Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolismin Langendorff-perfused Rat HeartsCHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINEACETYLTRANSFERASE*

Received for publication, December 9, 2014, and in revised form, January 30, 2015 Published, JBC Papers in Press, February 2, 2015, DOI 10.1074/jbc.M114.631549

Qingling Li‡, Shuang Deng‡, Rafael A. Ibarra‡, Vernon E. Anderson§, Henri Brunengraber‡, and Guo-Fang Zhang‡1

From the Departments of ‡Nutrition and §Biochemistry, Case Western Reserve University School of Medicine,Cleveland, Ohio 44106

Background: Multiple substrates generate acetyl-CoA used in heart citric acid cycle.Results: We observed unexpected metabolite labeling in hearts perfused with labeled substrates.Conclusion: A double substrate cycle operates between triose-phosphates, glucose 6-phosphate, and glycogen; a metabolictunnel operates between pyruvate dehydrogenase and carnitine acetyltransferase.Significance: This metabolomic � mass isotopomer strategy can be used to better characterize heart metabolism in disease models.

We developed an isotopic technique to assess mitochondrialacetyl-CoA turnover (≈citric acid flux) in perfused rat hearts.Hearts are perfused with buffer containing tracer [13C2,2H3]-acetate, which forms M5 � M4 � M3 acetyl-CoA. The buffermay also contain one or two labeled substrates, which generateM2 acetyl-CoA (e.g. [13C6]glucose or [1,2-13C2]palmitate)or/and M1 acetyl-CoA (e.g. [1-13C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA arecalculated from the uptake of the acetate tracer and the massisotopomer distribution of acetyl-CoA. The method was appliedto measurements of acetyl-CoA turnover under different condi-tions (glucose � palmitate � insulin � dichloroacetate). Thedata revealed (i) substrate cycling between glycogen and glu-cose-6-P and between glucose-6-P and triose phosphates, (ii)the release of small excess acetyl groups as acetylcarnitine andketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltrans-ferase. Because of this channeling, the labeling of acetylcarni-tine and ketone bodies released by the heart are not proxies ofthe labeling of mitochondrial acetyl-CoA.

The goal of the present project was to develop and exploit amultitracer technique to measure in the same perfused rathearts; (i) the total turnover of mitochondrial acetyl-CoA, i.e.citric acid cycle (CAC)2 flux and (ii) the absolute contribu-tion(s) of one or two sources of acetyl-CoA to total acetyl-CoA

turnover. The relative contributions of glucose and a fattyacid to the CAC can be measured by using a pair of labeledsubstrates that generate single-labeled (M1)3 and double-la-beled (M2) acetyl-CoA. Such substrate pairs could be[13C6]glucose � [1-13C]palmitate or [1-13C]glucose � [1,2-13C2]palmitate. The M1 and M2 enrichments of mitochondrialacetyl-CoA can be measured either (i) by cleaving citratewith ATP-citrate lyase � CoA and measuring the mass iso-topomer distributions (MID) of acetyl-CoA thus generatedby LC-MS/MS (1) or (ii) by measuring the labeling of C-4 andC-5 of glutamate by NMR (2). To convert these relative contri-butions of glucose and fatty acid to acetyl-CoA to absolutefluxes of acetyl-CoA from these substrates would require thesimultaneous tracing of total CAC flux with a tracer that doesnot perturb the productions of acetyl-CoA from glucose andfatty acids. Total CAC flux has been estimated by perfusinghearts with a high concentration of [13C2]acetate (2–11 mM)and modeling the labeling patterns of CAC intermediatesmeasured by NMR (2, 3). Because the heart has a high activity ofacetyl-CoA synthetase (4, 5), such protocols affect the genera-tion of acetyl-CoA from other substrates than [13C2]acetate.

In experiments with, for example [13C6]glucose � [1-13C]-palmitate, one would need a method to generate a knownamount of acetyl-CoA heavier than M2, and this only in mito-chondria. We hypothesized that this can be achieved by (i)infusing a low concentration of [13C2,2H3]acetate (M5) in theinflowing perfusate (Scheme 1), (ii) measuring the uptake of thetracer, (iii) measuring the labeling of tissue acetyl-CoA, and (iv)dividing the uptake of [13C2,2H3]acetate by the m5 enrichment ofheart acetyl-CoA. Because rat heart acetyl-CoA synthetase is pres-ent only in mitochondria (6, 7), the uptake of [13C2,2H3]acetateshould be equal to the amount of M5 acetyl-CoA formed inmitochondria. We also hypothesized that in perfusions with[13C6]glucose, the measurement of the MID of glucose-6-P andlactate would allow calculating the contributions of glycogen to

* This work was supported, in whole or in part, by National Institutes of HealthRoadmap Grant R33DK070291 and Case Mouse Metabolic and Phenotyp-ing Center (MMPC) Grant U24DK76174 (to H. B.). This work was also sup-ported by American Heart Association Grant 12GRNT12050453 (to G.-F. Z.)and by funds from the Cleveland Mt. Sinai Health Care Foundation.

1 To whom correspondence should be addressed: Dept. of Nutrition, CaseWestern Reserve University School of Medicine, 10900 Euclid AvenueW-G48, Cleveland, OH 44106. Tel.: 216-368-6548; Fax: 216-368-6560; Email:gxz35@case.edu.

2 The abbreviations used are: CAC, citric acid cycle; MID, mass isotopomerdistribution; CrAT, carnitine acetyltransferase; G6P, glucose 6-phosphate;OAT, 3-oxoacid-CoA transferase; PDH, pyruvate dehydrogenase; AcAc,acetoacetate.

3 Mass isotopomers are designated as M, M1, M2 . . . Mn, where n is the num-ber of heavy atoms in the molecule. Mol fractions of mass isotopomers aredesignated as m, m1, m2 . . . .

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 13, pp. 8121–8132, March 27, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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glycolysis and to the CAC. Lastly, we wanted to test the hypoth-eses that the concentrations and labeling patterns of acetylcar-nitine and/or ketone bodies released by the heart could be usedas (i) nondestructive proxies of the labeling pattern of mito-chondrial acetyl-CoA and (ii) an index of the production ofacetyl-CoA in excess of the rate of citrate synthesis. We con-ducted this study on non-working Langendorff-perfusedhearts, in anticipation to extending it to the working heartpreparation.

