The time-course development of the effects of ethanol ingestion on choline oxidase

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumim resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria is isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.     

Effect of ethanol intake on rat liver mitochondrial respiration and oxidative phosphorylation

The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats with a liquid isocaloric diet containing various concentrations of ethanol. We found that after feeding the liquid diet for 2 to 3 months, the body weight of rats did not show a significant difference between treated and control groups. However, the mitochondrial respiration rate decreased significantly with the increase of ethanol concentration in the diet. We found that when the rats were fed on 10.8% ethanol, the average succinate-supported State 3 respiration rate decreased from 54.5 to 44.8 nmol O2/min/mg and the glutamate-malate-supported State 3 respiration rate decreased from 38.8 to 23.6 nmol O2/min/mg as compared with the control. Interestingly, we noted that ethanol intake caused a more drastic effect on State 3 respiration than on State 4 respiration, irrespective of the substrate utilized by the mitochondria. In addition, the respiratory control and ADP/O ratios were found to decrease concomitantly with the increase of ethanol level in the diet. Moreover, we found that the effect of ethanol on both respiratory control and ADP/O ratios of liver mitochondria was more pronounced in glutamate-malate-supported respiration than succinate-supported respiration. These results clearly demonstrate that ethanol intake by the rat can cause impairment of liver mitochondrial respiration and oxidative phosphorylation, and that these effects are exerted through damage to mitochondrial membranes.     

Metabolic effects of ethanol in the rat liver.

The NADPH is one of the cofactors in ethanol metabolism. The aim of the study was to investigate the effect of ethanol on a NADPH generating enzyme (G6P-DH) and on some metabolic parameters of the liver. After a 2-day starvation period rats were fed a lipid free diet for three days. During this refeeding period the animals were divided into three groups; they received a single daily dose of 4 g per kg b.w. ethanol, isocaloric aqueous glucose solution or water by gastric tube. In response to ethanol the activity of hepatic G6P-DH decreased. The amount of triglyceride remained unchanged, certain changes occurred in the fatty acid composition of total lipid. The liver glycogen content was elevated. In female rats treated with ethanol the activity of glucose-6-phosphatase increased.     

Effects of ethanol-derived acetaldehyde on the phosphorylation potential and on the intramitochondrial redox state in intact rat liver

The role of acetaldehyde (AcH) in the ethanol-induced shift toward reduction of the cytosolic and mitochondrial free NAD⁺/free NADH ratios and its effect on the phosphorylation potential was investigated in livers of fed, intact rats given ethanol (1 g/kg ip). Calcium cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, was administered to block predominantly intramitochondrial NADH production from AcH oxidation. Compared with ethanol alone, cyanamide almost totally reversed the elevation of the β-OH-butyrate/acetoacetate ratio but only slightly reduced the lactate/ pyruvate ratio, which was calculated to be in near equilibrium with the hepatic ethanol/ AcH ratio after cyanamide. Ethanol or cyanamide alone had no effect on ATP, ADP, or P_i, but together they significantly decreased the $\text{ATP}\text{ADP}\text{· P}{}_{\mathrm{<mtext>i</mtext>}}$ ratio by increasing both ADP and P_i levels. No association between changes in the phosphorylation potential and the redox states was, however, observed. An ethanol-induced increase in AMP was abolished by cyanamide. The results demonstrate that the effect of ethanol on the mitochondrial redox state requires active AcH oxidation and suggest that moderate AcH accumulation likely to occur during alcohol-aversive drug treatment significantly lowers the cellular phosphorylation potential.     

Prevention by pyrazole of the effects of chronic ethanol administration on the redox states of the hepatic nicotinamide--adenine dinucleotide (phosphate) couples and on liver and brain tryptophan metabolism in the rat.

Chronic administration of pyrazole in the diet of rats does not cause toxicity and prevents the chronic effects of ethanol on: (1) the redox states of the hepatic NAD(P) couples; (2) liver tryptophan pyrrolase activity; (3) brain tryptophan and 5-hydroxytryptamine metabolism.     

