Cytosolic reducing power preserves glutamate in retinaJianhai Dua, Whitney Cleghorna, Laura Contrerasb,c, Jonathan D. Lintona, Guy C.-K. Chand, Andrei O. Chertova,Takeyori Sahekie, Viren Govindarajua, Martin Sadilekf, Jorgina Satrústeguib,c, and James B. Hurleya,g,1

Departments of aBiochemistry, dPharmacology, fChemistry, and gOphthalmology, University of Washington, Seattle, WA 98195; bDepartamento de BiologíaMolecular, Centro de Biología Molecular Severo Ochoa, and cCentro de Investigación Biomédica en Red de Enfermedades Raras, Universidad Autónoma deAutónoma de Madrid-Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain; and eInstitute of Resource Development and Analysis, KumamotoUniversity, 2-2-1 Honjo, Kumamoto 860-0811, Japan

Edited by Constance L. Cepko, Harvard Medical School/Howard Hughes Medical Institute, Boston, MA, and approved September 25, 2013 (received for reviewJune 13, 2013)

Glutamate in neurons is an important excitatory neurotransmitter,but it also is a key metabolite. We investigated how glutamate ina neural tissue is protected from catabolism. Flux analysis using13C-labeled fuels revealed that retinas use activities of the malateaspartate shuttle to protect >98% of their glutamate from oxida-tion in mitochondria. Isolation of glutamate from the oxidativepathway relies on cytosolic NADH/NAD+, which is influenced byextracellular glucose, lactate, and pyruvate.

Glutamate is especially important as a metabolite because it isrequired for the synthesis of glutathione, other amino acids,

and proteins. Glutamate also is a key intermediate in glutamine-dependent anaplerosis, now known to be a principal source ofcitric acid cycle intermediates in cancer cells (1).When it is released as a neurotransmitter at brain synapses,

glutamate that escapes from the synapse is taken up by astro-cytes. There it is converted to glutamine and is delivered back toneurons in a process called the “glutamate/glutamine cycle” (2).Uptake of glutamate and conversion to glutamine within astro-cytes stimulates glycolysis and synthesis of lactate. Astrocytesexport the lactate to neurons as fuel in a process called the“astrocyte neuron lactate shuttle” (ANLS) (3).Synaptic terminals of rod and cone photoreceptors have

characteristics that appear incompatible with the ANLS. Thephotoreceptor terminal is enriched with transporters for reup-take of glutamate (4), and it encapsulates the synapse. It is un-likely that much glutamate can escape the synapse before beingsequestered back into the photoreceptor. We initiated a study toevaluate the role of ANLS in retina. However, the unusualmetabolic features of retina revealed a surprising feature ofneuronal metabolism, that >98% of glutamate is protected fromcatabolism. We investigated this protection and show here thatthe protection is provided by activities associated with the met-abolic pathway known as the “malate aspartate shuttle” (MAS)(shown schematically in Fig. 1).MAS activity regenerates cytosolic NAD+ that is needed to

support glycolysis. To do so, it uses two important transporters totrap the reducing power from cytosolic NADH and shuttle it intothe mitochondrial matrix. One transporter is the neuronal as-partate/glutamate carrier (AGC1 or Aralar) (Fig. 1, orange cir-cle); the other transporter is the oxoglutarate carrier (OGC)(Fig. 1, light blue circle). AGC1 transports glutamate from thecytoplasm into the mitochondrial matrix in exchange for aspar-tate from the matrix (Fig. 1). OGC transports α-ketoglutaratefrom the matrix into the cytoplasm in exchange for malate fromthe cytoplasm (Fig. 1) (5). An important consequence of MASactivity is that it diverts metabolic flux in mitochondria awayfrom succinyl CoA, succinate, and fumarate (Fig. 1). Most im-portantly, glutamate that completes a MAS cycle functions asa catalyst for the importation of reducing power into the mito-chondria. The carbon atoms of glutamate are isolated from theoxidative pathway in the mitochondrial matrix. To determine theextent of that isolation in a neuronal tissue, we used 13C-labeledfuels to identify metabolic networks in mouse retinas and quantifytheir metabolic flux.

