Congenital Hypermetabolism and Uncoupled Oxidative Phosphorylation.

We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the ß subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism. (Funded by the National Institutes of Health.).

Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome.

Chronic ethanol feeding is known to negatively impact hepatic energy metabolism. Previous studies have indicated that the underlying lesion responsible for this may lie at the level of the mitoribosome. The aim of this study was to characterize the structure of the hepatic mitoribosome in alcoholic male rats and their isocalorically paired controls. Our experiments revealed that chronic ethanol feeding resulted in a significant depletion of both structural (death-associated protein 3) and functional [elongation factor thermo unstable (EF-Tu)] mitoribosomal proteins. In addition, significant increases were found in nucleotide elongation factor thermo stable (EF-Ts) and structural mitochondrial ribosomal protein L12 (MRPL12). The increase in MRPL12 was found to correlate with an increase in the levels of the 39S large mitoribosomal subunit. These changes were accompanied by decreased levels of nuclear- and mitochondrially encoded respiratory subunits, decreased amounts of intact respiratory complexes, decreased hepatic ATP levels, and depressed mitochondrial translation. Mathematical modeling of ethanol-mediated changes in EF-Tu and EF-Ts using prederived kinetic data predicted that the ethanol-mediated decrease in EF-Tu levels could completely account for the impaired mitochondrial protein synthesis. In conclusion, chronic ethanol feeding results in a depletion of mitochondrial EF-Tu levels within the liver that is mathematically predicted to be responsible for the impaired mitochondrial protein synthesis seen in alcoholic animals.

S-adenosyl-L-methionine co-administration prevents the ethanol-elicited dissociation of hepatic mitochondrial ribosomes in male rats.

BACKGROUND: Chronic ethanol feeding to male rats has been shown to result in decreased mitochondrial translation, depressed respiratory complex levels and mitochondrial respiration rates. In addition, ethanol consumption has been shown to result in an increased dissociation of mitoribosomes. S-adenosyl-L-methionine (SAM) is required for the assembly and subsequent stability of mitoribosomes and is depleted during chronic ethanol feeding. The ability of dietary SAM co-administration to prevent these ethanol-elicited lesions was investigated. METHODS: Male Sprague-Dawley rats were fed a nutritionally adequate liquid diet with ethanol comprising 36% of the calories according to a pair-fed design for 28 days. For some animals, SAM was supplemented in the diet at 200 mg/l. Liver mitochondria were prepared and mitoribosomes isolated. Respiration rates, ATP levels, respiratory complex levels, and the extent of mitoribosome dissociation were determined. RESULTS: Twenty-eight days of ethanol feeding were found to result in decreased SAM content, depressed respiration, and increased mitoribosome dissociation. No changes in mitochondrial protein content; levels of respiratory complexes I, III, and V; complex I activities; and ATP levels were detected. Co-administration of SAM in the diet was found to prevent ethanol-induced SAM depletion, respiration decreases and mitoribosome dissociation. CONCLUSIONS: Taken together, these findings suggest (1) that mitoribosome dissociation precedes respiratory complex depressions in alcoholic animals and (2) that dietary supplementation of SAM prevents some of the early mitochondrial lesions associated with chronic ethanol consumption.

Alcoholic liver disease and the mitochondrial ribosome: methods of analysis.

Chronic alcohol consumption has been shown to severely compromise mitochondrial protein synthesis. Hepatic mitochondria isolated from alcoholic animals contain decreased levels of respiratory complexes and display depressed respiration rates when compared to pair-fed controls. One underlying mechanism for this involves ethanol-elicited alterations in the structural and functional integrity of the mitochondrial ribosome. Ethanol feeding results in ribosomal changes that include decreased sedimentation rates, larger hydrodynamic volumes, increased levels of unassociated subunits and changes in the levels of specific ribosomal proteins. The methods presented in this chapter detail how to isolate mitochondrial ribosomes, determine ribosomal activity, separate ribosomes into nucleic acid and protein, and perform two-dimensional nonequilibrium pH gradient electrophoretic polyacrylamide gel electrophoresis to separate and subsequently identify mitochondrial ribosomal proteins.

A Ca2+-induced mitochondrial permeability transition causes complete release of rat liver endonuclease G activity from its exclusive location within the mitochondrial intermembrane space. Identification of a novel endo-exonuclease activity residing within the mitochondrial matrix.

