Effect of Chronic Ethanol Ingestion on Fatty Acid Oxidation by Hepatic Mitochondria*

To study possible factors in the pathogenesis of the ethanol-induced fatty liver, we investigated the effect of chronic ethanol consumption on the metabolism of fatty acids by isolated hepatic mitochondria. Chronic ethanol consumption resulted in decreased fatty acid oxidation, as evidenced by a reduction in oxygen uptake and CO, production associated with the oxidation of fatty acids. The State 3 rate of oxygen uptake was depressed to a greater extent than the State 4 or the uncoupler-stimulated rate; the respiratory control ratio was also decreased. Therefore, one site of action of chronic ethanol feeding is on oxidative phosphorylation. The reduction in fatty acid oxidation, in general, is not due to an effect on the activation or translocation of fatty acids into the mitochondria. There was no effect by ethanol feeding on the activity of palmitoyl coenzyme A synthetase, whereas carnitine palmitoyltransferase activity was increased. The use of an artificial system (formazan production) to study p oxidation in the absence of the electron transport chain is described. In the presence of fluorocitrate, which inhibits citric acid cycle and formazan were increased by chronic ethanol the level of acetyl-CoA is not by chronic


Effect of Chronic Ethanol Ingestion on Fatty Acid Oxidation by
To study possible factors in the pathogenesis of the ethanol-induced fatty liver, we investigated the effect of chronic ethanol consumption on the metabolism of fatty acids by isolated hepatic mitochondria. Chronic ethanol consumption resulted in decreased fatty acid oxidation, as evidenced by a reduction in oxygen uptake and CO, production associated with the oxidation of fatty acids. The State 3 rate of oxygen uptake was depressed to a greater extent than the State 4 or the uncoupler-stimulated rate; the respiratory control ratio was also decreased. Therefore, one site of action of chronic ethanol feeding is on oxidative phosphorylation.
The reduction in fatty acid oxidation, in general, is not due to an effect on the activation or translocation of fatty acids into the mitochondria. There was no effect by ethanol feeding on the activity of palmitoyl coenzyme A synthetase, whereas carnitine palmitoyltransferase activity was increased. The use of an artificial system (formazan production) to study p oxidation in the absence of the electron transport chain is described. In the presence of fluorocitrate, which inhibits citric acid cycle activity, ketogenesis and formazan production were increased by chronic ethanol consumption. Thus fl oxidation to the level of acetyl-CoA is not impaired by chronic ethanol consumption.
Total oxidation of fatty acids to CO, is depressed by chronic ethanol intoxication because of effects on oxidative phosphorylation or the citric acid cycle (or both). Neither nutritional deficiency, cofactor depletion, nor the presence of ethanol in uitro explains these effects. Several of the effects of chronic ethanol consumption on fatty acid oxidation are mimicked by acetaldehyde and acetate, products of ethanol oxidation. Chronic ethanol consumption leads to persistent impairment of mitochondrial oxidation of fatty acids to COz. However, oxidation of fatty acids to acetyl-CoA is not decreased by chronic ethanol consumption.
Chronic ethanol consumption in animals (l-4) and man (2,5,6) leads to fatty liver, in the absence of nutritional deficiencies.
Several mechanisms have been suggested for the ethanol-induced steatosis, including decreased fatty acid oxidation by mitochondria (7). The lipids deposited in the liver after chronic ethanol intoxication are principally of dietary origin (8), suggesting reduced hepatic oxidation of fatty acids. Ethanol metabolism has been reported to decrease '"CO, production from labeled palmitate and acetate in liver slices (7), which points to decreased /3 oxidation or reduced activity of the citric acid cycle (7) or both. The production of reducing equivalents by the oxidation of ethanol results in a lowering of the mitochondrial oxidation-reduction state, which causes reduction of oxalacetate to malate, thus decreasing activity of the citric acid cycle.
