Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation
Abstract
Jian LIU1, Jing-fang ZHANG2, Jin-zhi LU1, De-ling ZHANG1, Ke LI1, Ke SU1, Jing WANG1, Ye-min ZHANG1, Nian WANG1, Si-tu YANG1, Lang BU1, Jing-ping OU-YANG1, *
1Department of Pathophysiology, School of Basic Medical Sciences, Wuhan University, Hubei Provincial Key Laboratory of Allergy and Immune-Related Diseases, Wuhan 430071, China; 2Medical College, Jingchu University of Technology, Jingmen 448000, China
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-[3H]-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 µg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/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 β (CaMKKβ) 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.
Keywords: Astragalus polysaccharide; L6 myotubes; glucose uptake; AMP; AMPK; AS160; CaMKKβ; LKB1; type 2 diabetes mellitus
We are especially grateful to Dr Laurie J GOODYEAR (Joslin Diabetes Center, Boston, MA, USA), Dr Takahiro NAGASE (Kazusa DNA Research Institute, Chiba, Japan), and Dr Jun-ichi MIYAZAKI (Osaka University Medical School, Osaka, Japan) for providing valuable materials.
This work was supported by the National Natural Science Foundation of China (No 30771023).
* To whom correspondence should be addressed.
E-mail jpoy@163.com
Received 2012-04-26 Accepted 2012-08-05
Keywords:
1Department of Pathophysiology, School of Basic Medical Sciences, Wuhan University, Hubei Provincial Key Laboratory of Allergy and Immune-Related Diseases, Wuhan 430071, China; 2Medical College, Jingchu University of Technology, Jingmen 448000, China
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-[3H]-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 µg/mL) significantly increased glucose uptake in time- and concentration-dependent manners. The maximal glucose uptake was reached in the cells treated with APS (400 μg/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 β (CaMKKβ) 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.
Keywords: Astragalus polysaccharide; L6 myotubes; glucose uptake; AMP; AMPK; AS160; CaMKKβ; LKB1; type 2 diabetes mellitus
We are especially grateful to Dr Laurie J GOODYEAR (Joslin Diabetes Center, Boston, MA, USA), Dr Takahiro NAGASE (Kazusa DNA Research Institute, Chiba, Japan), and Dr Jun-ichi MIYAZAKI (Osaka University Medical School, Osaka, Japan) for providing valuable materials.
This work was supported by the National Natural Science Foundation of China (No 30771023).
* To whom correspondence should be addressed.
E-mail jpoy@163.com
Received 2012-04-26 Accepted 2012-08-05