Charakterisierung einer Mutante von Phodopus sungorus mit UCP3-Defizit im braunen Fettgewebe

Uncoupling proteins (UCPs) are mitochondrial carrier proteins of the inner mitochondrial membrane. Uncoupling protein 3 (UCP3) was already discovered in 1997, nevertheless, its function has been unknown till today. Within the present thesis hamsters were investigated, which lack the UCP3 protein in brown adipose tissue (BAT) due to a single nucleotide mutation within the UCP3 gene. Primary aim of this work was to gain new insight about UCP3 by characterisation of these hamsters. The anatomic examination of the hamsters showed that mutants are heavier and longer than wildtype hamsters. At the same time they have less body fat and a bigger amount of fat free body mass. The later is probably based on body length. The hamsters’ bigger habitus could be interpreted as consequence of a positive energy balance during growth. On the other hand the stable body composition throughout a longer time period points to a balanced energy balance in adult hamsters. The difference between young and adult animals can possibly be explained with a change of UCP3’s physiological impact with age. Moreover, an increased food intake of the mutants was measured. Hyperphagia and decreased body fat could refer to a higher energy need. Alternatively the hyperphagia of the mutant hamsters might indicate a poorer nutritional assimilation. It’s unknown though, if the hyperphagia is a result of increased energy consumption. To determine the mutants’ energy consumption, metabolic rate measurement would be needed. Primary cell culture was established to test if the UCP3 mutation in brown adipose tissue exists independently of systemic influences. First UCP3 mRNA expression was stimulated in wildtype cells. It was shown that UCP3 mRNA expression in brown adipose tissue is inducible via activation of core receptors PPAR and PPAR. This is a tissue specific difference between the regulation in brown adipose tissue and in skeletal muscle, where UCP3 expression is regulated differently from BAT by PPAR and . The activation of the UCP3 gene with transcriptional factors that regulate the fat metabolism support a potential function of UCP3 within the fat metabolism. In primary cell culture derived from mutant brown adipocytes no UCP3 mRNA could be detected. Nevertheless normal proliferation, differentiation and morphology of cells were observed. Also, electron microscopic photos did not show any abnormal morphology of mutant brown adipose tissue. Apparently UCP3 has no development or vitality promoting function. Despite normal PPARmRNA expression in mutant cells, it wasn’t possible to induce UCP3 expression with PPARactivators. This shows that the mutation in brown adipocytes works independent of extra cellular influences. Thus it is possible to use isolated brown adipocytes from mutant hamsters for future studies about the regulation of UCP3 mRNA expression. To examine whether UCP3 is capable to uncouple mitochondrial respiration, brown fat mitochondria were isolated and its respiration was measured. In brief, mitochondrial respiration is not affected by the lack of UCP3. Particularly in mutant mitochondria the proton leak was not reduced during uncoupled as well as coupled state-4-respiration. Also, the maximal respiration rate showed a normal mitochondrial efficiency in mutant mitochondria. In conclusion the comparison of mitochondria with and without UCP3 indicates that UCP3 does not function as an uncoupler of mitochondrial respiration. Since a function of UCP3 was postulated as a protector of mitochondria against radicals by means of uncoupling, the effect of radicals on mitochondrial respiration was examined. No activation of UCP3 and UCP1 by radicals was detected, neither in mutant nor in wildtype mitochondria. This is probably attributed to the cold acclimation of animals which led to higher concentrations of antioxidant enzymes in brown fat mitochondria. Phenotypic aspects of Djungarian hamsters with UCP3 deficiency examined so far are not definitely assignable to any hypothesises for the function of UCP3 suggested so far. Nevertheless it can be stated, that the unchanged size of the proton leak as well as the absence of obesity and the mutant’s hyperphagia contradict an uncoupling function for UCP3. Low body fat content and hyperphagia of adult hamsters could be interpreted as an indication of an impaired nutrient utilization and as a connection between UCP3 and fat metabolism. The control of UCP3 mRNA expression via PPARs points also in this direction as PPARs are involved in the regulation of fat metabolism. It is remarkable, that UCP3 deficient hamsters in contrast to UCP3 knockout mice display an altered phenotype whereas the mouse phenotype is largely unobtrusive. Thus, the loss of UCP3 in different species doesn’t have the same consequence.