Effects of Ethanol and Fat on the Transport of Reducing Equivalents into Rat Liver Mitochondria

TIIE JOURNAL OF BIOLOGKAL CHEMISTRY Vol. 248, No. 14,Issue of July 25,~~. 4977-4986, 1973

Printed in U.S.A.

Effects of Ethanol and Fat on the Transport of Reducing

Equivalents into Rat Liver Mitochondria *

(Received for publication, December 6, 1972)

ARTHUR I. CEDERBAUM, CHARLES S. LIEBER, ATTILA TOTH, DIANA S. BEATTIE, AND EMAIUUEL RUBIN

From the Departments of Biochemistry, Medicine, and Pathology, Mount Sinai School of iiIedi&e of the City Univemity of -\-ew York, New YOT1~ 10029, and Xection of Lice~+ Disease and lVutrition, Vetelmzs Adminis- tration Hospital, Brom, -Vew York 10468

SUMMARY

Chronic ethanol intoxication in rats accelerated the rate of ethanol metabolism, but decreased the activity of alcohol dehydrogenase. Chronic consumption of ethanol decreased the activities of cytochrome oxidase and succinic dehydrogenase in mitochondria and in cell-free homogenates, whereas total hepatic mitochondrial protein was not altered by ethanol feeding. Thus, total “mitochondrial activity” was decreased and cannot account for the increased rate of blood ethanol clearance. We therefore investigated the activities of mitochondrial enzymes and those of “shuttle” systems for the transport of reducing equivalents into mitochondria.

Reconstituted malate-aspartate, fatty acid, and cr-glycerophosphate shuttles were equally effective in transporting reducing equivalents into the mitochondria from ethanol-fed and control rats. The activities of enzymes involved in the shuttles, such as cytoplasmic and mitochondrial or-glycerophosphate dehydrogenase and glutamic oxalacetic transaminase, were either decreased or unchanged by chronic ethanol consumption. The permeability of the mitochondria to NADH and to substrate anions which participate in the shuttles was also not altered by ethanol feeding. It is therefore unlikely that the’ increased rates of ethanol oxidation produced by chronic ethanol consumption are caused by enhanced transport of reducing equivalents into the mitochondria.

A high fat diet alone (35 % of total calories) increased both the endogenous and total rates of shuttle activity, compared with the rates obtained with mitochondria from rats which had consumed a low fat diet. However, the rates of blood ethanol clearance were comparable in rats consuming high fat and low fat diets. Therefore, the transport of reducing equivalents into the mitochondria under these conditions does not appear to be rate-limiting for ethanol metabolism.

Chronic administration of ethanol to rats increases the rate of hepatic ethanol metabolism (1, 2). The factors responsible for

*This work was supported in part by United States Public Health Service Grants MH-20007. MH-15558. and AM-12511 and Training Grant 501 GM 00115.

this increase are controversial. The reaction of ethanol with alcohol dehydrogenase generates NADH in the cytoplasm; reoxidation of NADH may then be rate-limiting for the over-all metabolism of ethanol (3, 4). Since there appear to he no cytoplasmic processes which are sufficient tc reoxidize the NADH generated by ethanol metabolism, it is necessary for the reducing equivalents of KADH to enter the mitochondria for oxidation by the electron transport chain. In view of the virtual imper- meability of the mitochondria to NADH, several shuttle mechanisms have been proposed for the transport of reducing equivalents into the mitochondria (5-7). Thus, the rate of ethanol metabolism might theoretically br affected by several factors: the activity of enzymes responsible for converting ethanol to acetaldehydc (8), the activity of systems responsible for transporting reducing equivalents into the mitochondria (9), and the ability of the mitochondria to oxidize these reducing equivalents (9).

The effect of chronic ethanol administration on alcohol deli\-- drogenasc activity is disputed. Some have reported increased activity (8, IO), whereas others have found either no changr (9) or a decrease in activity (11). By contrast, the activity of the microsomal ethanol-oxidizing system is increased by chronic consumption of ethanol (I, 2). Estimates of the contribution of induced activity of the microsomal ethanol-osidizing system to the increase in the rate of ethanol metabolism after chronic ethanol consumption have varied from 14 (2) to 70% (12).

Chronic ethanol intoxication alters hepatic mitochondrial structure, as evidenced by ultrastructural examination (13) and reports of increased membrane permeability (14). Recently, it has been reported that mitochondria from mice chronically fed ethanol display increased utilization of NADH, which would suggest that ethanol alters the normally low permeability of the mitochondria to NADH (15, 16). Moreover, the contribution of shuttle mechanisms to ethanol metabolism might be enhanced in livers from chronically treated animals, since the transport of shuttle substrates into and out of the mitochondria may be augmented. Indeed, Rawat and Kuriyama (15, 16) indicated that the transport of NADH into the mitochondria by the malate-aspartate shuttle is increased by chronic ethanol administration.

The capacity of mitochondria to oxidize KADH may be a rate-controlling factor in ethanol metabolism. It was calculated that about 60% of the oxygen consumption in the liver may be

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utilized to reoxidize NADH formed from the oxidat.ion of ethanol (17). Videla and Israel (9) reported an increase in succinic dehydrogenase activit,y in livers from rats chronically treat’ed with et’hanol, and therefore attributed increased regeneration of lYAD+ to increased “mitochondrial activity” per g of tissue. On the other hand, Rubin et al. (13) observed that chronic administration of ethanol impairs mitochondrial function, as evidenced by reduced incorporation of [%]leucine into mitochondrial membrane proteins, decreased State 3 and State 4 respiration, and lowered contents of cytochromes a and b.

To evaluate the contribution of mitochondrial function to t’he increased rate of ethanol metabolism after chronic ethanol consumption, the activities and properties of three reconstit’uted shuttle systems were compared in hepatic mitochondria from rats given ethanol chronically and from pair-fed control rats. These results were compared with changes in rates of ethanol metabolism in viva.

EXPERIMEPU‘TAL PROCEDURE

Preparations

Mitocl~ondria-Mitochondria were prepared as described prc- viously (18). Albumin-bound fatty acids were prepared according to Bjiirntorp (19).