Chronic ethanol ingestion increases efficiency of oxidative phosphorylation in rat liver mitochondria

The efficiency of oxidative phosphorylation was compared between rats chronically fed with ethanol and controls. (i) Results showed that the liver mitochondria state 4 respiratory rate was strongly inhibited, while the corresponding proton-motive force was not affected; (ii) the cytochrome oxidase content and activity were decreased and (iii) the oxidative-phosphorylation yield was increased in the ethanol exposed group. Furthermore, oxidative phosphorylation at coupling site II was not affected by ethanol. Cytochrome oxidase inhibition by sodium-azide mimicked the effects of ethanol intoxication in control mitochondria. This indicates that the decrease in cytochrome oxidase activity induced by ethanol intoxication directly increases the efficiency of oxidative phosphorylation.     

The effects of linoleate hydroperoxide on respiration and oxidative phosphorylation of rat liver mitochondria.

Linoleate hydropepoxide, purified by silica gel chromatography and at concentrations 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300 microM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than by the free acid.     

The effect of methylene blue on the hepatocellular redox state and liver lipid content during chronic ethanol feeding in the rat.

Feeding of ethanol in a liquid diet to male Wistar rats caused decreases in the hepatic cytosolic and mitochondrial [NAD+]/[NADH] ratios. This redox-state change was attenuated after 16 days of feeding ethanol as 36% of the total energy intake. Supplementation of the ethanol-containing liquid diet with Methylene Blue largely prevented the ethanol-induced redox state changes, but did not significantly decrease the severity of the hepatic lipid accumulation that resulted from ethanol ingestion. Methylene Blue did not affect body-weight gain, ethanol intake or serum ethanol concentrations in ethanol-fed rats, nor did the compound influence the hepatic redox state or liver lipid content of appropriate pair-fed control animals. These findings suggest that the altered hepatic redox state that results from ethanol oxidation is not primarily responsible for the production of fatty liver after long-term ethanol feeding in the rat.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.     

Kinetics of glycerol uptake by the perfused rat liver Membrane transport, phosphorylation and effect on NAD redox level

The kinetics of glycerol uptake by the perfused rat liver were determined according to a model which includes membrane transport, intracellular phosphorylation and competitive inhibition of glycerol phosphorylation by l-glycerol 3-phosphate. The membrane transport obeys first-order kinetics at concentrations below 10 mM in the affluent medium. The K_m of the glycerol phosphorylation was was 10 μM and the K_i of the l-glycerol 3-phosphate inhibition was 50 μM. The maximum activity (V) was 3.70 μmoles/min per g liver wet wt. These results are similar to in vitro kinetics of the glycerol kinase, except that K_i was found to be somewhat lower in the intact organ. At low glycerol concentrations, gradient exists across the liver cell membrane.The increase in the lactate to pyruvate concentration ratio during glycerol metabolism is related to the actual concentration of l-glycerol 3-phosphate, not to the rate of glycerol uptake.     

Utilization and metabolic effects of acetaldehyde and ethanol in the perfused rat liver

1. The formation of the two 16-unsaturated alcohols 5alpha-androst-16-en-3alpha-ol and 5alpha-androst-16-en-3beta-ol from [5alpha-(3)H]5alpha-androst-16-en-3-one has been demonstrated in boar testis homogenates. 2. The optimum yield (23%) of the 3alpha-alcohol was obtained in the presence of NADPH, whereas that for the 3beta-alcohol (74%) was obtained when NADH was the added cofactor. 3. The two alcohols were not interconvertible. 4. Prolonged storage of boar testis tissue at -20 degrees C abolished the ability to form all androst-16-enes except androsta-4,16-dien-3-one from [4-(14)C]progesterone. 5. The production of 5alpha-androst-16-en-3-one and the two alcohols from [7alpha-(3)H]androsta-4,16-dien-3-one only occurred when fresh tissue was used, whereas reduction of [5alpha-(3)H]5alpha-androst-16-en-3-one was unaffected by storage of testis at -20 degrees C. 6. NADPH was the preferred cofactor for the reduction of androsta-4,16-dien-3-one. 7. The previously established conversion of androsta-5,16-dien-3beta-ol into androsta-4,16-dien-3-one was shown to be reversible, NADH and NADPH being equally effective cofactors. 8. Pathways of biosynthesis of 5alpha-androst-16-en-3alpha- and 3beta-ols, with the C(19) 3-oxo steroids as intermediates, are presented.     

Biochemical effects of PR toxin on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria.