The role of PLC, cPKC, L-type calcium channels and CAMKII in insulin-stimulated glucose transport in skeletal muscle

Insulin-stimulated glucose transport in skeletal muscle is mediated by a complex array of phosphorylation reactions. Although the distal components of this process have not been fully elucidated, it appears that a phospholipase C (PLC) dependent mechanism may be involved. Therefore, the purpose of this dissertation was to determine the interactions between PLC and several reputed downstream regulators of insulin action, namely, protein kinase C (PKC), L-type calcium channels and calcium/calmodulin dependent protein kinase II (CAMKII). Using an in vitro preparation of rat skeletal muscle in conjunction with 3H-3-O-methylglucose and C14 mannitol, glucose transport was determined in the presence of the PLC inhibitor U73122 and/or the conventional/novel PKC inhibitor GF109203X. U73122 (20 μM) and GF1029203X (1 μM) inhibited insulin stimulated (100 μU/ml) glucose transport by ∼32% and 41%, respectively. The combined effects of these two inhibitors were no greater than the effects of either compound alone. Western blot analysis revealed that insulin induced an ∼20% decrease in cytosolic PKC βII and a parallel increase in membrane associated PKC βII. The insulin induced redistribution of this enzyme was reversed in the presence of U73122 and GF109203X. Likewise, insulin stimulation caused an ∼30% increase in phosphorylated membrane associated PKC βII. This increase was attenuated by U73122 and GF109203X. To determine the relationship between PKC and L-Type calcium channels, glucose transport was measured in the presence of insulin, nifedipine (100 μM) and/or GF109203X. Insulin stimulated glucose transport was inhibited ∼36% by nifedipine. The combined effects of both nifedipine and GF109203X resulted in a ∼47% reduction in glucose transport. The final interaction examined was that of L-type calcium channels and CAMKII. Insulin stimulated glucose transport was attenuated to a similar degree by nifedipine (∼31%) and the CAMKII inhibitor KN62 (∼29%). The combined effects of these compounds had no further inhibitory effect on glucose transport. Utilizing Western blot analysis, insulin stimulation was found to result in an ∼35% increase in the phosphorylation of CAMKII. This increase was abrogated by KN62 but not nifedipine. In conclusion, a PLC - PKC βII signaling mechanism appears to be involved in the control of insulin stimulated glucose transport. L-type calcium channels, though involved in glucose transport, do not appear to be regulated by conventional PKC isozymes. Similarly, the involvement of CAMKII in glucose transport is not controlled, as hypothesized, by L-type calcium channels.