In contrast to acute ethanol intoxication, chronic ethanol ingestion is associated with striking ultrastructural changes in the mitochondria (9)(10)(11) and increased membrane fragility * This work was supported in part by United States Public Health Service Grants AA00287, AA00224, and AM12511. (12). In addition, selective mitochondrial functions such as oxygen consumption with a variety of substrates, oxidative phosphorylation, and energized Ca2+ uptake are impaired by chronic ethanol feeding (13). These findings suggested that chronically compromised integrity of the mitochondria, independent of the biochemical events associated with ethanol metabolism, might interfere with fatty acid oxidation. In this study we have continued our investigations into the pathogenesis of fatty liver by determining the effect of chronic ethanol ingestion on the metabolism of fatty acids by isolated hepatic mitochondria.  Lowry et al. (16). Bovine serum albumin was depleted of fatty acids by the charcoal treatment of Chen (17).

Preparations-Male
In this report total oxidation of fatty acids refers to oxidation to CO, and H,O, whereas  trols, permeability to anions or susceptibility to anion inhibitors was not altered, and P:O or respiratory control ratios with succinate or ascorbate were similar to those found with pair-fed controls (13,15). Various inhibitors of mitochondrial functions (oligomycin, atractyloside, rotenone, cyanide, etc.) were as We have previously reported that chronic ethanol feeding effective in mitochondria from ethanol-fed rats as in controls. Mitochondria from ethanol-fed rats contained cytochromes 6, did not cause biochemical disruption of the mitochondrial cl, a, and a3 (although the content of cytochromes was lower (11)) as well as classical mitochondrial marker enzymes such as membrane; mitochondria from ethanol-fed rats were impermesuccinic dehydrogenase, a-glycerophosphate oxidase, and cytochrome oxidase (although the specific activity of these en-able to NADH. showed comoarable ATPase activitv as conzymes was lower (15)). Indeed, by assaying the activity of these enzymes in mitochondria and in cell-free homogenates, the content of mitochondrial protein (milligrams per g wet weight) tally (19). Oxidation of Fatty Acids in Presence of Artificial Electron Accep-or the percentage of yield of mitochondria was the same for tars-We assayed fatty acid oxidation in the presence of artificial mitochondria from ethanol-fed rats as controls (15). It, thereelectron acceptors by a modification of the original method of Mii and fore, appears that chronic ethanol feeding did not grossly Green (20). The incubation medium contained, in a final volume of 1 disrupt the liver so that proteins isolated as mitochondrial ml, 0. Cyanide could effectively replace anaerobic conditions. At the end of the incubation, 0.1 ml of 6 N HCI was added to each test tube, followed by 1.5 ml of acetone and 4.5 ml of Ccl,. After centrifugation, the reduced formazan was retained in the Ccl, phase. The upper layer of acetone, which contained the methylene blue, was discarded.
The amount of formazan produced was calculated from the absorption at 485 nm. An optical density of 1.00 corresponded to a of the homogenate from ethanol-fed rats artifactually produces abnormal mitochondria, since such changes could hardly be expected to be selective.
Effect of Chronic Ethanol Consumption on Oxygen Uptake--The State 3 rates of oxygen consumption were comparable when control mitochondria oxidized palmitoyl-l-carnitine, palmitoyl-CoA, palmitate, oleate, or octanoate as the substrates (Table I). Although the rates in mitochondria from ethanol-fed animals were lower than in controls (see below), there were still no differences in rates between various substrates. We previously found that the rates of oxygen consumption in State 4 were depressed 10 to 20% in mitochondria from ethanol-fed rats, using NAD+-dependent substrates, succiconcentration of 83 I.LM formazan (20). All incubations were carried out in triplicate, each experiment having its own control, to which no fatty nate, a flavin-linked substrate, or ascorbate, which reduces acid was added. The rates in the absence of mitochondria were cytochrome c (13). In State 4, the rate of oxygen consumption negligible.
associated with the oxidation of all fatty acid substrates ~ahzitoyl-CoA Synthetase-The activation of palmitate by palmit-was also depressed 10 to 15% in mitochondria from ethanol- Effect of chronic ethanol consumption on oxygen uptake associated with fatty acids as substrates Oxygen uptake was assayed as described under "Materials and Methods" in the Dresence (State 3) or absence (State 4) of 1 mM ADP.

