Multiple signalling pathways involved in beta2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells.

British Journal of Pharmacology (2006) 147, 446–454 & 2006 Nature Publishing Group All rights reserved 0007 – 1188/06 $30.00 www.nature.com/bjp Multiple signalling pathways involved in b2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells 1,3 Julia Nevzorova, 1Bronwyn A. Evans, 2Tore Bengtsson & *,1Roger J. Summers 1 Department of Pharmacology, PO Box 13E, Monash University, Victoria 3800, Australia and 2The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden Keywords: Abbreviations: 1 b-adrenoceptor (AR) agonists increase 2-deoxy-[3H]-D-glucose uptake (GU) via b2-AR in rat L6 cells. The b-AR agonists, zinterol (b2-AR) and (
)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC50 ¼ 9.170.02 and 7.870.02). Cholera toxin (% max increase 141.872.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8713.5 and 196.4717.3%). 2 The adenylate cyclase inhibitor, 20 ,50 -dideoxyadenosine (50 mM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7 þ 35.0 to 21.6 þ 4.5 pmol well
1), or forskolin (10 mM) (230.1758.0 to 107.2726.3 pmol well
1), and partially inhibited zinterol-stimulated GU (217726.3 to 176.1720.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100 nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10 mM), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. 3 cAMP accumulation studies indicated that the b2-AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. 4 PTX pretreatment (100 ng ml
1) inhibited insulin or zinterol-stimulated but not 8Br-cAMP or dbcAMP-stimulated GU. The PI3K inhibitor, LY294002 (1 mM), inhibited insulin- (174.975.9 to 142.772.7%) and zinterol- (166.977.6 to 141.178.1%) but not 8 Br-cAMP-stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. 5 The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin-dependent pathway involving PI3K, (2) a b2-AR-mediated pathway involving both cAMP and PI3K, and (3) a receptor-independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate b2-AR-mediated GU. British Journal of Pharmacology (2006) 147, 446–454. doi:10.1038/sj.bjp.0706626; published online 16 January 2006 Glucose uptake; b-adrenoceptor (AR); skeletal muscle cells; cAMP; PI3K [3H]-2-DG, 2-deoxy-[3H]-D-glucose; CR, concentration–response curve; Gi proteins, inhibitory guanosine triphosphatases; GLUT, glucose transporter; GS proteins, stimulatory guanosine triphosphatases; GU, glucose uptake; pEC50, negative log EC50; PI3K, phosphatidyl inositol-3 kinase; pKB, negative log KB Introduction Several reports have demonstrated insulin-independent stimulation of glucose uptake (GU) in response to adrenoceptor (AR) stimulation, suggesting that ARs may represent an alternative way to increase GU in the absence of insulin. In the rat skeletal muscle cell line L6, GU is increased by activation of b2-ARs (Nevzorova et al., 2002), the predominant b-AR subtype expressed in these cells (Nagase et al., 2001; Nevzorova et al., 2002). b2-ARs are coupled to Gs and adenylate cyclase and utilise cAMP and protein kinase A (PKA) to produce the metabolic effects of catecholamines. Adrenergic agonists also increase GU in skeletal muscle, white adipose tissue and heart (Abe et al., 1993; Liu & Stock, *Author for correspondence; E-mail: 3 Current address: The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91 Stockholm, Sweden. 1995; Liu et al., 1996). In addition, adrenaline-stimulated translocation of the insulin-sensitive glucose transporter 4 (GLUT4) has been shown in skeletal muscle (Han & Bonen, 1998). In primary brown adipocytes, stimulation of b3-ARs increases GU in a cAMP-dependent manner (Shimizu et al., 1998; Chernogubova et al., 2004). In addition, b1- and to a smaller extent a1-ARs compensate for the lack of b3-AR in brown adipocytes from b3-AR knockout mice, and increase GU via cAMP/PKA/PI3K or PI3K/PKC respectively (Chernogubova et al., 2005). On the other hand, some studies show adrenaline-mediated inhibition of insulin-stimulated GU in skeletal muscle (Lee et al., 1997). In a number of cell types, it has been demonstrated that the inhibitory effect of cAMP on GU occurs in cells that express GLUT4, and possibly involves cAMP-dependent inhibition of intrinsic activity of the transporter (Lawrence et al., 1992; Piper et al., 1993; Reusch et al., 1993). In contrast, cAMP upregulates GLUT1 gene J. Nevzorova et al expression and protein synthesis in 3T3-L1 cells, L6 myoblasts and in a human choriocarcinoma cell line (Vinals et al., 1997; Ogura et al., 2000; Imamura et al., 2001; Fong et al., 2004; Chiou & Fong, 2005). Therefore, cAMP appears to be capable of both positive and negative modulation of GU with the effect being highly dependent on cell type. L6 cells are widely used to study insulin-dependent or independent mechanisms of GU and can be differentiated into skeletal muscle-like myotubes, that express the GLUT4 (Mitsumoto et al., 1991; Mitsumoto & Klip, 1992). Increases in GU can be produced in L6 cells by b-AR agonists independently of insulin (Tanishita et al., 1997), and we recently demonstrated in pharmacological and molecular studies that the b-AR-subtype involved in those responses is the b2-AR (Nevzorova et al., 2002). The signalling mechanisms mediating b2-AR stimulated GU in L6 cells have not been extensively studied. The present study examined the role of cAMP in GU, and also its involvement in b2-AR-mediated stimulation of GU in L6 myotubes. Methods Cell culture L6 cells were obtained from ATCC. The cells were grown and differentiated for 7 days as described previously (Tanishita et al., 1997; Nevzorova et al., 2002). For GU experiments, cells were grown and differentiated in 24-well cell culture dishes, and for cAMP experiments cells were grown and differentiated in 12-well or 96-well cell culture dishes. Cells were serumstarved overnight before each experiment. Measurement of cAMP using FlashPlate radioimmunoassay The assay is based on competition between unlabelled cAMP in the test samples and [125I]-cAMP for the anti-cAMP antibody embedded in the solid scintillant-coated wells of the 96-well plate. The cells were pretreated with antagonists or vehicle for 15 min, and then with agonists for further 30 min in 96-well plates containing phenol red-free medium with FBS (0.1%) in the presence of 3-isobutyl methyl-xanthine (IBMX; 2 mM). For time course experiments, the cells were (...truncated)