ResultsWe began by using gas chromatography/mass spectrometry (GC/MS) to analyze metabolites released into the medium frommouse retinas cultured in 5 mM glucose. Fig. 2A shows thatlactate and pyruvate accumulate in the culture medium at a ratioof ∼20:1. Release of monocarboxylates from retina is fast, four tofive times faster than from brain slices (Fig. 2 B and C). Incontrast, retinas release much less glutamate and glutamine thanbrain slices (Fig. 2 D and E).Next we analyzed metabolites within retinas. Retinas were

incubated with 5 mM glucose and were washed; then metaboliteswere extracted. The outer retina is composed mostly of photo-receptors, but metabolites in retinal homogenates also comefrom other retinal neurons and glia. We estimated the portion ofmetabolites from the outer retina by serial sectioning followed byGC/MS and found that more than half of the glutamate in theretina comes from the outer retina (Fig. 3).We then used GC/MS to measure the rates at which 13C from

uniformly labeled 13C (U-13C) glucose incorporates into retinalmetabolites. Fig. 4A shows the displacement of endogenous 12Cisotopomers by isotopomers in which two or more 12C atoms arereplaced by 13C (6). Glucose is taken up and oxidized by gly-colysis so quickly that half the endogenous pyruvate and lactatein the retina are replaced within 5 min.Table 1 summarizes the metabolic fluxes calculated from the

rate constants and the total level of each metabolite in the retina.Flux of 13C through citrate and α-ketoglutarate/glutamate is3–4% of the flux through lactate/pyruvate, as is consistent withprevious reports (7, 8). Remarkably, flux of 13C through succinateis nearly 100-fold slower. This result indicates that >98% ofα-ketoglutarate in mitochondria is removed from the matrix before

Significance

This report shows that the reducing power in the environmentinfluences oxidation of glutamate in a neuronal tissue. Gluta-mate is a neurotransmitter, and it is especially important asa metabolite because it is required for synthesis of glutathione,other amino acids, and proteins. Glutamate also is a key in-termediate in glutamine-dependent anaplerosis, now consid-ered to be a principal source of citric acid cycle intermediatesin cancer cells. Our analyses also show that the reducing powerin the environmental can influence glutamate oxidation incancer cells.

Author contributions: J.D., W.C., L.C., A.O.C., J.S., and J.B.H. designed research; J.D., W.C.,L.C., J.D.L., G.C.-K.C., A.O.C., V.G., J.S., and J.B.H. performed research; L.C., J.D.L., G.C.-K.C.,T.S., M.S., and J.S. contributed new reagents/analytic tools; J.D., W.C., L.C., A.O.C., J.S., andJ.B.H. analyzed data; and J.D., L.C., J.S., and J.B.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: jbhhh@uw.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311193110/-/DCSupplemental.

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it can be oxidized by α-ketoglutarate dehydrogenase. The mostlikely path for this removal is OGC activity (9–11).The data in Fig. 4A show that aspartate and malate accumulate

label faster than succinate and fumarate. A limitation of the type

of kinetic analysis shown in Fig. 4A is that it does not distinguishcitric acid cycle intermediates labeled by citrate synthase fromthose labeled by pyruvate carboxylase. To distinguish those path-ways, we analyzed the data in more detail. We quantified in-corporation of either two 13C atoms (“m2”) via the pyruvatedehydrogenase/citrate synthase route or three 13C atoms (“m3”)via pyruvate carboxylase. The pathways and the patterns of la-beling from U-13C–labeled glucose are shown schematically in Fig.S1. The measurements were made after a very short incubationtime (1 min) to ensure that much less than a single citric acid cyclecould occur. Analysis of the m2 isotopomers (Fig. 4B) showedthat the rate of succinate formation is <2% of the rate of glu-tamate formation, in agreement with the conclusions from thedata in Table 1. Analysis of the m3 isotopomers (Fig. 4C) showedthat the pyruvate carboxylase pathway is ∼25 times slower thanthe pyruvate dehydrogenase pathway.Retinas also consume fuels other than glucose. When gluta-

mine is the only fuel in the culture medium, it is effective inraising levels of α-ketoglutarate, succinate, fumarate, and malatein retinas (blue bars in Fig. 5A). However, when glucose is in-cluded with the glutamine, those retinal metabolites decrease,and glutamate increases (green bars). This effect of glucoseoccurs even when U-13C–labeled glutamine is used as fuel. Fig.S2 shows that incorporation of 13C from labeled glutamine intomitochondrial metabolites is suppressed by the presence of un-labeled glucose. Glucose decreases the incorporation of 13Catoms from glutamine into the citric acid cycle in both the“clockwise” direction (succinate, fumarate, malate) and in the“counterclockwise” direction [citrate via reversal of the isocitratedehydrogenase reaction (12–14)]. The unexpected effect of glu-cose led us to hypothesize that cytosolic reducing power, which isgenerated from glucose by glycolysis, has a dominant influenceon retinal metabolism.