Male Sprague-Dawley rats, weighing about 150 g, were fed a liquid diet (20) for 24 days, in which ethanol provided 36% of tot’al calories, protein IS%, and fat 35%. Pair-fed littermates were given a similar diet except that carbohydrate replaced ethanol isocalorically. In some experiments, mitochondria were isolated from rats maintained on Purina chow which, compared with the liquid diet, is low in fat (9 to 10% of total calories).

Reconstitution of Shuttles-The equilibrium of the alcohol dehydrogenase reaction favors formation of ethanol and NXD+ from acetaldehyde and NAl)H. Therefore, the rate of rt’hanol disappearance would be quit,e low in the absence of a shuttle mechanism to remove oue of the products of the reaction (NADH). Since dissociation and reoxidation of ?JAI>H bound to the enzyme probably represent the rate-limiting step in the alcohol dehydrogenasc reaction (3, 4), the rate of ethauol oxidation in these systems reflects the rate of passage of reducing equivalents into the mitochondria. Mitochondria (5 to 10 mg of protein) were suspended in a medium containing 300 m&r mannitol, 10 rnnf phosphate buffer (pH 7.4), 10 ml1 ‘Iris-HCl (pH 7.4), 10 mM KCl, 5 mM tiIgC&, 1 mM XDP, and I-I,0 to a final volume of 3.0 ml. B NADH-generating system was produced by the addition of 0.25 mnl XAD+, 6 rnM ethanol, and 16 units of alcohol dehydrogenase. The fatty acid shuttle was reconstituted by adding 1 m&z ATP, 0.2 InM coenzyme A, and 0.1 maI albuminbound fatty acid. The malate-aspartate shuttle was assembled by adding 3.3 mM aspartate, 1.33 rnM a-ketoglutarate, 3 unit.s of malate dehydrogenase, and 2 units of glutamic oxalacetic transaminase. The oc-glycerophosphate shuttle was formed by adding 1 mM dihydroxyacetone phosphate, 1 rnhf ATE’, and 1 unit of Lu-glycerophosphate dehydrogenase. The components of each shuttle system were added and the flasks were incubated at 30” for 2 min. The reaction was then initiated by the addition of ethanol. The flasks were sealed immediately and maintained at 30” in a Dubnoff metabolic shaker for 20 min. The reaction was terminated by the addition of trichloroacetic acid (final concentration, lo%), aliquots were removed, and the remaining ethanol concentration was determined by the method of Bonnichsen (21). Blank flasks contained trichloracetic acid, added before ethanol. Any disappearance of et’hanol from the

blank flasks presumably represents nonenzymatic events, including ethanol evaporation. The difference between the sam- ple value and that of the blank is the amount of ethanol oxidized. The endogenous rate, i.e. the rate of ethanol disappearance in the abscncc of the added shuttle components, was determined as above, except that fatty acid, a-ket’oglutarate, or dihydrosyace- tone phosphate was omitted.

Blood Ethanol Clearance

Rates of blood ethanol clearance were deterrnincd after ad- ministering intragastric ethanol (3 g per kg) following an over- night fast, as described previously (II). Acute ethanol intoxication was produced by giving ethanol, 5 g per kg of body weight, as a 50% solution (v/v) intragastrically. Control rats were given isocaloric glucose. The rats were killed 3 hours later and mitochondria were isolated.

Enzyme Assays-Succinic dehydrogenase activity was assayed by following the reduction of phenazine metho~ulfate, as described by King (22), with the use of mitochondria equivalent to 100 pg of protein or homogenate equivalent to 300 pg of total protein. The activity of cytochrorne oxidase was assayed according to Wharton and Tzagaloff (23). The reaction was initiated with mitochoudria (50 to 100 pg of protein) or homogenate containing 200 to 300 pg of protein. Alcohol dchydrogenase activity was assayed according to the method of Uonnichsen and Brink (24), with 105,000 x g supernat’ant containing 400 pg of protein. The activity of cytoplasmic oc-glycerop2losphate dehydrogcnase was determined in a manner similar to that for alcohol dehydrogenase, with an amount of 105,000 x g supernatant equivalent to 100 to 200 pg of protein. Mitochondrial oc-glycerophosphate oxidase activity was dctcrmined polarographically Lvith a Clark oxygen electrode. The polarograph chamber contained 300 mM mannitol, 10 mr\l KCI, 10 m&r KPi (pH 7.4)) 10 m&f Tris-HCl (pH 7.4), 5 m&f MgC12, and mitochondria (3 to 5 mg of protein) in a final volume of 3.0 ml. After the endogenous rate had been obtained, 20 rn>r a-glycerophosphate was added to initiate the reaction. GluOamic oxalacetic transaminase activity was measured as described by Stein et al. (25). To obtain maximal activity of the mitochondrial enzyme, the mitochondria were sonicated for 30 s (3- to 10-s bursts) with a lirslrlson LS-25 Sonifier.

Swelling Studies

Swelling of the mitochondria in isotonic solutions of several ammonium salts was assayed according to Chappell (26) and Chappell and Haarhoff (27), with the use of 100 rnM ammonium salts of malate, succinate, or glutamate, or 83 mivr ammonium salts of phosphate, citrate, or isocitratc, and about 400 pg of mitochondrial protein. The mitochondria were depleted of endogenous substrates by prior incubation for 5 min with 2 m&f ADP and 10 m&x KPi. Protein was determined by the procedure of Lowry et al. (28).

RESULTS

Rats chronically fed et,hanol continued to grow, but at a slightly lower rate than pair-fed control rats. The average weight gain was 2.13 + 0.18 g per day for the control animals, and 1.97 f 0.14 for rats treated with ethanol. The liver t,o body weight ratio in the rats fed ethanol \vas slightly greater t’han that in the controls, owing, in part, to hepatic fat accumulation in the ethanol-fed animals (20).

Ethanol Clearance and dlcohol Dehydrogenase A&i@-The rate of blood ethanol clearance was increased 44 to 477, in rats

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T I S.E.M.

