Studies on the Relationship between Ketogenesis and Pyruvate Oxidation in Isolated Rat Liver Mitochondria*

(Received for publication, June 27, 1977)

STEVEN C. DENNIS,~ MICHAEL DEBUYSERE, ROLAND SCHOLZ,~ AND MERLE S. OLSON

From the Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284

The regulation of the pyruvate dehydrogenase multien-

zyme complex was studied in the perfused rat liver and in isolated rat liver mitochondria. The rate of “CO, production from added [IJClpyruvate was utilized as a monitor of the pyruvate dehydrogenase activity in both preparations. In the perfused liver infusion of the medium chain length fatty acid octanoate resulted in an acceleration of pyruvate decar- boxylation at low (0.05 mu) perfusate pyruvate concentra- tions, while an inhibition of “CO, production was observed at high (5.0 rn>l) pyruvate concentrations. Mitochondrial experiments were performed to define metabolic conditions which mimic the differential effect of fatty acid on the flux through the pyruvate dehydrogenase reaction. A stimula- tion of pyruvate decarboxylation by fatty acid was observed in mitochondrial incubations maintained in State 4 in the absence of a source of oxalacetate, r,-malate, but only at low pyruvate concentrations. Changes in the intramitochondrial ATPlADP ratio likely were not involved in this regulatory process as there occurred no obligatory inverse relationship between the activity state of pyruvate dehydrogenase and the ATP/ADP ratio. Measurement of pyruvate dehydrogen- ase activity in the mitochondrial incubations indic%ed that the increase in the rate of pyruvate decarboxylation caused by octanoate addition was due at least in part to an intercon- version of the pyruvate dehydrogenase complex to its active form.

The stimulation of flux through pyruvate dehydrogenase and the interconversion of the enzyme complex to its active form by fatty acid in rat liver mitochondria likely involves a relationship between the process of ketogenesis and the regulation of pyruvate dehydrogenase by the substratelef- fector pyruvate. Consistent with our previous proposal (based on experimental results with the isolated perfused rat liver) it is suggested that the rapid efflux of intramito- chondrially generated acetoacetate resulting from fatty acid oxidation causes an acceleration of pyruvate entry into the mitochondrial matrix via the monocarboxylate trans-

* This research was supported by Grants HL-20544 and AM-19473 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of a travel grant from Welcome Trust. ij Present address, Institut fur Physiologische Chemie, Physikal-

ische Biochemie und Zellbiologie der Universitat Munchen, Munich, Federal Republic of Germany.

locator. .4s the mitochondrial pyruvate concentration is increased in exchange for acetoacetate pyruvate may in- crease the flux through the pyruvate dehydrogenase reac- tion due to an increased substrate supply and/or inhibitory effect by pyruvate on the pyruvate dehydrogenase kinase reaction leading to an increase in the active form of the enzyme complex.

Two different but interdependent mechanisms have a dem- onstrated involvement in the regulation of the pyruvate dehydrogenase multienzyme complex from mammalian tis- sues. A competitive feedback inhibition of the complex by the products of the reaction, NADH and acetyl-CoA, has been known for some time (l-4). More recently a covalent modifi- cation system has been elucidated in which phosphorylation and inactivation of the enzyme complex are mediated by a specific protein kinase, while dephosphorylation and reacti- vation of the inactive phosphoenzyme are catalyzed by a phosphoprotein phosphatase (5-7). Since this system of inter- conversion of pyruvate dehydrogenase between its active and inactive forms, besides being regulated by adenine nucleotides (8ll), pyruvate (8, 12-14), and mono- and divalent metal cations (6, 13-18), is subject to modulation by the oxidation- reduction state of the mitochondrial nicotinamide adenine dinucleotides (e.g. NADH/NAD+) and the acetyl-CoA/CoASH ratio (18-221, both types of control mechanisms (e.g. feedback inhibition by products and interconversion of active and inactive pyruvate dehydrogenase) have been implicated in the frequently observed inhibition of pyruvate oxidation during the oxidation of fatty acids.

This inhibitory effect of fatty acids has been observed in perfused organs (8, 23-27) and in isolated mitochondria (11, 19-22, 28, 29) and has been explained by some authors as a direct feedback effect of the products of the p oxidation sequence (NADH and acetyl-CoA) on the active form of pyruvate dehydrogenase (25, 29). However, others have pro- posed that elevated NADH/NAD’ and acetyl-CoA/CoASH and, in the case of long chain fatty acids (see Ref. 111, ATP/ ADP ratios represent determinants that alter negatively the

activity of the pyruvate dehydrogenase complex through its active/inactive interconversion system (18-22, 28).

In summary the recent literature is replete with reports which attempt to establish a hierarchy of metabolite effecters of this complex regulatory system and which attempt to assess

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2230 Regulation of Pyruuate Oxidation

the relative contributions of the two types of control mecha- nisms operative during the inhibition of pyruvate oxidation by simultaneous fatty acid oxidation.

Recently a further complexity has been imposed upon the delineation of the mechanism(s) for the effect of fatty acids on the regulation of the pyruvate dehydrogenase complex in liver. Scholz et al. (30) have observed a differential effect of fatty acid oxidation on the metabolic flux through the pyru- vate dehydrogenase reaction in the isolated perfused rat liver.

Whereas the characteristic inhibition of pyruvate dehydrogen- ase activity by long and medium chain fatty acids was observed when the perfusion medium contained high levels of pyruvate (e.g. 1 to 5 mM), at low (i.e. physiological) perfusate pyruvate concentrations (0.02 to 1.0 mM) up to a 3-fold stimulation of pyruvate dehydrogenase flux resulted from the infusion of fatty acid into the liver preparation. A proposal was advanced (30) that the stimulatory effect of fatty acids on pyruvate dehydrogenase flux at low perfusate pyruvate con- centrations was a result of an accelerated exchange of intra- mitochondrially generated acetoacetate for cytoplasmic pyru- vate during rapid ketogenesis. The acceleration of pyruvate entry then results in an inhibition of the pyruvate dehydro- genase kinase with subsequent conversion of inactive pyru- vate dehydrogenase to the active dephospho form.