Substrate
Palmitoyl-1-carnitine (11) Palmitoyl-CoA (6) Palmitate (11) Oleate (6) Octanoate (6)  The reduction in palmitate or oleate oxidation after ethanol feeding was also found in the absence of carnitine or ATP and in the presence of 0.1 mM CoA. Mitochondria obtained from rats 3 and 16 hours after acute ethanol intoxication displayed no change in the rate of fatty acid oxidation, compared to those isolated from animals given isocaloric glucose. The addition in vitro of up to 100 mM ethanol to mitochondria from chow-fed control rats had no effect on fatty acid oxidation.
State 3 octanoate oxidation was not as sensitive to ethanol feeding as was that of long chain fatty acids, the extent of inhibition being only one-half that found with the long chain fatty acids (Table I).
The greater reduction in ADP-stimulated oxygen uptake (State 3) after ethanol feeding, compared to that in State 4, suggests an effect of chronic ethanol treatment on the coupling process of oxidative phosphorylation.
We previously found that after ethanol feeding, oxidative phosphorylation was depressed with NAD+-dependent substrates, but not with succinate (13). The respiratory control ratio was also depressed in mitochondria from ethanol-fed animals oxidizing fatty acids as substrates (Fig. 1). This reduction (18 to 23%) was somewhat less than that previously observed with cu-ketoglutarate, glutamate, or P-hydroxybutyrate (about 30%), but more than that observed with succinate (9%). This intermediate value for the decrease in the respiratory control ratio with fatty acids as substrates (between that of NAD+-dependent substrates and flavin-linked substrates) may reflect the fact that reducing equivalents from the p oxidation of fatty acids enter the respiratory chain both at the level of NADH-dehydrogenase and at the cytochrome b-ubiquinone level (via the electron transfer flavoprotein).
The greater reduction of oxygen consumption under State 3 conditions, compared to State 4, may be due to the greater flux of electrons in the presence of ADP, which necessitates maximal activity of the respiratory chain. It has also been suggested that ethanol feeding causes decreased activity of the adenine nucleotide translocase system, thus inhibiting transport of ADP into the mitochondria (23). We,  Table I. N.S., not statistically significant. therefore, used an uncoupling agent to stimulate fatty acid oxidatidn, independent of the mitochondrial coupling apparatus. In the presence of dinitrophenol, the rate of oxygen consumption associated with the oxidation of palmitoyl-l-carnitine, palmitoyl-CoA, and palmitate was decreased 15 to 19% in mitochondria from ethanol-fed rats (Table II). This decrease was similar to that observed in the absence of the uncoupler (State 4). Thus, the stimulation by dinitrophenol was not altered by ethanol feeding (Table II). This contrasts with the greater reduction in ADP-stimulated oxygen consumption (29 to 32%, Table I) and the decrease in the respiratory control ratio (Fig. 1) found after ethanol ingestion. Whereas ADP was as effective as dinitrophenol in stimulating oxygen consumption in control mitochondria, it was not as effective as dinitrophenol in stimulating oxygen consumption in mitochondria from ethanol-fed rats (compare Fig. 1 and Table II). Chronic ethanol consumption apparently produces a defect in the respiratory chain, which is responsible for the slight decrease in fatty acid oxidation in State 4 or in the presence of an uncoupler (10 to 15%). Superimposed upon this effect is an inhibition of oxidative phosphorylation by ethanol feeding, which results in greater inhibition of ADP-stimulated respiration (30%).
Effect of Chronic Ethanol Consumption on CO, Production-Oxygen consumption associated with the oxidation of long chain fatty acids is reduced to a greater extent by L Effect ethanol consumption than that associated with the oxidation of medium chain fatty acids. It is also possible that ADP does not stimulate oxygen consumption in control mitochondria with octanoate to the same extent as with palmitate.