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Fig. 1. How the malate aspartate shuttle isolates glutamate. The gluta-mate/α-ketoglutarate cycle in retina isolates the carbon atoms of glutamatefrom the oxidative pathway inside mitochondria. In this report we demon-strate the influence that cytosolic reducing power and Aralar/AGC1 activityhave on this pathway. ASP, aspartate; OAA, oxaloacetate; MAL, malate;GLU, glutamate; AKG, α-ketoglutarate; OGC, oxoglutarate carrier, IPM,interphotoreceptor matrix.

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We tested this hypothesis directly by incubating retinas withmixtures of glucose, glutamine, pyruvate, or lactate. Glucose canreduce cytosolic NAD+ to NADH by fueling glycolysis. Lactatecan reduce cytosolic NAD+ to NADH via lactate dehydrogenaseactivity. Glutamine has no effect on cytosolic reducing power,whereas pyruvate oxidizes NADH to NAD+ via lactate de-hydrogenase. We confirmed these predictions by measuring totalNADH and NAD+ in retinas incubated with various fuel com-binations (Fig. 5B, Upper). We expect that cytosolic NADH andNAD+ equilibrate rapidly with intracellular lactate and pyruvate.Therefore the lactate/pyruvate ratio (Fig. 5B, Lower) more ac-curately reflects the average relative cytosolic NADH/NAD ratiounder the various incubation conditions.This influence of fuels on cytosolic NADH/NAD+ allowed us to

investigate relationships between cytosolic reducing power, aminoacids, and citric acid cycle intermediates. We focused on theglutamate/succinate ratio because it reflects the relative activitiesof two competing metabolic paths (Fig. 5C). Path 1 is oxidation ofα-ketoglutarate to succinyl CoA followed by conversion to succi-nate. Path 2 is removal of α-ketoglutarate from mitochondria byOGC followed by conversion to glutamate. Path 2 is part of theMAS, and it relies on reducing power from NADH.We predicted that high cytosolic reducing power would favor

path 2 over path 1, thereby increasing glutamate/succinate. Totest this prediction, we incubated retinas in pyruvate with orwithout glucose and evaluated the ratios of glutamate/succinateand lactate/pyruvate. We also used glutamine with or withoutglucose and glutamine with or without lactate. When glucoseor lactate was present, lactate/pyruvate increased, indicating

higher cytosolic NADH/NAD+. Fig. 5 D and E shows a directrelationship between glutamate/succinate and lactate/pyruvate.Fig. S3 shows the effects on the absolute levels of metabolites.We conclude from these data that high reducing power in thecytosol protects glutamate. This protection is especially im-portant for neurons, because glutamate is a neurotransmitterand is an essential precursor for the synthesis of glutathioneand proteins.To confirm this metabolic relationship between glutamate and

cytosolic reducing power, we examined the consequence of dis-rupting it. OGC-mediated exchange of mitochondrial α-keto-glutarate for cytosolic malate requires synthesis of cytosolicmalate from cytosolic oxaloacetate. Cytosolic oxaloacetate canbe generated by transport of matrix aspartate to the cytosol viaAralar/AGC1 followed by transamination (Fig. S4A). We dis-rupted this pathway by using retinas from Aralar/AGC1−/− mice.Aralar/AGC1 deficiency blocks a major path for NADH oxi-

dation in the cytoplasm, so we expected cytosolic NADH to ac-cumulate and drive the reduction of pyruvate to lactate. Consis-tent with this notion, we found that Aralar/AGC1 deficiencyincreases the lactate/pyruvate ratio (Fig. 5F). Consistent withour model, Aralar/AGC1 deficiency also lowers the glutamate/succinate ratio even at very high lactate/pyruvate ratios (Fig. 5F).The effect on other metabolites is summarized in Fig. S4B.We asked if the metabolic relationship between glutamate and

cytosolic reducing power occurs in cells other than the neuronsand glia in retinas. We incubated an immortal cancer cell line(HeLa) with a similar set of fuel combinations and analyzedlactate/pyruvate and glutamate/succinate ratios. Fig. S5 suggeststhat this metabolic relationship may be generalizable to othercells and tissues.