The present study represents a consideration of this differ- ential effect of fatty acid oxidation on pyruvate dehydrogenase activity in isolated liver mitochondria. Experiments were performed to attempt to support our contention (30) that the only plausible explanation of the enhanced metabolic flux through the pyruvate dehydrogenase reaction following fatty acid infusion in the liver involves an acceleration of pyruvate translocation across the mitochondrial membrane with subse- quent negative effects of pyruvate on the pyruvate dehydro- genase kinase.

MATERIALS AND METHODS

Livers, removed from female Sprague Dawley rats (200 to 250 g) which had been fed ad libitum, were employed for both the perfusion experiment and the isolated mitochondrial experiments.

Perfusion of livers (7 to 9 g wet weight) with a nonrecirculating Krebs-Henseleit medium (115 rnM sodium chloride, 25 mM sodium bicarbonate, 5.9 rnrvr potassium chloride, 1.2 rnM magnesium chlo- ride, 1.24 rnM potassium phosphate (monobasic), 1.2 rnM sodium sulfate, and 1 mM calcium chloride) at a flow rate of 33 to 35 mlimin was performed as described in detail elsewhere (31). Oxygen con- sumption by the liver was monitored continuously with a Clark-type electrode which was seated in a flow through chamber on the effluent side of the liver. Fractions of the perfusate for metabolite assays were collected for 1-min intervals. Radioactive carbon dioxide in the perfusate, produced from the decarboxylation of [l- “Clpyruvate and thus an index of pyruvate dehydrogenase activity (301, was released from 5 ml of perfusate by acidification (1 ml, 2 N hydrochloric acid) and trapped in phenylethylamine as described in detail below.

Isolation of mitochondria from rat liver was performed in a medium containing 75 rnM sucrose, 225 rnM mannitol, and 0.1 rnM ethylene glycol bis(2-aminoethyl)tetraacetate essentially according to the procedure of Schneider and Hogeboom (32). For mitochondrial protein determinations the biuret method of Gornall et al. (33) was employed.

Investigations into the effects of the products of octanoate metab- olism on pyruvate dehydrogenase flux in intact mitochondria were performed in 25.ml Erlenmeyer flasks sealed with rubber serum stoppers equipped with plastic center wells. Mitochondria (0.3 to 1 mg of protein) were added to incubation medium (1 or 2 ml) and the flasks were shaken at 37” for 3 or 5 min. The medium employed in these experiments was that described by Lopez Cardozo and Van Den Bergh (34) and was selected from a screening of several commonly used mitochondrial incubation media which when supple- mented with 5 rnM magnesium chloride and 20 mM glucose differed

only in that they contained much lower (5 mM) phosphate concentra- tions. This medium was found to be optimal in terms of the rapid rates of ketogenesis which were observed. It contained as basic ingredients: 15 rnM potassium chloride, 50 mM sucrose, 30 rnM glucose, 5 rnM magnesium chloride, 50 mM Trisichloride, pH 7.5, 2 mM ethylenediaminetetraacetic acid, and 30 mM potassium phos- phate, adjusted to pH 7.4. For reasons to be discussed under “Results,” 20 rnM Trisioxalate, pH 6.8, was included in all incuba- tions. To achieve State 3 conditions (351, the incubations were supplemented with 1 rnM ADP and approximately 5 units of hexoki- naseimg of mitochondrial protein. Malate and octanoate were added as indicated at concentrations of 5 rnM and 2 mM, respectively. Il- “ClPyruvate was present at a specific radioactivity of 209 dpminmol. At the end of 5 min, the incubations were terminated by the addition of 0.05 ml of 70% v/v perchloric acid. For control incubations perchloric acid was added prior to the addition of mitochondria. The “CO, liberated by pyruvate dehydrogenase activity was trapped in phenylethylamine injected into center wells which, after the flasks had been agitated at ambient temperature for 1 h, were transferred into scintillation vials containing 10 ml of Aquasol for scintillation counting. For the concomitant determination of ketogenesis in these experiments samples removed from the incubations were neutralized to pH 6.5 f 0.2 with 3 M potassium carbonate containing 0.5 M

triethanolamine. The levels of P-hydroxybutyrate and acetoacetate were determined using fluorometric enzymatic assays as described by Williamson and Corkey (36). Assays reported by these authors were employed for the enzymatic determinations of pyruvate, mal- ate, citrate, and the adenine nucleotides. For the measurements of intramitochondrial ATP and ADP, samples from State 4 incubation mixtures containing 8 to 9 mg of mitochondrial protein (in 0.45 ml) were layered on top of 0.5 ml of silicone oil (specific gravity, 1.054) which was in turn layered on top of 0.2 ml of 1.5 N perchloric acid. After centrifugation for 1 min at top speed in an Eppendorf microcen- trifuge, samples (0.15 ml) of the perchloric acid extracts were removed, neutralized with 3 M potassium carbonate containing 0.5 M triethanolamine, and assayed.

When the state of activity of the mitochondrial pyruvate dehydro- genase complex was investigated, samples were removed from the mitochondrial incubations which, apart from containing unlabeled pyruvate, were identical with the incubations described for the study of pyruvate dehydrogenase flux in the intact mitochondria. The incubations were stopped using the rapid freezing technique essentially as outlined by Taylor et al. (281. Samples (0.6 ml) of the incubation mixture were added to tubes containing 0.5 ml of a solution with the following composition: 2 rnM dithiothreitol, 12.5 rnM sodium fluoride, and 12.5 rnM EDTA in 10% (v/v) ethanol, pH 7.4, at 0”. Following sample addition the mixture was immersed in a dry ice-acetone bath until the pyruvate dehydrogenase activity was determined using an assay based on the decarboxylation of [I- “Clpyruvate. The pyruvate dehydrogenase assay was initiated by the addition of 1 ml of the mitochondrial sample which had been thawed on ice to 1 ml of the assay mixture which contained: 4 mM NAD+, 0.4 rnM CoASH, 0.8 mM thiamin pyrophosphate, 2 mM dithiothreitol, 5 mM oxalacetate, 0.02% (w/v) Lubrol WX, and 5 rnM [l-“Clpyruvate (240 dpm/nmol). The assay was performed in sealed Erlenmeyer flasks which contained plastic center wells. The assay was allowed to proceed for 5 min at 37” and the reaction was stopped by injection of 1 ml of 2 N hydrochloric acid into the flask. The ‘CO, released during the assay was trapped as described above.