However it does stimulate oxygen uptake comparably with both substrates in mitochondria from ethanol-fed rats. To corroborate these impressions, we studied the effect of ethanol feeding on CO, production from YZ-labeled palmitate and octanoate.
As shown in Fig. 2, CO, production from palmitate was reduced 37% in mitochondria from ethanol-fed rats, a value comparable to the 32% decrease in State 3 oxygen consumption.
CO, production from octanoate was reduced 20% by ethanol feeding, similar to the 17% decrease in State 3 oxygen consumption.
By either technique, palmitate oxidation was more sensitive to depression by ethanol feeding than was octanoate oxidation.
Effect of Chronic Ethanol Consumption on Palmitoyl-CoA Synthetase and Carnitine Palmitoyltransferase-Factors which might play a role in the differential inhibition of long chain and medium chain fatty acids include the fatty acid synthetase of the outer membrane, which activates long chain, but not medium chain fatty acids (24, 25), and carnitine palmitoyltransferase, which transports long chain fatty acids into the mitochondria (26, 27). We, therefore, investigated the effects of chronic ethanol feeding on the activities of these two enzymes. PalmitoylCoA synthetase activity was not affected by ethanol feeding (specific activity (micromoles of hydroxamate formed per hour per mg of protein) of 3.7 * 0.4 for control mitochondria and 3.5 * 0.5 for mitochondria from ethanol-fed rats). By contrast, carnitine palmitoyltransferase activity was stimulated 30% (specific activity (nanomoles of CoA released per min per mg of protein) of 27 * 1.4 for control mitochondria and 35 * 4 for mitochondria from ethanol-fed rats, p < 0.05). Thus, the depression of total fatty acid oxidation by ethanol feeding cannot be explained by an effect on the activities of these enzymes.
Effect of Chronic Ethanol Consumption on Ketogenesis--It seemed possible that chronic ethanol consumption might favor a flow of acetyl-CoA, derived from p oxidation, into ketogenesis rather than to total oxidation to CO,. Such a shift would cause COz production and 0, uptake to be reduced, but @ oxidation of fatty acids would not necessarily be depressed. In other studies a redistribution of acetyl-CoA was suggested to explain the inhibition of palmitate oxidation to CO, by acetate (28) feeding on ketone body production by hepatic mitochondria. Ethanol feeding had no effect on the endogenous rate of ketone body production (40.88 * 5.4 nmol of P-hydroxybutyrate and acetoacetate formed per 30 min per mg of protein for control mitochondria and 42.5 * 3.5 for mitochondria from ethanol-fed rats). The addition of palmitoyl-1-carnitine or palmitate increased ketone body production about 4-fold. There was a slight increase in ketone body production in mitochondria from ethanol-fed rats (+9 to +15%), but this did not reach statistical significance.
Similar results were obtained with lower concentrations of fatty acids, e.g. with 30 pM palmitoyl-l-carnitine, there was an 11% increase in ketogenesis after ethanol feeding.
Pande (29) has suggested that ketone body production in liver may be enhanced by the suppression of the citric acid cycle. We, therefore, investigated ketogenesis after inhibiting the activity of the citric acid cycle with fluorocitrate. With labeled palmitate and octanoate, 25 FM fluorocitrate inhibited CO, production 7x) to 80% in mitochondria from ethanol-fed rats and 80 to 90% in controls. In the presence of fluorocitrate, there was no difference in the endogenous rate of ketone body production between control mitochondria and those from ethanol-fed rats (Fig. 3). However, compared to the rates in the absence of fluorocitrate, this endogenous rate increased 70% in control mitochondria and 79% in those from animals given ethanol, suggesting diversion of acetyl-CoA into ketogenesis, a more accessible pathway. Fluorocitrate was reported to increase ketogenesis and decrease citrate formation from palmitoyl carnitine and malate in isolated mitochondria (30). Upon adding palmitoyl-l-carnitine or palmitate, ketogenesis was increased about 3-fold in control mitochondria, and about 4-fold in mitochondria from ethanol-fed rats (Fig. 3). The total rates of ketogenesis were greater in the presence of fluorocitrate than in its absence, with both mitochondrial preparations. Both the total and net rates of ketone body production were greater in mitochondria from ethanol-fed rats than controls, with either substrate. Thus, in the presence of fluorocitrate, ketogenesis was stimulated after ethanol feeding. This may explain, in part, the increase of serum ketone bodies which occurs after chronic ethanol feeding (31). The increase in ketone body production after ethanol ingestion may be due to increased activities of enzymes which participate in the formation of ketones or increased formation of acetyl-CoA via p oxidation (or both). The increase in ketone body production (and formazan formation, see below) suggests that ethanol feeding does not impair 0 oxidation of fatty acids to the level of acetyl-CoA.
