Communication Vol. 269, No. 44, Issue of November 4, pp. 27179-27182, 1994 THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

Reversibility of the Mitochondrial Isocitrate Dehydrogenase Reaction in the Perfused Rat Liver EVIDENCE FROM ISOTOPOMER ANALYSIS OF CITRIC ACID CYCLE INTERMEDIATES*

(Received for publication, August 16, 1994, and in revised form, September 12, 1994)

Christine Des Rosiersl, Charles A. FernandezS, France David% and Henri Brunengraberll(1 From the $Department of Nutrition, University of Montreal, Montreal, Quebec H3C 357, Canada and the Departments of Wutrition and Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106

The reversal of the mitochondrial isocitrate dehydro- genase reaction was investigated in rat livers perfused with [U-lsC,]glutamate or [U-'sC,lglutamine. The mass isotopomer distribution of citric acid cycle intermedi- ates extracted from the livers was determined by gas chromatography-mass spectrometry. Citrate was en- riched in an isotopomer containing five "C. The forma- tion of this isotopomer can only be explained by the reversal of the isocitrate dehydrogenase reaction. Cal- culation of kinetic parameters from the mass isoto- pomer data reveals a rapid interconversion of isocitrate and a-ketoglutarate. This interconversion results in an isotopic exchange between carbon 6 of citrate and mito- chondrial CO, that can affect the calculation of citric acid cycle kinetic parameters. Thus, the reversal of the isocitrate dehydrogenase reaction should be included in isotope labeling models of the citric acid cycle.

Isocitrate dehydrogenase (ICDH)' catalyzes the conversion of threo-q-isocitrate (ICIT) to a-ketoglutarate (aKG) as shown by Reaction 1.

threo-n,-Isocitrate + NAD(P)+ u aKG + HCO; + NAD(P)H +H+ REACTION 1

Liver contains three ICDH: one cytosolic and two mitochon- drial. The cytosolic ICDH uses NADP+ and generates NADPH for fatty acid and cholesterol syntheses (1). Liver mitochondria contain a NAD+-ICDH and a NADP+-ICDH (2), which are part of the citric acid cycle (CAC). In rat liver mitochondria, the NAD"1CDH is associated with other CAC enzymes: fumarase, malate dehydrogenase, citrate synthase, and aconitase (3). The

Medical Research Council of Canada, Grant DK35543 (to H. B.) from * This work was supported by Grant MA-9575 (to C. D. R.) from the

the National Institutes of Health, and a grant from the Nutrition De- velopment Fund of the Cleveland Mt. Sinai Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed.

a-ketoglutarate; CAC, citric acid cycle; ICIT, threo-ne-isocitrate; O M , The abbreviations used are: ICDH, isocitrate dehydrogenase; aKG,

oxaloacetate.

kinetic properties of the NAD"1CDH are consistent with its operation in vivo in the direction of aKG formation (1, 4). NAD+-ICDH is regulated by a variety of positive (Ca2+, ADP, citrate) and negative (ATP, NADH, NADPH) effectors (5). In contrast, NADP+-ICDH has no known allosteric effector and could operate in the direction of ICIT formation; its afflnity for NADPH is 100-fold greater than for NADP+ (61, and its K,,, for CO, (1.6 m ~ ) is in the range of the physiological concentration (1.5 m, Ref. 7).

There is a consensus view that NAD+-ICDH catalyzes the conversion of ICIT to aKG (4,8,9). However, the function of the mitochondrial NADP+-ICDH is uncertain (4, 8-11). Very re- cently, Sazanov and Jackson (12) proposed that, in the mito- chondrial matrix, a substrate cycle operates between ICIT and aKG, where NAD+-ICDH generates aKG and NADP+-ICDH regenerates ICIT. The NADPH used in the reverse reaction would be supplied by the H+h-anshydrogenase driven by the proton electrochemical gradient. The net balance of the ICIT z aKG cycle would be the dissipation of the gradient. The ICIT 2 aKG cycle provides a mechanism by which the flux through the CAC is (i) more tightly controlled by the modifiers of NAD+- ICDH and (ii) directly controlled by the energy state of the inner mitochondrial membrane. This hypothesis is supported by studies conducted in isolated liver mitochondria. However, the operation of an ICIT s aKG cycle has not been demon- strated in intact liver.

