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 CO2 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 beta 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 beta oxidation to the level of acetyl-CoA is not impaired by chronic ethanol consumption. Total oxidation of fatty acids to CO2 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 vitro 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 CO2. However, oxidation of fatty acids to acetyl-CoA is not decreased by chronic ethanol consumption.     

Effect of chronic ethanol ingestion on phosphate content of neurofilament proteins and neurofilament associated protein phosphatase in rat spinal cord

Rats were trained to drink alcohol solution by gradually increasing the ethanol content [2.5–15% (v/v)] in drinking water. After 11 months of alcohol (15% v/v) ingestion, animals were guillotined and the spinal cords were used for the preparation of neurofilaments (NF). NF triplet proteins were separated by SDS-PAGE and the phosphate contents of individual components were estimated. Results indicated a significant increase in phosphate content of 200 KD protein in alcohol fed rats (30.19±4.12 mol of phosphate/mole of protein: p<0.001) compared to control group (18.42 ±3.91 mol of phosphate/mole of protein). No significant change in the phosphate content of 150KD and 68KD components of NF were seen in experimental group. Further, the studies on NF associated protein phosphatase activity indicated a significant decrease in phosphatase activity among the alcohol fed rats (14.10±2.5 mU; p<0.001) against NF rich fraction as a substrate, as compared to control (20.15±2.15 mU). While the observed decrease in NF associated protein phosphatase would possibly explain the increase in phosphate content of NF proteins in alcohol fed rats, the precise mechanism of decrease in enzyme activity remains to be elucidated. Nevertheless, the change seen in phosphate content and NF associated protein phosphatase activity as a result of ethanol ingestion would possibly form the biochemical basis of some of the neuropathological changes seen in alcoholics.     

Dopamine receptor function after chronic ingestion of ethanol.

Investigations of physiologic, behavioral and biochemical correlates of CNS dopamine receptor function indicate that chronic ingestion of ethanol by mice results in “sub-sensitive” dopamine receptors.     

Effect of spontaneous ingestion of ethanol on brain dopamine metabolism

The effect of ethanol, either administered by gavage or voluntarily ingested, on brain dopamine (DA) metabolism was studied in alcohol-preferring and alcohol non-preferring rats. In alcohol non-preferring rats ethanol administration (2 g/kg) increased 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and reduced DA levels in the caudate nucleus and olfactory tubercle but was ineffective in the medial prefrontal cortex. In alcohol-preferring rats ethanol effect was greater than in non-preferring animals and ethanol influenced DA metabolism also in the medial prefrontal cortex. The effect of voluntary ethanol ingestion was studied in alcohol-preferring rats trained to consume their daily fluid intake within 2 hrs. Voluntary ingestion of ethanol (3.1 +/- 0.7 g/kg in 1 hr) increased DA metabolites and reduced DA levels in the caudate nucleus, olfactory tubercle and medial prefrontal cortex. The results suggest that voluntary ethanol ingestion increases the release of DA from nigro-striatal and meso-limbic DA neurons.     

Effects of chronic ethanol ingestion on steroid profiles in the rat testis

Testosterone, seven of its potential precursors, three of its metabolites and estradiol were analyzed in testes from rats given ethanol for 23 days in a nutritionally adequate liquid diet. The results were compared to those obtained with pair-fed control rats. The concentrations of pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were markedly lowered in four of the five rats given ethanol. The concentrations of the other 3 beta-hydroxy-delta 5 steroids and estradiol were unchanged, resulting in significantly increased ratios between 17-hydroxypregnenolone and 17-hydroxyprogesterone (P less than 0.025) and between androstenediol and testosterone (P less than 0.025) in the ethanol-treated rats. The results indicate that chronic ethanol administration reduces formation of testosterone by affecting a step prior to pregnenolone. There may also be an effect on the conversion of some 3 beta-hydroxy-delta 5 to the corresponding 3-oxo-delta 4 steroids. The levels of testosterone and three other steroids in testes of rats given the liquid diet were significantly lower than those in testes of animals fed a standard rat chow. This indicates a dietary influence on testicular steroid concentrations.     

Effects of chronic ethanol ingestion on glucose homeostasis in males and females

Liver glycogen levels were decreased about 61% in both males and females after chronic ethanol ingestion. In contrast, blood glucose concentration was decreased only in males. In an attempt to explain these data, six enzymes involved with the metabolism of glucose and glycogen were measured. Sex differences were found in the effects of ethanol on the activities of glucose-6-phosphate dehydrogenase, UDPG- pyrophosphorylase and phosphoglucomutase. Glucose-6-phosphate dehydrogenase and UDPG-pyrophosphorylase were decreased by ethanol in males 42 and 62%, respectively. Females were not changed. Phosphoglucomutase was decreased 29% in females, and males were not changed. Glucose-6-phosphatase was increased and hexokinase and phosphorylase $\text{a}$ were decreased about the same extent in both sexes by ethanol. The data suggest that both sexes have been altered in such a manner as to keep liver glucose in the unphosphorylated form and thus readily available to other tissues; however, the mechanism appears to be different between the sexes. The decrease in liver glycogen may be explained by an increased utilization of glucose by peripherial tissues; however, the lack of change in blood glucose in females argues against this.     