Oxidation of Fatty Acids in Presence of Artificial Electron Acceptors-The ethanol-induced decrease in fatty acid oxidation under State 4 conditions or in the presence of uncoupler points to some impairment of the respiratory chain. The additional reduction in ADP-stimulated fatty acid oxidation suggests impairment of coupled phosphorylation (13). Others have suggested that ADP entry into the mitochondria may be compromised (23). To verify the results described above, and to eliminate the influence of changes in the respiratory chain, we studied fatty acid oxidation under anaerobic conditions, with the use of artificial electron acceptors to reoxidize NADH.
Initial studies were concerned with characterization of the system in mitochondria from rats fed commercial chow. This diet contains 9 to 10% of total calories as fat, whereas the liquid diet contains 35%. Formazan production was linear for 60 min, and was proportional to concentration between 0.2 and 2 mg of mitochondrial protein per ml. At higher protein concentrations, it was difficult to extract the formazan from the protein precipitate.
Changing the concentrations of methylene blue, triphenyltetrazolium, NAD+, or ATP had no effect on the reaction. There were no significant differences in formazan production in mitochondria incubated under hypotonic or isotonic conditions, suggesting that the dyes had unrestricted access to the sites of p oxidation and citric acid cycle activity. The addition of 5 mM citrate increased formazan production from 12.9 to 26.2 nmol/hour/mg of protein, producing reducing equivalents via citric acid cycle activity. An inhibitor of citrate transport, 10 mM 1,2,3-benzene tricarboxylic acid (32) blocked the citrate-induced increase (rate = 14.3), which indicates that the integrity of the membranes and receptor sites was preserved. Table III shows that the rate of formazan production is increased upon adding fatty acids to the system. cy-Bromopalmitate, a competitive antagonist of palmitate metabolism (33), reduced the rate of formazan production in the presence of palmitate by 39%. The extent of inhibition may even be greater since oc-bromopalmitate itself may serve as a substrate (Table  III). The above studies show that the dyes effectively accept electrons from reducing equivalents produced by citric acid cycle activity or fatty acid oxidation (or both). Mitochondria isolated from rats fed ethanol for 24 days displayed a slightly higher rate of endogenous formazan production than their pair-fed controls (Table IV). Upon the addition of palmitate the total rate, as well as the net rate of formazan production, was higher in mitochondria from ethanol-fed rats. Comparable results were obtained in mitochondria from two pairs of rats, with the use of octanoate or oleate as substrates.
By contrast, there were no changes in formazan production after acute administration of ethanol (6 g/kg; 34.65 nmol of formazan/hour/mg for controls oxidizing palmitate, 34.98 for acute ethanol). Thus in the absence of a functional respiratory chain, mitochondria from ethanol-fed rats show a higher rate of formazan production, either because of increased @ oxidation, or increased activity of the citric acid cycle. To dissociate these two possibilities, we studied formazan production in the presence of fluorocitrate, which inhibits  Palmitate (5) 60. 16 * 4.40 51.04 * 5.40 -15 < 0.05 Oleate (6) 58. 30 + 2.72 49.29 + 6.00 -16 < 0.05 rather than to oxidation via the citric acid cycle. That such a diversion may occur is indicated by the demonstration that acetate depresses oxygen uptake and CO, production from palmitate, whereas ketogenesis is stimulated (28). We, therefore, examined some of the factors which may participate in the depression of fatty acid oxidation after ethanol feeding, including activation and translocation of fatty acids, /3 oxidation, ketone body production, and the activities of the citric acid cycle and the respiratory phosphorylating system.
