The hypothesis that nonshivering thermogenesis (NST) mediated by brown adipose tissue mitochondria (BATM) is linked to obesity and the regulation of body weight has been recently proposed. The molecular basis for this weight regulating ability is the occurrence of a nucleotide binding protein called uncoupling protein (UP) within the BATM inner membrane which imparts the unique capability of controlled uncoupling of respiration from normal rgulatory mechanisms thus permitting loss of energy as heat. The metabolic regulation of the UP and thus NST has not been ascertained. It is known that purine nucleotides will bind and inhibit the function of the UP. However, no activator of the UP function has been clearly established. We have recently purified and reconstituted the UP into a liposome system and observed faty acid activation of proton transport. In future studies we will attempt to purify the protein to homogeneity for use in the reconstituted system to determine its kinetic properties and mechanism of action by fatty acids. The UP will be extracted with Triton X-100 from mitochondria, purifed on hydroxylapatite and the Triton exchanged with octylglucoside for final reconstitution into a liposome system. Proton conductance will be assayed using the pH system as well as incorporation of cytochrome oxidase with the UP. Measurements of O2 consumption which would be more physiological could then be carried out. The mechanism of fatty acid activation of the UP will be studied using spin labeled fatty acids and measurement by electron spin resonance. Individual intracellular free fatty acids and their long chain thioesters will be determined by HPLC and thier concentrations related to activation of the UP. Hamsters will be the main source of BATM for purification and biochemical studies relating to mechanism of the UP. However, more physiological experiments will be carried out by reconstituting the UP from cold adapted rats and the ob/ob mouse which will provide the information as to whether the UP is non functional under certain conditions or merely decreased in concentration. The long term objectives of the proposal are to determine the mechanism of action of the UP in BATM and how certain aspects of the biochemical and molecular action of the protein might be extended to understanding basal energy expenditure in humans.