In order to calculate pyruvate dehydrogenase activities in terms of nanomoles of ‘“CO, liberated per min per mg of mitochondrial protein corrections had to be made for alterations in the specific radioactivity of the [l-‘“Clpyruvate in the assay due to dilution of the labeled pyruvate with varying amounts of unlabeled pyruvate carried over in the mitochondrial samples. Determinations of the unlabeled pyruvate were performed using an enzymatic pyruvate assay (36) in neutralized acid extracts of samples which were removed from the mitochondrial incubations simultaneously with the samples taken for the pyruvate dehydrogenase assay. The specific radioactivity of [l-“Clpyruvate was adjusted accordingly in each assay.

Pyruvate transport studies were performed essentially according to the technique of Harris and Manger (37). To a series of incubations (final volume 0.5 ml) containing the basic incubation medium described above, 20 rnM Tris/oxalate, pH 6.8, 2 mM sodium arsenite, 2 mM succinate/Tris, pH 6.8, 0.6 PCi of [:‘Hlwater, and an appropri- ate concentration of [l-‘4Clpyruvate f-2 x 10” dpmlnmol) were added to 6 to 8 mg of mitochondrial protein. Octanoate, when

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Regulation of Pyruvate Oxidation 2231

included, was present at a concentration of 2 mM. The reaction mixtures were incubated as before at 37” for 5 min at which time 0.45-ml samples were withdrawn and carefully layered on 0.5 ml of silicone oil (specific gravity 1.054) above 0.1 ml of 1.5 N perchloric acid in a microcentrifuge tube (1.5 ml) and spun for 1 min at top speed in an Eppendorf microcentrifuge. Samples from both above (0.02 ml) and below (0.05 ml) the oil were counted for 14C and 3H emissions in 10 ml of Aquasol scintillation mixture. In all experi- ments the sucrose-accessible space and, by difference from the mitochondrial water space, the matrix space were determined using [U-‘Clsucrose (2.5 @Zi) in place of substrate, both in the presence and absence of octanoate (2 mM). Intramitochondrial accumulations of pyruvate were calculated utilizing suitable quench corrections using a program written by one of us (S. D.) on a Monroe 1850 programmable calculator. In all computations the assumption was made that sucrose space was constant. Preliminary experiments (not shown) in which the effectiveness of sodium arsenite (2 mM) for inhibition of [l-llC]pyruvate decarboxylation under optimum condi- tions for pyruvate metabolism (State 3, presence of malate) was studied indicated that insignificant (10.01 nmol/mg of protein) pyruvate utilization was occurring in the presence of this inhibitor. Fortunately, ketogenesis from octanoate was largely unaffected by the presence of this compound. Rotenone (10 pM) commonly used for further mhibition of mitochondrial metabolism in studies of this nature (38-42) was found to have no further beneficial effect on the suppression of pyruvate metabolism, and because we were concerned about an adverse effect of rotenone on the rates of octanoate- supported ketogenesis this substance was omitted.

The experiments described in the various figures and tables are representative experiments each from a different mitochondrial preparation. Each experiment was repeated at least three times with only minimal variation in the results obtained. Individual measurements in each experiment, e.g. pyruvate dehydrogenase flux measurements, pyruvate dehydrogenase assays, or nucleotide determinations, were performed in duplicate and the data points represent the average of the two values obtained.

[l-‘4ClPyruvate, [U-‘%]sucrose, 13H1water, Aquasol, and phenyl- ethylamine were purchased from New England Nuclear. Silicone oil (specific gravity 1.054) was generously provided by the Dow Corning Co. Lyophilized hexokinase (EC 2.7.1.1) (type V) was obtained from Sigma and lactate dehydrogenase (EC 1.1.127) (muscle type) from Worthington. Other enzymes, P-hydroxybutyrate dehydrogenase (EC 1.1.1.30), malate dehydrogenase (EC 1.1.1.37), citrate lyase (EC

80-

T ; 60

02

80-

T k 60-

4.1.3.61, glucose-6-phosphate dehydrogenase (EC 1.1.1.491, pyruvate kinase (EC 2.7.1.40), and myokinase (EC 2.7.4.3) were supplied by the Boehringer Mannheim Corp. Serum stoppers and center wells were obtained from Kontes. The remaining chemicals and reagents were of the highest quality available and were purchased from commercial suppliers.

RESULTS

Most studies performed in complex metabolic systems con- cerning the effect of fatty acid oxidation on the regulation of pyruvate dehydrogenase have employed pyruvate concentra- tions in the 1 to 10 mM range. These investigations have consistently demonstrated that fatty acids lead to a restricted flux of carbon through the pyruvate dehydrogenase reaction largely due to a conversion of the multienzyme complex to its inactive, phosphorylated form. The observation of Scholz et al. (30) that long chain (oleate) or medium chain (octanoate) fatty acids or P-hydroxybutyrate caused up to a 3-fold enhancement of pyruvate dehydrogenase flux in the perfused rat liver at low perfusate pyruvate concentrations was the first indication that fatty acid oxidation may cause other than an inhibition or inactivation of the enzyme complex. As a starting point in an attempt to develop experimental support for a plausible mechanism for this fatty acid-mediated enhancement of pyru- vate dehydrogenase flux in the liver, the experiment shown in Fig. 1 was performed. In agreement with the observations of Scholz et al. (30) the infusion of octanoate into the isolated perfused liver resulted in reversible changes in all parameters monitored. At both low (0.05 mM) and high (4 mM) pyruvate concentrations oxygen consumption was considerably en- hanced and the rates of ketogenesis (i.e. total ketone bodies) were stimulated 18- to 21-fold. Associated with the markedly accelerated rates of ketogenesis was a pronounced shift in the P-hydroxybutyratelacetoacetate ratio from values in the ab- sence of octanoate of 0.3 to 0.4 to values in the presence of octanoate of around 2.3 at low perfusate pyruvate concentra-

/Total Ketones

G=-- Acetoacetote

Minutes of Perfusion Octanoote lmii m

[l-‘4c] Pyrwote I” I ” “,“’ 4.3mM 3

FIG. 1. Differential effects of octa- noate on pyruvate decarboxylation in perfused rat liver. Perfusion conditions and fraction manipulations were as de- scribed under “Materials and Meth- ods.”