DiscussionWe show in this report that an α-ketoglutarate/glutamate cir-cuit in retinas protects glutamate from oxidation. Glutamateexchanges into neuronal mitochondria via Aralar/AGC1, andα-ketoglutarate exchanges out via OGC (Fig. 1). OGC competeswith α-ketoglutarate dehydrogenase activity to remove α-keto-glutarate from the matrix before it can be oxidized. Protectionof glutamate by OGC activity ensures that glutamate is availablefor neurotransmission, synthesis of glutathione, and synthesis ofother amino acids and proteins.Our serial section analysis showed that more than half the

metabolites that we measure from whole-retina homogenatescomes from the photoreceptor layer, consistent with otherstudies that used a different type of method to map distributionsof amino acids in retina (15, 16). However, the experimentalapproach we used in our study does not distinguish the metab-olites from photoreceptors from the metabolites from otherneurons and from glia. The metabolic pathways we analyzed arenot identical in all cells in the retina because AGC1 is presentonly in neurons and not in Müller cells (11). Nevertheless, wefind from whole retina that only 1–2% of the carbon atoms thatflow through the α-ketoglutarate/glutamate pool undergo oxi-dation to succinate. This result is consistent with the ubiquitouspresence of OGC. OGC in neurons removes α-ketoglutaratefrom the matrix before it can be oxidized so that it can be used asa neurotransmitter. OGC activity in Müller cells protects theα-ketoglutarate/glutamate pool to ensure it can be used forglutamine synthesis, one of the primary functions of glia. OGC-mediated protection does not require AGC activity. It requiresonly that there be sufficient cytosolic malate to drive the ex-change. Because Müller cells lack AGC1, the source of carbonatoms for cytosolic malate may be aspartate from neurons (17).Further studies are underway to confirm this possibility in retina.The measurements we report here were made on isolated re-

tinas in culture so that we could control their fuel supply. It is im-portant to ask whether the metabolic relationships we identified

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Fig. 3. Distribution of metabolites in the retina. Most of the metabolites inthe retina are in photoreceptor inner segments and cell bodies. Rat retinasections were analyzed for (A) metabolites by GC/MS and (B) protein byquantitative immunoblots. Results are expressed as percent of total ion in-tensity for each metabolite in each section (n = 3). Rhodopsin and recoverinwere used as landmarks. GC, ganglion cell; PR, photoreceptor.

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also occur in vivo. To answer this question, we compared effectsof AGC1 inactivation from our in vitro experiments with thecorresponding effects under in vivo conditions. We comparedmetabolite levels from our study in which retinas were incubatedin culture with metabolite levels reported in a previous study thatused freshly isolated brain tissue (17). The effects of AGC1

inactivation were nearly identical under in vitro and in vivoconditions (Fig. S6). Another study showed that glutamate levelsare reduced by 29–48% in brains from AGC1−/− mice (18).Glutamine and GABA levels also are reduced substantially.These changes are consistent with our findings, but glutathionelevels increase slightly in the study by Llorente-Folch et al. (18).

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Fig. 4. Fluxes through glycolytic and citric acid cycle intermediates labeled with U-13C glucose. (A) Retinas were incubated with 5 mM U-13C glucose, and theamounts of each 12C metabolite not yet replaced by its 13C-labeled counterpart were determined at the indicated times. Continuous curves are double-exponential fits. Blue arrows highlight the predominant direction of each reaction. Data are from two sets of identical experiments. (B and C) Flux measured1.0 min after 12C-glucose was replaced with U-13C glucose. (B) The m2 isotopomers are derived from the glycolysis, pyruvate dehydrogenase, citrate synthasepathway (Fig. S1, Right). (C) The m3 isotopomers are derived from the glycolysis, pyruvate carboxylase pathway (Fig. S1, Left).

Table 1. Metabolic fluxes calculated from total metabolites per retina and the rate constants determined from thefirst 30 min of the data shown in Fig. 4

Total metabolites per retina, nmol Rate, min Flux per retina, nmol/min

Lactate + pyruvate 22.9 ± 1.1 0.26 ± 0.11 5.96 ± 2.5Citrate 1.1 ± 0.0 0.21 ± 0.07 0.23 ± 0.08α-Ketoglutarate + glutamate 11.1 ± 1.4 0.017 ± 0.005 0.19 ± 0.06Succinate 1.0 ± 0.1 0.003 ± 0.003 0.003 ± 0.003Fumarate + malate 1.3 ± 0.0 0.007 ± 0.012 0.009 ± 0.015

Total metabolites per retina, rate constants, and calculated fluxes are shown. Monocarboxylates were pooled because their rateconstants indicate they are in rapid equilibrium. Similarly, glutamate and α-ketoglutarate were pooled, and fumarate and malatewere pooled.

