Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Diet-Sensitive Sources of Reactive Oxygen Species in Liver Mitochondria: Role of Very Long Chain Acyl-CoA Dehydrogenases

High fat diets and accompanying hepatic steatosis are highly prevalent conditions. Previous work has shown that steatosis is accompanied by enhanced generation of reactive oxygen species (ROS), which may mediate further liver damage. Here we investigated mechanisms leading to enhanced ROS generation following high fat diets (HFD). We found that mitochondria from HFD livers present no differences in maximal respiratory rates and coupling, but generate more ROS specifically when fatty acids are used as substrates. Indeed, many acyl-CoA dehydrogenase isoforms were found to be more highly expressed in HFD livers, although only the very long chain acyl-CoA dehydrogenase (VLCAD) was more functionally active. Studies conducted with permeabilized mitochondria and different chain length acyl-CoA derivatives suggest that VLCAD is also a source of ROS production in mitochondria of HFD animals. This production is stimulated by the lack of NAD⁺. Overall, our studies uncover VLCAD as a novel, diet-sensitive, source of mitochondrial ROS.     

Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

The effect of 4-hydroxy-2, 3-trans-pent-en-1-al and maleylaldehyde on some mitochondrial functions

Low concentrations of HPE and MLA inhibited state 3 respiration of rat liver mitochondria in the presence of different NAD⁺-dependent substrates. MLA appeared to be more active than HPE. High aldehyde concentrations inhibited the state 3 respiration with succinate. The restraint of succinate oxidation by HPE and MLA and of glutamate plus malate oxidation by MLA correlated with the inhibition of succinate and glutamate dehydrogenase activites, respectively. HPE inhibited glutamate dehydrogenase at concentrations higher than those affecting glutamate oxidation. Malate dehydrogenase activity was slightly sensitive to HPE and MLA. Both aldehydes inhibited NADH oxidation by freeze-thawed mitochondria. These results suggest the existence of a site particularly sensitive to aldehydes in the electron transport chain between the specific NAD⁺-linked dehydrogenases and ubiquinone.     

Respiratory Properties of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Respiratory activities were compared among rice seedlings germinated in air for 6 days (aerobic seedlings), those germinated under water for 5 days (submerged seedlings), and those grown in air for 1 day after 5 days' submerged germination (air-adapted seedlings). The respiratory activity of the submerged seedlings increased rapidly on transfer to air and reached a plateau at 16 hours in air. Respiration of the submerged seedlings was as sensitive to cyanide as those of aerobic and air-adapted seedlings. 2,4-Dinitrophenol had no effect on the respiration of the submerged seedlings, but stimulated those of the other two types of seedlings. Mitochondria from three types of seedlings did not differ in the ADP/O ratio and the respiratory control ratio (RCR) when succinate was oxidized. However, mitochondria from submerged seedlings (submerged mitochondria) showed poor RCR of about unity when malate was oxidized. Both the rate of succinate oxidation and succinate dehydrogenase activity were low in submerged mitochondria, but increased during air adaptation. Although submerged mitochondria oxidized malate very slowly, this activity increased after exposure to air without any increase in malate dehydrogenase activity. When NAD⁺ was added to submerged mitochondria, oxidation of malate was restored to the level of the aerobic controls. Addition of NAD⁺ enhanced the state 3 rate in submerged mitochondria, and RCR recovered to nearly the same value as that of the aerobic controls. Similar effects of NAD⁺ on 2-oxoglutarate oxidation were observed. All these defects in submerged mitochondria were repaired during air adaptation. These results suggest that NAD⁺-linked substrate oxidation was low in submerged mitochondria because of NAD⁺ deficiency, and that the oxidation increased with an increasing level of NAD⁺ during air adaptation.     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Vitamin E deficiency in the rat. IV. Alteration in mitochondrial membrane and its relation to respiratory decline