0 Control a Chronic

Ethanol

., :. i : 1:‘; .: . . . ..:..... .

xl Ethanol Bk Clearonce /L

P<.OOl

CII B(

earance/Kg )dy Weight p<.oo1

200 TAIJLE I

Eflect of chronic ethanol ingestion OIL activities of succinic

175 dehydrogenase and cytochrome oxidase

The activities were assayed as described lmder “Experimental Proced\lre.” Specific activity of succinic dehydrogcnase is ex-

150 pressed as nanomoles of phenazine mcthosulfate reduced per min per mg of protein, and that of cytochrome oxidase is de-

k rived from the specific constant evaluat,ed according to Wharton 3

125 g and Tzagoloff (23). Total activity of homogenate is expressed

as activity per g of liver. The percentage yield of mitochondria

r-n was obtained by dividing the specific activity of the mitochon- S 9

dria by the specific activity of the homogenate. Mitochondrial

100 0 content is expressed as milligrams of mitochondrial protein per

1 g of liver and was obtained by dividing the total activity of the

d homogenate by the specific activity of the mitochondria.

75 $ Activity

Succinic dehydrogenase

Tot,al activity of homogenate

3 Specific activity of homog-

PIG. 1. Effect of chronic ethanol consumption on alcohol dehydrogenase activity and blood ethanol clearance. Alcohol dehydrogenase activity and blood ethanol clearance were measured as described under “Experimental Procedure.”

fed ethanol chronically (Fig. 1). IIomever, alcohol dehydro- gcuasc activity was decreased 20(;/, (p < 0.01) (Fig. 1). The discrepancy between the activity of alcohol dchydrogenasc in vitro and the rate of ethanol metabolism in tivo suggested that, theoretically, mitochondrial mechanisms for regeneration of cytoplasmic NAW might bc rate-limiting for ethanol metabolism.

Succinic Dehydrogenase and Cytochrome Ozidase Activity-

1\Iit,ochondria from ethanol-fed rats exhibited a small decrease in succinic dchydrogcnas!e activity and a larger decrease in cytochrome oxidasc activity (Table I). Rubin et al. (13) found that chronic ethanol treatment decreased mitochondrial succinosidase activity under State 3 and State 4 conditions. This might reflect decreased succinic dehydrogenase or cytochrome osidase activity. Total and specific activities of succinic dehydrogenase and cytochrome oxidase of the homogenate were also reduced in the ethanol-fed rats. Total mitochondrial protein per g of liver and t,he percentage yield of mitochondria were not affected by chronic ethanol feeding, whether calculated from the activities of succinic dehydrogenase or cytochrome oxidasc. The total prot.ein content of the liver was 201 and 208 mg per g of liver for the control rats and those fed ethanol, respectively. Thus, chronic ethanol consumption decreased rather than increased total hepatic mitochondrial activity. The reason for the accelerated ethanol metabolism under these conditions must be sought elsewhere. Addition of 175 to 700 mM ethanol in vitro to mitochondria from rats maintained on the low fat diet had no significant effect on the activities of succinic dchydrogenase and cytochrome oxidase. The specific activities of these enzymes in the mitochondria from rats fed the low fat diet, as well as the yield of mitochondrial protein, were similar to those in mitochondria from animals fed the high fat control diet.

Specific activity of mito chondria

Percentage yield of mitochondria

Mitochondrial content/g of

liver Cytochrome oxidase

Total activity of homogenate

Specific activity of homog-

Specific activity of mito-

chondria

Percentage yield of mitochondria

Mitochondrial content/g of

0 Mean f S.E.M. 6 n.s., not significant.

Control’~ Ethanol- treated”

z!z 440

14.43 f 1.71

12.20 f 1.03

44.18 38.10 It 9.33 + 6.58

32.GG + l.G9

32.02 f 1.40

G5.6 66.6

920 f 119

725 zt 100

f 0.61

zt 0.52

+ 1.81

f 1.50

zt 1.13

f 1.61

65.8 66.7

Effect

:0.001

Reconstituted Fatty Acid Shuttle-The cndogenous rate (no added fatty acid) for the fat.ty acid shuttle (Table II) was the same for mitochondrial preparations from ethanol-fed and control rats, which suggests that chronic ethanol consumption did not alter mitochondrial permeability to NADH. There was also no evidence for increased utilization of NADH in mitochondria from ethanol-fed rats, as reported by others (15, 16). However, the endogenous rate for mitochondria from animals fed the high fat control diet with or without ethanol was about 5-fold greater than that observed in mitochondria from animals fed the low fat diet (see below). Palmitate, oleate, or octanoate were equally effective in reconstituting the fatty acid shuttle in the two types of mitochondrial preparations (Table II). There

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TABLE II

Effect of chronic ethanol consumption on activity of fatty acid shuttle

Shuttle activity, expressed as nanomoles of ethanol oxidized

per min per mg of protein, was assayed as described under “Ex- perimental Procedure,” with the use of 5 to 10 mg of mitochondrial protein per flask. The endogenous rate is the activity in the absence of added albumin-bound fatty acid.

Addition

None (cndogenous)

Palmitate

Oleate

Octanoat,e

Carnitine

Citrate

Pa1mitat.e + carnitine

Palmitate + citrate

Joncentratior

0.1, + 3.:

0.1, + 3.;

Activity

Control’”

5.71 + 0.32

9.88 f 0.77

10.78 l 1.18

10.84 zt 1.24

8.72 zk 1.08

* 1.03

It 1.35

10.27 f 1.14

Effect

Ethanol- treated”

5.68 f 0.39

9.73 + O.G9

L!z 0.81

10.66 zk 0.9G

8.78 * 1.45

i 0.77

f 1.40

f 1.30

Effect

tl Mean + S.E.M.

were no significant differences in activity of the reconstituted

shuttle between mitochondria from ethanol-fed and pair-fed toll-

trol rats. Furthermore, several properties of the shuttle, e.g.

stimulation by carnitine or by citrate,l were similar in both

mitochondrial preparations. Thus, chronic ethanol consumption

did not enhance activity of the fatty acid shuttle.

