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Copyright 2004 The British Pharmacological Society

067P University of Bath
Summer Meeting July 2004

ß2-adrenoceptor mediated increases in glucose transport in l6 skeletal muscle cells involves coupling to both Gs and Gi

J Nevzorova, T Bengtsson, BA Evans & RJ Summers, Department of Pharmacology, Monash University, VIC 3800, Australia and Wenner-Gren Institute Stockholm University, S-106 91 Stockholm, Sweden

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Nevzorova J
Bengtsson T
Evans BA
Summers RJ

Stimulation of ß2-Adrenoceptors (AR) increases glucose transport (GT) in the rat skeletal muscle cell line L6 (Nevzorova et al., 2002). Classically, ß2-ARs couple to Gs to increase intracellular levels of cyclic AMP. In this project we examined the signalling mechanisms of ß2-AR-mediated increases in GT in differentiated L6 cells. GT was measured by uptake of 2-deoxy- [3H]-D- glucose after 180 min incubation with agonists and was increased by the selective ß2AR agonist zinterol (% max 218±5, pEC50 = 9.7±0.2, n=6) and the cell permeable cyclic AMP analogues 8-bromo-cAMP (% max 212±16, pEC500=4.1±0.4, n=4) and dibutyryl cyclic AMP (210±25, pEC50=4.6±0.5, n=4). The cell permeable adenylate cyclase inhibitor 2’,5’-dideoxyadenosine (50 µM) failed to inhibit zinterol stimulated GT (two-way ANOVA, p=0.35, n=4). The PKA inhibitor 4-cyano-3-methylisoquinoline (100nM) did not affect the maximal response to zinterol, but shifted the concentration-response curve to the left (pEC50=7.5±0.2 vs 8.8±0.2; two-way ANOVA, p<0.001, n=5). These results suggest that the cyclic AMP/PKA pathway is not the only mechanism involved in ß2-AR mediated GT. A number of studies show that continuous agonist stimulation of ß2-AR causes receptor desensitisation, following phosphorylation by protein kinase A (PKA) and G protein receptor kinases (GRK), which switches coupling to Gi and results in activation of other signalling pathways (Daaka et al., 1997; Zhu et al., 2001). In L6 cells, the levels of cyclic AMP were increased by zinterol (100nM) after 30 minutes, but were then decreased at times up to 180 min indicating b 2 -AR desensitization (table 1). In contrast, GT was increased after incubation with zinterol for 30 minutes and continued to rise to a significantly higher maximal response after 3 hours (one-way ANOVA, p<0.01, n=4; Table 1). Pertussis toxin reduced zinterol-stimulated GT (% max increase at 180 min, 183±6 vs 159±6, p<0.0001, n=6) indicating that Gi was involved in the process.

In conclusion, the study showed that GT in L6 myotubes can be increased by cyclic AMP analogues and Gs coupled receptors, and that ß2-AR mediated GT may also involve coupling to Gi that occurs following receptor desensitisation.

Table 1. Time-course of cyclic AMP and glucose transport (GT; 2-deoxy- [3H]-D- glucose uptake) in L6 myotubes following stimulation with zinterol (100nM).

Time (min)

0

30

60

90

120

150

180

Cyclic AMP (pmol/mg protein) n=4

35

±21

2105

±334

1538

±238

1049

±319

930

±378

894

±311

724

±257

GT (% basal) n=4

100

133±20

134±11

148±31

175±28

162±35

219±27

Daaka, Y et al (1997) Nature 390, 88-91
Nevzorova, J et al (2002) Br J Pharmacol., 137, 9-18
Zhu, WZ et al (2001) PNAS 98, 1607-1612