The respiratory control and rate of oxidation of exogenous NADH in vitro by liver mitochondria from vitamin E deficient rats were studied as a means of providing information concerning possible mitochondrial membrane alterations due to the deficiency.When mitochondria were aged at different temperatures for various periods of time, half-maximal inhibition of respiratory control occurred at lower temperatures and shorter aging periods in deficient mitochondria than in normal ones. Also, respiratory control was lost more rapidly in deficient mitochondria than in normal ones in the presence of either digitonin or low (hypotonic) concentrations of mannitol.Microsomes, both freshly prepared and boiled, dramatically lowered respiratory control and the effect was greater in the deficient mitochondria. Bovine serum albumin overcame the suppressed respiratory control, and exogenously added fatty acids mimiced the action of the microsomes.NADH oxidation by normal mitochondria proceeded slowly in isotonic media, while mitochondria of vitamin E deficient rats oxidized NADH much more rapidly. When mitochondria were subjected to ultrasonic disruption or incubated in hypotonic media, the rates of NADH oxidation by both types of mitochondria were similar.Respiratory decline associated with oxidation of β-hydroxybutyrate by the deficient mitochondria was decreased by including in the medium either a high concentration of NAD⁺, 0.5 mm oxalacetate, or 2 mm aspartate plus 1 mm α-ketoglutarate. This observation, plus the finding of similar activities of malate dehydrogenase and glutamic-oxalacetic transminase in normal and deficient livers, suggests that the action of each was due to an elevation of the mitochondrial NAD⁺/NADH ratio via a malate shuttle and cytoplasmic and mitochondrial glutamic-oxalacetate transaminase. It is postulated that the marked mitochondrial respiratory decline in the deficient rats is attributed to a limiting availability of NAD⁺ and a low ratio of NAD⁺ to NADH.     

On the mechanism of action of polyether XXVIII at site I of the electron-transfer chain in rat liver mitochondria

In rat liver mitochondria, the macrocyclic polyether, dibenzo-18-crown-6 (polyether XXVIII) inhibits the oxidation of NAD-dependent substrates, as stimulated by ADP, uncouplers and valinomycin plus K⁺. It does not inhibit the oxidation of succinate. It is concluded that polyether XXVIII inhibits electron transfer in the NADH-CoQ span of the respiratory chain. This is a process that is reversed by menadione. Inhibition of oxidation of NAD-dependent substrates in K⁺-depleted mitochondria induced by the polyether is reversed by concentrations of K⁺ higher than 60 mM, and also by Li⁺, a cation that does not complex with polyether XXVIII. As assayed by swelling mitochondria, reversal of the inhibition of electron transfer is accompanied by influx of monovalent cations. Polyether XXVIII also inhibits in submitochondrial particles the aerobic oxidation of NADH, but not that of succinate; this inhibition is also reversed by K⁺ at high concentrations, and Li⁺. The data are consistent with the hypothesis that a monovalent cation is required for maximal rates of electron transport in the NADH-CoQ span of the respiratory chain.     

Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism.     

Influence of cerebral ischemia and post-ischemic reperfusion on mitochondrial oxidative phosphorylation

Unilateral ischemia in the right cerebral hemisphere of the rat was induced by ligation of the right common carotid artery coupled with controlled hemorrhage to produce hypotension (25±8 mm/Hg). Where indicated after 30 min of ischemia, the withdrawn blood was reinfused to restore arterial pressure to normal. Mitochondria isolated from the ipsilateral hemisphere after 30 min of ischemia showed significantly lower respiratory rates than the organelles isolated from the contralateral side. Oxidation of NAD⁺-linked substrates was more sensitive to inhibition in ischemia (30%) than was of ferrocytochromec (12%), succinate oxidation being intermediate. The activities of membrane-bound dehydrogenases (both NADH and succinate-linked) were also significantly lowered. Ischemia did not affect the cytochrome content of mitochondria. Respiratory activity (NAD⁺-linked) of mitochondria isolated from the ipsilateral hemisphere was twice as sensitive to inhibition by fatty acid as was of preparations from the contralateral side. Mitochondria isolated from cerebral cortex after 90 min of post-ischemic reperfusion showed no significant improvement in the rate of substrate oxidation. Adenine nucleotide translocase activity and energy-dependent Ca²⁺ uptake, both of which decreased significantly in mitochondria isolated from the ischemic brain, showed little recovery, on reperfusion. These observations suggested the strong possibility that the deleterious effects of ischemia on mitochondrial respiratory function might be mediated by free fatty acids that are known to accumulate in large amounts in ischemic tissues. The pattern of inhibition of ATPase activity was consistent with this view.     

