Novel approach to the study of the regulation of hormone-sensitive lipase in rat adipocytes. Permeabilization of cells with digitonin.

Abstract

Use of a digitonin-permeabilized rat adipocyte preparation overcomes inherent problems which occur when currently used broken cell systems are utilized for studying the regulation of hormone-sensitive lipase. The effect of digitonin on plasma membrane permeability was concentration-dependent being nearly maximum at 20 micrograms/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular (86Rb) rubidium within 10 min and (c) immediate inhibition of glucose oxidation. Hormone-modulated rates of lipolysis were preserved in this preparation. Following maximal activation of lipolysis in adipocytes with catecholamines, the rate of lipolysis in intact cells and digitonin-treated cells was elevated 26-fold and 20-fold respectively, while the rate in homogenates from these cells was elevated only 2.8-fold. Insulin suppressed catecholamine-dependent activation of lipolysis by at least 90% when subsequently measured in intact cells and digitonin-treated cells. Insulin suppression was only 56% when measured in homogenates. The hormone-sensitive lipase in permeabilized cells, as opposed to intact cells and homogenates, was activated by cyclic AMP to a degree that approached activation by catecholamines. In homogenates, cyclic AMP (1.0 mM) plus ATP (0.25 mM) activated the lipase only 36%, while neither alone had any effect. In digitonin-permeabilized cells, however, exogenous cyclic AMP alone activated lipolysis in a concentration-dependent manner with 1 microM, 30 microM and 1.0 mM cyclic AMP activating lipolysis by 41%, 250% and 1300% respectively. In contrast, lipolysis in intact cells was activated by 0%, 25% and 250% by 1 microM, 30 microM and 1.0 mM cyclic AMP. Also in digitonin-treated preparations, ATP alone activated lipolysis 40%, but ATP plus cyclic AMP activated lipolysis to only 74% of the level due to cyclic AMP alone. These studies indicate that the permeabilized adipocyte preparation is an excellent system for investigating the mechanism of regulation of the hormone-sensitive lipase by permitting manipulation of the intracellular environment while preserving the physiological response of the lipase.