EXPERIMENTAL PROCEDURES

Materials—General chemicals, aniline, sodium [13C2,2H3]-acetate, sodium [13C2]acetate, [13C6]glucose, [1,2-13C2]palmiticacid, sodium [2H3]propionate, [2H9]pentanoic acid, and[2H9]carnitine were purchased from Sigma (Isotec). 1-(3-Di-methylaminopropyl)-3-ethylcarbodiimide hydrochloride wasbought from Acros Organics. A reference standard for theMID of [13C2,2H3]acetyl-CoA was synthesized by reacting[13C2,2H3]acetate with ATP � CoA � Mg2� � acetyl-CoA syn-thetase from yeast. ATP-citrate lyase was prepared from thelivers of rats that had been starved for 2 days then (re)fed for3 days with a high glucose diet. The enzyme, isolated fromthe Bio-Gel column (8), was precipitated with 50% ammo-nium sulfate, and aliquots of the suspension (2 units/0.1 ml)were kept frozen at �80 °C. Before use, the enzyme was dis-solved in 1 ml of 250 mM Tris-HCl (pH 8.7) containing 5 mM

dithiothreitol.Isolated Rat Heart Perfusions—The Institutional Animal

Care and Use Committee of Case Western Reserve Universityapproved all animal experiments. Hearts from ad libitum-fed male Sprague-Dawley rats (300 –350 g) were perfused inthe Langendorff mode at 37 °C with non-recirculating bicar-bonate buffer (119 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 0.05 mM

L-carnitine) containing 11 mM glucose and equilibrated with95% O2 � 5% CO2. The perfusate was pumped at a constant

flow (12 ml/min) through a 0.22-�m Millipore filter and awater-jacketed heating coil (Radnoti LLC, Monrovia, CA)equipped with a bubble trap before entering the aortic can-nula. The hearts beat spontaneously throughout theperfusion.

After a 15-min equilibration, the perfusions were switchedfor 30 min to bicarbonate buffer containing 3% dialyzed bovineserum albumin (fraction V, fatty acid-free; InterGen) and theconcentrations of substrates and tracers in the 8 groups of per-fusions listed in (Fig. 2). Each group of perfusions included 5hearts. At the end of the experiments the hearts were quick-frozen and stored at �80 °C until analysis. Another group ofhearts was perfused for 30 s to clear the blood and quick-frozento measure baseline glycogen concentrations.

Analytical Procedures—Some samples of perfusate weretreated with NaBH4 to reduce unstable pyruvate and aceto-acetate to stable (RS)-lactate and (RS)-�-hydroxybutyrate(9). The concentrations and MID of perfusate acetate, lac-tate, and �-hydroxybutyrate were assayed by ammonia neg-ative chemical ionization GC-MS of the pentafluorobenzylderivatives (10–12), with internal standards of [2H3]propionate,3-hydroxypropionate, and [2H9]pentanoate. The MID ofglucose in perfusate and in glycogen-glucose was assayed asthe pentaacetyl derivative (13). Heart glycogen was precipi-tated from heart perchloric acid extracts with ethanol, rinsedtwice with ethanol, and spiked with an internal standard ofsorbitol before acid hydrolysis and derivatization with aceticanhydride (13). The MID of acetyl-CoA, acetylcarnitine, andglucose-6-P (as aniline derivative prepared with 1-(3-dim-ethylaminopropyl)-3-ethylcarbodiimide-HCl) were assayedby LC-MS/MS (14, 15). The M1 or/and M2 labeling of theacetyl moiety of heart citrate, a probe of mitochondrialacetyl-CoA, was assayed by cleaving citrate with ATP-citratelyase isolated from rat liver (8) and analyzing acetyl-CoA byLC-MS/MS (1).

SCHEME 1. Main sources and fates of acetyl-CoA in rat heart. The M5 acetate tracer is [13C2,2H3]acetate. Note that in heart the activation of acetate is onlymitochondrial. BHB, (R)-�-hydroxybutyrate; CPT, carnitine-palmitoyl transferase system; TPS, triose phosphates.

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Calculations—All metabolic calculations are based on con-centrations and/or labeling patterns of metabolites in perfusateand freeze-clamped heart tissue. Raw MID were corrected fornatural enrichments with the Isocor software (16). Note that inthe following equations, mass isotopomers are identified as M1,M2 . . . M5, whereas the mol fractions of individual mass isoto-pomers are expressed as m1, m2 . . . m5.

Total acetyl-CoA turnover ��mol � min�1 � �g dry wt� � 1�

in hearts perfused with M2 �13C2�acetate �Group 1�

� ��mol M2 acetate uptake/min�/�m2 of acetyl-CoA�

� �total dry wt of heart� (Eq. 1)

Total acetyl-CoA turnover (�mol � min�1 � (g dry wt)�1) in

hearts perfused with [2H3, 13C2]acetate (G2, 3 . . . 8)

� (�mol M5 acetate uptake/min)/{[m5Ac-CoA � m4Ac-CoA

� m5Ac-CoA (m4acetate/m5acetate) � m3Ac-CoA

� m5Ac-CoA (m3acetate/m5acetate)] (total dry wt of heart)} (Eq. 2)

In the denominator of Equation 2, terms: �m5Ac-CoA(m4acetate/m5acetate) and �m5Ac-CoA(m3acetate/m5acetate) correct the m4and m3 enrichments of acetyl-CoA for the m4 and m3 compo-nents already present in tracer [13C2,2H3]acetate. The correctedm4 and m3 enrichments of acetyl-CoA compensate for the par-tial loss of 2H from M5 and M4 acetyl-CoA via enolization bycitrate synthase (see “Results”).