Effect of KCl-medium on succinate oxidation and hydrogen peroxide generation in mitochondria of sugar beet taproot

The effect of substitution of KCl for sucrose in the reaction medium on succinate oxidation and hydrogen peroxide generation was investigated in the mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.). In a sucrose-containing medium, oxidation of succinate was inhibited by oxaloacetate; this inhibition was especially pronounced upon a decrease in substrate concentration and eliminated in the presence of glutamate, which removed oxaloacetate in the course of transamination. Irrespective of succinate concentration, substitution of KCl for sucrose in the medium considerably enhanced suppression of succinate oxidation apparently as a result of slow activation of succinate dehydrogenase (SDH) by its substrate. In this case, mitochondria showed the symptoms of uncoupling, lower values of membrane potential (ΔΨ), respiratory control (RC), and ADP/O induced by electrophoretic transport of potassium via K⁺ channel of mitochondria. KCl-dependent suppression of succinate oxidation by taproot mitochondria was accompanied by a considerable inhibition of H₂O₂ production as compared with the sucrose-containing medium. These results indicate that in the presence of potassium ions, ΔΨ dissipates, suppression of succinate oxidation by oxaloacetate increases, and succinate-dependent generation of ROS in sugar beet mitochondria is inhibited. A possible physiological role of oxaloacetate-restricted SDH activity in the suppression of respiration of storage organs protecting mitochondria from oxidative stress is discussed.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca²⁺/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg²⁺-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD⁺ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD⁺ in the case of NAD⁺-linked substrates.     

MDH2 produced OAA is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD⁺ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD⁺ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the ‘respirasome free’ complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.     

Mechanisms of citrate oxidation by percoll-purified mitochondria from potato tuber

The mechanisms and accurate control of citrate oxidation by Percoll-purified potato (Solanum tuberosum) tuber mitochondria were characterized in various metabolic conditions by recording time course evolution of the citric acid cycle related intermediates and O₂ consumption. Intact potato tuber mitochondria showed good rates of citrate oxidation, provided that nonlimiting amounts of NAD⁺ and thiamine pyrophosphate were present in the matrix space. Addition of ATP increased initial oxidation rates, by activation of the energy-dependent net citrate uptake, and stimulated succinate and malate formation. When the intramitochondrial NADH to NAD⁺ ratio was high, α-ketoglutarate only was excreted from the matrix space. After addition of ADP, aspartate, or oxaloacetate, which decreased the NADH to NAD⁺ ratio, flux rates through the Krebs cycle dehydrogenases were strongly increased and α-ketoglutarate, succinate, and malate accumulated up to steady-state concentrations in the reaction medium. It was concluded that NADH to NAD⁺ ratio could be the primary signal for coordination of fluxes through electron transport chain or malate dehydrogenase and NAD⁺-linked Krebs cycle dehydrogenases. In addition, these results clearly showed that the tricarboxylic acid cycle could serve as an important source of carbon skeletons for extra-mitochondrial synthetic processes, according to supply and demand of metabolites.     

