title: Astragalus polysaccharide stimulates glucose uptake in L6 myotubes through AMPK activation and AS160/TBC1D4 phosphorylation.
abstract: OBJECTIVE
  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-[(3)H]-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 Ca(2+)/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.
  
  
  CONCLUSIONS
  Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.
authors.affiliation: Department of Pathophysiology, School of Basic Medical Sciences, Wuhan University, Hubei Provincial Key Laboratory of Allergy and Immune-Related Diseases, Wuhan, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Jing-fang
authors.initials: JF
authors.lastname: Zhang
authors.forename: Jin-zhi
authors.initials: JZ
authors.lastname: Lu
authors.forename: De-ling
authors.initials: DL
authors.lastname: Zhang
authors.forename: Ke
authors.initials: K
authors.lastname: Li
authors.forename: Ke
authors.initials: K
authors.lastname: Su
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Ye-min
authors.initials: YM
authors.lastname: Zhang
authors.forename: Nian
authors.initials: N
authors.lastname: Wang
authors.forename: Si-Tu
authors.initials: ST
authors.lastname: Yang
authors.forename: Lang
authors.initials: L
authors.lastname: Bu
authors.forename: Jing-ping
authors.initials: JP
authors.lastname: Ou-Yang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D027883:Astragalus Plant / Q000737:chemistry*; D001692:Biological Transport / Q000187:drug effects; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011134:Polysaccharides / Q000302:isolation & purification / Q000494:pharmacology*; D051381:Rats; D015854:Up-Regulation
chemical_list: D020690:GTPase-Activating Proteins; D011134:Polysaccharides; C533101:TBC1D4 protein, rat; D000255:Adenosine Triphosphate; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases; D005947:Glucose