The effect of chronic ethanol ingestion on protein metabolism in type-I- and type-II-fibre-rich skeletal muscles of the rat.

1. The effects of chronic ethanol feeding on muscles containing a predominance of either Type I (aerobic, slow-twitch) or Type II (anaerobic, fast-twitch) fibres were studied. Male Wistar rats, weighing approx. 90 g or 280 g, were pair-fed on a nutritionally complete liquid diet containing 36% of total energy as ethanol, or isovolumetric amounts of the same diet in which ethanol was replaced by isoenergetic glucose. After 6 weeks feeding, fractional rates of protein synthesis were measured with a flooding dose of L-[4-(3)H]-phenylalanine and muscles were analysed for protein, RNA and DNA. 2. Ethanol feeding decreased muscle weight, protein, RNA and DNA contents in both small and large rats. Type-II-fibre-rich muscles showed greater changes than did Type-I-fibre-rich muscles. Changes in protein paralleled decreases in DNA. 3. The capacity for protein synthesis (RNA/protein), fractional rates of protein synthesis and absolute rates of protein synthesis were decreased by ethanol feeding in both small and large rats. The amounts of protein synthesized relative to RNA and DNA were also decreased. Changes were less marked in Type-I than in Type-II-fibre-rich muscles. Loss of protein, RNA and DNA was greater in small rats, but protein synthesis was more markedly affected in large rats. 4. It was concluded that chronic ethanol feeding adversely affects protein metabolism in skeletal muscle. Fibre composition and animal size are also important factors in determining the pattern of response.     

Acute and chronic ethanol consumption differentially impact pathways limiting hepatic protein synthesis

This review identifies the various pathways responsible for modulating hepatic protein synthesis following acute and chronic alcohol intoxication and describes the mechanism(s) responsible for these changes. Alcohol intoxication induces a defect in global protein synthetic rates that is localized to impaired translation of mRNA at the level of peptide-chain initiation. Translation initiation is regulated at two steps: formation of the 43S preinitiation complex [controlled by eukaryotic initiation factors 2 (eIF2) and 2B (eIF2B)] and the binding of mRNA to the 40S ribosome (controlled by the eIF4F complex). To date, alcohol-induced alterations in eIF2 and eIF2B content and activity are best investigated. Ethanol decreases eIF2B activity when ingested either acutely or chronically. The reduced eIF2B activity most likely is a consequence of twofold increased phosphorylation of the alpha-subunit of eIF2 on Ser(51) following acute intoxication. The increase in eIF2alpha phosphorylation after chronic alcohol consumption is the same as that induced by acute ethanol intoxication, and protein synthesis is not further reduced by long-term alcohol ingestion despite additional reduced expression of initiation factors and elongation factors. eIF2alpha phosphorylation alone appears sufficient to maximally inhibit hepatic protein synthesis. Indeed, pretreatment with Salubrinal, an inhibitor of eIF2alpha(P) phosphatase, before ethanol treatment does not further inhibit protein synthesis or increase eIF2alpha phosphorylation, suggesting that acute ethanol intoxication causes maximal eIF2alpha phosphorylation elevation and hepatic protein synthesis inhibition. Ethanol-induced inhibition of hepatic protein synthesis is not rapidly reversed by cessation of ethanol consumption. In conclusion, sustained eIF2alpha phosphorylation is a hallmark of excessive alcohol intake leading to inhibition of protein synthesis. Enhanced phosphorylation of eIF2alpha represents a unique response of liver to alcohol intoxication, because the ethanol-induced elevation of eIF2alpha(P) is not observed in skeletal muscle or heart.     

Effect of ethanol ingestion on tryptophan metabolism in streptozotocin diabetic rats

Ingestion of ethanol by albino rats affected brain liver and plasma tryptophan contents in both normal and diabetic animals, although at different rates. Liver tryptophan was increased in both the groups, whereas tryptophan levels in brain and plasma of normal group were decreased and those of diabetic group were increased after the treatment. Similarly, while hepatic tryptophan dioxygenase activity was decreased in both the groups, activity of hepatic 3-hydroxykynureninase was increased only in normal rats and that of liver picolinic carboxylase was significantly decreased only in the diabetic group after ethanol administration.     

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA:cholesterol acyltransferase

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA: cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA: cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA: cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.     

The effect of chronic ethanol ingestion on synthesis and degradation of soluble, contractile and stromal protein fractions of skeletal muscles from immature and mature rats.