Flux of unlabeled and labeled free acetate

(�mol � min�1 � (g dry wt)�1) to acetyl-CoA

� (total �mol acetate uptake/min) � (perfusate flow

rate/min)/(total dry wt of heart) (Eq. 3)

Flux of exogenous glucose to the CAC (�mol acetyl � min�1

� (g dry wt)�1) in experiments with [13C6]glucose

(40% or 100% enriched) � (total acetyl-CoA turnover)

� (m2 acetyl-CoA)/ (fractional enrichment of perfusate

glucose) (Eq. 4)

Total glycolytic flux to CAC (from exogenous glucose

and tissue glycogen, in acetyl units) � [(total acetyl-CoA

turnover)] � (m2 acetyl-CoA enrichment from [13C6]glucose)

� (m6 enrichment of glucose)]/ [(fractional enrichment

of perfusate glucose) � (m6 enrichment of (G6P))] (Eq. 5)

Flux of glycogen to CAC � (total glycolytic flux to CAC)

� {1 � m6 enrichment ratio [(G6P in heart tissue)/

(glucose in perfusate)]} (Eq. 6)

Total glycolytic flux in C3 units � (total glycolytic flux

to CAC in acetyl units) � (production of

lactate � pyruvate) (Eq. 7)

Flux of exogenous [1,2-13C2] palmitate to the CAC

(in acetyl units) � [(total acetyl-CoA turnover) 8

(m2 acetyl-CoA)]/(total dry wt of heart) (Eq. 8)

Flux of exogenous [1-13C]octanoate to the CAC

(in acetyl units) � [(total acetyl-CoA turnover) 4

(m1 of acetyl-CoA)]/(total dry wt of heart) (Eq. 9)

Fractional contribution of glycogen to glycolytic flux

� [1 � (m6 G6P in heart tissue)/ (m6 glucose in perfusate)]

(Eq. 10)

Statistics—The statistical difference between groups of datawere tested using a paired t test (GraphPad Prism 3.0). Theexponential fitting was performed using Origin 9.1.

RESULTS

Testing the Concept Underlying the Measurement of TotalAcetyl-CoA Flux in Rat Heart—We first perfused a group ofhearts with 0.04 mM [13C2]acetate and 11 mM unlabeled glucose(Group 1). The uptake of M2 acetate was 0.71 0.15�mol�min�1�g dry wt�1 (Fig. 1, Group 1). We measured the m2enrichments of (i) acetyl-CoA in the heart extract (10.6 1.2%)and (ii) the acetyl moiety of citrate (9.8 1.7%, not significant).The m2 enrichment ratio (acetyl moiety of citrate)/acetyl-CoA)was 0.92 0.06; n � 5). Because the acetyl moiety of citratederives directly from mitochondrial acetyl-CoA, we took themeasured m2 enrichment of tissue acetyl-CoA as the enrich-ment of mitochondrial acetyl-CoA. The acetyl-CoA turnover,calculated by dividing the uptake of M2 acetate by the m2enrichment of heart acetyl-CoA, was 6.9 1.4 �mol�min�1�gdry wt�1 (Fig. 2, Group 1). This rate was compatible with themeasured O2 uptake of the same hearts, i.e. 22.8 3.7�mol�min�1�g dry wt�1, given that the oxidation of 1 �mol ofacetyl requires 3 �mol of O2 (17, 18). Tracer [13C2]acetate(infused at 0.04 mM into the perfusion line) contributed �10%to total acetyl-CoA turnover. We took the above data as dem-onstrating the feasibility of measuring acetyl-CoA turnover inthe perfused heart with a tracer of M2 [13C2]acetate.

Labeling Patterns of Heart Acetyl-CoA Labeled from[13C2,2H3]Acetate—Initial heart perfusion experiments with0.04 mM [13C2,2H3]acetate and 11 mM unlabeled glucose (Fig. 2,Group 2) revealed three unexpected features and constraints ofour strategy to measure acetyl-CoA turnover. The four panelsof Fig. 3 show the MID of (i) acetate in the inflowing perfusate,(ii) acetate in the outflowing perfusate, (iii) acetyl-CoA synthe-sized in vitro from M5 acetate with acetyl-CoA synthetase, and(iv) acetyl-CoA in the heart extract. The vertical scales of thefour panels were adjusted to equalize the visual heights of theM5 enrichment bars in all panels.

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First, although we infused pure tracer [13C2,2H3]acetate at arate calculated to induce a 0.04 mM concentration of tracer inthe inflowing perfusate, the total (M4 � M5) enrichment ofacetate in the inflowing perfusate was only 43% (Fig. 3A). Thisresults from the unavoidable contamination of distilled, deion-ized water (used to prepare the perfusate and reagents for theacetate assay) with ubiquitous traces of unlabeled acetate (10,19, 20). An unknown component of this dilution is the blank of

the assay of acetate concentration and enrichment. As a result,the actual total acetate concentration in the inflowing perfusatewas not 0.04 mM but between 0.04 and 0.04/0.43 � 0.09 mM.This concentration range is similar to the 0.1 mM physiologicalacetate concentration in plasma (19). The contamination doesnot affect the assay of the uptake of labeled acetate calculatedusing the mass spectrometric areas of M5 acetate and of the[2H9]pentanoate internal standard.

FIGURE 1. Uptake of acetate tracers by the perfused hearts. G1, G2 . . . G8 represent perfusion groups 1, 2 . . . 8 with various substrates indicated on the x-axislegend, where the concentrations of substrates in each group were 11 mM glucose, 40 �M acetate, 100 micro�nits of insulin, 0.4 mM palmitate, 0.1 mM

octanoate, and 2 mM dichloroacetate (DCA). In G1, the tracer was M2 [13C2]acetate; in G2– 8, the tracer was M5 [13C2,2H3]acetate. The uptakes were measuredin samples of effluent and influent perfusate collected just before and just after quick-freezing the hearts at the end of 30 min perfusions with tracer. Data arepresented as the mean S.D. (n � 5 in all groups). dw, dry weight.

FIGURE 2. Rates of acetyl-CoA turnover in rat hearts perfused with different mixes of unlabeled and labeled substrates (indicated at the bottom of thebars. G1, G2 . . . G8 represent perfusion groups 1, 2 . . . 8 with various substrates indicated on the x-axis legend, where the concentrations of substrates in eachgroup were 11 mM glucose, 40 �M acetate, 100 microunits of insulin, 0.4 mM palmitate, 0.1 mM octanoate, and 2 mM dichloroacetate (DCA), respectively. Eachfull bar indicates the total rate of acetyl-CoA turnover. Colored sections indicate the contributions of exogenous and endogenous fuels to acetyl-CoA turnover.The bottom beige sections refer to untraced endogenous, unlabeled substrates. Note that G1–3 were perfused with the same substrates but differently labeled.G6 –7 were perfused with another set of the same substrates but differently labeled. Data are presented as mean S.D. (n � 5 in all groups). dw, dry weight.

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Second, comparison between the MID of inflowing and out-flowing perfusates revealed a 1.4-fold isotopic dilution of M5enrichment without change in the M4/M5 labeling ratio (Fig. 3,A and B). Thus, the perfused hearts released unlabeled (or min-imally labeled) acetate, presumably from acetyl-CoA hydroly-sis. The occurrence of cycling between acetyl-CoA and acetatein mammalian cells is well known (21, 22).

