compared with the condition with no metformin added. All error bars indicate SD. SD, correlation coefficient R,and P values were calculated from 3 biologic replicates.

Fendt et al.





nutrient into the tricarboxylic acid cycle metabolite a-ketoglu-tarate by gas chromatography/mass spectrometry. In all celllines tested, metformin decreased glucose contribution toa-ketoglutarate in a dose-dependent manner, whereas gluta-mine contribution to a-ketoglutarate increased in a reciprocalpattern (Fig. 3A). As inmost cultured cells, glutamine accountedfor the majority of tricarboxylic acid cycle carbon in all 3 linestested (39). However, variability in the degree of glucose con-tribution was observed. Interestingly, the magnitude of glucosecarbon contribution to the tricarboxylic acid cycle was alsocorrelated with the sensitivity of the cell lines to metformin;DU145was the least sensitive tometforminandshowed the leastamount of glucose oxidation, PC3 cells had an intermediatephenotype, and the LNCaP cells displayed the most glucoseoxidation and were most sensitive to metformin (Fig. 3A).Inhibition of electron transport has been reported to switch

glutamine metabolism from an oxidative to a reductive path-way (40). Consistent with the inhibition of complex I bymetformin in these cells, metformin treatment led to anincrease in reductive glutamine metabolism. This was shownby an increase in theMþ 5mass isotopomer of citrate (Fig. 3B)and further confirmed by measuring the contribution of glu-tamine labeled with 13C only at the C5 position, to palmitate,which is lost if glutamine contributes oxidatively to fatty acids.Metformin increased the reductive contribution of glutamineto palmitate in a dose-dependent manner (Supplementary Fig.S2), as well as the overall contribution of carbon to the

tricarboxylic acid cycle. These data suggest that complex Iinhibition by metformin decreased glucose entry into thetricarboxylic acid cycle and subsequently increased glutamineanaplerosis via reductive carboxylation to citrate. Further-more, these phenomena track with sensitivity to metformin.

To further confirm that complex I inhibition by metforminresults in the switch from glucose toward additional glutaminefor fueling the tricarboxylic acid cycle, we measuredglutamine contribution to a-ketoglutarate and the reductiveglutamine contribution to palmitate in the presence of rote-none (Supplementary Fig. S3). Similar to metformin, rotenoneincreased the contribution of glutamine carbon to the tricar-boxylic acid cycle and reductive glutamine contribution topalmitate, providing further support for the role of complex Iinhibition in the antiproliferative effect for metformin inprostate cancer cells.

Decreased glutamine flux increases sensitivity tometformin

We next questioned whether increased glutamine metabo-lism is an adaptive response to complex I inhibition that mightattenuate the antiproliferative effect of metformin. To test thishypothesis, we took advantage of the small-molecule inhibitors968 and BPTES, both of which target glutaminase (refs. 15,16; Fig. 4A). Glutaminase catalyzes the conversion of glutamineto glutamate, and the increased activity of this enzyme is atleast partially responsible for elevated glutamine metabolism

A

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Figure 2. The metabolic responseto metformin is consistent withmitochondrial complex I inhibition.A, proliferation of cell lines treatedwith the complex I inhibitorrotenone. B, correlation betweenabsolute alteration in proliferationand glucose uptake rate ofmetformin (light gray)- or rotenone(dark gray)-treated cells comparedwith the condition with no drugadded. C, sensitivity of cell linestoward metformin (2.5 mmol/L) innormoxia compared with hypoxia.All error bars indicate SD. SD,correlation coefficient R, andP values were calculated from3 biologic replicates.






in cancer (16, 41). Consistent with increased glutamine metab-olism being an adaptive response following metformin treat-ment, we detected larger decrease in cell number with thesimultaneous administration of metformin and a glutaminaseinhibitor (968 or BPTES) than observed with either metforminor glutaminase inhibitor acting alone for all 3 cell lines (Fig. 4B;Supplementary Fig. S4A). The combination of rotenone and968 produced a similar effect (Fig. 4C). Finally, we testedwhether withdrawal of glutamine from the medium yieldsincreasedmetformin sensitivity. As none of the prostate cancercell lines proliferate in the absence of glutamine, we used Huh7liver cancer cells, which have been previously reported to growwithout glutamine (42). Indeed, Huh7 cells cultured withoutglutamine displayed a significantly increased metformin sen-sitivity (Supplementary Fig. S4B).