To assess the factors which contribute to the endogenous rate,

the effects of inhibitors of the citric acid cycle (fluorocitrate and malonate) were studied. Fluorocitrate stimulated the endog-

enous rate by IS’%, whereas malonate stimulated this rate by

52% in rats fed the low fat diet.1 The effects of fluorocitrate and malonate on the endogenous rates of the fatty acid shuttle in mitochondria from rats fed the high fat diet in this study were similar (Fig. 2). However, fluorocitrat.e significantly decreased the endogenous rate in mitochondria from ethanol-treated rats. Although malonate did not decrease the endogenous rate in rats

fed ethanol, neither was there any stimulation of this rate, as shown by control rats fed the high fat liquid diet alone. It is possible that stimulation by the dicarboxylate malonate “sparker”l was masked by inhibition of the citric acid cycle by malonate. Previous studies indicated that several intermediates of the citric acid cycle stimulated the fatty acid shuttle and the endogenous rate.l Since administration of ethanol has been reported to decrease the activity of the citric acid cycle and hepatic contents of oxalacetate, succinate, and a-ketoglutarate (29), it is possible that the contents of substrates of the citric acid cycle may be reduced in mitochondria from ethanol-fed rats. Ad- ditional reduction by the action of fluorocitrate (or malonate) may lead to inhibition of the endogenous rate. If sufficient

1 A. I. Cederbaum, C. S. Lieber, D. S. Beattie, and E. Rubin, manuscript in preparation.

I S.E.M. 0 Control 0 Chronic

Ethonol

uorocitrote

FIG. 2. Effect of 10 mM fluorocitrate and 10 m&r malonate on endogenous rate of ethanol oxidation, with the use of mitochondria from rats chronically fed ethanol and from pair-fed control rats. The incubation system and assays are described under “Ji>xperi- mental Procedure.” Statistical analysis refers to control versf(s ethanol-treated rats in all cases. J\‘.S., not significant.

intermediates of the citric acid cycle remain in control mitochondria after fluorocitrate addition, the endogenous rate might not be affected.

Malate-Asp&ate Shuttle--The activities of mitochondrial and

cytoplasmic glutamic oxalacetic transaminase were assayed to determine whether chronic ethanol feeding induces activity of enzymes which participate in the malate-aspartate shuttle. Ethanol feeding was shown previously to have no effect on malate dehydrogenase activity (30). The specific activity of mitochondrial and cytoplasmic transaminase was the same in preparat.ions from ethanol-fed and control rats (Table III). The responses of the enzyme to inhibitors or to an activator were also unaltered by ethanol treatment. Hydrazine sulfate, amino- oxyacetic acid, and cycloserine inhibited, whereas pyridoxal phosphate stimulated, mitochondrial and cytoplasmic transaminase from ethanol-fed rats to the same extent as that from control rats. The endogenous rate (absence of oc-ketoglutarate) of the malatc-aspartate shuttle was unaltered by ethanol consumption (Table IV). Moreover, this rate was comparable with that observed in experiments involving the fatty acid shuttle (Table II). After addition of ol-ketoglutarate to reconstitute the shuttle, there were no significant differences in the rates of ethanol oxidation with the use of mitochondria from ethanol-fed or pair- fed control rats. Further evidence for a lack of effect on the malate-aspartate shuttle was provided by studies of mit,ochondrial permeability. It was suggested that chronic ethanol treatment alters the permeability of the mitochondria to several substrate anions which participate in the malate-aspartate shuttle

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Eflect of chronic elhauol; consumptio~~ ou activity of glutamic Effect of chw~t~ic ethatLo1 admi/LisLratiotL on activity of malate- oxalacetic frannamina.se aspartate shutllc

Act,ivity was assayed as descrioed under “Experimental Pro- cedure.” The influence of inhihitors and of the stimulator was

tested by incltbat,ing them with the enzymes for 2 min before initiating the reaction with Ii.7 rnM a-ketoglutarato. Activit,ies are expressed as nanomoles of ?JAL)II oxidized per min per mg of

Shllttlc act,ivity, expressed as nsnomoles of ethanol oxidized per min per mg of protein, was assayed as described under “Ex- perirncntal Proccdlue,” with the llse of 5 to 10 mg of mitochon-

drial protein per flask. The rndogenorls rate was determined in the absence of cu.ketoghltarate. l?csldts show the effect of the inhibitors on the net, rate of the reaction, that is the shuttle- stimulated reaction.

protein.

Addition

hlil ochondrial glutamic ox- alacctic transaminase

activity None

Hydrazinc sulfate

TTydrazine sulfate

i\rnino-oxyacetic arid

Brnino-oxyacetic acid

Cycloserinc

Cycloscrinr

Pyridoxnl phosphate

I’yridoxal phosphate

Pyridoxnl phosphat c

Cytoplasmic: ghltnmic ox-

alacelic transaminase activity

SOlll?

I~ydrnxine sIllfate

IIydrazine sldfate

Anlillo-ox?-acetic acid

Amino-oxyawlic acid

Cycloserine

(iycloscrine

Pyridoxxl phosphate

l’yridoxxl phosphate

Pyridoxal phosphate

a Mean + S.F:.M.

