Control of Energy Metabolism in Hamster Brown Adipose Tissue

Isolated brown fat cells were prepared from hamsters fasted for 48 hours and were incubated in Krebs-bicarbonate buffer. Endogenous respiration was low and was stimulated 6-fold by succinate, 4-fold by α-glycerophosphate, 20-fold by oleate, up to 30-fold by epinephrine or norepinephrine, and 2- to 4-fold by uncoupling agents. The norepinephrine-stimulated respiration was inhibited in a concentration-dependent manner by oligomycin, arsenite, and uncoupling agents. Succinate respiration was relatively insensitive to inhibition by oligomycin or to stimulation by uncoupling agents. Malonate at concentrations which inhibited completely succinate respiration had no effect on norepinephrine-stimulated respiration. Malate and dihydroxyacetone phosphate were shown to be the end products of succinate and α-glycerophosphate respirations, respectively. The ratio of endogenous ATP to ADP in resting cells was 4.6. This ratio decreased by about one-half when respiration was initiated by norepinephrine or oleate. A submaximal concentration of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone decreased ATP levels by 60%, whereas subsequent addition of norepinephrine caused an elevation of the ATP content towards control values. Addition of oligomycin also caused a fall of ATP, which was restored almost to normal by addition of norepinephrine. Arsenite caused a small depletion of ATP, but subsequent addition of norepinephrine produced a rapid fall of the ATP content. A similar effect was obtained in the presence of arsenite and oligomycin. Addition of succinate to cells after prior additions of arsenite and norepinephrine was able to spark a large increase of oxygen uptake, possibly by providing ATP for fatty acid activation. The present results emphasize the role of substrate level phosphorylation for generation of high energy phosphate required for the activation of fatty acids to the coenzyme A derivatives. However, it is apparent that some degree of control at phosphorylation sites by phosphate acceptors limits respiration of brown fat in the resting state. Stimulation of the tissue by norepinephrine, which causes the liberation of free fatty acids from triglyceride, provides both substrate and messenger for the release of respiratory control. It is suggested that if fatty acids themselves are the primary uncoupling agent, brown fat mitochondria must be extremely sensitive to the release of respiratory control by these agents.