Brown adipose tissue mitochondria oxidizing fatty acids generate high levels of reactive oxygen species irrespective of the uncoupling protein-1 activity state

Mitochondria from brown adipose tissue (BATM) have a high enzymatic capacity for fatty acid oxidation and therefore are an ideal model to examine the sites of reactive oxygen species (ROS) generation during fatty acid oxidation. ROS generation by BATM (isolated from 3-week-old rats) was measured during acylcarnitine oxidation as release of H(2)O(2) into the medium and as inactivation of the matrix enzyme aconitase. The following results were obtained: (1) BATM release large amounts of H(2)O(2) in the coupled as well as in the uncoupled states, several times more than skeletal muscle mitochondria. (2) H(2)O(2) release is especially large with acylcarnitines of medium-chain fatty acids (e.g. octanoylcarnitine). (3) Reverse electron transport does not contribute in a significant extent to the overall ROS generation. (4) Despite the large release of H(2)O(2), the ROS-sensitive matrix enzyme aconitase is not inactivated during acylcarnitine oxidation. (5) In contrast to acylcarnitines, oxidation of α-glycerophosphate by BATM is characterized by large H(2)O(2) release and a pronounced aconitase inactivation. We hypothesize that acylcarnitine-supported ROS generation in BATM may be mainly associated with acyl-CoA dehydrogenase and electron transferring flavoprotein-ubiquinone reductase rather than with complexes of the respiratory chain.     

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance—ROS and PKCε activity—were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD⁺ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD⁺]/[NADH] ratio, indicating respiration-mediated control of the [NAD⁺]/[NADH] ratio. Supporting a prominent role for NAD⁺, increasing the concentration of NAD⁺ stimulated complete oxidation of fatty acids. Importantly, NAD⁺ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD⁺, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.     

Metabolism of N6,O2'-[3H]dibutyryl cyclic adenosine 3',5'-monophosphate and macromolecular interactions of the products perfused rat liver.

Mitochondria from liver, kidney, brain, and skeletal muscle metabolized acetaldehyde. Acetaldehyde oxidation by liver and kidney mitochondria was maximal at low levels of acetaldehyde and was sensitive to rotenone, suggesting the involvement of a NAD⁺-dependent aldehyde dehydrogenase with a high affinity for acetaldehyde. Acetaldehyde oxidation was stimulated 50% by ADP, suggesting that, in state 4, reoxidation of NADH is rate limiting for acetaldehyde oxidation. In state 4, acetaldehyde oxidation was decreased by NAD⁺-dependent substrates, as well as by succinate and ascorbate. The inhibition by the latter two substrates was prevented by ADP, dinitrophenol, valinomycin, and gramicidin, but not by oligomycin. Since these compounds are linked to energy transduction and utilization, the data suggest that the inhibition is mediated via energy-dependent reversed electron transport. In state 3, all of these substrates caused considerably less inhibition of acetaldehyde oxidation, suggesting that the activity of aldehyde dehydrogenase, and not of NADH reoxidation, is probably rate limiting for acetaldehyde oxidation. The ionophores valinomycin and gramicidin stimulated acetaldehyde oxidation to a greater extent than ADP. These ionophores also stimulated acetaldehyde oxidation in the presence of ADP. Stimulation by valinomycin occurred in the presence of monovalent cations transported by this ionophore, e.g., K⁺, Rb⁺, Cs⁺. Stimulation by gramicidin also occurred in the presence of these cations, but did not occur with Na⁺ or Li⁺. Na⁺ prevents the stimulation of acetaldehyde oxidation, which occurs in the presence of gramicidin and K⁺. The stimulation by valinomycin and gramicidin was energy dependent and required the presence of a permeant anion. In the absence of an ionophore, potassium phosphate had no effect on acetaldehyde oxidation. These data suggest that the oxidation of acetaldehyde by rat liver and kidney mitochondria is influenced by the oxidation-reduction state of the mitochondria and by the cationic environment. With brain and muscle mitochondria, the rate of acetaldehyde oxidation increased two- to threefold as the concentration of acetaldehyde was raised from 0.167 to 0.50 mm. Acetaldehyde oxidation in these mitochondria was also sensitive; to rotenone, indicating dependence on NAD⁺. ADP, valinomycin, gramicidin, and succinate, compounds which either increased or decreased the rate of acetaldehyde oxidation by liver and kidney mitochondria, had no effect on acetaldehyde oxidation by muscle or brain mitochondria. In state 4, mitochondria from Becker-transplantable hepatocellular carcinoma HC-252 oxidized acetaldehyde at the same rate as liver mitochondria. However, in the presence of ADP, dinitrophenol, valinomycin and gramicidin, the rate of acetaldehyde oxidation by the tumor mitochondria was two to three times greater than that of liver mitochondria, suggesting the presence of a more active; acetaldehyde-oxidizing system in tumor than in liver mitochondria.     