‘oncen ratior

Control”

870 zk 83

i 50 110

+ 58 795

* A4 li30

zt 127

1503 zk 8(i

1 (i50

+ 150 19TO

z!I 303

+tc, 00

xk 4 17

* 14 30

147 zt,w

f8 210

Ethanol- treateda

f 106 770

It 60 380

i 20 323

It 157

135 * 15

f 30 510

f 257 162'9

i-S 23

* 19 41

* 15 162

f 15 181

i 23 214

Effect

(16). These substrate anions rcquirc carrier systems to be transported into the mitoc+ondria (26, 27). Thcrc are several

inhibitiors available which can interact with these carriers and

block the transport of the substrate anions (31). If ct’hanol

Addition

None (endogenous)

wKetoglrltaratc + mersalyl

cr-Ketoglntarate + iodo- bcnxylmnlonatc

cu-Kctoglntarate + aven- sciolide

ol-Ketoglutarate + 1,2,3- benxerretric:~lboxylic: ‘acid

a-Ketoglntarntc + atrac-

t,yloside

COP entra- tion

0.0’

0.08, 11.75 pi 3.18

% 5.19

It 0.39 +220*: 18.-Z

* 2.13 I

-100 1 3.78

ii 0.78

-89 I 4.87

ii 1.52 I

-45 ’ 12.12

+ 1.99

20 12.77 ‘A 3.22

0.04: 12.05 -413

f 2.15 I ll.il

‘9 0.87

5.33 -t 0.29

& 1.57

k 1.80

k 2.77

a Mean f S.l~:.iVI.

h E;ffect on endogeno\ls rate.

Activity

l-ffect ~ Ethanol- on net rate treated5

Effect on net

trcatmcnt altered the pcrmcabilily of Ihc mitoc&ondria to these substrate anions, trausport inhibitors slloultl IIO~ bc a~ effective in mitoc>houdria from etllauol-treated nuirnals as in mitochondria

from control animals. To test’ this possibility, the effect of several transport itrhibitors on lhc malate-aspartate slluttlc was studied. For operation OC tile malatc-aqartatc shuttle, glutamate and rnalate must enter the mibochorldria, whik a-ketqlu-

tarate and aspart,at,e exit,. I\Ialat,e can cxchangc with phos-

phate, citrate, a-kctoglutarate, and other dicarboxylic acids (31). Mcrsalyl inhibits malatc-phosphate exchatlge (32) and 1,2,3- benzcnctricarboxylic acid malatc-cit’rale exchange (31), whereas iodobeuzylmalonic acid inhibits malate-phosphate, malate-dicarboxylate, and malate-tlicarbosylate exchange reactions (31).

Arcnaciolide is a specific inhibitor of glutamate transport (33). Chronic ethanol treatment neither altcrcd the permeability of the mitochondria to the anions studied nor affected the response to transport inhibitors (Table IV). 1,2,3-Ecnzenctricarboxylic acid decreased the activity of the malatc-aspartate shuttle 37 to 42c/, in mitochondria from rat,s fed the high fat diet, whereas it

had no effect in mitochondria lrom rats fed the low fat diet.l This suggests that the high fat diet, ilr contrast to the low fat diet, may enable accumulation of suficicnt cibratc within t’he mitochondria to permit an efficient citrate-malate exchange. The shuttles operate at maximal efficiency in the prescncc of ADP.’ Atractyloside, an inhibitor of mitochondrial adenine nucleotide translocase, inhibited the shuttle comparably in both

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mitochondrial preparations (Table IV). This indicates that chronic ethanol treatment did not change the permeability of the mitochondria to adenine nucleotides. Thus, the increase in ethanol metabolism produced by chronic ethanol ingestion is not caused by augmented activity of the malate-aspartate shuttle.

a-Glycerophosphate Shuttle-Hassinen (34) concluded that the effect of dihydroxyacetone on mitochondrial respiration, with the use of the a-glycerophosphate shuttle system (external NADH and extramitochondrial cr-glycerophosphate dehydrogenase), was negligible with control mitochondria but that it might be significant in mitochondria from thyrosin-treated rats. We found that the reconstituted shuttle was more effective in mitochondria derived from thyroxintreated rats than from untreated animals1 The above data suggest that the cr-glycerophosphate shuttle may be regulated by the activity of mitochondrial a-glycerophosphate dehydrogenase. The activities of both the cytoplasmic and mitochondrial a-glycerophosphate dehydrogenases were significantly decreased by chronic ethanol treatment (Fig. 3). It is therefore unlikely that enhanced activity of the cy-glycerophosphate shuttle contributes to the increased rate of ethanol oxidation. There was no significant difference in activity of the reconstituted cr-glycerophosphate shuttle between the two mitochondrial preparations (Fig. 4). Since mitochondrial oc-glycerophosphate dehydrogenase is located on the outer surface of the inner membrane (35), a-glycerophosphate need not penetrate the mitochondrial membrane to act as a substrate. Therefore, the permeability of the mitochondria to a-glycerophosphate should not be a consideration in evaluating over-all shuttle activity. The slight decrease in activity of the shuttle with mitochondria from ethanol-treated rats may be due to decreased activity of mitochondrial a-glycerophosphate dehydrogenase (Fig. 3).

0 Control

- Chronic Ethanol

:.. M chondriol

*Glycerophosphate Dehydrogenase

1 S.E.M.

elyceropnospnare Dehydrogenase

FIG. 3. Effect of chronic ethanol ingestion on activities of N- glycerophosphate dehydrogenase and a-glycerophosphate oxidase. Activities were assayed as described under “Experimental Pro- cedure.”

Mitochondrial Swelling-Since the mitochondria are permeable to NH8, the swelling of mitochondria in isotonic solutions of ammonium salts may be used to assay the penetration of anions into the mitochondria (26, 27). Mitochondria from control rats swelled to the same extent as those from ethanol-fed rats when suspended in the ammonium saltsof phosphate, malate, succinate, glutamate, citrate, or isocitrate (Fig. 5). Thus, ethanol consumption produced no change in permeability of the mitochondria to these anions. Addition of ethanol in vitro had no effect on swelling rates of control mitochondria suspended in ammonium salts. Mersalyl inhibited phosphatc-induced swelling to the same extent in both preparations. Both mitochondrial preparations required the addition of the same cofactors for optimal swelling rates; e.g. addition of phosphate was required for malate-induced swelling, and phosphate plus malate was rc- quired for citrate or isocitrate-induced swelling (27). These data, as well as those reported in Table IV, indicate that ethanol does not alter the pathways for anion entry into the mitochondria. Substitution of NH,+ by Kf reduced the swelling rates of both preparations by about 80 to 95%. Therefore, the normally low permeability of control mitochondria to K+ (36) was maintained in the mitochondria from rats fed ethanol.