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The ratio of reduced to oxidized glutathione decreases by halfin AGC1−/− brains, suggesting that an additional response toAGC1 inactivation might stimulate glutathione synthesis.Metabolic dysregulation can be a significant factor in diseases

that cause retinal degeneration and loss of vision (19). We showhere that the extent to which glutamate is protected depends onthe cytosolic reducing power, which in turn depends on extra-cellular lactate, pyruvate, and glucose. Consistent with this result,a recent study used a fluorescent NADH sensor to show thatextracellular lactate and pyruvate directly and rapidly influencecytosolic NADH/NAD+ (20). When retinas consume glucose,80–96% of the glucose is converted to lactate (8, 21). Lactate isreleased from retinas into the interphotoreceptor matrix (IPM)that bathes the retina and the neighboring retinal pigmentepithelium (22). Our findings suggest that the lactate/pyruvateratio in the IPM can influence retinal metabolism, function, andviability.Taken together, these results show that reducing power in the

environment influences glutamate levels in a neuronal tissue.Our preliminary analysis of HeLa cells shows that this influenceof extracellular fuels may extend to other types of cells and tis-sues. For example, metabolic adaptations that lead cancer cellsto prefer glutamine as a fuel (23) also may be influenced by themetabolic relationships we have described in this report.

MethodsReagents. U-13C glucose was obtained from Cambridge Isotope Labo-ratories, Inc. Other 13C tracers and reagents were from Sigma unlessotherwise specified.

Animals. All animal experiments were conducted in accordance with theguidelines of the University of Washington Animal Care and Use Committeeafter Institutional Animal Care and Use Committee approval and with pro-cedures approved in the Directive 86/609/EEC of the European Union and theEthics Committee of the Universidad Autónoma de Madrid. C57BL mice (6–8wk old) were purchased from The Jackson Laboratory. Aralar/AGC1+/− mice(24) were crossed to produce Aralar/AGC1−/− and Aralar/AGC1+/+ controllittermates in the laboratory of J.S.

Retina and Brain Tissue Culture. Mice were killed by cervical dislocation. Eyeswere removed, and retinas were separated from retinal pigment epithelium incold HBSS. All experiments in this report were performed under standard roomillumination. For brain tissues, after decapitation, thewhole brainwas removedquickly from the skull and was placed on wet filter paper with HBSS. Thecerebellum (consisting of the crus 1 ansiform, crus 2 ansiform, and paramedianlobules), hippocampus, and olfactory bulb were isolated. The retina and otherbrain tissue were cultured in Krebs–Ringer bicarbonate medium (25) with5 mM glucose or 5 mM other 13C tracers in a 37 °C, 5.0% CO2 incubator.

GC/MS Analysis of Metabolites. A single mouse retina was rinsed in cold 0.9%sodium chloride and snap frozen in liquid nitrogen. The retina was ho-mogenized in 120 μL of a 700:200:50 cold mixture of methanol/chloroform/

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Fig. 5. Cytosolic reducing power influences the distribution ofcitric acid cycle intermediates and amino acids. (A) Retinaswere incubated for 2 h with glucose alone, with glutaminealone, or with a mix of glucose and glutamine (5 mM each).Metabolites were extracted and quantified by GC/MS. Gluta-mine alone caused several-fold increases of α-ketoglutarate,succinate, fumarate, and malate. Addition of glucose coun-teracted this effect of glutamine. Remarkably, glucose andglutamine together also caused a nearly fourfold increase inthe total amount of glutamate in the retina (n = 3). (B) Dif-ferent types of fuel or fuel combinations affect NADH levelsand lactate/pyruvate ratios similarly. Each fuel was used at 5mM (n = 4–6). (C) Schematic of two competing pathways fromα ketoglutarate (α-KG). Path 1 produces succinate and occursindependently of NADH. Path 2 produces glutamate and isstimulated by NADH. (D) Glutamate/succinate correlates withthe lactate/pyruvate ratio. The reducing power of glucose andlactate increases both lactate/pyruvate and glutamate/succi-nate ratios (n = 3). (E) Summary of the relationship betweenthe lactate/pyruvate ratio and the glutamate/succinate ratio.Data are taken from the set of experiments shown in D. (F)Disruption of AGC1 activity in Aralar/AGC1−/− retinas disruptsthe relationship between the glutamate/succinate and lactate/pyruvate ratios. In the absence of AGC1 activity NADH accu-mulates in the cytoplasm. The elevated NADH level drives upthe lactate/pyruvate ratio. In striking contrast to the resultssummarized in E, the substantially elevated lactate/pyruvateratio in AGC1−/− retinas does not increase the glutamate/suc-cinate ratio. In fact, the glutamate/succinate ratio decreasesslightly. The effects of AGC1 inactivation on levels of othermetabolites are shown in Fig. S4B. Aralar/AGC1−/− retinas havenormal expression of photoreceptor and mitochondrial pro-teins (Fig. S7).