In the course of investigating (13) gluconeogenesis in isolated rat livers perfused with [U-13C,llactate and [U-13C,]pyruvate, mass isotopomer analysis of CAC intermediates suggested the reversal of the mitochondrial ICDH reaction in the intact liver. Briefly, the data were compatible with an isotopic exchange between C-6 of citrate and CO,. If that is the case, we reasoned that, in rat livers perfused with [U-13C,]glutamate or [U-'3C,]glutamine, reversal of the ICDH reaction would result in the formation of M+5 citrate. The present study was under- taken to test this hypothesis.

EXPERIMENTAL PROCEDURES Materials-Chemicals were obtained from Sigma-Aldrich and en-

zymes from Boehringer Mannheim. [U-13C,lGlutamate and [U-13C,]- glutamine (98%) were obtained from Cambridge Isotopes. The derivat- ization agent N-methyl-N-(t-butyldimethylsilyl~trifluoroacetamide was obtained from Regis Chemical Co. (Morton Grove, IL).

Perfusion Experiments-Livers from 24-h fasted male Sprague- Dawley rats (Charles River Laboratories) were perfused (14) with non- recirculating Krebs-Ringer bicarbonate buffer containing 4 m~ glucose, 1 mM lactate, 0.2 m~ pyruvate, 0.2 mM acetate, 0.2 mM octanoate, and either 0.5 m~ glutamate or 0.5 mM glutamine. After a 10-min equilibra- tion, glutamate or glutamine was replaced by [U-'3C,lglutamate or [U-'3C,lglutamine and the experiment continued for 20 min before freeze-clamping of the liver.

Analytical Procedures-'ho-gram samples of frozen livers were ex- tracted with 7 ml of 8% sulfosalicylic acid and 1 ml of 5 M hydroxyla- mine-HC1. After centrifugation, the extract was brought to pH 8 with KOH and incubated for 60 min at 65 "C to convert ketoacids to hydrox- amates. The solution was acidified to pH 1-2 with HCI, saturated with NaC1, and extracted three times for 10 min with 12 ml of ethyl acetate. The pooled extract was evaporated and the residue reacted with 50 pl of N-methyl-N-~t-butyldimethylsilyl~trifluoroacetamide at 60 "C for 1 h, to convert analytes to tert-butyldimethylsilyl derivatives. One or two pl were injected into a Hewlett Packard MS engine (HP 5890 gas chro- matograph and HP 5989 mass spectrometer) equipped with a HP-5 capillary column (25 or 50 m x 0.2 mm, inner diameter, 0.33 pm film thickness). Split ratio was 20/1 for lactate and succinate (splitless for all other compounds). Carrier gas was helium (1 ml/min), and column head

27179

27180 Reversibility of Isocitrate Dehydrogenase in Liver

Isotopomer distribution of metabolites labeled from W-I3C Jglutamate TABLE I

acetate, 0.2 mM octanoate, and 0.5 mM glutamate. After 10 min of equilibration, glutamate was replaced by [U-'3C]glutamate and the experiment Livers from 24-h-starved rats were perfused with non-recirculating buffer containing 4 m~ glucose, 1 m~ lactate, 0.2 m~ pyruvate, 0.2 m~

continued for 20 min. The isotopomer distribution of metabolites isolated from the freeze-clamped livers has been corrected for natural abundance of heavy isotopes (16, 17). The molar fraction of each isotopomer is expressed as a percentage (mean f S.D.). Influent glutamate was 94% M+5. Numbers printed in bold are used in the computations presented under "Discussion."

M M+l M+2 M+3 M+4 M+5 M+6

Citrate 82.9 * 2.4 0.9 * 0.6 1.9 * 0.4 2.2 f 0.3 2.1 f 0.4 aKG 71.3 f 1.8 0.4 f 0.2