Comparison of High Fat and Low Fat Diefs-The endogenous rate and the rates of the fatty acid and malate-aspartate shuttles were significantly higher in rats fed the high fat diets (both with and without ethanol) than in rats fed the low fat diet (Table 1’). The data indicate that a high fat diet increases the oxidation of reducing equivalents by isolated mitochondria. This suggests that, if the transport of reducing equivalents into the mitochondria is the rate-limiting step in ethanol metabolism, ethanol clearance rates in vivo should be higher in rats fed the high fat diet than in those fed low fat chow. However, the rates of blood

I: S.E.M.

0 Control

m Chronic Ethanol

Rate N.S.

hydroxyacetone

Phot!~?e

FIG. 4. Effect of chronic ethanol administration on activity of a-glycerophosphate shuttle. The shuttle was assayed as described under “Experimental Procedure.” Endogenous rate refers to activity in the absence of dihydroxyacetone phosphate. A’.LS’., not significant.

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- - --_-

lsocitrate

FIG. 5. Effect of chronic ethanol intoxication on swelling of mitochondria in isotonic solutions of ammonium salts. Swelling was measured as described under “Experimental Procedure.” The slight differences observed are not statistically significant. Control swelling rates, expressed as A O.D.,so per 5 min per mg of mitochondrial protein, were: phosphate, 0.420; malate, 0.272; snccinate, 0.139; glutamate, O.lGO; citrate, 0.073; and isocitrate, 0.068.

T,\IHX V

EjJect of high fat diet on endogenous rate, fatty acid n~td malate-

asparate shuttles, and blood ethmol clearance

The assays were carried out as described under “Experimental Procedure.” Rats were fed a diet containing either 35 or 10% of total calories as fat. Shuttle activity was expressed as nanomoles ethanol oxidized per min per mg of protein. Blood ethanol

clearance is expressed as micromoles of ethanol per min per liter. Clearance, calculated as micromoles of et,hanol per min per kg of body weight, was 105 i 11 for the high fat group and 104 f 3 for

the low fat group.

Reaction

Activity

High fat dieta 1 Low fat diet’ I P --. / I I-

Endogenous. Fatty acid shuttle.

Malate-aspartate shuttle. Blood ethanol clearance.

5.71 Y!E 0.321.17 + 0.41 <O.OOl 9.88 It 0.775.41 l 1.20 <O.OOl

17.08 x!z 1.577.96 f 1.87 <O.Ol 119 xk 13 120 f 5.6 11.S.b

“Mean f S.E.M. b n.s., not significant.

ethanol clearance were the same (Table V), which means t.hat transport of reducing equivalents into the mitochondria does not appear to be rate-limiting for ethanol metabolism.

ESf’ect of Acute Ethanol Administration on Shuttle Activity-

There was no significant difference, in any of the three shuttle activities, between mitochondria derived from rats given an acute dose of ethanol and mitochondria from control rats. Eth- anol oxidation rates for control rats were 9.80, 5.78, and 6.92 nmoles per min per mg of protein for the malate-aspart’ate, CL- glycerophosphate, and fatty acid shuttle, respectively. Cor- responding values for the acute ethanol-trea,ted rats were 10.40, 6.12, and 8.01 nmoles per min per mg of protein. Therefore, t,he immediate product,ion of reducing equivalents did not in- duce shuttle activity. Previous studies, in which addit’ion of ethanol to liver mitochondria in z&o enhanced substrate pene-

tration into the mitochondria (37, 38), were made with ethanol concentrations in the range of 0.3 to 1.2 M, concentrations incom- patible with life.

DISCUSSION

In rats fed a high fat diet without ethanol, the rate of blood ethanol clearance was about 90 ~molcs per min per kg of body weight, which corresponds t,o about 2.6 pmoles per min per g of liver. The reconstituted fatty acid shuttle is capable of transporting about 0.8 pmole per min per g of liver; the malate-aspartate shuttle, about 1.3; and the cr-glycerophosphate shuttle, about 1.2 (assuming 78 mg of mitochondrial protein per g of liver (39)). In animals fed similarly, the microsomal ethanol-oxidizing system is capable of metabolizing about 22 pmoles of ethanol per min per kg of body weight, or about 0.6 pmole per min per g of liver (11). Thus, assuming no other system for oxidizing ethanol, at least 2.0 pmoles of ethanol per min per g of liver are oxidized by the alcohol dehydrogenase reaction, which would necessitate transfer of reducing equivalents into the mitochondria. Presumably, most of the acetaldehyde is metabolized in the mitochondria (40). Therefore, it may be necessary for more than one shuttle mechanism to participate in the transport of reducing equivalents into the mitochondria to account for the rate of ethanol clearance observed in these rats. This is consistent with other indications that more than one shuttle may play a role in the transport of reducing equivalents into the mitochondria (41, 42). Ye found that the rates of ethanol oxidation were additive when any two shuttles were combined in vitro.*

In ethanol-fed rats, the rabe of et’hanol clearance corresponded to about 3.8 pmoles per min per g of liver, a finding which is similar to those of previous data (1,2). Although it is hazardous to extrapolate results obt’ained in vitro to physiological conditions, the simultaneous acbions of all three reconstituted shuttles could account for only 3.3 pmoles of the 3.8 pmoles of ethanol metabolized per min per g of liver. This may be a maximal value, since we find that the maximal capacity for the transport of reducing equivalents is reached by the combined actions of any two shutt1es.l Although there is some alcohol dehydrogenase activity present in organs other than the liver, activity of

the enzyme is by far greatest in the liver (43). In ethanol-fed rats, the microsomal ethanol-oxidizing system is capable of metabolizing ethanol at a rate of about 48 pmoles per min per kg of body weight, or about 1.4 pmoles per min per g of liver (11). Therefore, the combined activities of two shuttle syst’ems for transporting reducing equivalents gonerated by the alcohol dehydrogenase reaction into t’he mitochondria, plus the activity of the microsomal system, might account for the rate of ethanol metabolism observed in vivo in ethanol-fed rats.

