Molecular characterization of brown adipose tissue in a ‘protoendothermic’ mammal provides a novel approach to the understanding of uncoupling protein evolution

the presence of functional BAT, which appeared recruitable by cold acclimation. Molecular analysis confirmed the tissue specific expression of UCP1 in tenrecs (etUCP1). We cloned and stably transfected etUCP1 in HEK293 cells. Isolated mitochondria of etUCP1 HEK293 cells showed inducible proton conductance using palmitate, and GDP-sensitivity, similar to mitochondria containing mouse UCP1. For the search of functional differences, we established bioenergetic measurements in intact HEK293 cells using plate-based respirome-try, allowing high-throughput approaches for small molecule modulators. In the initial experiments, we show that a cell-permeable UCP1 activator allows direct specific activation of tenrec and mouse UCP1. Taken together, we show that E. telfairi possesses functional, UCP1-dependent brown adipose tissue, which may facilitate active rewarming from hypothermic states. Substantial evolutionary distance between tenrecs and modern mammals provides a new window to study the evolution of structure-function relationships of UCP1. The fruit-fly Drosophila melanogaster is widely used as a model organism to study human diseases, including those affecting mitochon-dria. Most mutant flies engineered to mimic mtDNA diseases die at the larval stages, yet a detailed bioenergetic characterization of developmental stage-specific features of Drosophila has never been carried out. We used muscular body-wall Drosophila larvae preparations to study respiration with the sensitive Seahorse â technology. This method allows us to study individual larvae and therefore whole tissue bioenergetics in situ. Larvae maintained a steady respiratory rate which was inhibited (i) by rotenone and antimycin A, demonstrating its mitochondrial origin and (ii) by 2-iodoacetate, suggesting that respiration is coupled to a high glycolytic flux. Unexpectedly, respiration could not be decreased by the F1FO ATPase inhibitor oligomycin nor increased by the uncoupler FCCP, suggesting that in Drosophila larvae mitochondria are uncoupled. Consistent with a developmental stage-specific uncoupling effect, the respiratory profile of embryonic multilineage Drosophila S2R + cells was instead essentially similar to that of mammalian cells in culture, as basal respiration could be inhibited by oligomycin and then stimulated by FCCP. Sequence homology analysis revealed the existence of four putative uncoupling proteins (UCPs) in Drosophila (UCP4a, UCP4b, UCP4c, and UCP5) that share 60-70% homology with their mammalian counterparts. Whether these are developmentally regulated is not known. We suspect that in the poikilotherm Drosophila uncoupling may be essential for thermogenesis in the pre-pupal stages. We are carrying out silencing of each UCP transcript in whole larvae, as well as genetic ablation of individual UCP genes, to address the potential role of UCP proteins in larval respiration.