Increased glutamine flux decreases metforminsensitivity

To test whether increased glutamine flux is sufficient tolimit the antiproliferative effect of metformin, we tookadvantage of the recently reported ability of mTOR topositively regulate net glutamine flux into the tricarboxylicacid cycle (43). mTOR was activated in these cells by a

knockdown of its negative regulator TSC2 (SupplementaryFig. S5A). Net glutamine flux into the tricarboxylic acid cycleincreased in the TSC2-knockdown cell lines by approximate-ly 2-fold (Fig. 5A). In line with the hypothesis that increasedglutamine metabolism mitigated metformin toxicity, cellswith activated mTOR were approximately 3-fold less sensi-tive to metformin when compared with control cells (Fig.5B). To further test the importance of glutamine conversionto a-ketoglutarate as an adaptive response to metformin, weused a cell-permeable form of a-ketoglutarate, dimethyl-ketoglutarate, in LNCaP cells following drug treatment. Inline with our previous findings, dimethyl-ketoglutarate sup-plementation in the presence of metformin increased cellnumber by 30% (Supplementary Fig. S5B). These resultssuggest that augmenting glutamine metabolism limited theantiproliferative effect of metformin.

Metabolic alterations triggered by metformin in vivomatch the in vitro response

To test whether our findings are relevant to tumors in vivo,we first established thatmetformin reaches prostate tumors inmice. Using the TRAMP prostate cancer mouse model, wedetermined that tumors of mice dosed 3 times with 500 mg/kg

A

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LNCaP

P = 0.02

P = 0.003

P = 0.007

P = 0.007

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Figure 3. Metformin decreasesentry of glucose carbon into thetricarboxylic acid cycle andincreases glutamine anaplerosis.A, glucose (top) and glutamine(bottom) contribution to thetricarboxylic acid cycle in prostatecancer cell lines measured by theincorporation of [U-13C]-labeledglucose or [U-13C]-labeledglutamine into a-ketoglutarate.B, the increase in glutaminecontribution leads to reductiveinstead of oxidative fueling asshown by the increase in M þ 5citrate from [U-13C]-labeledglutamine. All error bars indicateSD. SD and P values werecalculated from 4 replicatesderived from 2 independentexperiments.

Fendt et al.





(body weight) metformin had taken up between 23 and 103ng/mg (tissue protein) metformin into the prostate tumors.Metabolite analysis of both prostate cancer cell lines andprostate tumor tissue derived from TRAMP mice treated withvehicle or metformin showed that the most pronounced

alteration inmetabolite concentration as a result ofmetformintreatmentwas a fall in the citrate pool, which is associatedwithan activation of reductive glutamine metabolism (Fendt andcolleagues, submitted; Fig. 6A). This effect of metformin wasobserved both in cells and in tissues (Fig. 6A and B). This

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P = 0.008

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P = 0.07P = 0.33

P = 0.02

P = 0.0013

P = 0.008 P = 0.006

P = 0.0026

P = 0.004P = 0.013

P = 0.006

P = 0.006P = 0.006

P = 0.008

P = 0.026

P = 0.02

α-ketoglutarateGlutamine Glutamate

968 or BPTES

Glutamate dehydrogenaseGlutaminase

Gls1 Gdh1

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Figure 4. Decreased glutamine fluxincreases metformin sensitivity. A,enzymatic conversion steps fromglutamine to a-ketoglutarateincluding known inhibitors of thepathway. Relative cell count of celllines treated with a combination ofmetformin (B) or rotenone (C) withthe glutaminase inhibitor 968. Cellcounts were normalized to thecondition with no metformin andno 968 added. SD and P valueswere calculated from 3 biologicreplicates.