These studies indicate that t’he transfer of reducing equivalents into the mitochondria does not appear to be rate-limiting in the metabolism of ethanol, and that the acceleration of ethanol oxidation induced by chronic ethanol consumption is not explained by increased activity of the shuttles. The reconstituted shuttles are inhibited by 2,4-dinitrophenol, an uncoupler of osidative phosphorylation.’ Although one group reported that 2,4-dinitrophenol, prevents the increase in the mitochondrial /%hydroxy- butyrate to acetoacetate ratio caused by ethanol metabolism (44), which points to inhibit,ion of the transport of reducing equivalents into the mit’ochondria, another group found that this compound increases the rate of ethanol metabolism in rat liver slices and in vivo (9, 45). Increased electron transport activit’y caused by the addition of uncouplers or NH4C1 plus ornithine

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increased transport of reducing equivalents in perfused rat livers mitochondrial ar-glycerophosphate dehydrogenase is located on (41). Moreover, chronic ethanol consumption was reported to the exterior surface of the inner membrane (35). Hence, there change the mitochondrial oxidation-reduction potential, whereas is no requirement for a-glycerophosphate or dihydroxyacetone the cytoplasmic oxidation-reduction state remained unchanged phosphate to penetrate the inner membrane; indeed, the mito- (46). These data supports the evidence that the activity of chondria are impermeable to a-glycerophosphate (35). shuttle mechanisms is not rate-limiting in the oxidation of Our data contrast with those of Rawat and Kuriyama (15, 16)) ethanol. who reported increased permeability of mitochondria to NADH

The lack of correlation between ethanol metabolism on the and enhanced activity of the malate-aspartate shuttle after one hand, and alcohol dehydrogenase activity and the rates of ethanol feeding in mice. We find no change in the permeability transport of reducing equivalents into the mitochondria on the other, implies that a rate-limiting st,ep in ethanol oxidation may be the ability of the mitochondria to oxidize NADH and thereby to regenerate NAD+.

An increase in the total mitochondrial activity of the liver has been offered as an explanation by Videla and Israel (9) for accelerated ethanol metabolism; they reported an increase in the activity of succinic dehydrogenase after chronic ethanol feeding. By cont,rast, we find a decrease not only in the activity of succinic dehydrogenase, but also in that of cytochrome oxidase and a-glyc-

of the mitochondria to NADH when rats arc fed an ethanol- containing diet. Reed and Mezey (30) have also reported that the mitochondrial permeability barriers to NADH are not abol- ished by ethanol feeding, and that the inner membrane remains intact. The excessively high rates of NADII oxidation observed by Rawat and Kuriyama (15, 16) cannot be explained as a species difference between rat and mouse mitochondria, since Sactor and Dick (48) have shown that mouse mitochondria, when prepared intact, are impermeable to NADH. The abnormally high permeability to NADH, both in control mitochondria and

erophosphate dehydrogenase. WC also find no change in the in those from ethanol-treated animals in the studies by Rawat yield of mitochondrial protein per g of liver inintoxicated animals. and Kuriyama (15, 16), was probably caused by damage to the Mitochondrial protein per g of &sue (wet weight) was found to mitochondria. Their incubation medium was highly hypotonic, be unaltered after ethanol feeding by other investigators as well since no sucrose or potassium chloride was added to maintain (30), and Banks et al. (47) have reported a slight decrease in the isosmotic conditions. Mitochondrial slvelling is produced in yield of protein from the mitochondrial fraction. Thus, an in such a medium, especially in the presence of 20 mM phosphate. crease in mitochondrial activity does not explain the acceleration We also find no increase in the permeability of mitochondria of blood ethanol clearance in our experiments. The discrep- from ethanol-fed rats to several anions, which are components of ancies between our own and other data and those of Videla and the shuttles. Not only was the activity of the malate-aspartate Israel may be a result of several factors. In the experiments shuttle unchanged, but inhibitors of anion transport block the described here, the activity of the flavoprotein of succinic dehy- reconstituted malate-aspartate shuttle to the same extent in drogenase is measured directly by following the reduction of both groups of mitochondria, indicating that the pathways for phenazine methosulfate after the addition of succinate to isolated anion entry into the mitochondria arc not altered. We, there- mitochondria. Since we measure succinic dehydrogenase ac- fore, find no evidence that the contribution of the malate-aspar- tivity in freshly isolated mitochondria, the normal mitochondrial tate shuttle is increased by chronic ethanol consumption. permeability barriers are maintained. Videla and Israel (9) Rawat and Kuriyama (15, 16) found this increased contribution determined succinic dehydrogenase activity in liver homogenates by malate-aspartate shuttle only under State 3 conditions. It is by measuring the reduction of cytochrome c upon the addition difficult to understand why ADP (State 3 conditions) should of succinate, a method which requires participation of several change mitochondrial permeability to substrate anions which additional components of the respiratory chain. Furthermore, participate in the shuttle. I f chronic ethanol consumption did the diets, mode of ethanol administration, and the effects of indeed alter mitochondrial permeability, it would do so under chronic ethanol feeding were quite different in their study. For State 4 conditions as well. Rawat and Kuriyama (16) also re- example, t’heir control rats did not grow, whereas the ethanol-fed ported a 25% decrease in mitochondrial activity of glutamic rats showed a decrease in body weight. They also reported no oxalacetic transaminase after chronic ethanol ingestion. Since accumulation of triglycerides in the livers of the rats fed ethanol. those authors used untreated mitochondria, the activity of the Moreover, the protein content of the liver in their control rats mitochondrial transarninase which they found was about 20% (273 f 16 mg per g of liver) was considerably higher than that of total mitochondrial activity (25; Table III) ; the decrease in ethanol-fed rats (202 f 2), which raises the possibility of a which they found after ethanol was, therefore, only 5% of tot’al decrease rather than an increase in total mitochondrial mass in mitochondrial activity. In our procedure, mitochondria are the livers of their ethanol-fed rats. French (14) demonstrated sonicated, after which transaminase activity represents total an increase in succinic dehydrogenase activity after 30. to BO-min enzyme activity. No increase in the total activity of either the incubation. It is difficult to compare our results with those of mitochondrial or cytoplasmic transaminase was found after French because the methods used for the assay of succinic dehy- ethanol treatment. This finding is consistent with the un- drogenase are different, and we measured initial rates of reaction. changed activity of the malatc-aspartate shuttle.