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water with 0.1 mM methylsuccinate as an internal standard and was placedon ice for 15 min. The extracts were centrifuged at 16,000 × g for 15 min,and the supernatant was transferred into glass inserts. The extracts weredried under vacuum, derivatized with 25 μL of freshly prepared methylhy-droxylamine HCl in pyridine (20 mg/mL), and incubated at 37 °C for 90 min.The extracts were further derivatized with 25 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide at 70 °C for 30 min.

The medium metabolites were analyzed by drying 20 μL of medium andfollowing the procedure in the retina samples except for reduction of thederivatization volume to 20 μL total. The rat section samples were homog-enized in the mixture of methanol/chloroform/water and were derivatizedin 10 μL of methylhydroxylamine HCl in pyridine and 10 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide.

An Agilent 7890/5975C GC/MS system (Agilent Technologies) with anAgilent HP-5MS column (30 m × 0.25 mm × 0.25 μm film) column was usedfor GC separation and analysis of metabolites. Ultra–high-purity helium wasthe carrier gas at a constant flow rate of 1 mL/min. One microliter of samplewas injected in split-less mode by the autosampler. The temperature gradientstarted at 100 °C with a hold time of 4 min and then increased at a rate of 5 °C/min to 300 °C, where it was held for 5 min. The temperatures were set asfollows: inlet 250 °C, transfer line 280 °C, ion source 230 °C, and quadrupole150 °C. Mass spectra were collected from m/z 50–600 at a rate of 1.4 spectra/safter a 6.5-min solvent delay. The chromatograms were analyzed using AgilentChemstation software. Abundances of the following ions were extracted: m/z174–177 for pyruvate,m/z 261–264 for lactate,m/z 432–437 for glutamate,m/z431–436 for glutamine, m/z 418–422 for aspartate, m/z 287–291 for fumarate,m/z 289–293 for succinate, m/z 346–351 for α-ketoglutarate, m/z 419–423 formalate, m/z 591–597 for citrate, and m/z 260–263 for alanine. The measureddistribution of mass isotopomers was corrected the natural abundance ofisotopes using the software IsoCor (26, 27) and was defined with standardsand verified by mass after each experiment. Labeled metabolite data were

expressed using relative ion abundances or as a concentration (in microgramsper milligram) determined using an external calibration curve.

Protein Concentration Assay. After metabolites were extracted, tissue pelletswere dissolved in 200 μL of 0.1 M NaOH overnight at 37 °C. Protein con-centration was determined by the BCA assay kit (Thermo Fisher Scientific).

NADH Cycling Assay. Mouse retinas were extracted and rinsed once in HBSSbefore incubation in fuels (lactate, pyruvate, glucose, glutamine, and glu-cose + glutamine) for 1 h. Samples were homogenized in DMSO, lyophilized,and resuspended in the NADP/NADPH extraction buffer from the NADP/NADPH Assay Kit (Abcam). To measure NADH alone, samples were heated at60 °C for 20 min to degrade NAD+. NADH alone and total NADH/NAD+ werequantified using a cycling assay (28, 29). Measurements were taken everyminute for 20 min at 570 nm (25).

Statistical Analysis. Data are expressed as mean ± SD. Significance of dif-ferences between means was determined by unpaired two-tailed t tests orANOVA with an appropriate post hoc test. A P value <0.05 was considered tobe significant.

ACKNOWLEDGMENTS. We thank Ken Lindsay, Ian Sweet, and Jack Saari forhelpful comments and suggestions. This work was supported by GrantsEY06641, EY017863, and EY023346 from the National Institutes of Health (toJ.B.H.), and by Grants BFU2011-30456-C02-01/BMC fromMinisterio de Economíay Competitividad and S2010/BMD-2402 from Coumunidad Autónoma deMadrid (to J.S.). This work also was funded by the Centro de InvestigaciónBiomédica en Red de Enfermedades Raras, an initiative from the Instituto deSalud Carlos III, and an institutional grant from the Fundación Ramón Arecesto the Centro de Biología Molecular Severo Ochoa.

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