Abstract
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.
Highlights
Isolated brown fat cells were prepared from hamsters fasted for 48 hours and were incubated in Krebs-bicarbonate buffer
Fat Cells-The endogenous respiration of well prepared brown fat cells is normally low, being about 5% of the maximum rate elicited by norepinephrine
Only fatty acids, succinate, and a-glycerophosphate caused an appreciable stimulation of respiration above the endogenous rate
Summary
Brown Fat Cells-Isolated cells were prepared in Krebsphosphate buffer, pH 7.4 [33], containing 4$& bovine serum albumin, by collagenase treatment of minced brown fat from male hamsters (120 to 180 g), which had been fasted for 48 hours, with a slight modification of the method of Fain et al [19], as described by Prusiner et al [31]. The final triglyceride content of the cells was greatly diminished by resuspending the cells in 3 volumes of Krebs-phosphate-albumin buffer followed by 15 set of centrifugation in a clinical centrifuge and removal of the upper white layer of triglyceride by suction. This process was repeated until no distinct white layer formed. On the basis of a cytochrome c content of 40 mpmoles per g, fresh weight, of brown fat tissue [2], 1 ml of brown fat cell suspension used in the present work corresponded to about 20 mg, fresh weight, of tissue. Results of experiments are all reported per milliliter of original cell suspension, which was standardized for each preparation by adjustment of the packed cell volume
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