Third, the MID of tissue acetyl-CoA (Fig. 3D) was very dif-ferent from the MID of perfusate acetate (Fig. 3, A and B) and ofacetyl-CoA synthesized in vitro from [13C2,2H3]acetate andacetyl-CoA synthetase (Fig. 3C). Tissue acetyl-CoA showed sub-stantial proportions of the M4, M3, and M2 mass isotopomerscompared with M5. Thus, some process occurred after[13C2,2H3]acetate activation that resulted in partial multiplelosses of 2H from mitochondrial [13C2,2H3]acetyl-CoA, pre-sumably initially mostly M5 labeled, as in Fig. 3C. This evokedthe process of acetyl-CoA enolization on citrate synthasedescribed in 1965 by Eggerer (23). When [3H]acetyl-CoA wasincubated with citrate synthase, the specific activity of acetyl-CoA decreased with time. Enolization of acetyl-CoA, a step inthe citrate synthase reaction, results in the exchange of 3H fromthe methyl group of [3H]acetyl-CoA with H� from water.

We conducted a detailed in vitro study of the enolization of[13C2,2H3]acetyl-CoA on citrate synthase and on ATP-citratelyase (see the abstract in Li et al. (24)). The first part of the studydemonstrated multiple cycles of enolization of [13C2,2H3]-acetyl-CoA on citrate synthase, resulting in (i) time-dependentlosses of 2H atoms from acetyl-CoA and (ii) formation of citrateisotopomers with 0, 1, and 2 2H atoms (in addition to 2 13Catoms). The second part of the study demonstrated that thecleavage of deuterated citrate with ATP-citrate lyase is accom-panied by enolization and de-deuteration of acetyl-CoA gener-ated in the reaction. In our perfused heart experiments, enoliza-

tion of [13C2,2H3]acetyl-CoA resulted only in partial loss of 2Hbecause of the simultaneous citrate synthesis. Because of thetwo enolization processes, we could not use the labeling of theacetyl moiety of citrate as a proxy of the labeling of mitochon-drial acetyl-CoA in experiments with [13C2,2H3]acetate. This isbecause the M2 isotopomer of the acetyl-CoA derived fromcitrate cleavage would have two sources: (i) a fuel precursor (e.g.[13C6]glucose or [1,2-13C2]palmitate) and (ii) the complete de-deuteration (by ATP-citrate lyase during analysis) of the acetylmoiety of citrate labeled from the [13C2,2H3]acetate tracer.Thus, to calculate acetyl-CoA production from [1-13C]octanoate,[1,2-13C2]palmitate, or [13C6]glucose (in the presence of[13C2,2H3]acetate tracer; Groups 3– 8), we used the M1 or M2enrichment of acetyl-CoA in the heart extract as the MID ofmitochondrial acetyl-CoA labeled from these substrates. In allexperiments conducted with [13C2,2H3]acetate tracer (Groups2– 8), the total acetyl-CoA production was calculated fromEquation 2. The very small proportion of M2 mass isotopomerin acetyl-CoA from hearts perfused with [13C2,2H3]acetate (Fig.3C) shows that a very small fraction of the [13C2,2H3]acetyl-CoA formed in heart mitochondria had lost all their 2H atoms.Therefore, to calculate the turnover of acetyl-CoA, we used acomposite enrichment of tissue acetyl-CoA, i.e. m5 � theincreases in m4 and m3 enrichments resulting from partial lossof 2H from M5 acetyl-CoA via enolization (see Equation 2 in“Calculations” under “Experimental Procedures”).

Measuring the Turnover of Heart Acetyl-CoA with [13C2,2H3]-Acetate—We calculated the total turnover of acetyl-CoA in 7groups of hearts perfused with [13C2,2H3]acetate and with avariety of labeled and unlabeled substrates (Fig. 2; Groups 2– 8).Note that similar total acetyl-CoA turnover in (i) hearts fromGroups 1, 2, and 3 were perfused with 0.04 mM acetate � 11 mM

glucose differently labeled, and (ii) hearts from Groups 6 and

FIGURE 3. Relative distributions of labeled mass isotopomers of acetate and acetyl-CoA in hearts perfused with 0.04 mM [13C2,2H3]acetate � 11 mM

unlabeled glucose (Group 2). Panel A, inflowing acetate. Panel B, outflowing acetate. Panel C, acetyl-CoA synthesized in vitro from [13C2,2H3]acetate �acetyl-CoA synthetase (for comparison to tissue acetyl-CoA). Panel D, tissue acetyl-CoA. The vertical scales of the four panels were adjusted to equalize the visualheights of the M5 enrichment bars in all panels. Data are presented as the mean S.D. (n � 5).

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7 were perfused with 0.04 mM [13C2,2H3]acetate � 11 mM

glucose � 0.4 mM palmitate differently labeled. The rates of O2uptake were similar in Fig. 2 (Groups 1–3). The rates of acetyl-CoA turnover we measured were similar to values obtainedwith other techniques (18).

In the bars of Fig. 2, the lighter section at the bottom of eachbar correspond to sources of acetyl groups that were not tracedwith a labeled glucose or a labeled fatty acid (palmitate oroctanoate). This includes the conversion of traces of unlabeledacetate from the perfusate (the “Labeling Patterns of HeartAcetyl-CoA Labeled from [13C2,2H3]Acetate” under “Results”).The contribution of unlabeled acetate should be at most equalto that of labeled acetate if all unlabeled acetate was in theperfusate (rather than in the blank of the acetate assay; this isunlikely). In Fig. 2 the top section of each bar represents thecontribution of perfusate labeled acetate to the generation ofacetyl groups used in the CAC (�10%). In Fig. 2, where[13C6]glucose was present in the perfusate (Groups 3, 4, 5, 6, and8), the green and red represent the contributions of glucose andglycogen to the acetyl groups used in the CAC (see below),respectively. In Groups 7 and 8, the dark blue and light bluesections represent the contributions of palmitate and octano-ate, respectively.

