The International Journal of Biochemistry & Cell Biology 44 (2012) 1321–1330 Contents lists available at SciVerse ScienceDirect The International Journal of Biochemistry & Cell Biology journa l h o me page: www.elsevier.com/locate/biocel Acute exposure of L6 myotubes to cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid isomers stimulates glucose uptake by modulating Ca 2+ /calmodulin-dependent protein kinase II Suresh K. Mohankumar a,c , Carla G. Taylor a,b,c , Linda Siemens a,c , Peter Zahradka a,b,c,∗ a Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada b Physiology, University of Manitoba, Winnipeg, MB, Canada c Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface Hospital Research Centre, Winnipeg, MB, Canada R2H 2A6 a r t i c l e i n f o Article history: Received 13 February 2012 Received in revised form 2 May 2012 Accepted 8 May 2012 Keywords: Glucose uptake CLA isomers CaMKII AMPK Signal transduction L6 myotubes a b s t r a c t Conjugated linoleic acid (CLA), a dietary fat, has been considered beneficial in metabolic syndrome. Despite several findings indicating that CLA improves glucose clearance, little information is avail- able regarding the cellular dynamics of CLA on skeletal muscle. We sought to investigate the role of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) in cis-9, trans-11(c9,t11) and trans-10, cis-12 (t10,c12) CLA isomer-mediated glucose transport by L6 myotubes. t10,c12-CLA stimulated both intra- cellular Ca 2+ release (Ca i 2+ ) and CaMKII phosphorylation, whereas c9,t11-CLA showed only modest effects on both. Sequestering Ca i 2+ with BAPTA/AM abrogated the effect of both CLA isomers on Akt substrate-160 kDa (AS160) phosphorylation and glucose uptake by myotubes. Exposing myotubes to KN-93 or autocamtide 2-related inhibitory peptide to block CaMKII activity prevented both CLA isomers from inducing AS160 phosphorylation and glucose transport. Likewise, genetic knockdown of CaMKII in myotubes using siRNA completely abolished CLA isomer-mediated glucose uptake. These results indi- cate that CLA isomers require Ca i 2+ –CaMKII to mediate glucose uptake. Evidence that CaMKII blockers inhibit t10,c12-CLA-mediated AMP-activated protein kinase (AMPK) activation indicated that CaMKII acts upstream of AMPK in response to t10,c12-CLA. Lastly, CLA isomers stimulated the formation of reac- tive oxygen species but had no effect on stress-activated protein kinase/c-jun NH 2 -terminal kinase. These data establish that t10,c12-CLA acts via Ca i 2+ –CaMKII–AMPK–AS160 to stimulate skeletal muscle glucose transport, whereas the mechanism of c9,t11-CLA remains unclear. Given that impairments in muscle glu- cose utilisation are apparent in metabolic syndrome, delineating the molecular mechanisms by which CLA isomers mediate muscle glucose uptake may identify new approaches to manage this condition. © 2012 Elsevier Ltd. All rights reserved. Abbreviations: AIP, autocamtide 2-related inhibitory peptide; AMP, adenosine 5-monophosphate; AMPK, AMP-activated protein kinase; AS160, Akt substrate- 160 kDa; Ca i 2+ , intracellular calcium; CaMK, Ca 2+ /calmodulin-dependent protein kinase; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; CIP, calmodulin inhibitory peptide-control; CLA, conjugated linoleic acid; DCFH-DA, dichlorodi- hydrofluorescein diacetate; GLUT4, glucose transporter-4; HRP, horseradish peroxidase; H2O2, hydrogen peroxide; MAPK, mitogen-activated protein kinase; NF- B, nuclear factor B; PI3K, phosphatidylinositol-3 kinase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; SAPK/JNK, stress-activated protein kinase/c-jun NH2-terminal kinase; ROS, reactive oxygen species; SDS, sodium dode- cyl sulphate; SE, standard error; T2DM, type 2 diabetes mellitus; TBST, Tris-buffered saline-Tween. ∗ Corresponding author at: Canadian Centre for Agri-food Research in Health and Medicine, St. Boniface Hospital Research Centre, 351 Tache Avenue, Winnipeg, MB, Canada R2H 2A6. Tel.: +1 204 235 3507; fax: +1 204 237 4018. E-mail address: peterz@sbrc.ca (P. Zahradka). 1. Introduction Conjugated linoleic acid (CLA) isomers are lipids naturally present in our diet. They have gained considerable attention, since their consumption is associated with various health bene- fits (Belury, 2002; Bhattacharya et al., 2006). Linoleic acid, an 18 carbon-chain unsaturated fatty acid with two cis double bonds at the 9th and 12th carbons (cis-9, cis-12-octadecadienoate), is converted into various isomers of CLA in the rumen of rumi- nants (Kritchevsky, 2000). Cis-9, trans-11 (c9, t11)-CLA is the most abundant CLA isomer in dairy products and ruminant meats, while commercial dietary supplements contain a mixture of c9, t11-CLA and trans-10, cis-12 (t10,c12)-CLA. Recent findings have demonstrated isomer-specific effects of CLA on skeletal muscle glu- cose utilisation (Henriksen et al., 2003; Hommelberg et al., 2010; Mohankumar et al., in press; Qin et al., 2009; Ryder et al., 2001). We recently demonstrated that acute exposure of L6 myotubes to c9,t11 and t10,c12-CLA isomers elicited isomer-specific effects 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2012.05.005