Energy requirement of the transport of reducing equivalents from cytosol to mitochondriae in perfused rat liver

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Lifa Sciences, Vol. 15, pp .65- 72 Printed in the U.S .A .

ENERGY REQUIREMENT OF THE TRANSPORT OF REDUCING EQUIVALENTS FROM CYTOSOL TO MITOCHONDRIAE IN PERFUSED RAT LIVER Niels Krarup and Christian Olsen Institute of Physiology, University of Aarhus 8000 Aarhus C, Denmark (Received in final form 30 1~lay 1974)

The system transporting reducing equivalents across the mitochondrial membrane was investigated by following the flux of reducing equivalents from cytosol to mitochondriae, estimated from the ethanol elimination, and the redox potentials on both sides of the mitochondrial membrane, estimated from the lactate/pyruvate and ß-hydroxybutyrate/acetoacetate ratios in the effluent medium . -3 The power of the transport system was calculated to be 1 . Sx10 cal/min/g liver (wet wt .), which was about 1% of the metabolic rate . Uncoupling by 2,4 dinitrophenol increased the oxygen consumption 30%, but the ethanol elimination decreased despite s fall in the redox potential gradient, resulting in a 50X decrease in power of the transport system . This indicates that the transport of reducing equivalents from cytosol to mitochondriae is energy dependent . Although the oxidation of NADH by the respiratory chain takes place in the mitochondriae, the NAD+ -mediated redox potential is more reduced in this compartment than in the cytosol of the liver cell (1) . Transport of reducing equivalents from cytosol (e .g . from ethanol oxidation) to mitochondria is therefore uphill and may be energy dependent as indicated by experiments in which the oxidative phosphorylation was reduced (2) . Uncoupling of the oxidative phosphorylation causes a decrease in the NADH/NAD+ ratio in mitochondriae (3). The redox gradient across the mitochondrial membrane may therefore be decreased, and it might be expected that this would increase the transport rate of reducing equivalents . The energy level, however, is decreased after uncoupling, and as the transport is uphill, it might be reduced due to lack of energy . The results from preliminary experiments (Q) suggested that the transport of reducing equivalents across the mitochondrial membrane was impaired 65

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by DNP . The purpose of the present study was therefore to investigate furthey the effect of uncoupling on this transport. Materials and Methods Male Wistar rats fed ad libitum weighing about 180 g were used . Perfusion technique and medium were as described (5) with minor alterations (2) . The medium was recirculated and equilibrated with a gas mixture containing 75 % N2, 20 % 02, and 5 q C02 . After 45 min per¬usion ethanol was added to a concentration of about 5 mM in the medium . This concentration was maintained by continuous infusion of ethanol. 35 min later DNP was added to a concentration of 0.5 mM in the perfusion medium . 5 control experiments were performed without the addition of DNP . The flow was recorded continuously and related samples of the affluent and effluent perfusion medium were drawn . The concentrations of lactate (L), pyruvate (P), ß -hydroxybutyrate (ß -HB) and acetoacetate (Ac) were determined by an enzymatic, £luorometric micromethod (6) . Hemoglobin, oxygen saturation, glucose and ethanol were determinated as previously described (2) . P-values were obtained from t-tests based on paired comparisons, except for the ratios on which Wilcoxon's signed rank test was employed . From the L/P and ß -HB/Ac ratios the NAD+ -mediated redox potentials were calculated according to Scholz (7) : E cyt Emit

-204 - 30 .4 log L/P mV -270 - 30 .41og ß-HB/Ac mV

The difference between the cytoplasmic (E c~) and the mitochondrial

(Emit) redox potentials represents .the redox potential difference across the mitochondrial membrane . Results The oxygen uptake by the liver was stimulated as a result of the un-

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coupling by DNP. At the same time there was a 8-fold increase in the output of lactate from the liver (TABLE I) . The output of glucose was increased from 1 .03 ± 0.25 in the ethanol period to 2 :30 ± 0 .30 umoles/min/g wet wt . of liver . The ethanol elimination was reduced after addition of DNP to the medium . TABLE I Oxygen and Ethanol Consumption and Lactate Output by the Liver before and 20 min after Addition of 2 , 4-Dinitrophenol . Oxygen Uptake

Ethanol Uptake

Ethanol

1 .98 ± 0 .12

1 . 22 ± 0 .24

0 .21 ± 0 .12

Ethanol + DNP

2 .59 ± 0 .13

0 .87 ± 0.30

2 .02 ± 0 .36

Difference in %

30 < 0 .005

P

- 30 < 0 .05

Lactate Output

~

860 < 0 .005

Mean values ± S .E .M . from 6 experiments expressed in umoles/g wet wt . and liver/min.