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2232 Regulation of Pyruvate Oxidation

tions and 1.1 at high pyruvate concentrations. Since these ratios of p-hydroxybutyratelacetoacetate are usually indica- tive of the mitochondrial oxidation-reduction state (431, it is evident that octanoate infusion, particularly under conditions of limited pyruvate supply, resulted in an extensive shift toward reduction of the mitochondrial NADHINAD’ ratio. This observation is not without precedent. Several authors (44-461, from investigations of ketogenesis from starved rat livers perfused with octanoate in the absence of pyruvate, have reported very similar values (2.4 to 2.8) for the ratio of P-hydroxybutyrate to acetoacetate in effluent perfusates. To- tal rates of ketogenesis quoted by these groups were much higher (128 to 179 pmol/h/g wet weight), however, than the ketone body production rates seen in these experiments. At low pyruvate concentrations the appearance of ketone bodies occurred at a rate approaching 80 pmol/h/g wet weight and decreased with higher pyruvate concentration to 44 pmol/h/g wet weight. Some explanation for these apparently low rates of ketogenesis may lie in the fact that livers for these experi- ments were removed from female rats (see Ref. 47) which had not been fasted (see Refs. 45 and 481. Whether any significant reduction in rates of ketogenesis can be attributed to the presence of pyruvate, particularly in view of the differences in octanoate-supported ketogenesis and the reduction state of ketone bodies produced in the presence of high and low concentrations of pyruvate, remains to be seen. A suggestion that accelerated pyruvate decarboxylation at high pyruvate concentration results in an increased oxalacetate supply thus permitting a greater proportion of acetyl-CoA into the tricar- boxylate cycle would require further elucidation. More exten- sive consideration of the implications of rapid ketone body synthesis from octanoate on pyruvate metabolism will be made later in this study.

Of more immediate interest is the effect of octanoate on the rate of decarboxylation of [l-“Clpyruvate in this study. As was observed in the previous report of Scholz et al. (30) at low concentrations of pyruvate in the perfusate (0.05 mM) octano- ate infusion (0.5 mM) resulted in an approximate doubling of the rate of ‘*CO, production from [PC]pyruvate. At high perfusate pyruvate concentrations (5.0 mM) the classical inhi- bition (or inactivation) of pyruvate decarboxylation was ap- parent.

Clearly it would be desirable to investigate further this phenomenon of a fatty acid-mediated enhancement of pyru- vate decarboxylation in as intact a metabolic system as possible. However, routine experimental procedures for inves- tigating metabolite changes in various cellular compartments in intact tissues are only in the developmental stages. There- fore, in order to circumvent problems of compartmentation in the intact liver we elected to attempt to design complementary

experiments in isolated liver mitochondria to investigate the possible mechanism(s) involved in the regulation of the pyru- vate dehydrogenase complex under the metabolic conditions outlined above.

In both the perfused liver and in isolated liver mitochondria a problem exists with the use of “CO, evolution as a quanti- tative indicator of the pyruvate dehydrogenase activity. This problem is created by the presence of pyruvate carboxylase (EC 6.4.1.1) in these two metabolic systems which would carboxylate [l-lC lpyruvate forming oxalacetate which would enter the tricarboxylic acid cycle with resultant decarboxyla- tion and “CO, production in the isocitrate and a-ketoglutarate dehydrogenase reactions. The data shown in Table I repre- sents our attempt to minimize this pyruvate carboxylase

TABLE I

Effect of ox&k on malate and citrate ,6roduction in isolated rat lwer mitochondria

Incubations at 37” were performed with 1 ml of either standard incubation medium (see “Materials and Methods”) or medium in which the 15 rnM KC1 had been substituted for KHCO,,. Mitochondria (3.8 mg of protein) were added to initiate the incubations which contained 5 mM pyruvate and 10 rnM malonate. Samples for enzy- matic determinations of malate and citrate were removed and acid- quenched at 2-min intervals. Endogenous malate present in these mitochondria was negligible, whereas initial citrate content was 7.5 nmolimg of protein. Flux rates over the first 4 min of incubation were essentially linear.

Initial rates of metabolite production

Metabolite KC1 medium K HCO:, medium

Oxdate” oxa1ate

nmollmmlmg protein

Malate 2.15 0.20 14.3 0.46

Citrate 4.05 0.06 10.8 0.38

Malate plus cit- 6.20 0.26 25.1 0.84

rate

(1 Oxalate added as Tris salt, 20 mM.

problem in an isolated rat liver mitochondrial system. Mitochondria were incubated under State 4 conditions in

the absence of a 4-carbon source and in the presence of malonate to inhibit the tricarboxylic acid cycle at the succi- nate dehydrogenase step. Under these conditions the accumu- lation of L-malate and citrate were assumed to be dependent upon the anaplerotic activity of the mitochondrial pyruvate carboxylase. Support for this assumption is derived from the observation that the production rate of these two intermedi-

ates increased 4-fold to 25 nmol/min/mg of protein upon the addition of bicarbonate to the incubation. Values of 23 nmol/ minlmg of protein for the accumulation of tricarboxylic acid cycle intermediates under similar incubation conditions have been reported by Lopez-Cardozo and Van Den Bergh (49, 50). In State 3 in the absence of bicarbonate pyruvate carboxylase activity in intact liver mitochondria has been reported to be negligible (51, 52). However, in State 4 in the absence of bicarbonate a significant pyruvate carboxylation activity was implied from the rates of accumulation of L-malate and citrate (e.g. 6 nmol/min/mg of protein). A number of compounds have been screened as inhibitors of pyruvate carboxylase activity (53). Of the various possibilities oxalate (K, with respect to pyruvate, 1.1 to 1.4 x lo-” M (54)) seemed to be the most promising. As can be seen in Table I 20 mM oxalate added to intact liver mitochondria resulted in nearly a complete (97%) inhibition of the carboxylation of pyruvate as indicated by the rate of accumulation of L-malate and citrate. This concentra- tion of oxalate had no effect on the pyruvate dehydrogenase activity in these mitochondrial incubations. However, octano- ate oxidation and ketogenesis were inhibited by almost 50% (data not shown), a phenomenon possibly explained by the chelation of magnesium by oxalate (55) but which could not be relieved by further addition of magnesium. Nonetheless, the remaining experiments in the present study were performed in the presence of oxalate so that the “CO, production from [l-*lC]pyruvate could be employed as an accurate monitor of pyruvate dehydrogenase flux without the complication of having to correct for another decarboxylation process, e.g. the tricarboxylic acid cycle, in this mitochondrial system.

