Abstract
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. 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. 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. Our results demonstrate that APS stimulates glucose uptake in L6 myotubes through the AMP-AMPK-AS160 pathway, which may contribute to its hypoglycemic effect.
Highlights
Type 2 diabetes mellitus (T2DM) is one of the fastest growing public health problems
First, we show that Astragalus polysaccharide (APS) stimulates glucose uptake in L6 myotubes through AMPK, which has been previously reported www.nature.com/aps Liu J et al in our experiments using animal models of T2DM to test this hypothesis[16]
We determine that AMP is the major contributor to APS-induced AMPK activation in L6 myotubes, which is consistent with other reports showing that AMP may regulate AMPK activation through the inhibition of AMPK phosphatase under conditions of metabolic stress[25]
Summary
Type 2 diabetes mellitus (T2DM) is one of the fastest growing public health problems. The main defect due to this disease is impaired glucose homeostasis partly caused by insulin resistance. Glucose homeostasis is maintained when glucose production and utilization are in balance. The main site for glucose disposal[1], displays decreased glucose uptake when insulin resistance occurs in T2DM patients[2, 3]. In addition to the classical insulin signaling pathway, glucose disposal in skeletal muscle is regulated by the AMP-activated protein kinase (AMPK) signaling pathway[4]. AMPK, a heterotrimeric protein kinase comprising a catalytic α-subunit and regulatory β- and γ-subunits[5], is activated by phosphory-
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