The concentrations of pyruvate in the medium was reduced after addition of ethanol. The uptake of pyruvate by the liver was insignificant before as well as after addition of DNP to the medium . The production of ketone bodies was unaffected, but the output of ß-HB was reduced, and the output of Ac enhanced, as reflected by the significant decrease in the ß -HB/Ac ratio of the effluent medium (TABLE II) . The L/P ratio in the effluent medium was increased. From these ratios the redox potential in the mitochondriae and the cytosol was calculated . The mitochondrial compartment was the most reduced in the ethanol period as well as after addition of DNP . The calculated redox potential difference between cytosol and mitochondriae was reduced after DNP (TABLE II) .

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TABLE II L/P- and ß-HB/Ac-Ratios in the Effluent Medium and the Calculated Redox Potential Difference across the Mitochondrial Membrane before and 20 min after Addition of 2,4-Dinitrophenol . L/P

ß - HB/Ac

E mV

Ethanol

37 .3 C19 .0 - 71 .5)

2 .79 C1 .62 - 4 .72)

31 .3 (27 .6 - 36 .5)

Ethanol + DNP

52 .2 (27 .4 - 113 .3)

1.53 (0 .88 - 2 .55)

19 .4 C15.4 - 23 .9)

Difference in % P

40 < 0.025

45 < 0 .025

38 < 0 .025

Mean values with the ranges in the brackets from 6 experiments . The effects of DNP remained the same whether compaired to results from the preceding ethanol period (as in TABLE I and II) or to the control perfusions without addition of DNP (not included) . Discussion In accordance with the general findings , DNP increased the hepatic oxygen consumption. In an attempt to compensate the decrease in oxidative phosphorylation, the glycolysis increased, as reflected in the rise in glycose and lactate output (TABLE I) . In contrast to previous studies (S) the ethanol elimination was decreased after DNP. In the intact organism increased extrahepatic losses of ethanol may account for an increased elimination rate (9) . In rat liver slices ethanol elim ination was markedly increased by DNP, but only when the elimination rate was low (50 % of the present findings) . When the slices were respiring in pure oxygen, ethanol was eliminated at nearly the same rate as in the present experiments, and a slight, insignificant inhibition by DNP was seen . DNP did not inhibit the alcohol dehydrogenase (8) . The limiting step in the ethanol oxidation is claimed to be dissociation of the enzyme-NADH complex (10), and the decrease in ethanol elimination in the present experiments therefore agrees with the rise in L/P ratio, reflect-

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ing the cytosolic NADH/NAD ratio, as discussed below. The mitochondrial compartment was oxidized after DNP as indicated by the decrease in ß-HB/Ac ratio . To evaluate a possible effect of DNP on the system transporting reducing equivalents across the mitochondrial membrane, the potential gradient as well as the flux must be known . When ethanol is oxidized, the flux of electrons

from cytosol to mitochondriae equals two times the ethanol elimin-

ation under the following assumptions : a) all reducing equivalents produced in cytosol comes from the ethanol oxidation ; b) ethanol is oxidized to acetaldehyde in the cytosol, yielding one mole NADH per mole ethanol; c) reducing equivalents produced in the cytosol are not eliminated by the perfusion medium . ad s) . The increased glycolysis after DNP may lead to increased pyruvate oxidation in the mitochondriae, and in this case one mole NADH will be produced in cytosol per mole pyruvate oxidized . Assuming that the total in crease in oxygen consumption is used for pyruvate oxidation, it can be calculated that at most 0.26 umoles cytosolic NADH/g liver (wet wt .)/min is produced in the glycolysis . ad b) . Grunnet (11) has reported that 80 % of the acetaldehyde produced in ethanol metabolism is further oxidized in the mitochondriae. If ethanol is oxidized to acetate in cytosol, two moles NADH per mole ethanol are produced here . The flux of reducing equivalents across the mitochondrial membrane may therefore be underestimated, but probably to the same degree before and after DNP . ad c) . As the pyruvate uptake was insignificant during the experiments, the increase in lactate output after DNP does not represent elimination of reducing equivalents produced in the cytosol by the ethanol oxidation, but reducing equivalents from the glycolysis . Estimating changes in the NAD+ mediated redox potentials in the compartments from the L/P and ß-HB/Ac ratios assume unchanged pH . Uncoupl-

