Simulation of the uncoupling activity of fatty acids with the participation of ADP/ATP and aspartate/glutamate antiporters in liver mitochondria

It has been found that the protonophoric specific uncoupling activity of palmitic acid in rat liver mitochondria does not change as its concentration increases from 5 to 40 μM. Under these conditions, the component of the specific uncoupling activity that describes the participation in uncoupling of the ADP/ATP antiporter (sensitive to carboxyatractylate) increases, and the component of specific uncoupling activity that characterizes the participation in the uncoupling of the aspartate/glutamate antiporter (sensitive to glutamate) decreases by the same value. A kinetic model of the fatty acid-induced uncoupling activity with the participation of ADP/ATP and aspartate/glutamate antiporters has been developed. According to the model, these carriers can exist in two forms: active, i.e., participating in the uncoupling, and inactive. The interaction of a fatty acid with the regulator site of the ADP/ATP antiporter translates it from the inactive to the active form, while the interaction of a fatty acid with the regulator site of the aspartate/glutamate antiporter, on the contrary, translates it from the active form to inactive. The velocity of transport of a fatty acid anion by the antiporter from the internal monolayer of the inner membrane to the external monolayer is proportional to the product of the concentration of the fatty acid and the active form of this carrier. A good conformity of the model to experimentally obtained data is shown provided that (a) ADP/ATP and aspartate/glutamate antiporters, being completely in active state, transfer fatty acid anions with the same velocity; (b) the equilibrium dissociation constants of a complex of the carrier with the fatty acid in these antiporters are equal.     

Simulation of the uncoupling activity of fatty acids with the participation of ADP/ATP and aspartate/glutamate antiporters in liver mitochondria

It has been found that the protonophoric specific uncoupling activity of palmitic acid in rat liver mitochondria does not change as its concentration increases from 5 to 40 microM. Under these conditions, the component of the specific uncoupling activity, which describes the participation in uncoupling of the ADP/ATP antiporter (sensitive to carboxyatractylate), increases, and the component of specific uncoupling activity, which characterizes the participation in the uncoupling of the aspartate/glutamate antiporter (sensitive to glutamate), decreases by the same value. A kinetic model of the fatty acid-induced uncoupling activity with the participation of ADP/ATP and aspartate/glutamate antiporters has been developed. According to the model, these carriers can exist in two forms: an active, i.e., participating in the uncoupling, and an inactive. The interaction of a fatty acid with the regulator site of the ADP/ATP antiporter translates it from the inactive to the active form, while the interaction of a fatty acid with the regulator site of the aspartate/glutamate antiporter, on the contrary, translates it from the active form to inactive. The velocity of transport of a fatty acid anion by the antiporter from the internal monolayer of the internal membrane to the external monolayer is proportional to the product of the concentration of the fatty acid and the active form of this carrier. A good conformity of the model to experimentally obtained data is shown provided that (a) ADP/ATP and aspartate/glutamate antiporters, being completely in an active state, transfer fatty acid anions with the same velocity; (b) the equilibrium dissociation constants of a complex of the carrier with the fatty acid in these antiporters are equal.     

Uncoupling activity of fatty acids in liver mitochondria in the presence of substrates of ADP/ATP- and aspartate/glutamate antiporters is increased under oxidative stress

