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expressing UCP3 (15). Similar to previous reports (15,17), the UCP3-tg mice had lower body mass compared with wild-type littermates (22.4 previously validated antibody specifically detecting hUCP3 (code 1331) (19,20), together with our antibody detecting both hUCP3 and endogenous mouse and rat UCP3 (code 1338), we were able to delineate hUCP3 expression from endogenous UCP3 expression in the UCP3-tg mice. It was shown that in wild-type mice only expressing endogenous UCP3, a clear mitochondrial labeling was observed with the mice-specific antibody (1338), whereas using the anti- hUCP3 (1331) antibody, no label was detected. In UCP3-tg mice, the vast majority of the label was associated with the inner mitochondrial membrane and the cristae and did not result in extramitochondrial labeling (Figure 2). This may indicate that artificial uncoupling in UCP3 overexpression systems, as suggested previously (13,17,21), is not caused by extramitochondrial expression of UCP3. This is in con- trast with observations in yeast, where it was shown that expression of UCP3 resulted in the majority of UCP3 present in extramitochondrial compartments (14). A major difference between UCP3 expression in yeast and in the UCP3-tg mice is the in vivo condition and, thus, the pres- ence of all putative cofactors needed to induce or activate tron microscopical observations do not exclude that im- proper folding of UCP3 in the inner mitochondrial mem- brane resulted in pores in the inner membrane, thus leading to uncoupling in a way similar to protonophores (noninduc- ible uncoupling). Moreover, the mice in our study showed a 5.8-fold up-regulation, which apparently is much lower than other data available on UCP3-tg mice at the protein level (17). matters. In these mice, decreased state 4 respiration with similar state 3 respiration levels have been reported (23), indicating more tightly coupled mitochondria. Whole body oxygen uptake and fuel partitioning (reflected by respiratory exchange ratio) under resting conditions were not affected in UCP3-ko mice (22,23). Obviously, the lack of UCP3 was not compensated for by induction of any of the other un- coupling proteins known so far. The observation of more tightly coupled mitochondria in UCP3-ko mice did not result in apparent phenotypical changes (22,23). By creation of a double knock-out, lacking UCP1 and UCP3, it was shown that the phenotype observed after deletion of the UCP1 gene was not more pronounced by the additional gold-labeled subsarcolemmal mitochondria, without staining in other subcellular constituents like the nuclei (n). The right panel shows gold-labeled intramyofibrillar mitochondria with sparse background labeling on sarcomeres (s). The antibody used was tested to specifically recognize human UCP3. Using this antibody, it was shown that, in UCP3-tg mice, the vast majority of the label was restricted to mitochondria. |
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Study 