The initial consideration which was investigated using this mitochondrial system was establishment of conditions in

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Regulation of Pyruvate Oxidation 2233

which the addition of fatty acid octanoate to the isolated mitochondrial incubation resulted in a stimulation of pyruvate decarboxylation. A series of experiments was performed in which the rate of 14C02 production from [lJ%lpyruvate was measured under State 3 or State 4 conditions in the presence or absence of octanoate and L-malate at various concentrations of pyruvate added to the medium. The results of this series of incubations are shown in Fig. 2 and indicate that in State 3 octanoate suppressed pyruvate dehydrogenase activity under all conditions tested. The inhibition of pyruvate decarboxyla- tion was especially pronounced in the presence of Lmalate at high pyruvate concentrations. Subjection of the %O, produc- tion data collected in State 3 in the presence and absence of octanoate and/or L-malate to Michaelis-Menten kinetic analy- ses resulted in linear double reciprocal plots (data not shown) charactertistic of uncompetitive inhibition of pyruvate decar- boxylation likely the result of inhibition by the product(s) of octanoate metabolism. This observation is in accord with the proposals (18-22, 28) that fatty acid-mediated inhibition (in- activation) of pyruvate dehydrogenase is a result of covalent modification of the enzyme complex (by phosphorylation) resulting in its inactivation rather than a direct competitive feedback inhibition of the dephosphoenzyme at the active site. Uncompetitive inhibition (double reciprocal data not shown) of pyruvate decarboxylation by octanoate in the presence or absence of L-malate was observed in mitochondria maintained in State 4 only when pyruvate concentrations were relatively high (BO.19 mM). At lower concentrations of pyruvate, octa- noate was without effect in the presence of L-malate, while in the absence of Gmalate a relatively small but very consistent stimulation of pyruvate decarboxylation by octanoate was apparent but only at medium pyruvate concentrations at or below 0.2 rnM.

A time course of the octanoate-mediated stimulation of pyruvate dehydrogenase flux (data not shown) indicated that

the enhancement of pyruvate decarboxylation activity in the mitochondrial suspension increased with more prolonged in- cubation times. Incubation periods in the experiment depicted in Fig. 2 were limited by the fact that the mitochondria were maintained in State 3 which in prolonged incubations at low pyruvate concentrations would have resulted in a depletion of the substrate. With mitochondria incubated in State 4 the much greater stimulation in the rate of pyruvate decarboxyl- ation by octanoate when the incubations were continued for a longer time period is demonstrated in Fig. 3.

Outlined in Table II are the results of an experiment designed to determine whether the changes in mitochondrial pyruvate flux produced by octanoate metabolism were re- flected in altered activation states of the isolated pyruvate dehydrogenase complex. The results of this series of experi- ments indicated that in mitochondria maintained in State 4 conditions octanoate metabolism affected the activity of sub- sequently extracted pyruvate dehydrogenase in a manner entirely consistent with the previously observed actions (Figs. 2 and 3) of octanoate on pyruvate decarboxylation in intact mitochondria. Again, only at low pyruvate concentrations (0.1 mM) and in the absence of L-malate was a significant increase in pyruvate dehydrogenase activity in the presence of octano- ate observed. Furthermore, since samples of incubation prior to pyruvate dehydrogenase extraction and assay were diluted 4-fold to eliminate complications from end product inhibition, it may be assumed that octanoate inhibition at high pyruvate concentrations and stimulation in the absence of L-malate at low pyruvate concentrations of isolated pyruvate dehydrogen- ase activity reflect alterations by covalent modification in the activation state of this multienzyme complex. Thus, in the light of this data and the Michaelis-Menten kinetic analysis discussed above, it seems reasonable to suggest that the enhanced pyruvate flux seen under certain metabolic condi- tions during octanoate metabolism likely involves the inter-

.g i 7.5

1 State 4 plus Malate

f” ‘A Pe

Octanoate .

I State 3

FIG. 2. Differential effects of octa- noate on pyruvate dehydrogenase ac- tivity in intact liver mitochondria. In- cubations of rat liver mitochondria (0.35 mg of protein) were conducted for 3 min at 37”. In a final volume of 1 ml of standard incubation medium (see “Materials and Methods”), 20 mM oxa- late was present. When malate and/or octanoate was included they were at concentrations of 5 and 20 m& respec- tively. For State 3 conditions, incuba- tions were supplemented with 1 mM ADP and 5 units of hexokinase. [l- ‘%]Pyruvate (209 dpm/nmol) was added at the indicated concentrations. For further experimental details refer- ence may be made to the “Materials and Methods” section.

1 State 4 P w P Octanoate

0 Cl25 0.5 1.0

Pyruvate ( m M )

0 0.25 0.5 1.0

Pyruvate (mM)

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2234 Regulation of Pyruvate Oxidation

6

0 0 0’2 0’4 0’6

Pyruvote (mM)

FIG. 3. Octanoate stimulation of State 4 mitochondrial pyruvate decarboxylation. Rat liver mitochondria (0.65 mg of protein) were incubated under State 4 conditions in the absence of malate exactly as described in the legend to Fig. 2, except that the incubations were more prolonged (5 min).

TABLE II

Effect of fatty acid oxidation on pyruvate dehydrogenase activity of rat liver mitochondria in State 4

Mitochondria (1.9 mg of protein) were incubated for 5 min at 37” in a final volume of 1 ml of basic incubation medium containing 20 mM oxalate. Other additions were as shown in the table. Samples from the incubations removed for assays of pyruvate dehydrogenase activity were subjected to a protocol which is described in detail under “Materials and Methods.” The values expressed in this table are the means from duplicate experiments.