It is shown that upon oxidation of succinate in the presence of rotenone and antioxidant Trolox (or pyruvate) in liver mitochondria of mature rats (9–12-month old) the respiration stimulated by palmitate is suppressed by ADP (the substrate of ADP/ATP-antiporter) and aspartate (the substrate of aspartate/glutamate antiporter). However, it was found that in the presence of the oxidative agent tert-butylhydroperoxide neither ADP nor aspartate is effective even at their joint action. In the presence of ADP and aspartate, uncoupling activity of palmitate is minimal, since the lipid peroxidation is inhibited by Trolox or pyruvate, and rises as the accumulation rate of conjugated dienes increases, reaching the maximal value at the oxidative stress caused by tert-butylhydroperoxide. In liver mitochondria of senile rats (22–26-month old) at high intensity of lipid peroxidation, ADP and aspartate do not affect the uncoupling activity of palmitate (Samartsev and Kozhina, 2008, Biochemistry (Mosc.), vol. 73, no. 7, pp. 783–790). Comparative studies have shown that in liver mitochondria of mature and senile rats at the similar accumulation rate of the conjugated dienes in the presence of ADP and aspartate, the uncoupling activity of palmitate reaches the same level relative to the maximal activity. We conclude that an enhancement of free radical reactions and lipid peroxidation in liver mitochondria can result in an increase of protonophore uncoupling activity of fatty acids with the involvement of ADP/ATP- and aspartate/glutamate antiporters due to the suppression of the ability of physiological substrates of these carriers of ADP and aspartate to inhibit the uncoupling process.     

Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.     

Participation of the ATP/ADP antiporter and fatty acids in oxidative phosphorylation uncoupling in squirrel liver mitochondria during winter hibernation and awakening]

The parameters of energy coupling of mitochondria isolated from the livers of hibernating and awakening gophers were studied. The ATP/ADP-antiporter inhibitor carboxyatractylate slowed down the respiration rate, increased delta psi and decreased the ionic conductivity of the inner mitochondrial membrane as measured by the rate of the delta psi decline after addition of cyanide (in the presence of oligomycin and EGTA). A similar effect was produced by BSA, carboxyatractylate being fairly ineffective in the presence of BSA. In hibernating gophers the maximal rate of the uncoupled respiration and the ionic conductivity of the inner mitochondrial membrane were markedly decreased as compared with awakening gophers. The data obtained suggest that in awakening animals fatty acids induce the uncoupling of oxidative phosphorylation by the ATP/ADP-antiporter, this process being simultaneous with the activation of the respiratory chain.     

Fatty acid uncoupling of oxidative phosphorylation in rat liver mitochondria

Free fatty acids (FFA) are known to uncouple oxidative phosphorylation in mitochondria. However, their mechanism of action has not been elucidated as yet. In this study we have investigated in detail the patterns of uncoupling by the FFA oleate and palmitate in rat liver mitochondria and submitochondrial particles. The patterns of uncoupling by FFA were compared to uncoupling induced by the ionophores valinomycin (in the presence of K+) and gramicidin (in the presence of Na+) and the proton translocator carbonyl cyanide m-chlorophenylhydrazone (CCCP). The most striking difference in the pattern of uncoupling relates to the effect on the proton electrochemical potential gradient, delta mu H. Uncoupling by ionophores, particularly valinomycin, is associated with and most likely caused by a major reduction of delta mu H. In contrast, uncoupling by FFA is not associated with a significant reduction of delta mu H, indicating another mechanism of uncoupling. We suggest the use of the term decouplers for uncoupling agents such as FFA and general anesthetics that do not collapse the delta mu H [Rottenberg, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3313-3317]. The protonophore CCCP and to some extent the ionophore gramicidin indicate a mixed mode of uncoupling since their effect on delta mu H is moderate when compared to that of valinomycin. Another distinguishing feature of uncouplers that collapse the delta mu H is their ability to stimulate ADP-stimulated respiration (state 3) further. Decouplers such as FFA and general anesthetics do not stimulate state 3 respiration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of phosphate carrier as a part of complex with ADP/ATP and aspartate/glutamate antiporters in palmitic acid-induced uncoupling in liver mitochondria