the metabolism of two carbon fragments via the citric acid cycle. Under anaerobic conditions in the presence of fluorocitrate, formazan production upon the addition of palmitate presumably is a measure of p oxidation. Fluorocitrate depressed the endogenous rate in the controls by 41%, but only by 23% in mitochondria from ethanol-fed rats (Table IV). With palmitate as the substrate, the total rate of formazan production was increased 39% by ethanol ingestion, compared to pair-fed controls.
The net rate was also 28% greater in mitochondria from ethanol-treated animals (Table IV). Therefore, /3 oxidation does not appear to be decreased by chronic ethanol feeding since formazan production and ketogenesis were actually increased by ethanol feeding.

Effect
of Chronic Ethanol Consumption on Oxygen Uptake in Presence of Fluorocitrate-We previously found that acetaldehyde inhibits oxygen consumption in the presence and absence of fluorocitrate, indicating inhibition of the oxygen uptake which arises both from /3 oxidation and from citric acid cycle activity (33a). In the presence of fluorocitrate oxygen consumption reflects p oxidation of fatty acids to acetyl-CoA. Since /3 oxidation in the presence of fluorocitrate is stimulated by chronic ethanol consumption, oxygen uptake under these conditions should also be stimulated.
On the other hand, a lack of stimulation of oxygen uptake would imply inhibitory effects on the respiratory phosphorylation chain. In the presence of fluorocitrate, oxygen' uptake associated with the oxidation of fatty acids was slightly decreased in mitochondria from ethanol-fed rats (Table V). This suggests impairment of the respiratory phosphorylation chain by chronic ethanol consumption. However, the relative decrease in oxygen consumption was less than that found in the absence of fluorocitrate (compare with Table I). The greater inhibition of oxygen uptake in the absence of fluorocitrate suggests an effect of chronic ethanol feeding on citric acid cycle activity.

DISCUSSION
In this study fatty acid oxidation by isolated hepatic mitochondria was depressed after chronic ethanol feeding, suggesting that persistent changes in mitochondrial functions, in addition to the effects produced by metabolism of ethanol, may play a role in the production of fatty liver. The decreases in oxygen uptake and CO, production from fatty acids do not by themselves prove that fatty acid utilization is impaired, because acetyl-CoA derived from p oxidation may be diverted to other pathways, e.g. ketogenesis or fatty acid elongation, We previously found that the State 3 oxidation of several NAD+-dependent substrates was depressed after ethanol feeding much more than uncoupler-stimulated or State 4 oxygen consumption.
These data suggested that in addition to a defect in the respiratory chain produced by chronic ethanol consumption there is also an inhibitory effect on oxidative phosphorylation (13). In this study, similar results were obtained in studies of fatty acid oxidation. Thus, State 4 or uncoupler-stimulated oxygen uptake was decreased 10 to 15% by ethanol feeding, whereas the State 3 rate was reduced about 30%. That oxidative phosphorylation is impaired is further suggested by the decrease in the respiratory control ratio, whereas the stimulation of fatty acid oxidation by dinitrophenol was not altered by ethanol feeding. In the presence of fluorocitrate, oxygen uptake'associated with fatty acid oxidation was also depressed after ethanol feeding. Thus in the absence of activity of the citric acid cycle, the inhibitory effect of ethanol feeding on oxidative phosphorylation results in a net inhibition of oxygen uptake. There are additional reports indicating that chronic ethanol consumption decreases oxygen consumption by hepatic mitochondria (23,34,35). The decrease in CO, production from labeled palmitate or octanoate suggests the possibility of an inhibitory effect of chronic ethanol feeding on the activity of the citric acid cycle. The ethanol-induced decrease in oxygen uptake was greater in the presence of intact citric acid cycle activity than when fluorocitrate inhibited the cycle. Thus, inhibition of oxygen uptake by ethanol feeding involves depression of oxygen consumption which arises from citric acid cycle activity. The fact that formazan production and ketogenesis are stimulated, but total oxidation of fatty acids is depressed, suggests that acetyl-CoA is diverted from the citric acid cycle to other pathways, e.g. ketogenesis.