F’yruvate dehydrogenase activity

Additions Pyruvate, 0.1 IrIM Pyruvate, 1 nm

Malate, 5 Malate, 5 InM nn.3

nmollminlmg protein None 2.3 4.9 6.9 11.0 Octanoate. 2 rnM 2.7 9.9 2.4 8.8

conversion system of the pyruvate dehydrogenase complex. In either event, from the experiments described in Figs. 2

and 3 and Table II, it is apparent that rat liver mitochondria, incubated in State 4 in the absence of malate and at low medium pyruvate concentrations, represent a suitable meta- bolic situation in which to investigate possible mechanisms for this stimulatory effect of fatty acid oxidation on the activation state of the pyruvate dehydrogenase multienzyme complex in this mitochondrial system.

In perfused rat livers to which oleate (56, 57) or octanoate (46) were infused and in uiuo during states of accelerated fat oxidation such as fasting or diabetes (58, 591, a decrease in the ratio of ATP/ADP has been reported. Since a direct correlation of the ATPIADP ratio with the pyruvate dehydrogenase kinase activity and thus an inverse relationship between the ATPIADP ratio and the pyruvate dehydrogenase activity have been indicated frequently (8-11, 14, 281, a decrease in the ATP/ADP ratio would have the effect of stimulating the pyruvate dehydrogenase activity in this mitochondrial sys- tem. However, as shown in Table III, the effects of intramito- chondrial octanoate activation (60) and metabolism on the adenine nucleotide levels in isolated liver mitochondria incu- bated in State 4 indicated changes in the ATPIADP ratio which were inconsistent with the enhancement by octanoate of pyruvate dehydrogenase activity being mediated via this parameter. Although at both high and low pyruvate concen-

TABLE III

Effect of octanoate metabolism in State 4 on intramitochondrial adenine nucleotide levels

In addition to the basic ingredients the incubation medium contained 20 rnM oxalate and 9.7 mg of mitochondrial protein in a final volume of 0.5 ml. Other additions were as indicated in the table. Samples for adenine nucleotide assays were removed after 5 min at 37” and spun through silicone oil (specific gravity 1.054) into perchloric acid. Details of manipulations can be seen under “Mate- rials and Methods.”

Adenine nucleotide levels”

Additions Adenine nu- cleotides

Pyruvate, 0.1 nm Pyruvate, 1 rm

Malate, Malate, 5rnM 5rnM

nmollmg protein

None ATP 2.18 0.65 2.23 1.47 ADP 0.21 1.28 0.23 0.57 AMP 0.70 1.00 0.69 0.99 ATPIADP 10.38 0.51 9.70 2.58

Octanoate, ATP 1.25 0.76 1.25 1.14 2rnM ADP 0.23 0.41 0.29 0.25

AMP 1.63 1.71 1.61 1.70 ATPIADP 5.43 1.85 4.31 4.56

n Total adenine nucleotides were 3.05 rl: 0.1 nmobmg of mitochon- drial protein.

trations in the presence of L-malate, octanoate addition re- sulted in a reduction in the ATP/ADP ratio, under these incubation conditions there occurred no marked stimulation of pyruvate decarboxylation by octanoate (Fig. 2, Table IV). Conversely, in the absence of L-malate at low pyruvate concen- trations where an octanoate-mediated stimulation of pyruvate decarboxylation was observed (Figs. 2 and 3, Table IV) the addition of octanoate was associated with a markedly elevated ATPIADP ratio which again is not consistent with an ATP/ ADP-mediated activation of pyruvate dehydrogenase via the kinase-phosphatase system.

Hence, it is evident that another regulatory factor must be primary in this enhancement of pyruvate dehydrogenase activity by fatty acid. Of the end products of ,6 oxidation of fatty acids NADH and acetyl-CoA have demonstrated effects as activators of the pyruvate dehydrogenase kinase. Elevation of these two species upon initiation of rapid fatty acid oxida- tion should result in an inhibition of pyruvate decarboxylation in this system (see introduction to the text). In liver a frequent consequence of the initiation of rapid /3 oxidation of fatty acids is the synthesis of /3-hydroxybutyrate and acetoacetate. While no direct effects of the ketone bodies per se have been demon- strated on the isolated pyruvate dehydrogenase multienzyme complex or its two regulatory enzymes, acetoacetate has been shown by Papa and Paradies (38) to be an excellent anionic exchange species for pyruvate on the monocarboxylate ex- changer in the inner mitochondrial membrane of liver mito- chondria. Therefore, it seems plausible that under conditions where pyruvate entry into the mitochondria might be limit- ing, e.g. at low medium pyruvate concentrations, any process which could accelerate the exchange or translocation of pyru- vate across the membrane would lead to an enhancement of the pyruvate dehydrogenase flux through two mechanisms: (al through increased substrate (pyruvatel supply, and (b) through the documented ability of pyruvate to inhibit the pyruvate dehydrogenase kinase with subsequent activation of the multienzyme complex (8, 12-14). In either event or both together, initiation of rapid synthesis of intramitochondrial

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Regulation of Pyruvate Oxidation 2235

TABLE IV

Effect of octanoate on W02, acetoacetate, and P-hydroxybutyrate production in rat liver mitochondria under various metabolic conditions Mitochondria (0.8 mg of protein) were incubated for 5 min at 37” in a final volume of 1 ml of basic incubation mixture containing 20 mM

oxalate and the indicated concentrations of [1-‘Qpyruvate (209 dpm/nmol). A full description of the procedures employed can be found under “Materials and Methods.” Values in the table involve the assumption that rates of metabolite production between zero time controls and 5 min were linear. They represent the means of duplicate determinations.

Rates of metabolite production

Additions Metabolite Pyruvate, 0.1 nlM Pyruvate, 1.0 mu

State 3 State 4 State 3 State 4

Malate” Malate Malate Malate

None ‘TO, Acetoacetate P-Hydroxybutyrate

Octanoate, 2.0 mM ‘TO, Acetoacetate P-Hydroxybutyrate

n Malate was added at 5.0 mM.

17.8 13.3 0.5 4.1

<0.04 0.6

6.5 9.1 2.1 5.5 0.6 0.7

acetoacetate which could exchange for extramitochondrial pyruvate would represent an intriguing possibility to explain the fatty acid-mediated increase in the pyruvate dehydrogen- ase activity.