10.8 1.4 -0.8 2 0.4

Succinate 81.1 * 1.4 1.5 * 0.1 1.4 f 0.1 1.5 0.1 24.0 1.7 Fumarate

0.8 f 0.1 94.5 f 1.0

1.3 2 0.1 1.0 f 0.04 15.8 1.2

Malate 0.1 f 0.9 0.8 f 0.9 1.8 * 0.7 2.8 f 0.5

Lactate 93.5 f 0.6 99.1 f 0.2

0.0 f 0.2 1.3 * 0.1 2.1 * 0.1 3.1 f 0.3 0.2 f 0.2

Pyruvate 0.4 0.1 0.3 * 0.03

97.9 * 0.5 0.2 * 0.1 0.6 0.1 1.3 0.5

pressure was 200 kilopascals. Three column temperature programs were used. For lactate, pyruvate, succinate, aKG, and malate, the pro- gram was: 50-m column, 100 "C for 1 min, increase 5 Wmin until 205 "C, 35 "C/min until 275 "C, 20 min at 275 "C. For citrate, the pro- gram was: 25-m column, 100 "C for 4 min, increase 5 "C/min until 275 "C, 5 min at 275 "C. For fumarate, the program was: 2 min at 80 "C, increase by 5 Wmin until 185 "C, 25 min at 185 "C, increase 35 Wmin until 300 "C, 5 min at 300 "C. Very long chromatographic runs were necessary to avoid interferences at all m / z monitored. Ions monitored were m / z 261-264 (lactate), 274-277 (pyruvate), 289-293 (succinate), 287-291 (fumarate), 419423 (malate), 446-451 (aKG), and 459465 (citrate). The enrichments of influent [U-'3C5]glutamate and [U-'3C51glutamine were measured as described in Ref. 15. Areas under each fragmentogram were determined by computer integration and corrected for naturally occurring heavy isotopes (16, 17).

RESULTS AND DISCUSSION Table I shows data from four liver perfusions with

[U-'3C,]glutamate. The mass isotopomer distributions of liver CAC intermediates, lactate, and pyruvate have been corrected for naturally occurring heavy isotopes (16, 17). Influent [U-13C,]glutamate was 94% enriched in M+5 isotopomer. The corresponding M+5 enrichment of aKG was 24%. This 4-fold dilution reflects the influx of unlabeled carbon in the CAC. The very low enrichments of aKG and citrate in M+l to M+4 isoto- pomers show that unlabeled carbon enters the CAC both as acetyl-coA and oxaloacetate (OAA). In livers perfused under similar conditions with [3-'3C11actate + [3-'3C]pyruvate (14, E ) , we showed that very little acetyl-coA is formed from pyruvate. Most of the acetyl-coA derives from octanoate.

The striking finding in this study is that citrate is 10.8% enriched in M+5 isotopomer. The M+5 enrichment of citrate amounts to 10.8/24, i.e. 45% of the M+5 enrichment of aKG.

One perfusion was conducted with [U-'3C,lglutamine. The distribution of the heaviest isotopomers of the compounds as- sayed was: M+5 glutamine (94%), M+5 citrate (4.5%), M+5 aKG (13.6%), M+4 succinate (18.2%), M+4 fumarate (4.0%), M+4 malate (3.6%), M+3 lactate (0.3%), and M+3 pyruvate (0.7%).

There are only two pathways by which M+5 glutamate or M+5 glutamine can yield M+5 citrate. The first involves M+5 glutamate + M+5 aKG -j M+4 succinate + M+4 OAA + M+3 pyruvate + M+2 acetyl-coA. Some of the M+4 OAA could be- come M+3 in the pyruvate cycle: M+4 OAA + M+3 phos- phoenolpyruvate 4 M+3 pyruvate + M+3 OAA. Then, M+3 OAA could combine with M+2 acetyl-coA to form M+5 citrate. However, one would expect to find even more M+6 citrate re- sulting from the condensation of M+4 OAA with M+2 acetyl- CoA. No M+6 citrate was found. This first mechanism can thus be excluded because of (i) absence of M+6 citrate, (ii) very low enrichments in M+4 malate and M+3 pyruvate, and (iii) very low enrichments in M+l to M+4 isotopomers of citrate and aKG.

The second and only mechanism that can explain the forma- tion of M+5 citrate from M+5 aKG is the reversal of reactions catalyzed by aconitase and ICDH. Aconitase is fully reversible (18). However, the sequence of the ICDH and aKG dehydrogen- ase reactions is considered irreversible in intact cells, given the cyclical and continuous nature of the CAC. This would not be the case when the mitochondrial [NADH]/[NAD+l ratio is el- evated, as is the case during ethanol oxidation in the liver. Then, the flux through the CAC can be decreased to almost zero (19). However, under our conditions, livers were perfused with medium containing a redox buffer made up of 1 mM lactate and 0.2 mM pyruvate. This [lactate]/[pyruvate] ratio of 5, which is at the oxidized end of the physiological range, imposed a similarly oxidized [NADHl/[NAD+] ratio in the liver cytosol. In other perfusions with similar substrates, we measured a [R-p- hydroxybutyratel/[acetoacetatel ratio of 1.1 (14, 151, which is also fairly oxidized. Since the [R-@-hydroxybutyrate]/ [acetoacetate] ratio reflects the mitochondrial [NADH]/[NAD+l ratio, we can exclude an inhibition of aKG dehydrogenase and NAD+-ICDH by a redox shift.

