guanosine-diphosphate and malic-acid

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.

Topics: Adenosine Triphosphate; Adipose Tissue, Brown; Animals; Cell Respiration; Fatty Acids; Guanosine Diphosphate; Hydrogen Peroxide; Malates; Male; Mitochondria; Oleic Acid; Pyruvic Acid; Rats; Rats, Wistar; Rotenone; Succinic Acid; Thermogenesis; Uncoupling Agents

The roles of mild uncoupling caused by free fatty acids (mediated by plant uncoupling mitochondrial protein (PUMP) and ATP/ADP carrier (AAC)) and non-coupled respiration (alternative oxidase (AO)) on H(2)O(2) formation by plant mitochondria were examined. Both laurate and oleate prevent H(2)O(2) formation dependent on the oxidation of succinate. Conversely, these free fatty acids (FFA) only slightly affect that dependent on malate plus glutamate oxidation. Carboxyatractylate (CAtr), an inhibitor of AAC, completely inhibits oleate- or laurate-stimulated oxygen consumption linked to succinate oxidation, while GDP, an inhibitor of PUMP, caused only a 30% inhibition. In agreement, CAtr completely restores the oleate-inhibited H(2)O(2) formation, while GDP induces only a 30% restoration. Both oleate and laurate cause a mild uncoupling of the electrical potential (generated by succinate), which is then followed by a complete collapse with a sigmoidal kinetic. FFA also inhibit the succinate-dependent reverse electron transfer. Diamide, an inhibitor of AO, favors the malate plus glutamate-dependent H(2)O(2) formation, while pyruvate (a stimulator of AO) inhibits it. These results show that the succinate-dependent H(2)O(2) formation occurs at the level of Complex I by a reverse electron transport. This generation appears to be prevented by mild uncoupling mediated by FFA. The anionic form of FFA appears to be shuttled by AAC rather than PUMP. The malate plus glutamate-dependent H(2)O(2) formation is, conversely, mainly prevented by non-coupled respiration (AO).

Topics: Atractyloside; Carrier Proteins; Glutamic Acid; Guanosine Diphosphate; Hydrogen Peroxide; Ion Channels; Lauric Acids; Malates; Membrane Proteins; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Oleic Acid; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Pisum sativum; Succinic Acid; Uncoupling Agents; Uncoupling Protein 1

In this study, oxygen consumption and H(2)O(2) release rate by succinate or pyruvate/malate supplemented mitochondria isolated from skeletal muscle of trained and untrained rats were investigated. The overall mitochondrial antioxidant capacity and the effect of preincubation of mitochondria with GDP, an inhibitor of uncoupling proteins UCP1 and UCP2, on both succinate-supported H(2)O(2) release and membrane potential were also determined. The results indicate that training does not affect mitochondrial oxygen consumption with both complex-I- and complex II-linked substrates. Succinate-supported H(2)O(2) release was lower in trained than in untrained rats both in State 4 and State 3. Even the antimycin A-stimulated release was lower in trained rats. When pyruvate/malate were used as substrates, H(2)O(2) release rate was lower in trained rats only in the presence of antimycin A. The increase of mitochondrial protein content (determined by the ratio between cytochrome oxidase activities in homogenates and mitochondria) in trained muscle was such that the succinate-supported H(2)O(2) release per g of tissue was not significantly different in trained and untrained rats, while that supported by pyruvate/malate was higher in trained than in untrained animals. The lack of training-induced changes in overall antioxidant capacity of mitochondria indicates that the decrease in mitochondrial H(2)O(2) release cannot be attributed to a greater capacity of mitochondria to scavenge the reactive oxygen intermediates derived from univalent O(2) reduction by respiratory chain components. In contrast, the above decrease seems to depend on the drop induced by training in mitochondrial membrane potential. These training effects are not due to an increased level of mitochondrial uncoupling protein, because in the presence of GDP the increase in both membrane potential and H(2)O(2) release was greater in untrained than in trained rats.

Topics: Adenosine Diphosphate; Animals; Antimycin A; Antioxidants; Electron Transport Complex IV; Free Radicals; Guanosine Diphosphate; Hydrogen Peroxide; Malates; Male; Membrane Potentials; Mitochondria, Muscle; Muscle, Skeletal; Oxygen; Physical Conditioning, Animal; Physical Endurance; Pyruvic Acid; Rats; Rats, Wistar; Reactive Oxygen Species; Rhodamine 123; Succinic Acid; Uncoupling Agents

(1) Rabbit liver mitochondria can convert exogenous phosphoenolpyruvate to malate. (2) Malate production is dependent on phosphoenolpyruvate and HCO3- and is stimulated by CN- or malonate alone and especially in combination. (3) Malate production is inhibited 70% by 3-mercaptopicolinate, a specific inhibitor of phosphoenolpyruvate carboxykinase, and 50-60% by 1,2,3-benzenetricarboxylate, an inhibitor of the tricarboxylate transporter. (4) Rat liver mitochondria incubated with phosphoenolpyruvate under identical conditions do not produce malate. (5) Malate production from phosphoenolpyruvate is stimulated by exogenous GDP or IDP but not by ADP. (6) Data support the conclusion that malate is being produced from oxalacetate generated by reversal of mitochondrial phosphoenolpyruvate carboxykinase. A possible role for this enzyme in hepatic lipogenesis is suggested.

Topics: Animals; Benzene Derivatives; Bicarbonates; Cyanides; Guanosine Diphosphate; Inosine Diphosphate; Lipids; Malates; Male; Malonates; Mitochondria, Liver; NAD; Phosphoenolpyruvate; Phosphoenolpyruvate Carboxykinase (GTP); Picolinic Acids; Rabbits; Rats; Sodium; Sodium Bicarbonate; Tricarboxylic Acids