In Table IV the results of an experiment are presented in which the rates of ketogenesis and pyruvate dehydrogenase are compared in the presence and absence of octanoate under a variety of experimental conditions. A number of observa- tions are pertinent in this experiment. First, the degree of reduction of the P-hydroxybutyratelacetoacetate ratio pro- duced in the presence of octanoate should be compared with those in the liver perfusion experiment shown in Fig. 1. Only the ratios of P-hydroxybutyratelacetoacetate in the mitochon- drial incubations in the presence of L-malate in State 4 approached the ratios seen in the perfused liver experiment. This observation, together with the fact that the differential effects of octanoate on the pyruvate decarboxylation seen in the perfused liver could only be stimulated in the mitochon- drial system in State 4 (Table IV, Fig. 2), is consistent with the hypothesis (61) that the in uiuo metabolic situation in the liver resembles an ADP-controlled State 4. Second, support for the general assumption (see Ref. 50) that ketogenic rates are dependent upon excess of the rates of acetyl-CoA production over utilization by citrate synthase which in turn is controlled by oxalacetate availability (34) is seen in the diminished rates of ketogenesis in the presence of L-malate. Finally, and of primary interest, is the effect of Lmalate on the stimulation of pyruvate dehydrogenase activity by octanoate in mitochon- dria incubated in State 4 at low pyruvate concentrations. When a source of oxalacetate was included in these incuba- tions, the increase in the rates of ketogenesis upon addition of octanoate was negligible. Similarly, the increase in pyruvate dehydrogenase flux, possibly explicable in terms of the data presented in Table III, was also relatively small. In the absence of cmalate, however, the inclusion of octanoate in the incubation resulted in the maximum stimulation in the pyruvate decarboxylation activity and associated with this stimulation of pyruvate dehydrogenase was a large increase in acetoacetate production. Hence, a primary conclusion from this experiment may be that the correlation between the increased acetoacetate production and the increased pyruvate dehydrogenase activity resulting from octanoate addition, although not absolute proof, is certainly consistent with the

1.3 0.5 0.2

n?rw1lm1nlmg protern

2.8 31.3 1.8 0.7 0.7 10.04

15.5 5.4 9.3 4.8 0.4 3.9 0.2 0.2 0.4

1.9 4.4 9.8 11.4 4.6 6.9 0.7 3.9 2.5 5.5 1.1 4.4 0.4 0.6 0.9 0.4 0.5 0.6

o r , 1 : ::‘, 2;:: 0 2.5 5 7.5 IO

Pyruvote (mM 1

FIG. 4. Mitochondria (7 mg of protein) were incubated again for 5 min at 37” with a range of [l-Wlpyruvate concentrations (specific radioactivity 209 dpm/nmol) exactly as specified under “Materials and Methods.” The matrical and sucrose-accessible spaces of the mitochondria under these conditions were calculated (both in the presence and absence of octanoate) to be 0.5 5 0.08 and 1.82 t 0.04 &mg of protein, respectively. The results expressed as nanomoles of pyruvate accumulated/mg of protein have been corrected for metabolite adherence in the sucrose-accessible space. 0, Control plus succinate (2 mm); 0, presence of octanoate (2 mM); W, presence of octanoate (2 mM) plus succinate (2 mM).

suggestion that the elevated ketone body eMux promoting pyruvate accumulation in the mitochondrial compartment could be the factor responsible for the increased pyruvate dehydrogenase activity.

Further support for this suggestion was obtained by inves- tigating the effect of initiation of rapid ketogenic rates from octanoate on pyruvate accumulation in isolated liver mito- chondria (see Fig. 4). It can be seen that there occurred a significant accumulation of pyruvate by the mitochondria and this accumulation was enhanced in the presence of octanoate is compared to the control in which octanoate was omitted. The omission of succinate, added as an energy source in the control incubations, which should accelerate fatty acid oxida- tion (62) and decrease the reduction state of the ketone bodies produced (Fig. 4) had no significant effect on the enhancement of pyruvate uptake in the mitochondria to which octanoate was added.

DISCUSSION

It is evident from our previous studies of pyruvate dehydro-

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2236 Regulation of Pyruvate Oxidation

genase regulation in the isolated perfused rat liver (30) and the results reported in this study that fatty acid addition can have a differential effect on the pyruvate dehydrogenase flu-x depending upon the metabolic conditions of the liver prepara- tion under consideration, especially the availability of pyru- vate. In the presence of oxalate to inhibit pyruvate carboxyl- ase so that “CO, evolution from [l-‘Clpyruvate could be employed as a relatively unambiguous monitor of the pyru- vate dehydrogenase flux in this system (Table I), a similar differential effect of octanoate addition was observed in iso- lated mitochondria as long as a careful definition of the metabolic conditions of the preparation was made. When the mitochondria were maintained in State 4, in the absence of the dicarboxylic acid, L-malate, and in the presence of low concentrations of pyruvate, octanoate inclusion in the incuba- tion resulted in a stimulation of pyruvate dehydrogenase activity (Figs. 2 and 3, Tables II and IV). Under otherwise the same conditions but in the presence of higher concentrations of pyruvate, the presence of octanoate resulted in a depression in the activity of pyruvate dehydrogenase activity. An inhibi- tion of pyruvate dehydrogenase flux by fatty acid was appar- ent in all State 3 incubations. Consequently, it is evident that the interaction of the processes of pyruvate decarboxylation and fatty acid oxidation involves an intricate balance between the often stated inhibitory effects produced by the end products (NADH and acetyl-CoA) of both processes (see Ref. 20) and a stimulatory effect of the fatty acid which must predominate at limiting pyruvate concentrations. This intricate balance noted above can only be detected in the in viuo situation, e.g. the perfused liver, at low perfusate pyruvate concentrations, or in mitochondria incubated in State 4 in the absence of a readily available source of oxalacetate.