In the metabolic sequence aKG + succinate + fumarate + malate, most of the dilution in the enrichment in fully labeled isotopomers (M+5 for citrate and aKG, M+4 for the others) occurs at the level of fumarate, about 5-fold. The strong dilution of fumarate is caused by the influx of unlabeled OAA derived from pyruvate. OAA interconverts with malate and fumarate through the very active and reversible malate dehydrogenase and fumarase reactions. The identical M+4 enrichments of fu- marate and malate show that the reversible fumarase reaction is sufficiently rapid to achieve isotopic equilibrium. The enrich- ment of M+4 succinate (16%) is lower than that of M+5 aKG but higher than that of M+4 malate (2.8%). This confirms that the succinate dehydrogenase is reversible (20), although it does not reach isotopic equilibrium.

The mass isotopomer distributions of the various metabolites in Table I allow calculation of certain relative input fluxes. Since M+5 aKG is the only source of M+5 citrate, the balance of M+5 mass isotopomers of citrate and adjacent metabolites yields the formula shown by Equation 1, where FC,,,,,, is the fractional contribution of aKG to citrate via the reversal of the ICDH reaction.

(FC,K~, , , ) ' (MF,+, .K~) = (MFM+6CIT) (Eq. 1)

MF,,, and MFM+5 CIT are the mole fractions of M+5 aKG and M+5 citrate, respectively. Using the mole fractions of Table I, one calculates a 45% fractional contribution of aKG t o citrate, via the reversal of ICDH; the remaining 55% of citrate mol- ecules come from OAA. These percentages can be introduced in the balance of M+4 mass isotopomers of citrate and adjacent metabolites, using similar notations (Equation 2).

Reversibility of Isocitrate Dehydrogenase in Liver 27181

0.55.(MFM+40,) + O . ~ ~ . ( M F M + ~ , , K G ) = ( M F M + ~ c , T ) (Eq. 2)

Solving this equation yields the mole fraction of mitochondrial M+4 OAA, i .e. 2.6%. This enrichment is comparable to what we measured for M+4 malate (3.1%, Table I) and fumarate (2.8%), suggesting that the latter enrichments are representative of mitochondrial metabolites. One can thus consider mitochon- drial OAA, malate, and fumarate as a single pool whose aver- age M+4 enrichment is 2.8%, for which M+4 succinate is the only source of M+4 isotopomers. The M+4 isotopomer balance around mitochondrial OAA, malate (MAL), and fumarate (FUM) yields Equation 3.

(FC~UC-F""ALI~AA).(MFM+~SUC) = (MFM+,FLJ~~~IIOAA) = 0.028 (Eq. 3)

From this equation, the fractional contribution of mitochon- drial succinate to the pool of fumarate, malate, and OAA is 18%. Consequently, the fractional contribution of pyruvate to this pool is 82%. The M+4 isotopomer balance around succinate (SUC) yields Equation 4.

(FCaKG+SUC)'(MFM+5 aKG) + (FcFUh-SUC)' (Eq. 4)

( M F M + 4 F ~ ~ O A A ) = ( M F M + ~ SUC) = 0.16

From this equation, the fractional contribution of aKG to succinate is 61%, Consequently the fractional contribution of mitochondrial fumarate to succinate is 39%. The flux ratio (pyruvate (PYR) carboxylase)/(CAC) equals Equation 5, where the CAC flux is equal to the irreversible flux from aKG to succinate.

PC/CAC = (FCpm-FwO,Y

[FC,.KG+S,C)'(FCSUC-FU"AUOAA)I = 7.5 (Eq. 5)

Such a high ratio has also reported previously by others (21-24) and by us (15).

Since the measured M+5 molar enrichment of influent glu- tamate is 94%, a M+5 mass isotopomer balance around aKG yields Equation 6.

