The investigators seek to understand the role of facultative and obligatory thermogenesis in the regulation of body weight and to find mechanisms that will reduce the development of obesity by increasing thermogenesis. Nonshivering thermogenesis, the major physiological function of brown fat, is based on the brown fat specific expression of the mitochondrial uncoupling protein (UCP1), a proton carrier in the inner mitochondrial membrane, that uncouples oxidative phosphorylation to produce heat. Genetic manipulations in mice which increase UCP1 in transgenic mice reduce obesity. Mice in which the Ucp1 gene has been inactivated by gene targeting are extremely sensitive to cold; however, paradoxically, they do not develop obesity. The result suggests that alternative thermogenic mechanisms exist that compensate for the loss of UCP1. The investigators have hypothesized that a newly discovered homologue of Ucp1, called Ucp2, is a mechanism for obligatory thermogenesis that can restore energy balance in mice with a defective Ucp1. Experiments are proposed to characterize the expression of Ucp2 in wild-type mice in homozygous for the Ucp1 knockout with respect to its changes during development, among tissues and as a consequence of feeding high fat/high sucrose diets. To establish whether the UCP2 molecule is thermogenic, a transgenic mouse will be constructed using the double replacement technique in ES cells that will insert the Ucp2 cDNA into the Ucp1 gene. This will bring the Ucp2 gene under the control of the sympathetic nervous system and will enable us to determine whether the UCP2 molecular can protect mice deficient in UCP1 from the cold exposure. A third specific aim will inactivate the Ucp2 gene with the Cre-loxP bacteriophage system of recombination. Since Ucp2 is a ubiquitously expressed gene, this system for inactivating a gene will enable us to inactivate Ucp2 in those tissues for which Cre recombinase is selectively expressed. With this genetic system the unambiguous evaluation of the role of UPC2 in tissue energy balance can be achieved. The fourth specific aim seeks to produce a new model for the ablation of brown fat with the cell specific expression of diphtheria toxin. This new model will be generated by gene replacement techniques to insert the diphtheria toxin gene into the Ucp1 gene in order to maximize the cell specificity of its expression and test unequivocally the hypothesis that the ablation of brown fat leads to hyperphagia.
