Signalling pathways mediating glucose uptake in skeletal muscle following β2-adrenoceptor activation

Masaaki Sato1,3, Nodi Dehvari1, Anette Oberg1, Olof Dallner1,2, Anna Sandstrom1, Jessica Olsen1, Robert Csikasz1, Roger Summers3, Dana Hutchinson3, Tore Bengtsson1. 1Stockholm University, SE-106 91 Stockholm, Sweden, 2Rockefeller University, NY 10065, USA, 3Monash University, Parkville Vic 3052, Australia

Although activation of β2-adrenoceptors (ARs) in vitro has been shown to stimulate glucose uptake in skeletal muscle, the signalling pathways involved are unclear (1). In vivo, sympathetic effects on glucose homeostasis are influenced by glucose outflow from the liver, insulin release from the pancreas, as well as glucose uptake into peripheral tissues such as white fat, brown fat and muscle. Previously we demonstrated that β2-ARs increase glucose uptake in L6 muscle cells by an AMPK-independent pathway. Although both insulin- and isoprenaline-stimulated glucose uptake in L6 cells was inhibited by LY294002 (10μM), suggesting a role for PI-3-kinase, this inhibitor also inhibits other members of the PI-3-kinase/PIKK family. Akt is downstream of PI-3-kinase, but was clearly not phosphorylated at T308 or S473 following β2-AR stimulation, whereas insulin increased Akt phosphorylation at both sites. The Akt inhibitor X (10μM) inhibited insulin-stimulated (1μM) glucose uptake (% control, 176.2±14.3 vs 128.8±5.7**, n=8) and Akt phosphorylation but not isoprenaline-mediated (1μM) glucose uptake (175.6±18.0 vs 157.7±15.1, NS, n=8). AS160 was also phosphorylated following insulin but not isoprenaline treatment, in an Akt-dependent manner. Thus β2-AR-stimulated glucose uptake does not utilize major components of the insulin pathway. The cell-permeable cAMP analogues 8-bromoadenosine 3′,5′-cAMP (8-Br-cAMP, 1mM), N6,2′-O-dibutyryladenosine 3′,5′-cAMP (db-cAMP, 1mM), and cAMP (2mM) increased glucose uptake, whereas the impermeable analogue 8- Hydroxyadenosine- 3\\\', 5\\\'- cAMP (8-OH, 1mM) had no effect. The general PI-3-kinase/PIKK inhibitor PI-103 reduced isoprenaline-mediated glucose uptake (pIC50 6.30±0.96), as did the selective mTOR inhibitors Torin-1 (pIC50 9.14±0.20) and KU0063794 (KU, pIC50 7.83±0.23). Isoprenaline caused phosphorylation of mTOR at S2481, and insulin at both S2448 and S2481. Akt inhibition decreased insulin-induced phosphorylation of mTOR at S2448 but not isoprenaline-induced phosphorylation of mTOR at S2481 indicating that Akt is not upstream of mTOR S2481. Administration of 8-Br-cAMP (1mM) or the PKA-selective cAMP-analogue 6-Benz-cAMP (1mM) caused phosphorylation of mTOR at S2481, and was also blocked by KU. Short-term (30min) treatment with the mTORC1 inhibitor rapamycin had no effect on isoprenaline and 8-Br-cAMP-stimulated glucose uptake whereas long-term exposure (48h) inhibits mTORC2 assembly and inhibited isoprenaline-stimulated glucose uptake, suggesting involvement of mTORC2. siRNAs for Rictor markedly depleted Rictor protein and abolished isoprenaline-stimulated glucose-uptake (% control, 105.0±15.1*, n=4) whereas Raptor siRNA (169.5±9.4, n=3) or control siRNAs (166.2±14.0, n=4) had no effect. This clearly demonstrated that mTORC2 is the key regulator of β2-AR-mediated glucose uptake in skeletal muscle. GLUT4 translocation was examined by immunohistochemistry in non-permeabilized L6 cells and was observed following β2-AR stimulation in native L6 myoblasts or myotubes, cells transiently transfected with GLUT4mycGFP or stably transfected with GLUT4myc. GLUT4 translocation in response to isoprenaline was abolished by mTOR inhibition. β-AR-mediated glucose uptake is therefore dependent on mTORC2, changes in actin reorganization and on GLUT4 translocation.

P<0.05; ** P<0.01; *** P<0.001

(1) Nevzorova J et al, Brit J Pharmacol 147:446,2006