Adrenaline increases glucose transport via a Rap1-p38MAPK pathway in rat vascular smooth muscle cells

Abstract

Background and purpose: Adrenaline has been implicated in the pathogenesis of atherosclerosis. However, little is known regarding the role of adrenaline in glucose transport in VSMC.

Experimental approach: In this study, we examined the effects of adrenaline on glucose uptake in rat VSMC. We also examined the downstream signaling pathway from the beta-adrenoceptor to glucose uptake, using a pharmacological approach. To investigate the downstream action of adenylate cyclase, we studied the effects of GGTI-298, an inhibitor of geranylgeranylation of GTPases, including Rap1. To confirm the involvement of Rap1, we silenced Rap1 by siRNA.

Key results: Adrenaline induced glucose uptake in a dose-dependent manner. The adrenaline-induced glucose uptake was inhibited by L-propranolol, (a selective beta-adrenoceptor antagonist), but not by prazosin (a selective alpha(1)-adrenoceptor antagonist) or UK14304 (a selective alpha(2)-adrenoceptor antagonist), suggesting the involvement of beta-adrenoceptors in glucose transport. Long-term treatment with cholera toxin, which resulted in sequestration of G(s) proteins, prevented the adrenaline-induced glucose uptake. Forskolin, a direct activator of adenylate cyclase, was found to mimic the effects of adrenaline. Adrenaline-induced glucose uptake was inhibited by GGTI-298, not by H89 (a selective inhibitor of PKA). Silencing of Rap1 by siRNA attenuated the adrenaline-induced glucose uptake. Adrenaline-induced glucose uptake was inhibited by SB203580 (a selective inhibitor of p38MAPK) and adrenaline-induced p38MAPK activation was inhibited by GGTI-298 and siRNA against Rap1.

Conclusions and implications: These findings suggest that adrenaline-induced glucose transport is mediated by beta-adrenoceptors, G(s), adenylate cyclase, Rap1, and p38MAPK in vascular smooth muscle cells.

Figure 1

Effects of adrenaline on 2-DG uptake in VSMC. VSMC grown in 24-well plates were serum-starved for 24 h. (a) The cells were stimulated for 1 h with various concentrations of adrenaline (Adr). (b) The cells were pretreated with adrenoceptor antagonists (L-propranolol (prop), prazosin (praz), UK14304 (UK) all at 10 μM) or vehicle and then stimulated with adrenaline (10 μM) for 1 h. (b) The cells were pretreated with L-propranolol (10 μM) or vehicle and then stimulated for 1 h with isoprenaline (Iso, 10 μM). After stimulation, uptake of 2-DG by the VSMC was measured. Each value represents the mean±s.d. of three independent experiments in triplicate. ^*P<0.05.

Figure 2

The role of G_s in adrenaline-induced 2-DG uptake in VSMC. VSMC were incubated with or without cholera toxin (CTX, 100 ng ml⁻¹) for 72 h in DMEM. (a) Western blot analysis with anti-G_s antibody or anti-α-smooth muscle (SM) actin antibody. (b) The cells were stimulated with adrenaline (Adr, 10 μM) for 1 h and then uptake of 2-DG was measured. (c) VSMC were incubated with forskolin (FSK, 50 μM), dibutyryl cAMP (dbcAMP, 100 μM) or vehicle for 30 min. Then uptake of 2-DG by the VSMC was measured. Each value represents the mean±s.d. of three independent experiments in triplicate. ^*P<0.05.

Figure 3

Effects of PKA inhibitors on adrenaline-induced 2-DG uptake and PKA activation in VSMC. Serum-starved VSMC were incubated with H89 (1 μM), KT5720 (1 μM) or vehicle for 30 min. (a) The cells were stimulated with adrenaline (Adr, 10 μM) for 1 h and then uptake of 2-DG by the VSMC was measured. (b) The cells were stimulated with adrenaline (10 μM) for 10 min and PKA activity was analyzed. Each value represents the mean±s.d. of three independent experiments in triplicate. ^*P<0.05.

Figure 4

Effects of GGTI-298 in 2-DG uptake in VSMC. (a) Serum-starved VSMC were incubated with GGTI-298 (10 μM) or vehicle for 30 min. The cells were then stimulated with adrenaline (Adr, 10 μM) for 1 h and then uptake of 2-DG by the VSMC was measured. Each value represents the mean±s.d. of three independent experiments in triplicate. (b) VSMC were incubated with GGTI-298 (10 μM) or vehicle for 30 min and then stimulated with adrenaline (Adr, 10 μM) for 10 min. The cells were lysed and Rap1 activity was measured using GST-RalGDS Rap1 binding domain. (c) VSMC were incubated with GGTI-298 (10 μM) or vehicle for 30 min and then stimulated with adrenaline (10 μM) for10 min. The cells were lysed and Rap1 activity was measured as in (b). (d) Serum-starved VSMC were stimulated with 8-pCPT-2′-O-Me-cAMP (8-CPT, 1 mM) for 30 min and then uptake of 2-DG by the VSMC was measured. Each value represents the mean±s.d. of three independent experiments in triplicate. ^*P<0.05.

Figure 5

The role of Rap1 in 2-DG uptake in VSMC. (a) Forty-eight hours after transfection with the siRNA against Rap1, cell lysates were prepared and subjected to SDS-PAGE and immunoblotting with and anti-Rap1 antibody, anti-Rac antibody or anti-α-smooth muscle (SM) actin antibody. (b) Rap1-silenced VSMC were serum-starved for 4 h and then stimulated with adrenaline (Adr, 10 μM) for 1 h. Then uptake of 2-DG by the VSMC was measured. Each value represents the mean±s.d. of three independent experiments in triplicate. ^*P<0.05.

Figure 6

The role of p38MAPK in adrenaline-induced 2-DG uptake in VSMC. (a) Serum-starved VSMC were incubated with SB203580 (SB, 10 μM), PD98059 (PD, 20 μM) and U0126 (U, 1 μM), or vehicle for 30 min and then stimulated with adrenaline (Adr, 10 μM). Then uptake of 2-DG by the VSMC was measured. Each value represents the mean±s.d. of three independent experiments in triplicate. (b) Serum-starved VSMC were incubated with PD98059 (20 μM), U0126 (1 μM) or vehicle for 30 min and then stimulated with adrenaline (10 μM) for 5 min. ERK phosphorylation was analyzed by immunoblotting with anti-phospho-specific ERK antibody. (c) Serum-starved VSMC were incubated with or without GGTI-298 (GG, 10 μM) for 30 min. The cells were then stimulated with adrenaline (10 μM) for 10 min. p38MAPK activity was measured as described in Methods. (d) Forty-eight hours after transfection with the siRNA against Rap1, VSMC were stimulated with adrenaline (10 μM) for 10 min. Then p38MAPK activity was measured as in (c). ^*P<0.05.

Figure 7

Schematic model summarizing our findings. G_s, cAMP, Rap1 and subsequent p38MAPK activation play a role in adrenaline-mediated glucose uptake in VSMC.