Glucagon-like peptide 1 activates protein kinase C through Ca2+-dependent activation of phospholipase C in insulin-secreting cells

Although the stimulatory effect of glucagon-like peptide 1(GLP1), a cAMP-generating agonist, on Ca2+ signal and insulin secretion is well established, the underlying mechanisms remain to be fully elucidated. We recently discovered that Ca2+ influx alone can activate conventional protein kinase C (PKC) as well as novel PKC in insulin-secreting (INS-1) cells. Building on this earlier finding, here we examined whether GLP-1-evoked Ca2+ signaling can activate PKC alpha and PKC epsilon at a substimulatory concentration of glucose (3 mM) in INS-1 cells. We first showed that GLP-1 translocated endogenous PKC alpha and PKC epsilon from the cytosol to the plasma membrane. Next, we assessed the phosphorylation state of the PKC substrate, myristoylated alanine-rich C kinase substrate ( MARCKS), by using MARCKS-GFP. GLP-1 translocated MARCKS-GFP to the cytosol in a Ca2+-dependent manner, and the GLP-1-evoked translocation of MARCKS-GFP was blocked by PKC inhibitors, either a broad PKC inhibitor, bisindolylmaleimide I, or a PKC epsilon inhibitor peptide, antennapedia peptide-fused pseudosubstrate PKC epsilon-(149-164) (antp-PKC epsilon) and a conventional PKC inhibitor, Go-6976. Furthermore, forskolin-induced translocation of MARCKS-GFP was almost completely inhibited by U73122, a putative inhibitor of phospholipase C. These observations were verified in two different ways by demonstrating 1) forskolin-induced translocation of the GFP-tagged C1 domain of PKC gamma and 2) translocation of PKC alpha-DsRed and PKC alpha-GFP. In addition, PKC inhibitors reduced forskolin-induced insulin secretion in both INS-1 cells and rat islets. Thus, GLP-1 can activate PKC alpha and PKC epsilon, and these GLP-1-activated PKCs may contribute considerably to insulin secretion at a substimulatory concentration of glucose.