Fig. 4 shows the complete MID of acetyl-CoA in hearts per-fused with tracer [13C2,2H3]acetate � 11 mM 100% labeled[13C6]glucose � 0.1 mM [1-13C]octanoate (Group 8). The M0acetyl-CoA derived from unlabeled endogenous sources. TheM1 and M2 acetyl-CoA derived from [1-13C]octanoate and[13C6]glucose, respectively. The M5, M4, and M3 acetyl-CoAderived from tracer [13C2,2H3]acetate. This figure demon-strates the feasibility of measuring in the same hearts the totalturnover of acetyl-CoA (�CAC cycle) as well as the contribu-tions to acetyl-CoA of two labeled substrates and of totalendogenous unlabeled substrates. Note that in this case onlyone of the four acetyl-CoAs derived from [1-13C]octanoate waslabeled. Therefore, to calculate the contribution of [1-13C]-octanoate to acetyl-CoA turnover, one must multiply the M1enrichment of acetyl-CoA by four (Equation 9). Likewise, tocalculate the contribution of [1,2-13C2]palmitate to acetyl-CoAturnover (Fig. 4, Group 8), one must multiply the M2 enrich-ment of acetyl-CoA by eight (Equation 8).

Labeling of Glucose-6-P and Glycogen from [13C6]Glucose—We assayed the concentration of glycogen in hearts perfusedfor 30 min with [13C6]glucose (Fig. 5, Groups 3, 4, 5, 6, and 8). Atthe zero time control, we assayed the glycogen concentration inhearts perfused for 30 s with 11 mM glucose before freeze-clamping (Fig. 5, left bar). In 3 of the 5 groups of hearts perfusedfor 60 min with 11 mM [13C6]glucose, there was statisticallysignificant net accumulation of glycogen (1.8 –2 �mol ofglucose�min�1�g dry wt�1). This increase occurred in heartsperfused with insulin, as expected from the known effect ofinsulin in the heart (25). This increase also occurred in thepresence of 0.4 mM palmitate, which contributed 85% to acetyl-CoA turnover (Fig. 2, Group 7) and spared glucose catabolism(Fig. 2, Group 6) compared with perfused without palmitate(Fig. 2, Group 3).

In perfusions with 40% or 100% [13C6]glucose, we measuredthe MID of G6P and glucose from glycogen in attempts to cal-culate (i) the contributions of glycogen to glycolysis and toacetyl groups used in the CAC and (ii) the contribution of per-fusate glucose to tissue glycogen. We reasoned that a lower M6enrichment of G6P compared with perfusate glucose wouldreflect a glycogenolytic flux because the tissue glycogen wasunlabeled at the beginning of the perfusions. Also, the presenceof M6 isotopomer in glucose from glycogen would reflect somedegree of glycogen synthesis from perfusate glucose.

Fig. 6 shows the MID of inflowing perfusate glucose, tissueG6P, and glucose from tissue glycogen. Note that the first fourgroups (Groups 3– 6) shown in each mass isotopomer clusterwere perfused with 40% [13C6]glucose, whereas the last group(Group 8) was perfused with 100% [13C6]glucose. The small M5components in perfusate glucose result from the presence of�1% 12C in 13C used to synthesize commercial [13C6]glucose.This explains why the M5 isotopomer of glucose is �6 –7% thatof the M6 isotopomer.

In two groups there were significant decreases in the propor-tion of the M6 isotopomer of G6P compared with perfusateglucose (Fig. 6B, Groups 6 and 8). Also in Group 8 (perfusedwith 100% [13C6]glucose), 20% of G6P was unlabeled, whereasthere was no unlabeled glucose in the perfusate (Fig. 6A). Thedata reflect substantial glycogenolysis concomitant with netglycogen accretion (Fig. 5). The latter was confirmed by thepresence of M6 isotopomer in glucose from glycogen (Fig. 6C).Such cycling between glucose and glycogen had been previ-ously described in perfused hearts (25). However unexpectedly,we detected small proportions of M3 isotopomers in G6P andin glycogen (Fig. 6, B and C, insets), whereas there was nodetectable M3 isotopomer in perfusate glucose (influent (Fig.6A, inset) or effluent (not shown)). We interpret the data asreflecting the occurrence of two substrate cycles linking at G6P:G6P 3 triose phosphates 3 G6P 3 glycogen 3 G6P (see“Discussion”).

Rates of Glycolysis—Because we could not measure the sin-gle-pass uptake of glucose across the heart, we estimated ratesof glycolysis in perfusions with [13C6]glucose from the sum of (i)release of unlabeled � labeled lactate � pyruvate and (ii) therate of glucose conversion to acetyl-CoA (Fig. 7). This slightlyunderestimates glycolysis because we could not estimate therate of carboxylation of glucose-derived pyruvate in hearts per-

FIGURE 4. MID of acetyl-CoA in hearts perfused with tracer M5 acetate �11 mM 100% labeled [13C6]glucose � 0. 1 mM [1-13C]octanoate (Group 8).Data are presented as mean S.D. (n � 5).

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fused with [13C6]glucose. The enrichment ratio (m3 lactate infinal effluent perfusate)/(m6 G6P in heart) was within 5–10% ofunity in all cases (not shown). This ratio was barely increasedwhen the m2 and m1 enrichments of lactate were included inthe numerator of the labeling ratio. Thus, as expected, glucoseand glycogen were the only substantial sources of lactate. Also,there was very low cycling between pyruvate, oxaloacetate, andphosphoenolpyruvate (as reflected by the presence of M2 andM1 isotopomers of lactate). Fig. 7 shows the distribution of thecalculated rates of glycolysis between lactate release and con-version to acetyl-CoA. The calculated rates of glycolysis (Fig. 7)varied up to 5-fold between the groups as expected; the lowestrate occurred in the presence of 0.4 mM palmitate.

Testing for Non-CAC Fates of Acetyl-CoA—Our calculationof the CAC flux assumes that the only fate of heart mitochon-drial acetyl-CoA is oxidation in the CAC. Potential other fatesof mitochondrial acetyl groups are releases as acetylcarnitineand ketone bodies, i.e. �-hydroxybutyrate � acetoacetate(Scheme 1). Such releases would occur through the reversiblereactions catalyzed by (i) carnitine acetyl transferase (CrAT)and (ii) (S-3)-hydroxyacyl-CoA dehydrogenase � 3-oxoacid-CoA transferase � (R)-3-hydroxybutyrate dehydrogenase. Thisraises the question, Do the releases of acetyl carnitine andketone bodies represent substantial losses of the [13C2,2H3]-acetate label and/or of total acetyl-CoA turnover in the hearts? Toanswer this question we measured the release of acetyl carnitineand ketone bodies. The loss of label via acetylcarnitine and ketonebodies amount to at most 0.1% and 0.9%, respectively, of theuptake of acetate tracer (not shown). The total release of acetylgroups from the hearts (labeled � unlabeled) as acetylcarnitine �ketone bodies ranged from 0.17 to 2.3% of the acetyl-CoA turnover(not shown). Thus, the release of acetyl groups by hearts perfusedunder our conditions does not substantially underestimate calcu-lations of acetyl-CoA turnover.

