Characterization of the beta-adrenoceptor subtype involved in mediation of glucose transport in L6 cells

Abstract

1. The receptor that mediates the increase in glucose transport (GT) in response to beta-adrenoceptor (beta-AR) agonists was characterized in the rat skeletal muscle cell line L6, using the 2-deoxy-[(3)H]-D-glucose assay. 2. The beta(3)-AR agonist BRL37344 (pEC(50) = 6.89 +/- 0.21), the beta-AR agonist isoprenaline (pEC(50) = 8.99 +/ -0.24) and the beta(2)-AR agonist zinterol (pEC(50) = 9.74 +/- 0.15) increased GT as did insulin (pEC(50) = 6.93 +/- 0.15). The highly selective beta(3)-AR agonist CL316243 only weakly stimulated GT. 3. The pK(B) values calculated from the shift of the pEC(50) values of the agonists in the presence of the beta(1)-AR selective antagonist CGP 20712A or the beta(3)-AR selective antagonist SR 59230A were not indicative of activation of beta(1)- or beta(3)-ARs. Only (-)-propranolol and the beta(2)-AR selective antagonist ICI 118551 caused marked rightward shifts of CR curves to isoprenaline (pK(B) = 10.2 +/- 0.2 and 9.6 +/- 0.3), zinterol (pK(B) = 9.0 +/- 0.1 and 9.4 +/- 0.3) and BRL 37344 (pK(B) = 9.4 +/- 0.3 and 8.4 +/- .2), indicating participation of beta(2)-ARs. 4. The pharmacological analysis was supported by reverse transcription and polymerase chain reaction analysis of L6 mRNA, which showed high levels of expression of beta(2)-AR but not beta(1)- or beta(3)-AR in these cells. 5. Forskolin and dibutyryl cyclic AMP produced negligible increases in GT while the phosphatidylinositol-3 kinase inhibitor, wortmannin, significantly decreased both insulin- and zinterol-stimulated GT, suggesting a possible interaction between the insulin and beta(2)-AR pathways. 6. This study demonstrates that beta(2)-ARs mediate the increase in GT in L6 cells to beta-AR agonists, including the beta(3)-AR selective agonist BRL 37344. This effect does not appear to be directly related to increases in cyclic AMP but requires P13K.

Figure 1

(a) Undifferentiated L6 cells. Scale bar=45 μm. (b) L6 myotubes differentiated for 7 days. (c) CR curves for accumulation of [³H]-2-DG were constructed for insulin, BRL 37344, CL 316243, zinterol and isoprenaline, in L6 myotubes. The graph shows mean±s.e.mean, n=6–10.

Figure 2

[³H]-2-DG transport in response to BRL 37344, zinterol and isoprenaline in the absence or presence of 10⁻⁷ M of the β₃-AR selective antagonist SR 59230A (a, b and c), or the β₁-AR selective antagonist CGP 20712A (d, e and f) in L6 myotubes. The graphs show mean±s.e. of the mean, n=4–5.

Figure 3

[³H]-2-DG transport in response to BRL 37344, zinterol and isoprenaline in the absence or presence of 10⁻⁷ M of the β₂-AR selective antagonist ICI 118551 (a, b and c) or the non-selective β-AR antagonist propranolol (d, e, and f) in L6 myotubes. The graphs show mean±s.e.mean, n=4–6.

Figure 4

The figure shows β₁- (441 bp), β₂- (468 bp) and β₃-AR (473 bp) PCR products from rat heart, cerebellum and WAT, and β₂-AR PCR product from L6 myotubes. Levels of β₁-, β₂- and β₃-ARs expression were measured using RT/PCR analysis.

Figure 5

(a,b) [³H]-2-DG transport in response to insulin and zinterol in the absence or presence of PI3K inhibitor wortmannin (10⁻⁷ M) in L6 myotubes. (c) CR curves for accumulation of [³H]-2-DG by forskolin and dibutyryl cyclic AMP in L6 myotubes. The graphs show mean±s.e.mean, n=4–6.

Figure 6

[³H]-2-DG transport in response to 10⁻⁵ M of BRL 37344, zinterol and isoprenaline in the absence or presence of insulin (10⁻⁵ M). The histograms are mean±s.e.mean, n=4–5.