In preliminary experiments we found that CO, production from various "C-labeled citric acid cycle intermediates was decreased 15 to 25% in mitochondria from ethanol-fed rats.' In liver cells metabolizing ethanol, Ontko (36) suggested that inhibition of the citric acid cycle 'A. I. Cederbaum, C. S. Lieber, and E. Rubin, unpublished observations. occurred at the level of cu-ketoglutarate dehydrogenase, with a minor site of inhibition at the span beyond succinate. In the perfused liver, sites of inhibition during ethanol oxidation were identified at the citrate synthetase and isocitrate dehydrogenase steps (37). The inhibitions observed in those studies involve active metabolism of ethanol, with a consequent alteration of the oxidation-reduction state of the mitochondria. Further studies are in progress to study directly the possibility that persistent changes caused by chronic ethanol feeding may also alter citric acid cycle activity, independent of the changes caused by the oxidation of ethanol.
In these studies we did not examine the possible utilization of acetyl-CoA in fatty acid elongation, since the cofactors necessary for significant rates of elongation were not present. In the absence of NADPH, elongation is a sluggish process (less than 0.01 nmol of acetyl-CoA incorporated per min per mg (38, 39)) compared to the rates of ketogenesis or total oxidation to CO,. The finding that the 20:4 to 18:2 fatty acid ratio is depressed after ethanol feeding (40, 41) suggests that, if anything, fatty acid elongation may be decreased by ethanol feeding.
Ethanol feeding induced a slight increase in ketone body production and formazan formation, which was augmented considerably in the presence of fluorocitrate. These data suggest that mitochondrial p oxidation to the level of acetyl-CoA is increased, rather than decreased by chronic ethanol ingestion. This is consistent with the increased blood levels of ketone bodies and increased acetoacetate formation by liver slices from rats chronically fed ethanol (31). In view of the fact that formazan production and ketogenesis are increased rather than decreased in mitochondria from ethanol-fed rats, the decrease in CO, production from fatty acids may be caused either by depression of citric acid cycle activity or by impaired oxidative phosphorylation.
Mitochondria from rats fed a high fat diet (35% of total calories) display higher endogenous and total rates of p oxidation (formazan production) than mitochondria from rats fed a chow diet in which fat provides 9 to 10% of total calories. /3 oxidation may be induced by a high fat diet and is further enhanced by ethanol feeding. The increase in carnitine palmitoyltransferase activity after ethanol feeding may contribute to the increased rate of /3 oxidation, by transporting fatty acids to the site of /3 oxidation. However, for reasons described below, it is unlikely' that this enzyme normally is rate-limiting for fatty acid oxidation.
It is possible that the increase in p oxidation and ketone body production provides a mechanism for disposing of excess fatty acids.
The decreased rate of fatty acid oxidation cannot be attributed to the presence of ethanol in the tissue, since the washing procedures would have removed any contaminating ethanol. Furthermore, addition of ethanol in uitro had no effect on fatty acid oxidation.
The addition of ATP, CoA, or NAD+ did not prevent the reduction in fatty acid oxidation after ethanol feeding, suggesting that nucleotide or cofactor depletion is not a factor. Acetaldehyde (0.6 to 3.0 mM) inhibited the oxidation of fatty acids by rat liver mitochondria, as assayed by oxygen consumption and CO, production (33a). Oxygen uptake was depressed by acetaldehyde in the presence and absence of fluorocitrate.
ADP-stimulated oxygen uptake was more sensitive to inhibition by acetaldehyde than was uncouplerstimulated oxygen uptake, and, as a consequence, acetaldehyde depressed the respiratory control ratio associated with the oxidation of fatty acids. Thus, there are many similarities in