In liver mitochondria, the phosphate carrier is involved in protonophoric uncoupling effect of fatty acids together with ADP/ATP and aspartate/glutamate antiporters (Samartsev et al. 2003. Biochemistry (Moscow). 68, 618–629). Liver mitochondria depleted of endogenous oxidation substrates (exhausted mitochondria) have been used in the present work. In these mitochondria, like in the intact liver mitochondria, the specific inhibitor of ADP/ATP antiporter (carboxyatractylate) and the substrate of aspartate/glutamate antiporter (aspartate) suppress the uncoupling activity of palmitic acid. It is shown that in exhausted mitochondria the substrate of phosphate carrier (inorganic phosphate) and its nonspecific inhibitor mersalyl partially suppress palmitic acid-induced uncoupling due to decrease in the component of uncoupling activity sensitive to carboxyatractylate and aspartate. In the presence of inorganic phosphate or mersalyl, carboxyatractylate and aspartate added separately subsequent to palmitic acid do not suppress its uncoupling activity. They are effective only when added jointly. In the presence of thiourea or pyruvate, such effects of inorganic phosphate and mersalyl are not observed. It is supposed that in the presence of inorganic phosphate or mersalyl and under the condition of oxidation of critical SH-groups in mitochondria, the phosphate carrier, ADP/ATP antiporter, and aspartate/glutamate antiporter are involved in uncoupling function together with the general fatty acid pool as an uncoupling complex. The role of phosphate carrier in this complex may consist in facilitation of lateral transfer of the fatty acid molecules from one antiporter to another.     

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.     

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 M) in the presence of 3 mM MgCl₂ had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.     

Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg²⁺ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl₂ and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg²⁺ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg²⁺ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.     

Uncoupling of oxidative phosphorylation by fatty acids and detergents suppressed by ATP/ADP antiporter inhibitors

The effects of ATP/ADP-antiporter inhibitors on the uncoupling of oxidative phosphorylation by palmitic acid, detergents and protonophore FCCP in liver mitochondria were studied. The uncoupling activity of these compounds was estimated by their stimulating effect on succinate oxidation and H+ conductivity of the inner mitochondrial membrane in the presence of oligomycin. Carboxyatractylate and pyridoxal 5-phosphate suppressed the uncoupling effects of palmitic acid and anionic detergents but had no effect on the uncoupling action of the nonionic detergent Triton X-100, the cationic detergent CTAB and FCCP. The data obtained are discussed in terms of the putative role of the ATP/ADP-antiporter in the electrophoretic transport of hydrophobic anions from the mitochondria.     

Role of the ADP/ATP-antiporter in fatty acid-induced uncoupling of Ca2+-loaded rat liver mitochondria

We show that Ca2+ loading of mitochondria substantially augments the myristate-induced decrease in the transmembrane electric potential difference (deltapsi). Such a Ca2+ action is without effect on the respiration rate and is not accompanied by the high-amplitude swelling when low concentrations of Ca2+ and myristate are used. The myristate-induced deltapsi decrease is prevented and reversed by cyclosporin A (CsA); the decrease is prevented and transiently reversed by nigericin. To explain these effects, we suggest that myristate induces opening of the mitochondrial permeability transition pore at a low-conductance state. Addition of carboxyatractylate (CAtr) after myristate induces the CsA-sensitive uncoupling, but when added after myristate and CsA, CAtr produces a decrease in deltapsi, if the interval between myristate and CsA addition is sufficiently long. The CAtr effect is completely reversed by EGTA and transiently reversed by nigericin. This suggests that the ADP/ATP-antiporter participates in the CsA-sensitive uncoupling when present as a pore complex constituent. ADP/ATP-antiporter that does not take part in the pore complex formation is involved in the CsA-insensitive uncoupling.     

