We investigated the role of leptin in regulating energy metabolism through induction of UCP1based brown fat thermogenesis by comparing phenotypes of energy balance in ob/ob and double mutant ob/ob.Ucp1-/-mice. Measurements of adiposity and lean body mass (NMR), energy expenditure (indirect calorimetry), body weight, food intake and core body temperature were determined in the two mutant stocks of 3-month-old mice maintained at an initial ambient temperature of 28°C for 21 days, then at 21°C for

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... 16 days and finally with leptin administration for 8 days at 21°C. No phenotypic differences between ob/ob and ob/ob.Ucp1-/-mice were detected suggesting that UCP1-based thermogenesis is not essential for the regulation of adiposity in ob/ob mice at temperatures between 21 and 28°C. Although both Ucp1-/-and ob/ob mice can survive in extreme cold at 4°C, provided they are adapted to the cold by gradually lowering ambient temperature, ob/ob.Ucp1-/-mice could not adapt and survive at temperatures below 12°C, unless they were administered leptin. As the ambient temperature was reduced from 20 to 16°C ob/ob.Ucp1-/-mice treated with leptin have elevated levels of circulating T3 that correlate with elevated SERCA 2a mRNA levels in gastrocnemius muscle. Furthermore, ob/ob.Ucp1-/-mice, treated with T3, were able to maintain body temperature and stimulate SERCA 2a expression when the ambient temperature was gradually reduced to 4°C. Thus, in the absence of UCP1, leptin-induced thermogenesis protects body temperature in part through its action on the thyroid hormone axis. 6 euthanized by cervical dislocation and tissue samples collected and stored at -80°C. Serum was obtained and stored at -20°C. All animal experiments were approved by the Pennington Biomedical Research Center Institutional Animal Care and Use Committee in accordance with NIH guidelines for the care and use of laboratory animals. Phenotypes of energy balance: Body temperature was measured with a rectal thermoprobe (TH-8, Physitemp Instruments Inc., NJ) and body composition was analyzed by nuclear magnetic resonance (Bruker,TX). Energy expenditure was evaluated by indirect calorimetry (Oxymax, Columbus Instruments, Ohio). Animals with ad libitum access to food (specified above) were singly housed in air-proof plastic metabolic cages two days prior to initiation of an experiment. Cages were connected to the oxygen (O 2 ) and carbon dioxide (CO 2 ) sensors and placed in an incubator enabling precise temperature control. Alpha-Dri (Shepherd Specialty Papers Inc., Watertown, TN) was used as the nestling material during the indirect calorimetry experiment. Calibration of gas sensors and body weight corrections were performed daily. The Oxymax Flow Max system allowed 16 individually housed animals to be monitored simultaneously. Energy expenditure and respiratory exchange ratio were measured over a 60-second period in 40-minute intervals. Physical activity was measured by interruption of infrared beams that sensed movement in Y and Z directions and data were expressed as counts per hour. For a Y count a mouse has to move more than 0.5 inch in a horizontal direction and for a Z count the mouse has to raise its body 1.5 inch above the chamber floor.