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ing results in decreased pH in mitochondriae (12) . Enhanced glycolysis after DNP (cf. TABLE I) indicates a fall in pH in the cytosol too . As the L/P ratio increases and the ß-HB/Ac ratio decreases after DNP, the change in mitochondrial redox potential may be underestimated and the change in the cytosolic overestimated . If the diminished transport of reducing equivalents should be counterbalanced by a rise in the redox potential difference across the mitochondrial membrane, one has to assume a fall of at least 1 .0 pH unit in the cytosol . This seems not reasonable, especially as the pH in the effluent medium was only decreased 0 .15 pH units after DNP . When the potential gradient across the mitochondrial membrane and the flux of reducing equivalents across the membrane are estimated, the power of the transport system can be calculated . TABLE III Power of the Transport of Reducing Equivalents from Cytosol to Mitochondriae (A), its Relation to the Energy Gain from Oxidation of the Transported (B), and to the Total Energy Consumption of the Liver (C). Power cal/min/g liver Ethanol

1 . 8 " 10-3

Ethanol 0 0 .8 " 10 -3 + DNP

Power/ ATP gain

Power/ metabolic rate

3 , g%

0.8 % .3 %

Power : F " z " pE " 2n " 0 .239 cal/min/g liver (wet wt .) ; F, the Faraday nnm~r, 96500 coulombs/mole ; z, electron charge ; DE, potential gradient across the mitochondrial membrane in Volts (from TABLE II); n, ethanol elimination rate, mole/min/g liver (wet wt .) from TABLE 1 ; 0.239, conversion factor from joule to cal . ATP sin: 3 moles ATP per NADH, ATP represents 12 .4 kcal/mole . eta o is rate : from the oxygen consumption (TABLE I), assuming a caloric coe icient o oxygen of 5 kcal/1 .

It appears that the power is reduced about 50 % after addition of DNP . In the control state at least 3 .8% of the energy (TABLE III) produced in the respiratory chain from cytosolic NADH is used for the transport . This frac-

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tion is an underestimate, as the efficiency of the transport system is hardly 100 % . After uncoupling the ATP generation is impaired, and the energy need for the transport may consume a larger fraction of the ATP gained from cytosolic NADH . It is therefore more economical to oxidize substrates freely available in the mitochondriae, i .e . accelerate the Krebs cycle . It has in fact been demonstrated that the slow production of C02 when ethanol is metabolized, is nearly doubled after addition of DNP (3) . The energy needed for the transport of reducing equivalents is only a small fraction of the metabolic rate . Even with an efficiency of the transport system of only 20 %, ethanol consumption would increase the metabolic rate of the liver only by 4 %, which is probably too small to be detected with the methods ordinarily used . After DNP an even lesser fraction of the oxygen is used for the transport of reducing equivalents . It is concluded that the power of the system transporting reducing equivalents from cytosol to mitochondriae is decreased by DNP. This may be due to lack of energy caused by the uncoupling . A direct effect of DNP on the transport system can, however, not be excluded .

Acknowledgements We are indebted to assoc . professor J . A . Larsen for helpful suggestions during the preparation of the manuscript . Excellent technical assistance from L . Hansen, B . Hogsberg, A .-M . Krogh, L . B . Nielsen and I . Thierry Andersen is thankfully acknowledged . References 1 . H . A . KREBS and R . L . VEECH in : The Ener Level and Metabolic Control in Mitochondria , eds . S . Papa, . ager, uag Tarie o an ater, p . 3, Adriatica Éditrice, Bari (1959) . 2 . C . OLSEN and N . KRARUP, Acta pharmacol . et toxicol . , 34 232-240 (1974) .

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3. P . B . VENDSBORG and P . SCHAMBYE, Acta pharmacol . et toxicol . 28 113-123 (1970) . 4. N . KRARUP and C . OLSEN, Abstr. 8th Meet . Fed . Europ . biochem. Soc . 1129 (1972) . 5 . H . HEMS, B . D. ROSS, M . N . BERRY and H . A . KREBS, Biochem . J . l01 284-292(1956) " 6 . C . OLSEN, Clin . Chim . Acta 33 293-300 (1971) . 7 . R . SCHOLZ in : Stoffwechsel der isoliert erfundierten Leber, eds . , pringer- er ag, er in, eidelberg, W. Staib and R . c o z, p. (1968) . 8 . L . VIDELA and Y . ISRAEL, Biochem . J . 118 275-281 (1970) . 9 . P . L . EWING, Quart . J . Studies Alc . 1 4$3-500 (191.,0) . 10 . H . THEORELL and B . CHANCE, Acta Chem . Scand. 5 1127-1144(1951) . 11 . N . GRUNIJET, Eur . L. Biochem . 35 236-243 (1973) . 12 . E . CARAFOLI and C . S . ROSSI in : Mitochondria . Structure and Function, eds . L . Ernster and Z . Drahota, pp .~~3ca emic ress