BIOCHEMICAL STUDIES OF THERMOGENESIS

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The objective of this study is to elucidate the mechanism of non-shivering thermogenesis. Norepinephrine stimulation of brown fat respiration is mainly responsible for this type of thermogenesis. One explanation of hormone action involves stimulation of (Na+,k+) ATPase in the cell membrane followed by increased mitochondrial respiration coupled to the synthesis of ATP. An alternative hypothesis suggests that brown fat respiration is uncoupled from ATP synthesis by norepinephrine. Support for this theory comes from the discovery of protein uniquely present in the membranes of brown fat mitochondria which can uncouple respiration from ATP synthesis. The protein which is an hydroxyl ion carrier is inactive in the presence of ATP and ADP. Since the binding constant of the channel for these nucleotides is in the MuM range, control of coupling via physiological fluctuations of cytosolic nucleotides seemed unlikely. Data from the present study suggest that the uncoupling protein is regulated by cytosolic ATP. Most of the adenine nucleotides of brown fat cells are compartmented within the mitochondria. The Ki of ATP for inhibition of respiration is not in the MuM region but is about 1 mM because of the presence of free Mg++ in the cell and an excess of the uncoupling protein in the mitochondrial membranes. When the mitochondria are coupled, the electrogenic character of the adenine nucleotide transporter excludes ATP from the mitochondrial matrix so that most of the tissue ATP is cytosolic. Addition of nonrepinephrine causes a small decrease in ATP. This may partially activate the uncoupling protein, allowing some ATP into the matrix, further lowering cytosolic ATP and continuously amplifying the initial drop. The specific aims of the proposed study are 1) to develop methods of rapid tissue fractionation so that cytosolic ATP may be directly measured, 2) to study the effect of hormone on cytosolic ATP in cells from cold- and warm-adapted animals and from genetically obese animals and their lean controls where large variations in hormonal responsiveness exist, 3) to attempt to find out what causes the initial drop in total tissue ATP levels since we believe this initial drop in ATP is the trigger for the hormone induced increase in respiration, 4) to examine each of the steps of brown fat hormone activation in the genetically obese mouse in an attempt to identify the metabolic lesion responsible for its insensitivity to norepinephrine.