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver.

In isolated hepatic mitochondria, the oxidation of NAD⁺-dependent substrates was decreased after chronic consumption of ethanol or by the addition of acetaldehyde in vitro. Reversed electron transport from succinate to NAD^?, which requires transfer of electrons through the NADH dehydrogenase complex and energy transduction through coupling site 1, was depressed by ethanol feeding and by acetaldehyde in vitro, whereas NADH formation from glutamate, which is mediated directly by substrate oxidation and is not energy-dependent, was slightly increased. By contrast, reactions involving the terminal portion of the phosphorylation chain, e.g., ATP-³²P exchange or dinitrophenolstimulated ATPase activity, were not affected. Adenine nucleotide translocase activity was not altered by chronic consumption of ethanol or the addition of acetaldehyde in vitro. These data suggest that the NADH-ubiquinone oxidoreductase complex of the respiratory chain, a segment which contains several iron-sulfur centers which participate in electron transport and energy transduction, may be impaired by chronic consumption of ethanol and is especially sensitive to inhibition by acetaldehyde in vitro. Neither energy coupling sites II or III, nor the terminal reactions of oxidative phosphorylation share this sensitivity. CO₂ production from various labeled intermediates of the citric acid cycle was depressed after chronic consumption of ethanol and after the addition of acetaldehyde. Acetate had no effect on these reactions, indicating that the inhibition by acetaldehyde is not mediated via acetate. Impairment of the activities of the respiratory chain and the citric acid cycle, or both, may explain the decreases in oxygen uptake and CO₂ production from citric acid cycle intermediates and fatty acids, as well as the increase in ketone body production, found in mitochondria from ethanolfed rats.     

The physical and chemical properties of a chicken egg white glycoprotein purified by nondenaturing methodology

The oxidation of ethanol by the liver produces acetaldehyde, which is a highly reactive compound. Low concentrations of acetaldehyde inhibited mitochondrial respiration with glutamate, β-hydroxybutyrate, or α-ketoglutarate as substrates, but not with succinate or ascorbate. High concentrations led to respiratory inhibition with all substrates. Inhibition of succinate- and ascorbate-linked oxidation by acetaldehyde correlates with the inhibition of the activities of succinic dehydrogenase and cytochrome oxidase. A site more sensitive to acetaldehyde appears to be localized prior to the NADH-ubiquinone oxidoreductase segment of the respiratory chain. Acetaldehyde inhibits energy production by the mitochondria, as evidenced by its inhibition of respiratory control, oxidative phosphorylation, the rate of phosphorylation, and the ATP-³²P exchange reaction. Energy utilization is also inhibited, in view of the decrease in both substrate- and ATP-supported Ca²⁺ uptake, and the reduction in Ca²⁺-stimulated oxygen uptake and ATPase activity. The malate-aspartate, α-glycerophosphate, and fatty acid shuttles for the transfer of reducing equivalents, and oxidation by mitochondria, were highly sensitive to acetaldehyde. Acetaldehyde also inhibited the uptake of anions which participate in the shuttles. The inhibition of the shuttles is apparently caused by interference with NAD⁺-dependent state 3 respiration and anion entry and efflux. Ethanol (6–80 mm) had no significant effect on oxygen consumption, anion uptake, or mitochondrial energy production and utilization. The data suggest that acetaldehyde may be implicated in some of the toxic effects caused by chronic ethanol consumption.