Combined effects of insulin and dexamethasone on cyclic AMP phosphodiesterase 3 and glycogen metabolism in cultured rat hepatocytes

Primary cultures of rat hepatocytes were used to study the combined effects of Insulin and dexamethasone on cyclic AMP phosphodiesterase 3 (PDE 3) and glycogen metabolism. PDE activity was measured in extracts obtained by hypotonic shock treatment of the particulate fraction from cultured hepatocytes. PDE 3 was identified by inhibition with ICI 118233, Western blotting, immunoprecipitation of the activity with the use of a new PDE 3B-specific anti-peptide antibody and stimulation of the activity after adding Insulin, glucagon and okadaic acid to the culture medium. Specific PDE inhibitors were always used to identify the measured PDE activities. Hypotonic extracts contained 30% PDE 3 and 50% PDE 4. Both PDE types show a nearly constant level during cultivation up to 48 h. Long-term exposure of dexamethasone alone has no effect on PDE 3 activity, whereas, in combination with Insulin, the Insulin stimulation of PDE 3 activity was found to be increased between 48 and 72 h of cultivation. Additionally, db-cAMP was able to stimulate PDE 3. A possible effect of Insulin or db-cAMP on PDE 3B expression could not be found. On the other hand, activation of PDE 3B after 48 h of culturing decreased rapidly after removal of Insulin or db-cAMP from the culture medium. Insulin-stimulated incorporation of 14C-glucose into glycogen was inhibited by PDE 3- and PDE 4-specific inhibitors as well as by the unspecific PDE inhibitor IMBX. Inhibitions by PDE 3- and PDE 4-specific inhibitors were found to be additive and reached the same extent as with IMBX. Summarising our results, we can conclude that PDE 3 and PDE 4 effectively control the hepatic glycogen metabolism. Insulin effects on PDE activity and glycogen metabolism require the presence of dexamethasone. Insulin-stimulated PDE seems to play an important role in realising Insulin effects on hepatic glycogen metabolism.