Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes

Abstract

Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (˙NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since ˙NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to ˙NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS−/− mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS−/− mice suggesting that increased mitochondrial sensitivity to ˙NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of ˙NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.

Binge alcohol promotes hypoxic liver injury through a CYP2E1–HIF-1α-dependent apoptosis pathway in mice and humans

“…Particularly, significant relationships among BAC, CYP2E1, HIF-1α andiNOS provide evidence about the involvement of HIF-1α in binge alcohol-induced iNOS induction and subsequent events such as protein nitration under hypoxia, at least partially, in binge alcohol-exposed humans and mice. Although not conducted in this study, it is likely that increased protein nitration, that is likely mediated by the increased CYP2E1 and iNOS protein levels, in different sub-cellular fractions might be also involved in inactivation of their functions, contributing to mitochondrial dysfunction, endoplasmic reticulum stress, and apoptosis in alcoholexposure, as recently demonstrated [17,52,53]. …”

“…In hypoxia, HIF-1α can stimulate iNOS expression [13]. It has been also shown that hepatic Kupffer cell activation following ethanol exposure increased iNOS expression [52]. The NO produced from iNOS might have damaged the mitochondrial respiratory chain, contributing to hypoxia, as described [52].…”

Micromolar changes in lysophosphatidylcholine concentration cause minor effects on mitochondrial permeability but major alterations in function

“…Primary mouse hepatocytes were then plated on a collagen-coated 96-well Seahorse (Seahorse Bioscience) plate at a density of 5000 cells/well. The cells were allowed to adhere overnight and then treated with 50 μM oleate and indicated leels of LPC, 10 μg/mL oligomycin, 3 μM carbonyl cyanide 4-(trifluoromethoxy) phenyhydrazone (FCCP) and 4 μM antimycin A/1 μM rotenone to measure oxidation changes and construct mitochondrial bioenergetics profiles [19-21]. Basal oxygen consumption rate (OCR) was established prior to injection of oleate and LPC.…”

Role of CYP2E1 in Mitochondrial Dysfunction and Hepatic Injury by Alcohol and Non-Alcoholic Substances

“…In addition, iNOS -null mice were partially protected from hypoxia-induced mitochondrial dysfunction, whereas WT mice exhibited increased mitochondrial dysfunction following ethanol exposure. Under hypoxia, HIF-1α also regulates iNOS [74, 76]. Further, CYP2E1 has been involved in the induction of hypoxia and the resultant nitroxidative stress, as evidenced by the results observed with Cyp2e1 -null mice where they exhibited lower levels of hypoxia, HIF-1α, fatty liver, and apoptosis, implying a role of CYP2E1 in mediating ethanol-induced hypoxic liver damage [66, 77, 78].…”