Open AccessNovel approach to the study of the regulation of hormone‐sensitive lipase in rat adipocytes
Author(s) -
MOONEY Robert A.,
EBERSOHL Richard D.,
McDONALD Jay M.
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07783.x
Subject(s) - lipolysis , digitonin , adipocyte , medicine , lipase , endocrinology , hormone sensitive lipase , chemistry , insulin , biochemistry , biology , enzyme , adipose tissue
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 μg/ml as assessed by (a) leakage of 85% cellular lactate dehydrogenase after 30 min, (b) the efflux of 72% preloaded cellular ( 86 Rb) 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 μM, 30 μM 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 μM, 30 μM and 1.0 mM cyclic AMP. Also in digition‐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.