Ethanol in vitro does not affect the oxidative processes of hepatocyte mitochondria upon glucose, pyruvate, or monomethyl succinate oxidation

Most of the alcohol that enters the body is metabolized in the liver. In the case when the rate of ethanol oxidation is lower than the rate of its intake, the accumulation of ethanol leads to liver cell damage. Since reduced forms of NADH are formed in ethanol oxidation, mitochondrial respiration plays an important role in alcohol metabolism by regenerating NAD+, which regulates the level of both ethanol oxidation enzymes and is necessary for ethanol-acetaldehyde metabolism. The aim of the study was to investigate the effect of glucose, pyruvate and monomethyl succinate on the oxidative processes of hepatocyte mitochondria under the influence of ethanol in vitro. Experiments were performed on male Wistar rats weighing 220–250 g. Isolation of hepatocytes was carried out by the two-stage Seglen method. The oxygen consumption rate was determined with a Clark electrode. Hepatocytes were incubated for 60 min with ethanol (50 mM) in a medium with glucose (10 mmol/L), pyruvate, or monomethyl succinate (2 mmol/L each. An Olympus IX73 fluorescent microscope with a DP-74 digital camera was used to study mitochondrial membrane potential and NADH autofluorescence. Ethanol did not affect the basal and maximal FCCP-stimulated respiration of hepatocytes, membrane potential, and NADH autofluorescence upon glucose, pyruvate, or monomethyl succinate oxidation. Monomethyl succinate increased basal and FCCP-uncoupled respiration, both in the presence and absence of ethanol. The analysis confirmed the influence of the oxidative substrates on the NADH autofluorescence of hepatocytes. The presence of monomethyl succinate ameliorated the FCCP-induced reduction of NADH autofluorescence of hepatocytes. When mitochondrial complex I was inhibited with rotenone, pyruvate caused a decrease of NADH-autofluorescence of hepatocytes, compared to glucose or monomethyl succinate presence. Ethanol in vitro does not cause hypermetabolic state in mitochondria of isolated hepatocytes irrespectively of pyruvate or monomethyl succinate presence.