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Figure 5. Increased glutamine fluxdecreases metformin sensitivity.A, glutamine flux of TSC2-knockdown cell lines relative tocontrol cell line. Control cells forknockdown were infected with anempty plasmid. B, metforminsensitivity of TSC2-knockdowncell lines, which exhibit increasedglutamine flux based on cellcounts. All error bars indicate SD.SD and P values were calculatedfrom at least 3 biologic replicates.






finding shows that at least some of the metabolic effects ofmetformin in vitro are also observed in tumors in vivo.

Finally, we assessed the extent to which reductive glutaminemetabolism was impacted along with glucose metabolism bythe presence of metformin in vivo. To trace glutamine metab-olism in tumors, 3 consecutive intraperitoneal injections of

uniformly labeled 13C-glutamine were administered to TRAMPmice treated with metformin or vehicle. Following tumorharvest the enrichment of M þ 5 citrate was measured inprostate tumors as a surrogate for reductive glutamine metab-olism. We found a significant increased enrichment of M þ 5citrate in different parts of the prostate from metformintreated animals (Fig. 6C). This increase in M þ 5 citrateenrichment was accompanied by a decrease in the relativecitrate pool size (Fig. 6D). In conclusion, these data show thatthe metabolic effects of metformin in tumor cells in vitroextend to similar responses in tumors in vivo.

DiscussionThe oral anti-diabetic drug metformin is beneficial for at

least a subset of patients with cancer, but whether metformincan directly affect metabolism in tumor cells was not known.We systematically dissected the metabolic alterations metfor-min triggers in prostate cancer cell lines through complex Iinhibition and found that this results in an adaptive increase inglutamine metabolism (Fig. 7). It is possible that in addition tothe adaptive response in glutaminemetabolism described herein response to metformin, energy homeostasis regulation viaAMPK following metformin treatment further diminishes theeffect of the drug as shown recently for non–small lungcarcinoma (44). Our finding of an adaptive response in gluta-mine metabolism also complements a recent study showingthat cell lines grown on glutamine as the major carbon sourceare sensitive tometformin (45). The finding of this study can beexplained by our data, as we show that metformin leads toreductive glutamine metabolism and consequently leads to adramatic decrease in ATP production by the tricarboxylic acidcycle. Importantly, we additionally show that inhibition ofglutamine metabolism can promote metformin toxicity, sug-gesting that therapy with an agent that limits glutaminemetabolism may potentiate the effect of metformin.

Our in vivomodel showed that mice tolerate metformin at adose of 500 mg/kg/d, considerably higher that than the con-ventional anti-diabetic dose in humans of�20mg/kg/d, whichled to accumulation of the drug to�60 ng/mg (tissue protein)in cancer tissue. This exposure level was observed to have invivo metabolic effects on prostate tissues that matched our invitro data.We show for the first time that this is correlated witheffects on cellular carbon metabolism, specifically a depen-dency on reductive glutamine metabolism. This finding sug-gests that high-dose biguanide exposure might be a method totherapeutically induce metabolic stress in a subset of tumors,either alone or in rational combinations. It is important torecognize, however, that the effect of conventional anti-dia-betic metformin doses currently being examined for antineo-plastic activity in clinical trials may have a smaller effect onmetabolism. Our work suggests that a dose escalation study ofbiguanides may be warranted to determine whether the doserange withmore profound direct effects on cancer metabolismcan safely be administered to humans.

Because the antiproliferative effect ofmetformin tracks withits ability to inhibit complex I, and this effect is mitigated byincreased glutamine metabolism, it is possible that cells with ahigh dependence on oxidative glucose metabolism and low

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Figure 6. Metabolic alterations triggered by metformin in vivo match thein vitro response. A, relative citrate pool size in prostate cancer cell linesnormalized to the condition with no metformin added. SD and P valuesare calculated from 4 biologic replicates. B, citrate pool size in prostatetissue from TRAMP mice treated with (4 animals) or without (3 animals)metformin (left). One to two different parts of tissue were analyzed peranimal. Average of citrate pool size for TRAMP mice treated with orwithout metformin (right). Enrichment of M þ 5 citrate (C), which isindicative for reductively used glutamine, and relative citrate pool size (D)of prostate tissue from TRAMP mice treated with (1 animal) or without (2animals) metformin. Three to four different parts of the prostate wereanalyzed per animal. All error bars indicate SD.