Labeling Pattern of Acetylcarnitine and Ketone BodiesReleased by the Hearts—We tested whether the labeling patternof acetylcarnitine and/or ketone bodies released by the per-

fused heart could be used as a continuous proxy of the labelingpattern of acetyl-CoA in hearts perfused with labeled substratesprecursors of M1 or M2 acetyl-CoA ([1-13C]octanoate, [1,2-13C2]palmitate, [13C6]glucose, or [13C2]acetate). Fig. 8A showsthat the isotopomer labeling ratios (mx acetyl carnitine)/(mxacetyl-CoA) varied over a 30-fold range between groups (from0.18 to 5.7). We concluded that the labeling pattern of effluentacetylcarnitine is not a proxy of the labeling pattern of mito-chondrial acetyl-CoA in the heart. Because of the instability ofacetoacetate, the samples of effluent perfusate were treated withNaBH4 to convert acetoacetate to stable (RS)-�-hydroxybutyrate(9). Thus, the MID of ketone bodies was assayed as the MID of total�-hydroxybutyrate. Because ketone body molecules contain twoacetyl groups, we observed, as expected, M2 � M4 or M1 � M2isotopomers from substrates generating M2 or M1 acetyl-CoA,respectively. We calculated an approximate average enrichment ofthe acetyls of ketone bodies by dividing the enrichment of thelower mass ketone body isotopomer by two. This was only anapproximation because of the incomplete isotopic equilibration ofthe two acetyls of AcAc-CoA by AcAc-CoA thiolase in heart (Ref.26, originally described in liver by Huth et al. (27)). We then com-pared this estimate of ketone body acetyl enrichment with thecorresponding enrichment of acetyl-CoA. In fact, the enrichmentratio (acetyl of ketone bodies)/acetyl-CoA) was lower than 1.0 inmost cases (Fig. 8B). We concluded that the labeling pattern ofeffluent ketone bodies is not a proxy of the labeling pattern ofmitochondrial acetyl-CoA in the heart. However, the labeling pat-terns of acetylcarnitine and ketone bodies provide useful informa-tion on the mechanisms by which excess acetyl groups generatedin heart mitochondria are disposed (see “Discussion”).

DISCUSSION

Substrate Cycling between Trioses-P, G6P, and Glycogen—The presence of M3 isotopomers of G6P ((1–3%) Fig. 6, B and C,insets) in hearts perfused with 40% [13C6]glucose reveals a par-tial isotopic equilibration between G6P and triose phosphates.Because perfusate glucose is 40% M6 and 60% M, the MID of

FIGURE 5. Concentrations of glycogen in hearts at the end of the perfusions. The control group is an additional group of hearts perfused for only 30 s withbuffer containing 11 mM unlabeled glucose. G3, G4, G5, G6, and G8 represent perfusion groups 3, 4, 5, 6, and 8 with various substrates indicated on the x-axislegend, where the concentrations of substrates in each group were 11 mM glucose, 40 �M acetate, 100 microunits of insulin, 0.4 mM palmitate, 0.1 mM

octanoate, and 2 mM dichloroacetate (DCA), respectively. Data are presented as the mean S.D. (n � 5 in all groups).

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triose phosphates should be �40% M3 and 60% M. This is com-patible with the measured MID of lactate in the effluent perfu-sate (not shown). If there were total isotopic equilibration

between G6P and triose phosphates, the M, M3, and M6enrichments of G6P would be 36, 48, and 16%, respectively.This is not the case (Fig. 6B). The limited isotopic equilibrationof G6P and triose phosphates must occur via substrate cyclingbetween fructose 6-phosphate and fructose 1,6-diphosphate,coupled to some degree of reversibility of the fructose-1,6-diphosphate aldolase reaction. Because this cycling involvestwo reactions, one of which uses ATP, this process is a truesubstrate cycle, not a simple partial isotopic equilibration viareversible reactions (28, 29). The cycling between fructose6-phosphate and fructose 1,6-diphosphate would not in itselflead to the formation of M3 G6P were it not coupled to somedegree of reversibility of fructose-1,6-diphosphate aldolase.

The same considerations apply to the presence of M3 andM6 isotopomers of glucose in heart glycogen (Fig. 6C). The M6isotopomer of glycogen glucose and the decrease in the M6enrichment of G6P compared with inflowing glucose reflect asubstrate cycle between G6P and glycogen. The M3 isotopomerin glycogen reflects the coupling of the G6P/glycogen cycle tothe G6P/triose phosphates cycle. Thus, our data demonstratesubstrate cycling between triose phosphates and glycogen atthe cost of two ATP per cycle.

Channeling of Acetyl Groups from Pyruvate Dehydrogenase(PDH) to CrAT—We had hoped that the labeling pattern ofacetylcarnitine released by hearts could be used as a continuousproxy of the labeling pattern of acetyl-CoA in hearts perfusedwith labeled substrates as precursors of M1 or M2 acetyl-CoA([1-13C]octanoate, [1,2-13C2]palmitate, [13C6]glucose, or [13C2]-acetate). This was a reasonable expectation because (i) acetyl-CoA is the direct precursor of acetylcarnitine via a rapid revers-ible reaction (30), (ii) with all labeled substrates we used, labeled

FIGURE 6. Comparisons of the MID of glucose in inflowing perfusate (A),tissue G6P (B), and tissue glycogen glucose (C). The MID of glucose in theeffluent perfusate (not shown) was identical with that of the influent perfus-ate. Note the presence of M3 mass isotopomers in B and C, insets). Data arepresented as the mean S.D. (n � 5 in all groups).

FIGURE 7. Distribution of the rates of glycolysis between lactate � pyru-vate production and conversion to acetyl-CoA in 5 groups of hearts per-fused with [13C6]glucose. G3, G4, G5, G6, and G8 represent perfusion groups3, 4, 5, 6, and 8 with various substrates indicated on the x-axis legend, wherethe concentrations of substrates in each group were 11 mM glucose, 40 �M

acetate, 100 microunits of insulin, 0.4 mM palmitate, 0.1 mM octanoate, and 2mM dichloroacetate (DCA), respectively. Data are presented as the mean S.D. (n � 5 in all groups). dw, dry weight.

