Destruction and Restoration of the Insulin Effector System of Isolated Fat Cells

Abstract

Abstract Incubation of fat cells with trypsin (1 mg per ml) for 1 min at 37° in the presence of serum albumin (20 mg per ml) caused a marked stimulation of glucose transport (an insulinlike effect). When the incubation was continued for 15 min, however, the insulin-like effect disappeared and insulin itself (added after neutralization of the enzyme with soybean trypsin inhibitor) failed to stimulate glucose transport and inhibit lipolysis in the cells. Cells rendered unresponsive to insulin still maintained normal levels of ATP, lactate dehydrogenase, and malate dehydrogenase, and retained the capacity to oxidize glucose rapidly. The glucose transport system of treated cells maintained the normal sensitivity to specific inhibitors and to variations of the external glucose concentration. The lipolytic system of the cell remained highly responsive to epinephrine and adrenocorticotropic hormone, but the responsiveness of the system to glucagon was abolished entirely. When trypsin-treated cells were kept at 37° for more than 30 min after inactivation of the enzyme, the insulin responsiveness returned progressively, reaching 60 to 80% of the control in 60 min. This recovery was inhibited by 0.1 mm puromycin or 0.01 mm cycloheximide. Recovered activity could be abolished again by a second trypsin treatment. In contrast, the responsiveness to glucagon was not restored in 2 hours. These results indicate that trypsin modifies rather selectively the effector systems for insulin and glucagon without affecting other metabolic systems of the cell, including the effector systems for epinephrine and adrenocorticotropic hormone. It is suggested that an essential part of the insulin-effector system is a rapidly renewable peptide element located on the surface of the cell, and that an initial action of trypsin on the system mimics the effect of insulin while subsequent proteolytic modification of the system renders the cells unresponsive to the hormone.