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

Abstract

1. Beta-adrenoceptor (AR) agonists increase 2-deoxy-[3H]-D-glucose uptake (GU) via beta2-AR in rat L6 cells. The beta-AR agonists, zinterol (beta2-AR) and (-)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC50=9.1+/-0.02 and 7.8+/-0.02). Cholera toxin (% max increase 141.8+/-2.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8+/-13.5 and 196.4+/-17.3%). 2. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (50 microM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7+35.0 to 21.6+4.5 pmol well(-1)), or forskolin (10 microM) (230.1+/-58.0 to 107.2+/-26.3 pmol well(-1)), and partially inhibited zinterol-stimulated GU (217+/-26.3 to 176.1+/-20.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100 nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10 microM), 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 beta2-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 microM), inhibited insulin- (174.9+/-5.9 to 142.7+/-2.7%) and zinterol- (166.9+/-7.6 to 141.1+/-8.1%) 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 beta2-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 beta2-AR-mediated GU.

Figure 1

cAMP accumulation in L6 myotubes following exposure to insulin, or the β-AR agonists (−)-isoprenaline or zinterol. In (a) the (−)-isoprenaline CR curve is markedly shifted to the right by the β₂-AR antagonist ICI 118551 (100 nM) (two-way ANOVA, P<0.0001, n=4), but is not affected by the β₁-AR antagonist CGP 20712A (100 nM) (P=0.3, n=2). In (b) the β₂-AR agonist zinterol significantly increased cAMP levels in a concentration-dependent manner (one-way ANOVA, P<0.0001, n=4), whereas insulin did not significantly affect cAMP levels (P=1.0, n=4).

Figure 2

[³H]-2-DG uptake (GU) in L6 myotubes was increased (a) by insulin (10 μM) (Student's t-test, P<0.01, n=6) or zinterol (100 nM) (P<0.05, n=6), as well as (b) by the direct Gs activator, cholera toxin (ChT, 1 μg ml⁻¹) (P<0.01, n=3) and the cell-permeable cAMP analogues, 8-Br-cAMP (1 mM) (P<0.01, n=4) or dbcAMP (1 mM) (P<0.05, n=4). In (c) the adenylate cyclase inhibitor, ddA (50 μM) partially inhibited GU stimulated by zinterol (P=0.02, n=6), but had no significant effect on either basal (P=0.79, n=5) or insulin (1 μM)-stimulated GU (P=0.59, n=2). In (d and e) cAMP levels in response to zinterol (100 nM) or forskolin (10 μM) were significantly inhibited by ddA (P<0.05, n=3–5).

Figure 3

The selective PDE4 inhibitor, rolipram (10 μM), (a) enhanced the [³H]-2-DG (GU) response to zinterol, causing higher responses at low concentrations of zinterol (two-way ANOVA and Bonferroni post-test, P<0.01, n=4). pEC₅₀ values in the absence (8.7±0.5) or presence (9.3±0.5) of rolipram was not significantly different (Student's t-test, P=0.4, n=4). In (b) rolipram had no significant effect on insulin responses (two-way ANOVA, P=0.06, n=4). In (c) and (d) cAMP accumulation in response to zinterol (100 nM) or forskolin (10 μM) was significantly augmented by the presence of rolipram (10 μM), (Student' t-test, P<0.001, n=3–5).

Figure 4

The selective PKA inhibitor, 4CM, shifted the [³H]-2-DG (GU) CR curve to zinterol (a) to the left at 60 nM (two-way ANOVA, P<0.01, n=5) and 100 nM (P<0.001, n=5). pEC₅₀ values for zinterol were increased from 7.5±0.2 to 8.6±0.3 and 8.8±0.2 in the presence of 4CM at 60 nM and 100 nM, respectively (one-way ANOVA, P<0.0001, n=5). In (b) 4CM had no significant effect on the insulin CR curve (two-way ANOVA, P=0.2 and P=0.4, n=3) or pEC₅₀ values (one-way ANOVA, P=1.0, n=3). In (c) inhibition of zinterol-induced phosphorylation of acetyl Co-A carboxylase (ACC) at Ser79 by 4CM demonstrating that PKA was inhibited by the concentrations used. The blot was obtained using protein from L6 myotubes and shows: (1) pACC in unstimulated cells, (2) pACC in cells treated with zinterol (100 nM), (3) pACC in cells treated with zinterol+4CM (100 nM).

Figure 5

Zinterol-stimulated [³H]-2-DG uptake (GU) (a) was significantly reduced in L6 cells treated with PTX (100 ng ml⁻¹ for 16 h), (P<0.0001, n=6). PTX pretreatment also inhibited insulin-stimulated GU (b) (P<0.05, n=5) but had no inhibitory effect on GU responses to either 8-bromo cyclic AMP (c) (P=0.5, n=5) or dibutyryl cyclic AMP (d) (P=0.5, n=3). Statistical significance was tested using two-way ANOVA.

Figure 6

In (a) [³H]-2-DG uptake (GU) in response to insulin (1 μM) or zinterol (1 μM) was significantly inhibited by the presence of PI3K inhibitor LY294002 (1 μM) (Student's t-test, P=0.01 and P<0.001, n=4–5), whereas the response to 8Br-cAMP (1 mM) was not affected by LY294002 (P=0.9, n=5). In (b) Western blot analysis showed that phosphorylation of Akt at Ser473 occurred in response to insulin (10 μM; 5 min stimulation) but not zinterol (100 nM; 3 h stimulation). Total Akt was detected in all samples. The labels are: basal (1), zinterol (2) and insulin (3). Blots from three separate experiments are shown.

Figure 7

CR curves for (a) zinterol (two-way ANOVA, P<0.0001, n=4) and (b) insulin (P<0.05, n=4) showing [³H]-2-DG uptake (GU) responses at 30 and 180 min, together with responses to (a) zinterol (1 μM) or (b) insulin (10 μM) at different time points showing the progressive increase in GU with time. In contrast, (c) zinterol (100 nM) produced a marked rise in cAMP accumulation after 30 min (Student's t-test, ^***P<0.0001, n=4) but then a progressive decrease at times up to 180 min (one-way ANOVA, ^###P<0.0001, n=4). The desensitisation of the cAMP response does not result from a desensitisation of adenylate cyclase since forskolin-stimulated cAMP accumulation (d) was (one-way ANOVA, ^**P<0.01, n=3) maximal by 60 min, and was maintained up to 180 min.

Figure 8

cAMP accumulation in response to zinterol in the presence or absence of (a) the PKA inhibitor, 4CM (100 nM), at 30, 60 and 180 min. 4CM did not affect zinterol-stimulated cAMP accumulation, or the desensitisation associated with continuous receptor stimulation (two-way ANOVA, P=0.8, n=3) suggesting that desensitisation did not follow PKA phosphorylation of the β₂-AR. In (b) the cAMP time course in response to zinterol following pretreatment with pertussis toxin (PTX, 100 ng ml⁻¹; 16 h). Zinterol-mediated increases in cAMP were significantly lower after PTX pretreatment (two-way ANOVA, P<0.05, n=2).