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Department of Nutritional Sciences,
*
Department of Psychology and
Department of Biology, Rutgers University, New Brunswick, NJ 08901-0231
2To whom correspondence should be addressed. E-mail: fisher{at}aesop.rutgers.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • blood alcohol • blood glucose • blood lactate • liver fat • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) made it possible to study alcohol dependence in rats and produced high blood alcohol levels and substantial liver fat accumulation. Rao and associates (2
–4
) suggested that the fatty liver and high blood alcohol levels in rats fed the Lieber-DeCarli diet were the result of nutritional inadequacies rather than alcohol per se. Work from this laboratory (5
,6
) helped clarify this issue by showing that high carbohydrate, low fat diets ameliorated the effects of alcohol compared with high fat, low carbohydrate diets but did not prevent withdrawal seizures or elevated blood alcohol and liver fat levels when alcohol was provided in excess of 30% of dietary energy. Several other groups have also reported beneficial effects of high carbohydrate, low fat diets on ethanol-induced fatty livers (7
–10
). Some of these studies suggested that in rats consuming a low carbohydrate (high fat) diet, ethanol induces high levels of cytochrome P-450 2E1 (CYP2E1) which, in turn, generates reactive oxygen species and other free radicals from ethanol and fatty acid metabolism. These substances are then thought to be responsible for the liver damage and fat accumulation (9
,10
). However, Badger’s group (9
), as well as Tsukamoto et al. (11
), concluded that high carbohydrate diets will not prevent ethanol-induced fatty liver. Questions remain concerning whether high carbohydrate diets can reverse the deleterious effects of ethanol on the liver and whether protective effects of carbohydrate might be secondary to altered absorption of alcohol or induction of metabolic enzymes. The present experiments were undertaken to study more extensively the relationship of isocaloric high fat and high carbohydrate diets to ethanol utilization when ethanol is administered either chronically or acutely. Furthermore, the effect of switching rats from high fat to high carbohydrate diets with the uninterrupted consumption of a high level of ethanol was also studied. Liver fat and plasma ethanol, glucose and lactate were measured.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Male, Long-Evans rats, weighing 120–130 g, were obtained from Charles River Laboratories (Wilmington, MA). The rats were housed individually in a room at constant temperature (21–23°C) with a 12-h light:dark cycle. In all experiments in which blood was taken at the end of the experiment for ethanol, glucose and lactate analyses, the cycle was reversed, i.e., light from 1900 to 0700 h, and dark from 0700 to 1900 h. All experiments were conducted as part of an approved protocol for animal care by the Office of Research and Sponsored Programs, Rutgers University.
Experimental diet.
This was an improved version of the original Lieber-DeCarli diet (1
), given as a liquid suspension as previously reported by Mirovsky et al. (12
). Each liter of this diet contained the following ingredients common to all diets (referred to as "premix" in Table 1
) (g): casein, 42.0; DL-methionine, 0.6; salt mix (13
), 7.3; vitamin mix (13
,14
), 2.1; choline bitartrate, 0.4; cellulose, 1.0; xanthan gum, 3.0; sodium saccharin, 0.254; sucrose, 25.0. These ingredients, common to the four diets used in these experiments, were combined with varying amounts of corn oil, maltodextrin, ethanol and water as shown in Table 1
. Fresh mixtures of liquid diet were prepared daily and were offered in glass or plastic bottles designed to minimize spillage.
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In this experiment, 8 rats per treatment group, were fed high carbohydrate, low fat and high fat, low carbohydrate diets with or without alcohol for 2 wk. Measurements after killing included plasma ethanol, glucose and lactate and liver fat. The rats were given fresh food at 0700 h (when the lights went off) on the final day. This ensured food and alcohol consumption before killing 2 h later.
Experiment 2.
In this experiment, 16 rats consumed the high fat, low carbohydrate, ethanol-containing diet for 13 d. Of the 16 rats, 8 continued to consume this diet, and the other 8, of similar body weight, were switched to the low fat, high carbohydrate, ethanol-containing diet for 11 d. Plasma and liver metabolites in the 2 groups were compared.
Experiment 3.
Rats were fed ethanol-free, high carbohydrate, low fat and high fat, low carbohydrate diets and were administered a single, acute, intraperitoneal injection of ethanol (2 g/kg body) at the end of a 2-wk feeding period and 30, 60 and 120 min before killing.
Blood and liver analyses.
Trunk blood was taken after decapitation of the rats for the determination of plasma alcohol, glucose and lactate concentrations. For alcohol and lactate, blood was collected into heparinized capillary tubes which were then centrifuged at 500 x g for 25 min. Alcohol was determined in plasma by an enzyme assay (Sigma 333-UV, St. Louis, MO), and lactate was measured using Sigma procedure 735. Glucose was measured in whole blood applied to the One-Touch II Blood Glucose Monitor from Lifescan (Milpitas, CA).
