Funktionsanalyse der mitochondrialen Transportproteine UCP2, UCP3, UCPx und SOUP

In the first part of the present work a possible function of UCP2, UCP3 and UCPx, as proton transporters or uncoupling proteins were analysed. For the analysis a new assay was set up, to show a possible influence of transient expressed uncoupling proteins on mitochondrial respiration of HEK293 cells. Because UCP1 is up to now the only funcional characterized memeber of the protein family, the assay was tested with UCP1 transfected HEK293 cells. The expression and incorporation of UCP1 and UCP3 into the mitochondria were proved by western blot analysis. The measurement of O2 consumption showed less coupling of respiration in UCP1 transfected cells compared to control cells after inhibition of the ATP synthase with oligomycin. After addition of the fatty acid palmitate the respiration of UCP1 transfected cells were significantly increased, whereas control cells showed no increase of respiration in presence of fatty acids. By this the expression of functional UCP1 has been confirmed. UCP2, UCP3 and UCPx transfected cells neither showed an increased of uncoupled respiration in presence of oligomycin nor could be activated by addition of palmitate. A with UCP1 comparable uncoupling function of UCP2, UCP3 and UCPx could not be confirmed. Additionally a possible regulative or inhibitory influence of the mitochondrial folate transporter SOUP on UCP induced uncoupling were investigated after coexpression of SOUP with either UCPx or UCP1. Coexpression of SOUP and UCPx did not effect the mitochondrial respiration. Also the coexpression of SOUP and UCP1 did not influence UCP1 induced uncoupled respiration. In the second part of the present work a possible regulative function of UCP3 in fatty acid metabolism was analysed in hamsters which show a tissue specific lack of UCP3 in brown adipose tissue due to an unknown mutation. For the analysis wildtype and mutant hamsters were fasted for 48 h, exposed to 5°C for seven days or kept at 23°C with food ad libitum (control group). Geneexpression of UCP3 and keyenzymes of fatty acid metabolism were analysed in wildtype and mutant hamsters to confirm a possible impairement of fatty acid metabolism due to the lack of UCP3. Beyond UCP3 the mRNA expression of the mitochondrial thioesterase I (MTE-I) and the carnitine palmitoyl transferase I (CPT-I) were analysed. In brown adipose tissue of mutant hamsters no UCP3 mRNA or protein were detectable by northern and western blot analysis. In wildtype hamsters the UCP3 mRNA expression in brown adipose tissue were similar in fasted, cold exposed and control animals. The UCP3 protein content in cold exposed hamsters showed a 3-fold increase, in fasted animals protein content were slightly deacreased compared to controls. Analysis of MTE-I mRNA expression in wildtypes and mutants revealed no coupled regulation of MTE-I and UCP3 expression in brown adipose tissue. Therefore both proteins are not functionally coupled, but their expression in this tissue depends obviously on fatty acid metabolism. Expression of CPT-I mRNA in brown adipose tissue of wildtype hamsters and mutants was similar. Therefore there seemst to be no impairement of regulation or an inhibition of fatty acid import into mitochondria of mutant hamsters. The capacity of fatty acid oxidation in brown adipose tissue of fasted, cold exposed or control wildtype and mutant hamsters were compared by measuring the CO2 production of tissue samples in vitro with oleate as substrate. Additionally β-oxidation were analysed by measuring the O2 consumption of isolated mitochondria of cold exposed animals of both phenotypes with palmitoyl carnitine as substrate. All measurements revealed no impairement of fatty acid oxidation due to the lack of UCP3 neither in tissue samples nor in isolated mitochondria. In conclusion, according to gene expression studies and in vitro measurements of fatty acid oxidation, no direct functional relation or regulative influence of UCP3 in fatty acid metabolism could be confirmed.