An investigation into the nature of this octanoate-mediated stimulation of pyruvate decarboxylation indicated that, with the exception of a minor enhancement of pyruvate dehydro- genase activity by fatty acid in the presence of L-malate in State 4 (Tables II and IV), there was no consistent obligatory inverse relationship between the activity state of the pyruvate dehydrogenase complex and the intramitochondrial ATP/ADP ratio (Table III). However, a contention of primary importance

in our studies is that the activation of pyruvate dehydrogenase under conditions of limiting pyruvate concentrations likely is a result of an accelerated rate of pyruvate entry into the mitochondrial compartment in exchange for intramitochon- drially generated acetoacetate. An insight into whether this stimulation of pyruvate dehydrogenase activity was the result of an interconversion of the enzyme complex to its active form (8, 12-14) or whether the increased rates of pyruvate decarbox- ylation were simply a consequence of increased substrate availability can be obtained from a consideration of the data in Table II. From this study of the effect of octanoate metabo- lism on the activation state of the liver mitochondrial pyru- vate dehydrogenase it was apparent that, in mitochondrial incubations under State 4 conditions in the absence of L-

malate and at low concentrations of pyruvate where fatty acid consistently caused stimulation of pyruvate dehydrogenase flux, the presence of octanoate resulted in a significant 2-fold increase in the active (dephospho) form of pyruvate dehydro- genase. From a consideration of the kinetic properties of the two pyruvate-dependent processes which could determine py- ruvate dehydrogenase flux (e.g. pyruvate as substrate uersus pyruvate as negative effector of the pyruvate dehydrogenase kinase), activation of pyruvate dehydrogenase through inter- conversion is in complete accord with the findings expressed

in Fig. 4. The K,, for pyruvate of the active form of the liver- derived pyruvate dehydrogenase has been shown to be be- tween 20 and 30 pM (20), while half-maximal activation of the pyruvate dehydrogenase complex via inhibition of the kinase is achieved at 0.4 mM pyruvate (8). Under metabolic condi- tions where pyruvate decarboxylation was stimulated by in- clusion of fatty acid, i.e. pyruvate concentrations ~0.6 mM

(Fig. 31, intramitochondrial pyruvate concentrations were observed to be in the region of 0.4 mM in the presence of octanoate and 0.24 mM in the absence of fatty acid (see Fig. 4). In view of these findings it would seem reasonable to speculate that with the possible exception of extremely low concentra- tions of added pyruvate the stimulation of pyruvate decarbox- ylation by fatty acids is primarily a result of an interconver- sion of the multienzyme complex to its active form mediated by an inhibition of the kinase reaction by elevated intramito- chondrial pyruvate levels.

In extrahepatic tissues the reverse relationship has been proposed to exist (39). The characterization of the apparent similarity between the translocation of pyruvate and P-hy- droxybutyrate into liver mitochondria lead to the proposal that these two compounds share a monocarboxylate carrier and that the counter-exchange of these metabolites in periph- eral tissues would explain the sparing effect of ketone bodies on glucose utilization since the expulsion of pyruvate from the mitochondrial compartment would lead to a restriction in the pyruvate dehydrogenase complex.

In the present study a ketone body efflux in exchange for pyruvate entry into the mitochondria with subsequent activa- tion of pyruvate dehydrogenase is suggested. However, a stimulation of pyruvate dehydrogenase activity under ketotic conditions where carbohydrate supply is limited is difficult to rationalize in a physiological context. In terms of metabolic economy it would be more advantageous for pyruvate oxida- tion to be suppressed by fatt,y acid oxidation. Such a suppres- sion of pyruvate oxidation would spare pyruvate for utilization in the gluconeogenic pathway while the NADH and acetyl- CoA resultant from p oxidation could serve as an energy source. It is our present contention that the observed activa- tion of pyruvate dehydrogenase by a fatty acid-mediated acceleration of pyruvate transport into the mitochondrial compartment may be a somewhat detrimental but unavoida- ble consequence of a more general control mechanism which is designed to increase the supply of mitochondrial pyruvate for the pyruvate carboxylase reaction with subsequent gluco- neogenesis. In rat livers perfused with low concentrations of pyruvate (0.05 mM) octanoate infusion caused a 2-fold increase in the rate of glucose production (e.g. 50 to 100 pmol/h/g wet weight) despite a marked stimulation (e.g. 2- to 3-fold in the pyruvate decarboxylation (30)). The K,,, of the pyruvate car- boxylase for pyruvate is relatively high (0.44 mM (53)) which suggests that at low pyruvate concentrations, a primary form of regulation could be substrate supply. Furthermore, it has been suggested (63, 64) that pyruvate entry into liver mito- chondria is a control point for gluconeogenesis. In fact, from a consideration of the kinetic parameters of the pyruvate trans- locase (K,,, for pyruvate, 0.15 mM; V,,, at 37”, 42 nmol/min/ mg of protein), the rates of gluconeogenesis from lactate in livers perfused with glucagon or fatty acids (e.g. 100 to 200 pmol/h/g wet weight (65, 6611, and the concentrations of pyruvate in livers under these conditions (e.g. 200 to 400 nmol/g wet weight (67)), Halestrap (64) concluded that pre- dicted rates of pyruvate transport into liver mitochondria for carboxylation were inadequate to account for the measured

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Regulation of Pyruuate Oxidation 2237

rates of glucose production. It is tempting to speculate that, at least, in the case of the commonly observed fatty acid-me- diated stimulation of gluconeogenesis (671, accelerated pyru- vate entry into the mitochondria as a result of the rapid efflux of ketone bodies could resolve this apparent inconsistency.

A further point of information regarding the proposal that the stimulatory effect of fatty acid on the rate of pyruvate decarboxylation is mediated via a pyruvate transport mecha- nism is that we have been unable to demonstrate a similar effect in either the perfused heart or in cardiac mitochondria. At any and all perfusate pyruvate concentrations, octanoate or P-hydroxybutyrate infusions cause a substantial inhibition

of ‘CO, evolution from infused [1-‘4C1pyruvate. This result is consistent with our proposal for the liver as cardiac tissue has little, if any, ability to produce ketone bodies upon fatty acid addition and, in addition, the assessment of pyruvate dehydro- genase flux in the perfused heart is uncomplicated due to the virtual absence of pyruvate carboxylase in this organ.

In conclusion, the possibility must be considered that the rates of decarboxylation or carboxylation of pyruvate in the liver might be regulated by alterations in the activity of the mitochondrial monocarboxylate transporter in certain physio- logical circumstances such as accelerated lipolysis with sub- sequent ketogenesis.

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S C Dennis, M DeBuysere, R Scholz and M S Olsonisolated rat liver mitochondria.

Studies on the relationship between ketogenesis and pyruvate oxidation in

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