The whole liver was removed, blotted to remove blood, weighed and frozen in liquid nitrogen and stored at -70°C until fat analysis. Duplicate pieces of 0.25 g frozen liver were cut up and homogenized with 10 mL of a 2:1 chloroform/methanol solution. The homogenate was vortexed for 15 s every 5 min for a total of 30 min. The homogenate was then filtered and the filter paper and filtrate washed with 5 mL chloroform/methanol solution. Then, 0.84 g/L KCl (0.2 x volume of solution) was added, followed by further vortexing and subsequent resting for 30 min. After removal of the top (water) layer, the bottom layer was poured into aluminum dishes for evaporation of solvent and weighing of the remaining fat.
Statistical analyses.
Two-way ANOVA was used to compare the data for Experiments 1 and 3, followed by post-hoc comparison of individual means by Fisher’s partial least square difference. Experiment 2 was analyzed by one-way ANOVA. Results are means ± SEM. Differences were considered significant at P 
0.05. In Experiment 3, because the lactate values for the 30- and 60-min groups within each diet treatment did not differ, they were combined for analysis to yield a larger sample size.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Daily energy intake by rats consuming ethanol was less than in their diet-matched controls; this was reflected in sharply lower weight gains (Table 2)
. The high fat, low carbohydrate, ethanol-fed group actually lost 12 g during the 2-wk experimental period.
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The liver fat concentration was highest in the high fat, low carbohydrate, ethanol-fed group, and lowest in the high carbohydrate, low fat, ethanol-free controls. The high carbohydrate, low fat, ethanol-supplemented rats had liver fat levels that did not differ from the rats fed the high fat, low carbohydrate, ethanol-free diet.
Experiment 2.
Energy and ethanol intakes and weight gain were greater in rats fed the high carbohydrate, low fat diet for the last 11 d of the experiment than in rats fed the high fat, low carbohydrate diet for the full 24 d (Table 3)
. The plasma ethanol concentration was lower in the rats that had the diet switched compared with the rats fed the high fat, low carbohydrate, ethanol-containing diet throughout. The liver fat concentration of the switched group was lower (P = 0.0001) than that of the rats that had been fed the high fat, low carbohydrate, ethanol-containing diet throughout. The importance of these findings is accentuated by the fact that the rats switched to the high carbohydrate, low fat diet consumed significantly more alcohol during the 11 d that they consumed this diet than the rats that continued to be fed the high fat, low carbohydrate diet.
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The rats fed the high fat, low carbohydrate diet consumed less energy than those fed the high carbohydrate, low fat diet (273 ± 4 vs. 302 ± 4 kJ/d; P = 0.001). Concomitantly, the high fat, low carbohydrate diet-fed rats gained less weight, 58 ± 3 g/2 wk, than the high carbohydrate, low fat diet-fed rats, 70 ± 2 g/2 wk (P = 0.0016).
At 2 h after alcohol administration, the dietary carbohydrate effect on plasma ethanol was evident (Table 4)
. At 30 and 60 min postalcohol administration, the diet groups did not differ but at 120 min, rats fed the high fat, low carbohydrate diet had plasma ethanol levels 26% lower than at 60 min. By comparison, the plasma ethanol concentration in rats fed the high carbohydrate, low fat die decreased 58% between 60 and 120 min. Plasma glucose was not affected by diet or time after ethanol injection.
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, lactate concentrations were lower in the alcohol-injected rats fed the high fat, low carbohydrate diet, compared with those fed the high carbohydrate, low fat diet. These differences were noted at 120 min after alcohol injection, but existed also (Table 4)
in the comparison of the combined 30 + 60 min values for the two diet treatments (P < 0.05). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and that of others (3
), that alcohol-consuming rats have much lower blood alcohol levels when given high carbohydrate diets than isocaloric, high fat diets. Sankaran et al. (15
) suggested that lower blood alcohol levels were essentially the result of an increase in total nonalcohol energy. Although this may be a contributing factor, the acute injection study (Table 4)
showed that a difference in metabolism due to type of diet is the main factor. The ethanol injections could not have induced the changes observed after only 2 h. They must have been produced by the preinjection consumption of the high carbohydrate and high fat diets.
This study extended our earlier observations to liver fat of chronically ethanol-administered rats. Liver fat was very low in rats fed high carbohydrate, low fat compared with high fat, low carbohydrate diets. Remarkably, in Experiment 1, the high carbohydrate, ethanol-fed rats had liver fat concentrations no higher than those in rats fed a high fat, low carbohydrate, control diet without ethanol. The results of Experiment 2 further accentuate the positive role of high carbohydrate intake in chronically alcohol-fed rats. The deleterious high blood ethanol and elevated liver fat values were lowered within 11 d of switching from a high fat to a high carbohydrate diet, even while consuming more alcohol after the switch (Table 3)
.
