Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation

Aim: To establish the mechanism responsible for the stimulation of glucose uptake by Astragalus polysaccharide (APS), extracted from Astragalus membranaceus Bunge, in L6 myotubes in vitro. Methods: APS-stimulated glucose uptake in L6 myotubes was measured using the 2-deoxy-[H-3]-D-glucose method. The adenine nucleotide contents in the cells were measured by HPLC. The phosphorylation of AMP-activated protein kinase (AMPK) and Akt substrate of 160 kDa (AS160) was examined using Western blot analysis. The cells transfected with 4P mutant AS160 (AS160-4P) were constructed using gene transfer approach. Results: Treatment of L6 myotubes with APS (100-1600 mu g/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 mu g/mL) for 36 h. The APS-stimulated glucose uptake was significantly attenuated by pretreatment with Compound C, a selective AMPK inhibitor or in the cells overexpressing AS160-4P. Treatment of L6 myotubes with APS strongly promoted the activation of AMPK. We further demonstrated that either Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKK beta) or liver kinase B1 (LKB1) mediated APS-induced activation of AMPK in L6 myotubes, and the increased cellular AMP: ATP ratio was also involved. Treatment of L6 myotubes with APS robustly enhanced the phosphorylation of AS160, which was significantly attenuated by pretreatment with Compound C. Conclusion: Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.