EVIDENCE THAT PROTEIN-KINASE-C MAY NOT BE INVOLVED IN THE INSULIN ACTION ON CAMP PHOSPHODIESTERASE - STUDIES WITH ELECTROPORATED RAT ADIPOCYTES THAT WERE HIGHLY RESPONSIVE TO INSULIN

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mm NaCl, 118.0 mm KCl, 0.38 mm CaCl2, 1.00 mm EGTA, 1.19 mm Mg2SO4, 1.19 mm KH2PO4, 25.0 mm Hepes/K, 20 mg/ml bovine serum albumin, and 3 mm pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 μF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nm and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nm. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mm, and that in electroporated cells was 0.075 mm. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high. © 1991.