A protective effect of carbohydrate vis-à-vis fatty liver disease has been reported by several research teams (7
,9
,10
) and was also reviewed by Lieber (16
). Much of the focus in the cited studies concerns the role played by CYP2E1, an enzyme system that appears to be strongly induced by ethanol in animals fed low carbohydrate diets. The liver damage has been proposed to be caused by the CYP2E1 metabolism of ethanol, which yields reactive oxygen species and other free radicals. Support for this comes from a study by Morimoto et al. (17
), who observed that CYP2E1 inhibitors partially ameliorated changes in hepatic fatty acid composition of rats that chronically consumed ethanol and high fat diets. On the other hand, Kono et al. (18
) recently reported in CYP2E1 knockout (-/-) mice that CYP2E1 is not involved in early, alcohol-induced liver injury. Our findings, although not directly related to CYP2E1, do cast some doubt on the importance of this hypothesis. It would be hard to explain the reversal in liver fat accumulation and blood ethanol level upon switching from the high fat to the high carbohydrate diet. In particular, the sharp reduction in blood ethanol in rats fed the high carbohydrate diet only 2 h after a single alcohol injection is not likely due to the induction of CYP2E1 by ethanol.
Tsutsumi and Takase (19
), in reviewing possible causes of alcohol-induced fatty liver, mention a mechanism proposed by Lieber, in which ethanol becomes "a preferred fuel for the liver and displaces fat as a source of energy, contributing to fat accumulation." The present study casts doubt on this explanation because the rats with the fatty livers also had much higher blood alcohol levels when fed high fat rather than high carbohydrate diets (Experiment 1, Table 2
).
Blood glucose levels were significantly lower in rats fed the high fat, ethanol-containing diet (Table 2)
, whereas rats fed the high fat diet after ethanol injection had lower lactate levels (Table 4)
. These results suggest that ethanol administration exacerbates a diet-related inhibition of glucose metabolism that may be related to the accumulations of ethanol in blood and, in turn, fat in the liver.
The current measures of glucose and lactate in plasma, although indirect indications of liver function, are generally consistent with more direct measures. Walker and Gordon (20
) found a profound depletion of liver glycogen in rats after chronic ethanol feeding, a finding that is reflected in altered expression of glucose transporters (21
). In the current study, the lowered levels of blood glucose after chronic ethanol intake are consistent with the low availability of liver glycogen. Chronic feeding of ethanol to rats has previously been found to lower levels of lactate and pyruvate in subsequently isolated hepatocytes (22
). A likely explanation for this is an inhibition of glycolysis. A decrease in glycolytic compared with gluconeogenic enzyme activity has been demonstrated in rats following acute ethanol administration (23
). In the current study, the differences in steady-state lactate production in liver were not apparent in blood, possibly due to other compensatory mechanisms. However, lactate was lowered after acute ethanol injection.
In summary, this study has advanced our understanding of alcohol-induced fatty liver in rats by showing the following: 1) an ethanol-containing high carbohydrate, low fat diet produces liver fat levels no higher than those in rats fed an isocaloric, high fat diet without ethanol; 2) the elevated liver fat accumulation in rats fed a high fat, ethanol-containing diet can be reversed by switching to a high carbohydrate diet without altering the ethanol intake of the rats; 3) blood ethanol, after a single intraperitoneal injection of ethanol, was reduced by more than half within 2 h in rats prefed a high carbohydrate, low fat compared with a high fat, low carbohydrate diet. This last finding should put to rest explanations involving a differential ethanol absorption rate by rats fed such diets. It also makes it unlikely that the differences are due to an ethanol effect on enzyme induction within such a short time after ethanol ingestion.
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FOOTNOTES |
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Manuscript received 21 November 2001. Initial review completed 14 April 2002. Revision accepted 24 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Lieber, C. S., Jones, D. P. & DeCarli, L. M. (1965) Effects of prolonged ethanol intake: production of fatty liver despite adequate diets. J. Clin. Invest. 44:1009-10021.
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15. Sankaran, H., Baba, G. C., Deveney, C. W. & Rao, G. A. (1991) Enteral macronutrients abolish high blood alcohol levels in chronic alcoholic rats. Nutr. Res. 11:217-222.
16. Lieber, C. S. (1997) Cytochrome P-450 2E1: its physiological and pathological role. Physiol. Rev. 77:517-544.
17. Morimoto, M., Reitz, R. C., Morin, R. J., Nguyen, K., Ingelman-Sundberg, M. & French, S. W. (1995) CYP 2E1 inhibitors partially ameliorate the changes in hepatic fatty acid composition induced in rats by chronic administration of ethanol and a high fat diet. J. Nutr. 125:2953-2964.
18. Kono, H., Bradford, B. U., Yin, M., Sulik, K. K., Koop, D. R., Peters, J. M., Gonzalez, J., McDonald, T., Dikalova, A., Kadiiska, M. B., Mason, R. P. & Thurman, R. G. (1999) CYP 2E1 is not involved in early alcohol-induced liver injury. Am. J. Physiol. 277:G1259-G1267.
19. Tsutsumi, M. & Takase, S. (2001) Effect of fenofibrate on fatty liver in rats treated with alcohol. Alcohol. Clin. Exp. Res. 25:75S-79S.[Medline]
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23. Duruibe, V. & Tejwani, G. A. (1981) The effect of ethanol on the activities of the key gluconeogenic and glycolytic enzymes of rat liver. Mol. Pharmacol. 20:621-630.
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