Acetoacetate as regulator of palmitic acid-induced uncoupling involving liver mitochondrial ADP/ATP antiporter and aspartate/glutamate antiporter

The effect of acetoacetate on palmitate-induced uncoupling with the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter has been studied in liver mitochondria. The incubation of mitochondria with acetoacetate during succinate oxidation in the presence of rotenone, oligomycin, and EGTA suppresses the accumulation of conjugated dienes. This is considered as a display of antioxidant effect of acetoacetate. Under these conditions, acetoacetate does not influence the respiration of mitochondria in the absence or presence of palmitate but eliminates the ability of carboxyatractylate or aspartate separately to suppress the uncoupling effect of this fatty acid. The action of acetoacetate is eliminated by β-hydroxybutyrate or thiourea, but not by the antioxidant Trolox. In the absence of acetoacetate, the palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter); in its presence, it is limited by a stage insensitive to the effect of these agents. In the presence of Trolox, ADP suppresses the uncoupling action of palmitate to the same degree as carboxyatractylate. Under these conditions, acetoacetate eliminates the recoupling effects of ADP and aspartate, including their joint action. This effect of acetoacetate is eliminated by β-hydroxybutyrate or thiourea. It is supposed that the stimulating effect of acetoacetate is caused both by increase in the rate of transfer of fatty acid anion from the inner monolayer of the membrane to the outer one, which involves the ADP/ATP antiporter and aspartate/glutamate antiporter, and by elimination of the ability of ADP to inhibit this transport. Under conditions of excessive production of reactive oxygen species in mitochondria at a high membrane potential and in the presence of small amounts of fatty acids, such effect of acetoacetate can be considered as one of the mechanisms of antioxidant protection.     

Temperature dependence of rat liver mitochondrial respiration with uncoupling of oxidative phosphorylation by fatty acids. Influence of inorganic phosphate

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22°C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22°C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22°C and from 56 to 67 kJ/mol in the range from 22 to 37°C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.     

Reversible effects of fatty acids on respiration, oxidative phosphorylation, and heat production of rat liver mitochondria.

1. Oleic acid at low concentrations (0--70 nmol/mg protein) stimulated mitochondrial state 4 respiration 4-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -112 to -208 kJ/O and completely inhibited ATP synthesis without significant effect on the Mg-ATPase activity of mitochondria. 2. Similar effects on mitochondrial respiratory activities were observed with other fatty acids. 3. Bovine serum albumin (BSA) protected mitochondria from the effects of oleic acid irrespective of the order of addition of oleic acid and BSA to mitochondria. The capacity of BSA to bind oleic acid was calculated to be 3.6--7.1 (mean, 4.9) mol of oleic acid/mol of BSA. 4. The response time of mitochondrial respiration to added oleic acid or BSA was 20--25 s.     

Oxidative stress as regulatory factor for fatty-acid-induced uncoupling involving liver mitochondrial ADP/ATP and aspartate/glutamate antiporters of old rats

Palmitate-induced uncoupling, which involves ADP/ATP and aspartate/glutamate antiporters, has been studied in liver mitochondria of old rats (22-26 months) under conditions of lipid peroxidation and inhibition of oxidative stress by antioxidants--thiourea, Trolox, and ionol. It has been shown that in liver mitochondria of old rats in the absence of antioxidants and under conditions of overproduction of conjugated dienes, the protonophoric uncoupling activity of palmitate is not suppressed by either carboxyatractylate or aspartate used separately. However, the combination of carboxyatractylate and aspartate decreased uncoupling activity of palmitate by 81%. In this case, palmitate-induced uncoupling is limited by a stage insensitive to both carboxyatractylate and aspartate. In the presence of antioxidants, the palmitate-induced protonophoric uncoupling activity is suppressed by either carboxyatractylate or aspartate used separately. Under these conditions, palmitate-induced uncoupling is limited by a stage sensitive to carboxyatractylate (ADP/ATP antiporter) or aspartate (aspartate/glutamate antiporter). In the absence of antioxidants, the uncoupling activity of palmitate is not suppressed by ADP either in the absence or in the presence of aspartate. However, in the presence of thiourea, Trolox, or ionol ADP decreased the uncoupling activity of palmitate by 38%. It is concluded that in liver mitochondria of old rats the development of oxidative stress in the presence of physiological substrates of ADP/ATP and aspartate/glutamate antiporters (ADP and aspartate) results in an increase of the protonophoric uncoupling activity of palmitate.