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acetyl-CoA is formed in mitochondria, (iii) there is essentiallyno CrAT in the cytosol of rat heart (31), (iv) the non-recirculat-ing perfusate contained 0.05 mM carnitine to prevent carnitinedepletion of the perfused hearts, and (v) the hearts were per-

fused under steady state conditions. However, in half thegroups, the isotopomer labeling ratios (mx acetylcarnitine)/(mx acetyl-CoA) were very different from 1.0 (Fig. 8A). Also, fora given substrate mix (e.g. glucose � palmitate), the M2 labelingratio was different when the M2 label derived from[13C6]glucose versus [1,2-13C2]palmitate ((5.7 versus 0.74) Fig.8, Groups 6 and 7)). Fig. 8 (red symbols) shows that in the 5groups of hearts perfused with M6 glucose under differentsteady state conditions, the (M2 acetylcarnitine)/(M2 acetyl-CoA) ratio decreased from 5.7 to 1.0 when the contribution ofM6 glucose to acetyl-CoA increased from 10 to 80%. The datapoints were well fitted to a decaying exponential with an extrap-olated ratio of 7.9 at zero glucose contribution to acetyl-CoAturnover. Thus, at low PDH flux, labeled acetyl-CoA derivedfrom M6 glucose via PDH is preferentially channeled to CrAT,and acetylcarnitine is more labeled than the total pool of mito-chondrial acetyl-CoA. As the contribution of PDH to acetyl-CoA increases, there is a progressive isotopic equilibrationbetween acetylcarnitine and acetyl-CoA labeled from M6glucose.

In Fig. 8A, the data points in green, purple, and blue show thelabeling ratios (mx acetylcarnitine)/(mx acetyl-CoA) in perfu-sions with [13C2]acetate (Group 1), [1-13C]octanoate (Group 8),and [1,2-13C2]palmitate (Group 7) as a function of the contri-butions of these substrates to total acetyl-CoA turnover. When80 –95% of the label derived from M2 palmitate (Group 7),there was incomplete isotopic equilibration between acetylcar-nitine and acetyl-CoA. Even under these conditions, the lowflux of unlabeled acetyl-CoA derived from PDH was still pref-erentially channeled to CrAT. In addition, when the tracer wasM2 acetate (green symbols), which contributed only �10% tothe acetyl-CoA flux, the (M2 acetylcarnitine)/(M2 acetyl-CoA)was �0.2. Thus, the labeling of acetylcarnitine from M2 acetatewas much less than the contribution of acetate to acetyl-CoA.This is the opposite of what was observed with M6 glucose (redsymbols). Thus, although the streaming to CrAT of acetyl-CoAformed from M2 acetate was impeded, the streaming of acetylformed from M6 glucose was favored.

We view these data as evidence of metabolic channeling ofacetyl groups from PDH to CrAT. The concept of metabolicchanneling was developed by Srere (32, 33). Metabolic channel-ing results from associations of enzyme molecules forming ametabolic tunnel (or metabolon) through which the product ofone enzymatic reaction is transmitted to the active site of asecond enzyme in a sequence.

Schroeder et al. (34) perfused rat hearts with non-recirculat-ing bicarbonate buffer containing 11 mM glucose � 2.5 mM

unlabeled pyruvate. The latter was twice transiently replacedfor 2-min periods by 2.5 mM hyperpolarized [2-13C]pyruvate.The authors monitored by NMR the labeling kinetics of acetyl-carnitine and of citrate. The labeling of citrate was taken as aproxy of the labeling of acetyl-CoA. From the kinetics of acetyl-carnitine and citrate labeling, they concluded that under largePDH flux, about one-half of acetyl groups derived from PDHwere transiently stored as acetylcarnitine before being re-con-verted to acetyl-CoA and irreversibly incorporated in citrate.The authors carefully acknowledged that their rate constants“reflect the rate of 13C accumulation in each metabolite pool,

FIGURE 8. Labeling pattern of acetylcarnitine and ketone bodies releasedby the hearts in relation to the labeling of mitochondrial acetyl-CoA. A,labeling ratio (acetylcarnitine)/(acetyl-CoA). B, labeling ratio (average acetylof ketone bodies)/(acetyl-CoA). C, labeling ratio (acetylcarnitine)/(averageacetyl of ketone bodies). Groups are identified by numbers (1– 8). Colorsreflect the substrates generating labeled acetyl-CoA: [13C6]glucose (red), [1,2-13C2]acetate (green), [13C2]palmitate (blue), [1-13C]octanoate (red and purple).Note that Group 8 generated 2 clusters of points because it was perfused with[13C6]glucose � [1-13C]octanoate, which generated M2 and M1 acetyl-CoA,respectively. In panel A data from all 5 groups perfused with [13C6]glucose(red symbols) were fitted to a decaying exponential (y � 0.89 �7.0exp(�0.061x%); r2 � 0.93).

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not necessarily the overall amount of production of eachmetabolite.” We view their data on apparent return of label ofacetyl groups from acetylcarnitine to acetyl-CoA as reflecting acontinuous isotopic exchange rather than a net flux. Schroederet al. (34) data illustrate that even under a supra-physiologicalsupply of acetyl-CoA via PDH, the CrAT reaction is reversible.The reversibility of the CrAT reaction presumably spared thepool of carnitine in these hearts perfused without carnitine.These non-steady state isotopic hyperpolarization NMR exper-iments allowed a glimpse into the CrAT kinetics of the intactheart perfused under metabolic steady state conditions. Oursteady state isotopic data show that preferential labeling of ace-tylcarnitine via PDH occurs in the absence of pyruvate overloadand especially at low PDH flux. Thus, Schroeder et al. (34)experiments and our experiments provide complementaryinformation on the channeling of acetyl groups through thePDH-CrAT metabolic tunnel.