Our finding that chronic ethanol ingestion results in a decrease Our data indicate that neither alcohol dehydrogcnase activity in mitochondrial oc-glycerophosphate dehydrogenase activity is nor the transfer of reducing equivalents into the mitochondria is at variance with some reports of unchanged (30) or increased rate-limiting for ethanol metabolism. This suggests that the activity (15, 16). It is usually assumed that the activity of the capacity of the mitochondria to reoxidize NADH might limit mitochondrial a-glycerophosphate dehydrogenase is the rate- ethanol metabolism. Adaptation of the microsomal ethanol- limiting step in the a-glycerophosphate shuttle; we find a slight oxidizing system may play a role in the acceleration of ethanol decrease of the reconstituted shuttle when mitochondria from metabolism after chronic ethanol consumption, but it may not ethanol-fed rats are used. Furthermore, the explanation that account for the entire increase in the rate of ethanol blood clear- the alleged increase in activity reflects increased permeability of ante. Therefore, other factors, such as extrahepatic metabolisrn the mitochondria to cr-glycerophosphate (15) is not tenable, since of ethanol (lo), coenzyme availability, and transhydrogenation

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from the NADH-alcohol dchydrogenase complex to NBDPf, which would accelerate both the microsomal ethanol-oxidizing system and the alcohol dehydrogenase pathways (ll), should be considered further.

Additional experiments are required for an understanding of the effects induced by the high fat diet. There were no differences between mitochondria from rats fed the low fat diet ad bibitum and those fed a restricted amount of chow containing the same number of calories consumed by rats fed the high fat diet. Rats fed a high fat diet show higher endogenous rates and fatty acid and malate-aspartate shuttle rates than do rats fed a low fat diet. The endogenous rate is comprised of direct NADH pene- tratiou into the mitochondria and the activity of an endogenous shuttle which transports reducing equivalents into the mitochondria. The only functional endogenous shuttle currently understood is the fatty acid shuttle, which contains both the elongation system and the p oxidation system within the mitochondria; these may utilize endogenous free fatty acids supplied by the high fat diet or phospholiyids liberated by mitochondrial phospholipases. The malate-aspartate or oc-glycerophosphate shuttles cannot contribute to the endogenous rate in isolated mitochondria, since extramitochondrial enzymes and cofactors must be added to reconstitute these shuttles. Since it is gen- crally agreed that mitochondria arc impermeable to NADH, the differences observed are probably caused by increased activity of t,he cndogenous fatty acid shuttle. Indeed, we have found an increase in the rate of p oxidation by mitochondria from rats fed the high fat diet.2 That induction of activity may occur is also evidenced by the higher rates of ethanol oxidation upon addition of fatty acid to mitochondria from rats fed the high fat diet (9.88 nmoles per min per mg of protein) compared wit,h mitochondria from rats fed the low fat diet (5.41, Table V). Similarly, the rate of ethanol oxidation with the malate-aspartate shuttle is significantly greater after a high fat diet (17.08) than after a low fat diet (7.96).

It has already been shown that the level of dietary fat can af- fect the ability of several iuducers, including ethanol, to stimulate microsomal enzyme activity (49). The quantity of fat in the diet may regulate the activity of acetyl-CoA carboxylase and /I- hydrosy-P-methylglutaryl-CoA reductase, key enzymes in the pathways of fatty acid synthesis and cholesterol synthesis, respectively (50). It has been shown that feeding of fat to animals decreases de no00 synthesis of fatty acids from acetyl-CoA (51, 52). Since regulatory mechanisms for control of fatty acid synthesis are present in rat liver, it is possible that increased fatty acid oxidation represents another mechanism by which the liver may adjust to high levels of fat.

Note Added in Proof-Oxidatiou of et,hanol occurred in the absence of mitochondria when t,he a-glycerophosphate shuttle was reconst,ituted with dihydroxyacetone phosphate, apparently because it reacts with NADH t’o form a-glycerophosphate and NAT)‘. This rate was about 15y0 of that observed in the presence of the mitochondria. When the malate-aspartate shuttle was reconstituted with cr-ketoglutarate plus aspartate, the product of the reaction, oxalacetate, allowed direct oxidation of NADH in the presence of malat,c tlehydrogenase. The rate of ethanol oxidation using only the extramitochondrial components was 50 to BOrr/, of that found in the presence of the mitochondria. No oxidation of ethanol occurred in the absence of mitochondria when the ol-glycerophosphat,e shuttle was re-

2 A. Toth, C. S. Lieber, D. S. Beattie, A. I. Cederbaum, and E., Rubin, manuscript in preparation.

constituted with ac-glycerophosphate or the malate-aspartate shuttle with glutamate plus malate. We found no differences in the activities of the shuttles between mitochondria derived from ethanol-fed rats and their pair-fed controls, regardless of which components were used to reconstitute the malate-aspartate or the ar-glycerophosphate shuttles. The extramitochondrial system of the fatty acid shuttle oxidized no ethanol without mitochondria.

Acknowledgment-We thank Dr. W. I3. Turner, Imperial Chemical Industries, Ltd., Macclesfield, Cheshire, United King- dom, for his generous gift of avenaciolide.

9. 10. 11. 12.

14. 15.

19. 20. 21.

22. 23.

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RubinArthur I. Cederbaum, Charles S. Lieber, Attila Toth, Diana S. Beattie and Emanuel

Liver MitochondriaEffects of Ethanol and Fat on the Transport of Reducing Equivalents into Rat

1973, 248:4977-4986.J. Biol. Chem.

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Effects of Ethanol and Fat on the Transport of Reducing Equivalents into Rat Liver Mitochondria

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