Article

Diabetologia

, Volume 52, Issue 5, pp 901-911

First online:

Functional involvement of protein kinase C-βII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells

  • D. S. ChappellAffiliated withDepartment of Molecular Medicine, University of South Florida
  • , N. A. PatelAffiliated withDepartment of Molecular Medicine, University of South FloridaThe Research Service, James A. Haley Veterans Hospital
  • , K. JiangAffiliated withDepartment of Molecular Medicine, University of South Florida
  • , P. LiAffiliated withDepartment of Molecular Medicine, University of South Florida
  • , J. E. WatsonAffiliated withThe Research Service, James A. Haley Veterans Hospital
  • , D. M. ByersAffiliated withAtlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University
  • , D. R. CooperAffiliated withDepartment of Molecular Medicine, University of South FloridaThe Research Service, James A. Haley Veterans Hospital Email author 

Abstract

Aims/hypothesis

Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCβII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements.

Methods

We examined phosphoPKCβII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCβII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCβII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCβII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353.

Results

Insulin increased phosphoPKCβII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCβII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells.

Conclusions/interpretation

The results suggest that PKCβII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Keywords

F-actin Glucose transporter 4 Insulin-stimulated glucose uptake L6 myocytes MARCKS Phospholipase D1 PKCβ