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

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 PKCbetaII 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 phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII 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, PKCbetaII, 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 phosphoPKCbetaII(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 PKCbetaII 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 PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Fig. 1

a Insulin increased phosphoPKCβII. L6 myotubes were serum-starved for 6 h prior to pretreatment with PP1 (10 μmol/l), LY294002 (20 or 10 μmol/l) for 30 min followed by serum (10%) or insulin (10 μIU/ml) for 30 min. Whole-cell lysates were separated by SDS-PAGE (10%) and transferred to membranes for western blot analysis as indicated with anti-phosphoPKCβII, phosphoAkt, phosphoERK1/2 or actin. Blots were scanned and quantitated using UnScanIt gel digitising software (Silk Scientific, Orem, UT, USA). b, c Relative densitometric units (Den. U) for phosphoPKCβII S660 and T641 and for phosphoAkt T308 and S473. Black bars show units for pPKCβII-S660 (b) and pAkt-T308 (c) and grey bars show units for p-PKCβII-T641 (b) and pAkt-S473 (c). The experiment was repeated three times. Significant inhibition (*p<0.05) of insulin effects by inhibitors on pPKCβII and pAkt sites, as determined using Prism4 (GraphPad Software, La Jolla, CA, USA) by one-way ANOVA. p, phospho

Fig. 2

Cytosolic MARCKS phosphorylation was blocked by PKCβII inhibition. L6 cells were grown as described in Methods. a–d Cytosol; e–i membrane. Subcellular fractions were separated by SDS-PAGE, transferred to nitrocellulose and probed using phosphoMARCKS (p-MARCKS), MARCKS, PLD1 or actin antibodies as indicated. The experiment is representative of three that showed similar results on immunoblots (IB). Densitometric scans (Den. U), determined as described in Fig. 1, are shown above each blot/panel. *p<0.05, unpaired t test

Fig. 3

Insulin-stimulated glucose uptake was reduced by inhibiting either PKCβII (CG, CG53353) or CaCaM (W13) and mimicked by addition of a calcium ionophore (A23187). a L6 myotubes were grown as described and 2-deoxy-D-[1,2-³H]glucose uptake was then determined as outlined in Methods. The experiment was performed in duplicate and repeated on two occasions with similar results. Data are expressed as percentage of maximal insulin response. *p<0.05 for inhibition of ISGT in the presence of inhibitors, Student’s t test. b Cells were transfected with two different PKCβII siRNAs or a scrambled control siRNA as described in Methods. Forty-eight hours later, cells were exposed to insulin (10 μIU/ml) for 30 min. Cell lysates were analysed by immunoblotting (IB) for PKCβII, pMARCKS and MARCKS as described in the Methods. The siRNA lanes represent two separate experiments with different siRNA preparations. Lanes were scanned as described in the legend of Fig. 1 and results are shown in the graphs (c, d). Ctr, control; Den. U, densitometric units; Ins, insulin

Fig. 4

Insulin-stimulated increase in 2-deoxyglucose uptake in L6 cells was blocked by MARCKS siRNA. a L6 cells were grown to 80% confluence and allowed to fuse into myotubes for 4 days. Cells were transfected with siRNA for MARCKS purchased from Ambion as described. Forty-eight hours later, cells were treated with insulin for 30 min. Half of the cells were lysed to perform western blot analysis (a), in which enolase was used to access loading of the gel and half were assayed for 2-deoxy-D-[1,2-³H]glucose uptake (b). The uptake assay was performed in triplicate on two occasions with similar results. Dark grey bars show basal uptake and light grey bars show insulin-stimulated uptake. Results are mean±SEM. *p<0.05 compared with scrambled siRNA for insulin response, Student’s t test

Fig. 5

PKCβII, GLUT4 and MARCKS bound actin in an insulin-dependent manner. L6 myotubes were treated for 30 min with and without 10 μIU/ml insulin, treated with cross-linking agent and then immunoprecipitated (IP) with anti-actin antibody. The bound proteins were then extracted by magnetic separation and treated with dithiothreitol to break cross-links. a Proteins were separated by SDS-PAGE and subjected to immunoblotting (IB) for actin, PKCβII, GLUT4, MARCKS and the insulin receptor. b–f Densitometric analysis (as described in the legend of Fig. 1) for the relevant proteins in (a) are shown beneath the blots. The experiments were repeated on two occasions in separate experiments with similar results. Ins, insulin; cyt, cytosol fraction; memb, membrane fraction

Fig. 6

Transient transfection of MARCKS serine-152-alanine mutant blocked insulin-stimulated 2-deoxyglucose uptake. L6 myotubes at 80% confluence were transiently transfected with pcDNA3, S152A MARCKS or wild-type MARCKS as described in the Methods for 48 h. 2-Deoxyglucose uptake was measured after 10 μIU/ml insulin treatment for 30 min. Duplicate wells were harvested for western blot analysis of phosphoMARCKS, MARCKS and actin (b). Transfection was performed in three separate experiments run in duplicate for 2-deoxyglucose uptake. Light grey shaded bars show basal uptake and dark grey bars show insulin-stimulated uptake. Results are mean± SEM. *p<0.05 compared with pcDNA3 for insulin response, Student’s t test

Fig. 7

a GLUT4 in the plasma membrane was reduced by inhibiting PKCβII activity. L6 cells were grown as described in the Methods. Prior to insulin treatment (10 μIU/ml for 30 min), cells were treated for 30 min with or without 1 μmol/l CG53353 or DMSO. Cells were then treated with KCN to halt metabolic activity and trypsin to cleave exofacial proteins. GLUT4 transporters inserted into the plasma membrane would be susceptible to proteolysis of the exofacial loops by trypsin. Cells were then lysed, membranes isolated and proteins were separated by SDS-PAGE, transferred to nitrocellulose and probed using anti-GLUT4 antibodies recognising the intracellular region of the protein. This detects the full-length (near membrane) and the cleaved (inserted in plasma membrane) protein. The experiment was performed on three occasions with similar results. b The change in the lower molecular mass, fully cleaved fragment determined by scanning as described in the legend of Fig. 1