Flavoprotein-linked substrate oxidation in preparations of hamster brown adipocytes. A discrimination between internally and externally oxidized substrates.

Noradrenaline-stimulated oxidative metabolism in isolated hamster brown fat cells is very reproducible between different cell preparations, 565 +/- 81 (S.D.) nmol O/min per 10(6) cells (n = 25). In contrast, the oxygen consumption rate induced by the addition of succinate or sn-glycerol 3-phosphate strongly varies between different cell preparation, although these substances have been reported to be potent substrates for isolated hamster brown fat cells. By filtration and by successive washings we demonstrate that the flavoprotein-linked substrate oxidation is mainly dependent on extracellular succinate and sn-glycerol 3-phosphate-oxidizing enzymes. These enzymes originate from damaged and broken cells and are present in different amounts in different cell preparations. In discriminating between intra- and extracellular succinate oxidation 5,5'- dithiobis(2-nitrobenzoate) is used as an inhibitor of the extracellular portion. This application of 5,5'-dithiobis(2-nitrobenzoate) ought to be useful also in other cell or tissue preparations. Added succinate can, however, be oxidized by the intact brown adipocyte but at very low rate, probably as a result of a limited transport rate over the membrane(s). In the presence of noradrenaline, added succinate can potentiate the noradrenaline-inducible oxygen consumption by catalytically increasing the oxidative capacity of the citric acid cycle. Our conclusions is that the only effectors which significantly increase oxidative metabolism in intact isolated hamster brown fat cells are catecholamines and free fatty acids. Provided the cells are uncoupled, also pyruvate can function as substrate for these cells.