Endonuclease G, a protein historically thought to be involved in mitochondrial DNA (mtDNA) replication, repair, recombination and degradation, has recently been reported to be involved in nuclear DNA degradation during the apoptotic process. As a result, its involvement in mtDNA homeostasis has been called into question and has necessitated detailed analyses of its precise location within the mitochondrion. Data is presented localizing rat liver endonuclease G activity exclusively to the mitochondrial intermembrane space with no activity associated with either the interior face of the inner mitochondrial membrane or with the mitochondrial matrix. Additionally, it is shown that endonuclease G can be selectively released from the mitochondrion via induction of a Ca2+-induced mitochondrial permeability transition and that, upon its release, a further nuclease activity loosely associated with the interior face of the inner mitochondrial membrane and distinct in its properties from that of endonuclease G can be detected.

Ethanol feeding enhances age-related deterioration of the rat hepatic mitochondrion.

Chronic ethanol feeding damages the hepatic mitochondrion by increasing mitochondrial DNA (mtDNA) oxidation, lowering mtDNA yields and impairing mitochondrial respiration. These effects are also seen during aging. By employing a 21-day chronic feeding regimen, we investigated the effects of ethanol consumption on mtDNA content and mitochondrial respiration in 2-, 12-, and 24-mo-old male rats. Aging resulted in decreased mtDNA content, increased mtDNA damage (as indicated by inhibition of Taq polymerase progression), and a decline in state 3 respiration; effects that were further exacerbated by ethanol feeding. Additionally, ethanol consumption caused an increase in the levels of citrate synthase while not impacting mitochondrial protein content. In conclusion, ethanol and aging combine to cause deterioration in the structural and functional integrity of the hepatic mitochondrion. The additive effects of aging and ethanol feeding may have serious consequences for hepatic energy metabolism in aged animals, and their detrimental combination may serve as one of the molecular mechanisms underlying the progression of alcoholic liver disease.

Alcohol and mitochondria: a dysfunctional relationship.

Mitochondria are intimately involved in the generation of and defense against reactive oxygen species (ROS). Mitochondria are themselves targets of oxidative stress and also contribute to mechanisms by which oxidative stress-related signals control cell fate. Ethanol promotes oxidative stress, both by increasing ROS formation and by decreasing cellular defense mechanisms. These effects of ethanol are prominent in the liver, the major site of ethanol metabolism in the body. The question remains to what extent this contributes to ethanol-dependent tissue damage or the susceptibility of cells to other stressors. In this review, we consider how mitochondrial actions of ethanol influence oxidative stress management of liver cells. Mitochondrial electron transport constitutes the major intracellular source of ROS, and ethanol treatment imposes conditions that promote ROS formation by mitochondria, the effects of which may be enhanced by a decrease in mitochondrial oxidative stress defenses. A significant target of ethanol-related increases in oxidative stress is mitochondrial DNA. Ethanol-induced damage to mitochondrial DNA, if not adequately repaired, impairs mitochondrial function, which further increases oxidative stress in the cell, leading to a vicious cycle of accumulating cell damage that is more apparent with advancing age. Uncontrolled mitochondrial formation of ROS promotes the inappropriate activation of the mitochondrial permeability transition, increasing the sensitivity of cells to other pro-apoptotic or damage signals. In combination with ethanol-induced defects in mitochondrial function, these alterations may promote both apoptotic and necrotic cell death in response to otherwise benign or beneficial challenges and contribute to the onset or progression of alcohol-induced liver diseases.

Effects of alcohol and oxidative stress on liver pathology: the role of the mitochondrion.

This article represents the proceedings of a symposium at the 2001 Research Society on Alcoholism meeting in Montreal, Canada. The chairs were Alan Cahill and Carol C. Cunningham. The presentations were (1) Mitochondrial regulation of ethanol-induced hepatocyte apoptosis: possible involvement of proapoptotic Bcl-2 family protein Bax, by Masayuki Adachi and Hiromasa Ishii; (2) Effects of ethanol on mitochondrial reactive oxygen species production and oxidative protein modification, by Shannon M. Bailey; (3) Acute ethanol binges elicit widespread oxidative mitochondrial DNA damage and depletion: protective effects of antioxidants and inhibitors of ethanol metabolism, by Bernard Fromenty; and (4) Effects of chronic ethanol consumption upon hepatic mtDNA oxidative modification and depletion, by Alan Cahill and Adrian Davies.