Roles Of UCPs In The Change Of Brain Mitochondrial Energy Synthesis And Oxygen Consumption From Rat Exposed To High Altitude Hypoxia

Mitochondrion is an organelle with a main function of ATP production. When electrons passing through the electron transport chain, protons are pumped across the inner mitochondrial membrane from the matrix to the intermembrane space. Thus an proton gradient (called proton motive,Δp) which provides energy for ATP syntheses by F0F1-ATPase has been created. The whole process is called the oxidative phosphorylation. However, the protons may return to the matrix not only by the F0F1-ATPase but by an alternative way which is called proton leak. Proton leak leads to decrease of proton gradient, uncoupling of oxidative phosphorylation, decrease of ATP syntheses and efficiency of oxygen utilization. Uncoupling proteins(UCPs) have been verified as a protein superfamily located in the inner mitochondrial menbrane associated with the proton leak phenomenon. There are increases of uncoupling oxygen consumption and decreases of mitochondria membrane potential (MMP) and ATP synthesis in brain mitochondria during rat exposed to simulated high altitude hypoxia. The newly discovered UCP4 and UCP5 which expressed predominately in brain of mammalian may contribute largely to cerebral anoxia or other brain disorders in high altitude environment. The functions are still in debate. Long-chain free fatty acids have protonophoric action by stimulating the uncoupling capability of UCPs. To realize the roles of UCPs on mitochondrial energy produce and efficiency of oxygen utilization in rat brain during hypoxic exposure, we observed the effects of palmitic acid (long chain free acid with hexadeco-carbon) on the activity of UCPs and the expression of UCP4 and UCP5 mRNA and protein and the roles of UCPs in mitochondria oxygen consumption and energy produce during animal hypoxia exposure.MethodsTwo models were set up to discovery the roles of palmitic acid on the activity and expression of cerebral UCPs after hypoxia exposure. Adult male SD rats were exposed to a hypobaric chamber simulated 5000m high altitude for 23h every day for 0 (control), 3 (acute group)and 30 d (chronic group) respectively. Rats were sacrificed by decapitation. blood was collected and brain was removed. Serum was seperated and brain mitochondria were isolated by centrifugation program. Free fatty acid content was tested by fatty acid content kit. Mitochondria oxidative respiratory function was measured by Clark oxygen electrode. Proton leak was measured by TPMP+ electrode together with Clark oxygen electrode. The UCPs activity and content was detected by [3H]-GTP binding method. Mitochondrial membrane potential was detected by Rhodamine123 method. The content of adenine nucleotide pool (ATP,ADP,AMP) in mitochondria was measured by high performance liquid chromatography(HPLC). The F0F1-ATPase's activity was determined by oligomycin-inhibitor method. The mRNA and protein expression of UCP4 and UCP5 in rat brain was determined after incubation by RT-PCR and Western-blot analysis respectivly.Results1. Hypoxia exposure increased the content of free fatty acid in serum, brain homogenate and mitochondria matrix especially during acute hypoxia to a extent of 51.36%, 243.35% and 69.49% respectively.2. The activity of mitochondrial UCPs from rat brain significantly increased during hypoxia exposure especially in acute hypoxia group, which Kd decreased 41.24% and Bmax increased 1.56-fold respectively. Also UCPs activity greatly enhanced after the interfere of palmitic acid in vitro in all groups.3.The content of free fatty acid in serum,brain homogenate and mitochondria matrix is negatively correlated with Kd value, and positively correlated with Bmax.(brain homogenate FFA content is most highly correlated with Kd and Bmax ) 4. There were significant decreases in ST3, RCR, P/O and MMP of mitochondria fromhypoxia-exposed rat brain, especially in acute hypoxia group. But ST4 and proton leak were significantly higher than control group. Plamitic acid lead to significant increase in mitochondrial oxygen consumption and proton leak, but decrease in RCR, P/O and MMP in vitro in all groups.5. The activity of F0F1-ATPase, ATP content and the ratios of ATP/ADP and ATP/(ATP+ADP+AMP) were decreased in hypoxia-exposed rat brain mitochondria. Plamitic acid resulted in significant decrease in (ATP+ADP+AMP),(ATP+ADP)content,energy charge of control and acute group, increased in ATP/ADP ratio and decreased in ATP/(ATP+ADP+AMP) in each group.6. The message and protein of UCP4 and UCP5 are deteced after exposion to high altitude hypoxia. UCP4, UCP5 mRNA and protein level greatly enhanced with the acute group at the highest. Incubated with palimitic acid , the UCP4, UCP5 mRNA and protein level in brain were up-regulated, interesting, the palmitic acid had the least potential ability to regulate the transcription and translation of acute group.Conclusion1. Mitochondria phosphorylation efficiency can be harmed after incubated with palmitic acid in vitro, [徐晓维9]and this effect depends on PA's concentration and incubation time. The uncoupling phenomenon promoted by palmitic acid is acquired through a directly enhancement on UCPs activity as well as by up-regulating UCP4,UCP5 gene expression2. Hypoxia exposure attenuated the direct effect of palmitic acid on UCPs activity which lead to the proton leak and uncoupling.3. The enhancement of UCPs activity after hypoxia exposure is associated with a change in metabolism of fatty acids.The key links in hypoxia induced energy failure can be summarized as hypoxia—free fatty acid released—UCPs activity enhanced—uncoupling—ATP synthesis shortage.4. We deduce UCP4,UCP5mRNA and protein up-regulation after hypoxia exposure is associated with the change of fatty acid metabolism. Conclusion summaryHypobaric hypoxia exposure can increase the content of FFA in serum, brain and brain mitochondria, UCPs activity as well as proton leak, and a decreases in mitochondrial MMP, oxidative phosphorylation rate and energy production rate. Plamitic acid can aggravate the deficiency of energy production when exposed to hypoxia by futher activate UCPs and up-regulate the expression of UCP4 and UCP5.