Glucose-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in isolated rat pancreatic islets.
In order to further evaluate the role of protein kinase C activation in glucose-induced insulin secretion, the extent of phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) was examined in freshly isolated rat pancreatic islets prelabeled with [32P]orthophosphate. The islets were incubated with either 2.75 mM glucose alone, 2.75 mM glucose + 1 microM phorbol myristate acetate, 20 mM glucose, or 20 mM glucose + 50 nM staurosporine. After stimulation, the homogenized islets were processed by immunoprecipitation with a specific polyclonal anti-MARCKS antibody, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Densitometric analysis of autoradiograms revealed that phorbol myristate acetate caused a 3.78 +/- 0.97-fold increase in MARCKS phosphorylation over control. In the islets exposed to 20 mM glucose, an increase of 3.43 +/- 0.46-fold over control was observed. In islets exposed to G20 + 50 nM staurosporine, MARCKS phosphorylation was inhibited by 90 +/- 4% compared with control islets exposed to 20 mM glucose alone. Islets similarly treated (but incubated without 32P) were examined by immunocytochemistry using an alpha-PKC-specific monoclonal antibody and visualized by confocal immunofluorescence microscopy. The alpha-PKC redistributed from the cytosol to the plasma membrane in the beta-cells of islets exposed to 20 mM glucose. In separate experiments, unlabeled but similarly treated islets were shown to respond with a 5-7-fold increase in insulin secretion in static incubation. Thus, when freshly isolated rat pancreatic islets are exposed to stimulatory glucose concentrations, they exhibit both a translocation of alpha-PKC and a significant increase in the extent of phosphorylation of MARCKS protein. These data suggest that alpha-PKC is activated during glucose-induced insulin secretion.
Calle
R
R
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Ganesan
S
S
Smallwood
J I
JI
Rasmussen
H
H
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D047908:Intracellular Signaling Peptides and Proteins; D007515:Islets of Langerhans / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D019311:Staurosporine
D000470:Alkaloids; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D019311:Staurosporine; D005947:Glucose