Fendt et al.





dependence on glutamine metabolism may be most sensitiveto this drug. Whether these finding will extend to non–prostatecancer cases remains to be determined. Importantly, theseresults do not rule out non-cancer cell–autonomous effects inpatients.However, a direct effect onprostate cancer cellsmaybebeneficial. Prostate cancer cells, compared with nontrans-formed prostate epithelium, reactivate aconitase activity lead-ing to increased citrate oxidation (46) and display high rates ofde novo lipid synthesis (47, 48). Since we found that cell linesdisplaying an oxidative metabolism were most sensitive tometformin, the reactivation of citrate oxidation in prostatetumor tissue may predict a beneficial therapeutic effect ofmetformin in prostate cancer therapy. Furthermore, cells adaptto metformin treatment by increasing reductive glutaminemetabolism, which directly effects the carbon source selectionfor fatty acid synthesis. Thus, glutaminase inhibitors, adminis-trated in combination with metformin, might specificallyenhance the beneficial effects ofmetformin in cancer treatment.

Disclosure of Potential Conflicts of InterestM.G. Vander Heiden and L.C. Cantley have ownership interest (including

patents) from and are consultants/advisory board members of Agios Pharma-ceuticals. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S.-M. Fendt, E.L. Bell, G.J. Wirth, A. Patnaik, L.C.Cantley, A.F. Olumi, M.G. Vander Heiden, G. StephanopoulosDevelopment of methodology: S.-M. Fendt, E.L. Bell, S.M. Davidson, B. Fiske,M. Schwab, A. Csibi, A. Patnaik, M.G. Vander Heiden, G. Stephanopoulos

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.-M. Fendt, E.L. Bell, M.A. Keibler, S.M. Davidson, G.J.Wirth, B. Fiske, J.R. Mayers, M. Schwab, G. Bellinger, A. Patnaik, L.P. Guarente, J.Blenis, A.F. OlumiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.-M. Fendt, E.L. Bell, S.M. Davidson, J.R. Mayers, M.N. Pollak, A.F. Olumi, M.G. Vander Heiden, G. StephanopoulosWriting, review, and/or revision of themanuscript: S.-M. Fendt, E.L. Bell, M.A. Keibler, S.M. Davidson, B. Fiske, J.R. Mayers, M. Schwab, A. Patnaik, L.C.Cantley, A.F. Olumi, M.G. Vander Heiden, G. StephanopoulosAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.-M. Fendt, G. Bellinger, M.-J. Blouin, J.Blenis, A.F. OlumiStudy supervision: S.-M. Fendt, A. Patnaik, L.C. Cantley, A.F. Olumi, M.G.Vander Heiden, G. Stephanopoulos

AcknowledgmentsThe authors thank Agios Pharmaceuticals for providing BPTES.

Grant SupportS.-M. Fendt is supported by the German Research Foundation (DFG), grant

FE1185. L. Guarente is supported by the NIH and the Glenn Foundation forMedical Research. A. Csibi is supported by a fellowship from the LAM Founda-tion. M.G. Vander Heiden acknowledges NIH grant support from 5-P50-090381-09 and 5-P30-CA14051-39 and support from the Burrough's Wellcome Fund, theSmith Family, and theDamonRunyonCancer Research Foundation. The authorsalso received grants 1R01DK075850-01 and 1R01CA160458-01A1 from the NIH(G. Stephanopoulos).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received January 9, 2013; revised April 22, 2013; accepted May 5, 2013;published OnlineFirst May 17, 2013.

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Glucose Glucose Glucose

Pyruvate Pyruvate Pyruvate

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Glutamine Glutamine Glutamine

Metformin Metformin

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α-Ketoglutarate α-Ketoglutarateα-Ketoglutarate

Respiratory chain Respiratory chain Respiratory chain

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2013;73:4429-4438. Published OnlineFirst May 17, 2013.Cancer Res Sarah-Maria Fendt, Eric L. Bell, Mark A. Keibler, et al. MetabolismDependency of Prostate Cancer Cells on Reductive Glutamine Metformin Decreases Glucose Oxidation and Increases the

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