Both insulin and glucagon from the pancreas of the holocephalan cartilaginous fish Callorhynchus milii (elephantfish) have been isolated and purified. Two reverse-phase h.p.l.c. steps enabled recovery of sufficient material for gas-phase sequencing of the intact chains as well as peptide digestion products. The elephantfish insulin sequence shows 14 differences from pig insulin, including two unusual substitutions, Val-A14 and Gln-B30, though none of these is thought likely to influence receptor binding significantly. The insulin B-chain contains 31 residues, one more than mammalian insulins, but markedly less than that of the closely related ratfish with which it otherwise exhibits high sequence similarity. Elephantfish and pig glucagons differ at only four positions, but there are six changes from the ratfish glucagon-36 (normal glucagon contains 29 residues) sequence. It is apparent that different prohormone proteolytic processing mechanisms operate in the two holocephalan species.     

Effect of ethanol on the ultrastructure of pancreatic acini

Histological and ultrastructural changes of the acinar parenchyma of the pancreas under the influence of ethanol were studied on Wistar rats. Lipid inclusions in the cytoplasm of the acinar cells and mitochondrial changes were referred to the functional ones. The opinion of some investigators on the cytotoxic influence of alcohol on the pancreas was not confirmed by the author. The results of this investigation indicate that the leading role of alcohol in the etiology of acute pancreatitis is doubtful.     

Effect of prolonged ethanol ingestion on hepatic lipogenesis and related enzyme activities

1. Hepatic lipogenesis in vivo and the activities of enzymes associated with fatty acid synthesis in the liver were studied in rats fed for 21 days on liquid diets containing ethanol. 2. The ethanol-fed rats developed a moderate hepatic triacylglycerol accumulation during this period. When carbohydrate was replaced by ethanol in the diet, the rate of fatty acid synthesis was slower in the ethanol-fed rats on low-, medium- and high-fat diets than in the appropriate controls. However, when the fat/carbohydrate ratio was kept the same in the ethanol-fed and control rats, ethanol had no influence on the rate of fatty acid synthesis. 3. Glucose 6-phosphate dehydrogenase activity was lower in the ethanol-fed group. ;Malic' enzyme activity did not change during the ethanol treatment when the fat/carbohydrate ratio was kept unchanged. 4. The ATP citrate lyase activity was lower in the ethanol-fed rats on all diets, whereas acetyl-CoA synthetase activity was independent of the composition of the control diet, but was lower in the ethanol-fed rats, in which the concentration of the active form of pyruvate dehydrogenase was also lower. 5. It is concluded that hepatic fatty acid synthesis does not play any major role in ethanol-induced triacylglycerol accumulation. Careful design of the diets is necessary to reveal the specific effects of ethanol on the enzymes associated with lipogenesis.     

Effect of maternal ethanol consumption on foetal and neonatal rat hepatic protein synthesis.

Effects of maternal ethanol consumption were investigated on the rates of protein synthehsis by livers of foetal and neonatal rats both in vivo and in vitro, and on the activities of enzymes involved in protein synthesis and degradation. The rates of general protein synthesis by ribosomes in vitro studied by measuring the incorporation of [14C]leucine into ribosomal protein showed that maternal ethanol consumption resulted in an inhibition of the rates of protein synthesis by both foetal and neonatal livers from the ethanol-fed group. The rates of incorporation of intravenously injected [14C]leucine into hepatic proteins were also significantly lower in the foetal, neonatal and adult livers from the ethanol-fed group. Incubation of adult-rat liver slices with ethanol resulted in an inhibition of the incorporation of [14C]leucine into hepatic proteins; however, this effect was not observed in the foetal liver slices. This effect of externally added ethanol was at least partially prevented by the addition of pyrazole to the adult liver slices. Pyrazole addition to foetal liver slices was without significant effect on the rates of protein synthesis. Cross-mixing experiments showed that the capacity of both hepatic ribosomes and pH5 enzyme fractions to synthesize proteins was decreased in the foetal liver from the ethanol-fed group. Maternal ethanol consumption resulted in a decrease in hepatic total RNA content, RNA/DNA ratio and ribosomal protein content in the foetal liver. Foetal hepatic DNA content was not significantly affected. Ethanol consumption resulted in a significant decrease in proteolytic activity and the activity of tryptophan oxygenase in the foetal, neonatal and adult livers. It is possible that the mechanisms of inhibition of protein synthesis observed here in the foetal liver after maternal ethanol consumption may be responsible for at least some of the changes observed in 'foetal alcohol syndrome'.     

Effect of ethanol on protein synthesis in the tissues of various organs of guinea pigs

Long-term administration (for three months) of ethanol has been studied for its effect on the composition and synthesis of electrophoretic fractions of soluble proteins of the liver, brain, heart, kidneys and adrenals tissues in the guinea pigs. The obtained data permit supposing that the quantitative redistribution of the fractions under study as influenced by ethanol is due to their aggregation. Most of the studied tissues demonstrate a decrease in specific radioactivity of total proteins and 5 of 8-9 recorded electrophoretic fractions. Only one fraction in the heart and brain has been characterized by an increase of the protein synthesis level. The authors' results and data from literature make it possible to conclude that while analyzing pathogenesis of the chronic intoxication by ethanol and alcoholism the quantitative redistribution of soluble proteins and suppression of their synthesis in various tissues should be taken into account.