Abstract
1. The effect of insulin on K+ currents was studied with enzymatically dispersed ventricular myocytes from insulin-deficient (type I) diabetic rats. Diabetic conditions were induced by a single intravenous injection of streptozotocin (100 mg kg-1) given 8-13 days before the experiments. Measurements of plasma glucose and insulin levels confirmed the diabetic status of the animals. 2. A Ca2+-independent transient outward K+ current, It, and a slowly inactivating, quasi-steady-state current, Iss, which are depressed in diabetic myocytes, could be restored by exposure to 1, 10 or 100 nM insulin. This was only observed after a delay of 5-6 h, although an insulin exposure of only 1 h was sufficient to initiate its stimulatory action on It and Iss. The stimulatory effect of insulin on these K+ currents was prevented by 2 microM cycloheximide, which in itself had no direct effect on these currents. 3. Disruption of the actin microfilament network with 1 microM cytochalasin D (CD) also prevented the stimulatory effect of 100 nM insulin on both It and Iss. Since CD was added 1 h after insulin, inhibitory effects on insulin signalling were ruled out. Adding CD (1 microM) 5-9 h after insulin, when currents were already augmented, had no effect (up to 50 min exposure). Incubating control cells for 6-10 h with 1 microM CD had no effect on any of the currents measured. 4. Stabilization of the actin network by pre-exposure to 2.5 microM phalloidin restored the stimulatory effect of insulin, in the continued presence of CD, ruling out any effects of CD on components other than the cytoskeleton. 5. The stimulatory effect of insulin was also prevented by incubating cells with insulin in the presence of the microtubule-disrupting agent colchicine (5 microM). 6. These results suggest that the insulin-mediated augmentation of K+ currents in diabetic myocytes requires protein synthesis, possibly of K+ channels, as well as an intact cytoskeleton. The possibility that newly formed channels translocate to the plasma membrane in a process dependent on different elements of the cytoskeleton is discussed.