We did not find in the literature-specific reports on the asso-ciation of PDH and CrAT molecules in mitochondria. How-ever, the following reports point to interplay between CrATand PDH. First, chronic carnitine treatment stimulates thePDH complex in human muscle mitochondria (35). Second,muscle-specific deletion of CrAT in mice and human myocytesdecreases glucose tolerance and metabolic flexibility (36).Third, obesity and lipid stress inhibit PDH and CrAT in musclemitochondria (37). Very active enzymes that catalyze reversiblereactions are generally not viewed as regulating carbon fluxthrough pathways. However, as a component of a metabolictunnel with irreversible PDH, CrAT may contribute to the reg-ulation of glucose metabolism in heart as we have shown andpossibly in muscle.

Labeling of Ketone Bodies Released by the Perfused Hearts—The average labeling of acetyl groups of ketone bodies releasedby the hearts was, in most cases, lower than the labeling ofacetyl-CoA (Fig. 8B). This, although the acetyl-CoA and ketonebody pools are linked by the reversible reactions catalyzed byAcAc-CoA thiolase and OAT. This results from the dilution ofAcAc-CoA labeling by the �-oxidation of endogenous unla-beled fatty acids (Scheme 1). Also, the �-oxidation of [1,2-13C2]palmitate forms unlabeled AcAc-CoA after six cycles of�-oxidation, the first of which releases labeled acetyl-CoA. Asecond cause is the incomplete isotopic equilibration of the twoacetyls of AcAc-CoA via the thiolase (26, 27). For these reasons,the labeling of ketone bodies released by the perfused heartcannot be used as a proxy for the labeling of mitochondrialacetyl-CoA.

Release of Acetyl Groups from the Perfused Hearts in Excess ofCAC Flux—The perfused hearts release small amounts of acetylgroups in the form of ketone bodies and acetylcarnitine. Thisrelease amounts to at most 2% of the turnover of acetyl-CoA(not shown). We view this release as a double “bleeding valve” ofacetyl groups, which are produced in slight excess of the CACflux. This is akin to the safety valve on a steam engine. Therelease of acetyl groups reflects the slight excess of (i) the rate ofproduction of mitochondrial acetyl-CoA from all sources over(ii) the irreversible flux through the citrate synthase reaction.The latter flux is directly linked to the respiratory chain flux.Note that the equilibrium position of the CrAT reaction is close

to 1.0 (37). In addition, the equilibrium constant of the OATreaction is far toward acetoacetate (38). Also, the heart is themammalian organ with the highest OAT activity (48 units/g inthe direction AcAc-CoA to acetoacetate (39)). Thus, the CrATand OAT reactions can rapidly dispose of any acetyl-CoA gen-erated in excess of the citrate synthase flux. By this mechanism,any excess acetyl is disposed of without CoA trapping, whichcould perturb many metabolic processes (40).

One could view the mechanisms of acetyl group release viaCrAT and OAT as parallel. The channeling of acetyl from PDHto CrAT would dispose of excess carbohydrate carbon, and theOAT reaction would dispose of excess fatty acid carbon. In fact,our data (panels A, B, and C of Fig. 8) reveal an integrated andflexible mechanism of acetyl release via CrAT and OAT. Evenwhen labeled acetyl groups generated from M6 glucose viaPDH are channeled to CrAT, ketone bodies are labeled(Fig. 8B). Conversely, labeled acetyl groups generated from[1-13C]octanoate or [13C2]palmitate are found in ketone bodiesand in acetylcarnitine (Fig. 8). The link between the two mech-anisms of excess acetyl group disposal is the reversible reactioncatalyzed by acetoacetyl-CoA thiolase (Scheme 1). Thus, thethree reversible reactions catalyzed by CrAT, OAT, and AcAc-CoA thiolase dispose of excess acetyl available to the CAC whilesparing CoA.

Because the hearts were perfused with nonrecirculatingbuffer containing zero ketone bodies, the small release ofketone bodies we report represents a small net ketogenesis.However, in vivo there are always some ketone bodies inplasma. These are taken up by muscle and heart. If the pool ofmuscle or heart AcAc-CoA becomes labeled from an exoge-nous fatty acid, the organ releases labeled ketone bodies whilebeing a net sink of labeled � unlabeled ketone bodies (26). Bythe same isotopic exchange we have called pseudoketogenesis(26, 41, 42), the muscle OAT reaction dilutes the enrichment ofinfused labeled ketone bodies, leading to an artifactual overes-timation of ketogenesis (41).

Because the acetylcarnitine concentration in heart is �20times greater than that of acetyl-CoA, the pool of acetylcarni-tine is often viewed as a buffer of acetyl groups. Note, however,that the size of the acetylcarnitine pool in live rat hearts (0.9�mol�g dry wt�1 (34)) is small compared with the rate of acetyl-CoA turnover (4.5 to 7 �mol�min�1�g dry wt�1 (Fig. 2)). Thus,although the release of acetylcarnitine from the heart is a valveon acetyl production, the flux through this valve is limited bythe pool of carnitine in the heart. In contrast, the release ofacetyl groups as ketone bodies is not limited and is favored bythe equilibrium of the OAT.

We acknowledge that physiological citrate cataplerosis (notmeasured in this study) contributes somewhat to the release ofacetyl groups from the heart (up to 2% of CAC flux in perfusedworking rat hearts (43). Total cataplerosis in live pig heartsamounts to �2% that of CAC turnover (44). Because the rathearts we perfused were in steady state of beats/min and O2uptake, we assume that cataplerosis was compensated byanaplerosis from pyruvate (44). Similarly, cataplerosis affectsthe rates of total acetyl-CoA flux calculated by others from theproduction of labeled CO2 (45).

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In conclusion, the association of targeted metabolomics andmass isotopomer analysis has allowed us to estimate the rate ofCAC flux in the perfused heart and the contributions of fuels toenergy. It also led to the identification of substrate cyclesbetween triose phosphates glucose-6-P and glycogen. Further-more, it identified a metabolic channeling between PDH andCrAT. Lastly, it characterized physiological bleeding valvemechanisms on the supply of acetyl groups to the CAC.Although this study has been conducted in nonworking hearts,its protocol can be adapted to the working hearts. This willallow in depth studies of metabolic interconnections in diseasemodels.

Acknowledgments—We thank the Case Mouse Metabolic and Phe-notyping Center (MMPC) for helping with the heart perfusionexperiments and analytical studies.

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and Guo-Fang ZhangQingling Li, Shuang Deng, Rafael A. Ibarra, Vernon E. Anderson, Henri BrunengraberPYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASELangendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in

doi: 10.1074/jbc.M114.631549 originally published online February 2, 20152015, 290:8121-8132.J. Biol. Chem. 

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