(FCGLU-aKG)'(MFM+5 GLU) + (FCCIT-mKG)' 0%. 6)

(MFM+5 CIT) = (MFM+5 aKG)

Thus, the fractional contributions of citrate and glutamate to aKG are 84 and 16%, respectively.

Liver pyruvate contains 1.3% of M+3 isotopomer, which must arise from the action of pyruvate kinase on M+3 phosphoenol- pyruvate formed from [U-'3C,10AA and [1,2,3-'3C,10AA. As- suming that the M+4 and M+3 enrichments of malate repre- sent that of the O M precursor of phosphoenolpyruvate, the M+3 enrichment of phosphoenolpyruvate (PEP) is shown by Equation 7.

(MF,,, = (MF,,, -) + 0.5.(MFM+, -) = 4.2% (Eq. 7) In this equation, the factor 0.5 reflects the assumption of com- plete randomization of OAA. The calculated M+3 enrichment of phosphoenolpyruvate is 3 times higher than the measured M+3 enrichment of tissue pyruvate. This is due to the continuous influx of unlabeled lactate and pyruvate in the inflowing perfusate.

The above calculations, summarized in Fig. 1, illustrate the power of mass isotopomer distribution analysis. Such compu- tations could not have been achieved with radioactive tracers or with singly labeled 13C-substrates.

One important consequence of the reversibility of the ICDH reaction in the intact liver is its impact on the labeling pattern of citrate. The reversal of the ICDH reaction causes an isotopic exchange between C-6 of citrate and mitochondrial CO,. Con- sequently, in experiments with tracers such as [l-l4C1pyruvate

PY R I 7.5

FIG. 1. Relative rates of citric acid cycle reactions. Rates of various CAC reactions, including the reversal of the combined aconitase + ICDH reactions, were calculated from the data of Table I using the equations developed under "Results and Discussion." Rates are ex- pressed relative to the net flux through the CAC, i.e. the rate of the aKG dehydrogenase reaction. Abbreviations: CIT, citrate; SUC, succinate; GLU, glutamate; PYR, pyruvate; OAA, oxaloacetate; MAL, malate; FUM, fumarate.

or [l-13C]pyruvate, the specific activity or enrichment of C-6 of citrate will be decreased. In contrast, labeled bicarbonate will be incorporated into C-6 of citrate and into other metabolites following [6-14C]- or [6-l3C]citrate cleavage to [1-l4C1- or [1-13C]OAA. Such a scheme was proposed by Heath and Rose (25) in their study of Hl4CO; fixation in liver metabolites, by DAdamo and Haft (26) to explain the labeling of hepatic lipids and glucose from [2-14C]glutamate and [5-14C]glutamate and by Kellehe? to account for the incorporation of [5-14Clglutamine into lipids and CO, by rat hepatoma cells.

Kelleher (27) developed equations to determine rate con- stants of various CAC reactions from the distribution of 14C on carbons of citrate labeled from [14C]acetate, -pyruvate, or -suc- cinate. This model, which includes reversible reactions between citrate and aKG, could be used with the corresponding ['3C]substrates. However, this would require a technique for measuring the 13C enrichment of each carbon of citrate. Be- cause of the chemical symmetry of its molecule, citrate must first be cleaved into its acetyl-coA and OAA moieties before measuring the labeling pattern of these moieties by NMR of gas chromatography-mass spectrometry. An alternative approach is to use uniformly labeled 13C-substrates and to apply mass isotopomer analysis to the calculation of CAC and gluconeogen- esis parameters (28, 29). However, a reversible ICDH reaction would also affect the profiles of mass and positional isoto- pomers of citrate, OAA, phosphoenolpyruvate, and glucose. Therefore, incompatible parameters could be calculated from the isotopomer profiles of these metabolites. To avoid such problems, models of the CAC based on isotopomer analysis should include the reversal of the ICDH reaction.

Models of the CAC that incorporate a reversible ICDH reac- tion would help to test some of the assumptions of the Sazanov and Jackson hypothesis (12) on the functions of the NAD'- and NADP+-ICDH in liver mitochondria. Our demonstration of a rapid interconversion of ICIT and a-ketoglutarate is consistent with this hypothesis. However, under our conditions, mass iso- topomer distribution analysis does not differentiate substrate cycling between ICIT and aKG from the reversibility of the ICDH reaction(s).

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