| Diabetes mellitus is a kind of endocrine and metabolic disease caused by the interaction of genetic and environmental factors, it severely endangers human health and life. With the development of the social economy, the epidemic of population aging, and the changes of lifestyle, the global prevalence of diabetes mellitus is increasing rapidly year by year. At present, there are about347million diabetic patients in the world. China is one of the three countries which have the most diabetic patients. At present, at least92.4million people suffer from diabetes mellitus in China, and this data accounts for25%of the global diabetes population. Type2diabetes mellitus (T2DM) accounts for more than90%of all diabetes in China. T2DM is becoming the third noncommunicable disease that threaten human health and life along with cancer, cardiovascular and cerebrovascular diseases. Therefore, it is of great importance to investigate the pathogenesis and find the effective drug for T2DM.Astragalus polysaccharide (APS) is the polysaccharide component of an aquenous extract of the dried roots of Astragalus membranaceus (Fisch.) Bge. or Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Fam. Leguminosae). In our previous study, we found that treatment with APS lowers the level of blood sugar and improves the state of insulin resistance in animal models of T2DM. Enhanced activity of AMP-activated protein kinase (AMPK) is involved in the hypoglycemic effect of APS, but the exact underlying mechanism involved remains elusive.Here, with the L6myotube as the target cell, we demonstrate that APS significantly stimulates glucose uptake. The aim of this study is to investigate the mechanism by which APS enhances AMPK activity and the effects of APS on the downstream signaling induced by AMPK activation. Furthermore, we clarify the mechanism by which APS lowers the level of blood sugar. The research was divided into two parts as follows.Part I The effects and mechanisms of Astragalus polysaccharide on the AMPK activationAim:AMPK is considered as a potential therapeutic target of T2DM because of its stimulation of glucose uptake in muscle cells. APS was extracted from Astragalus membranaceus Bunge and identified as its main active component. Previously, we found that APS alleviated glucose toxicity via activation of AMPK in skeletal muscle in T2DM animal models. In this study, with the L6myotube as the target cell, we observe the effects of APS on the AMPK activation and attempt to elucidate the underlying molecular mechanism involved in this effect.Methods:(1) The3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay was performed to test the cytotoxicity of APS in L6myotubes;(2) L6myotubes were incubated with400μg/mL APS for24h, the expressions of p-AMPK (Thrl72), AMPK, p-ACC (Ser79), and ACC were detected by Western blot analysis and the ratios of p-AMPK/AMPK and p-ACC/ACC were calculated;(3) L6myotubes were incubated with400μg/mL APS for24h, the AMPK activity was measured using the HMRSAMSGLHLVKRR (SAMS) peptide;(4) L6myotubes were incubated with different treatments (400μg/mL APS for24h,200μmol/L rosiglitazone for30min, or2mmol/L metformin for3h), the rosiglitazone group was regarded as the positive control and the metformin group was the negative control, the adenine nucleotides in L6myotubes were extracted using cold perchloric acid, the AMP, ADP, and ATP contents in these cells were measured by high performance liquid chromatography (HPLC) and the ratios of AMP:ATP and ADP:ATP were calculated;(5) L6myotubes and HeLa cells (lacking LKB1) were treated with400μg/mL APS for24h,1μmol/L ionomycin for30min, or0.5mmol/L AICAR for30min after preincubation in the presence or absence of10μg/mL STO-609(the selective CaMKKβ inhibitor) for1h, the expressions of p-AMPK (Thr172), AMPK, p-ACC (Ser79), and ACC were detected by Western blot analysis and the ratios of p-AMPK/AMPK and p-ACC/ACC were calculated.Results:(1) Different concentration of APS (100,200,400,800and1600μg/mL) had little cytotoxicity in L6myotubes, no significant differences of cellular proliferation were found between the control and APS treated L6myotubes;(2) The expressions of p-AMPK (Thr172) and p-ACC (Ser79), the ratios of p-AMPK/AMPK and p-ACC/ACC, and the AMPK activity were all higher in APS treated group than control group;(3) Incubation with APS caused marked increases in the levels of AMP and the calculated AMP:ATP ratio compared with untreated control cells;(4) APS caused significant increases in the expressions of p-AMPK (Thr172) and p-ACC (Ser79), and the ratios of p-AMPK/AMPK and p-ACC/ACC in L6myotubes and HeLa cells. Preincubation with STO-609did not abolish the enhanced phosphorylation of AMPK and ACC by APS in L6myotubes, however, preincubation of HeLa cells with STO-609abrogated the increased expressions of p-AMPK (Thr172) and p-ACC (Ser79) by APS.Conclusions:(1) APS strongly promoted the activation of AMPK;(2) APS increased the cellular AMP levels and the AMP:ATP ratio;(3) APS-induced AMPK activation could be mediated by either LKB1or CaMKKβ. Part Ⅱ The action of Astragalus polysaccharide on glucose uptake in L6myotubes and its effect on AS160phosphorylationAim:Akt substrate of160kDa (AS160) has been thought to be a key molecule linking the AMPK signaling pathway to glucose transporter4(GLUT4) translocation and subsequent glucose uptake in skeletal muscle. Astragalus polysaccharide (APS), an important bioactive constituent of Astragalus membranaceus Bunge, has recently been found to stimulate glucose uptake through activation of AMPK in skeletal muscle cells. Here, with the L6myotube as the target cell, we observe the effects of APS on glucose uptake and further attempt to investigate whether APS-stimulated glucose uptake is mediated by AS160.Methods:(1) L6myotubes were treated with400μg/mL APS for different lengths of time (6to48h) or with various concentrations of APS (100to1600μg/mL) for24h, the glucose uptake in these cells was measured using the2-deoxy-[3H]-D-glucose method;(2) After being preincubated with1μmol/L Compound C (the AMPK inhibitor) for30min or10μg/mL STO-609(the selective CaMKK(3inhibitor) for1h, L6myotubes were treated with400μg/mL APS for24h, the glucose uptake in these cells was measured using the2-deoxy-[3H]-D-glucose method;(3) L6myotubes were incubated with400μg/mL APS for24h, the expressions of p-AS160and AS160were detected by Western blot analysis and the p-AS160/AS160ratio was calculated;(4) The wild-type AS160(AS160-WT) DNA and4P mutant AS160(AS160-4P) DNA constructs were amplified in Escherichia coli TOP10cells and purified using an endotoxin-free Plasmid Midi Kit. The accuracy of the DNA sequences was confirmed using the high-throughput DNA sequencing service;(5) AS160-WT plasmid, AS160-4P plasmid, or empty pCAGGS vector were treated on L6myoblasts by transient transfection, the expressions of AS160and Myc-Tag in transfected L6myoblasts were detected by Western blot analysis;(6) L6myoblasts transfected with AS160-WT or AS160-4P plasmids were treated with400μg/mL APS for24h, the glucose uptake in these cells was measured using the2-deoxy-[3H]-D-glucose method;(7) L6myotubes were treated with400μg/mL APS for24h,1μmol/L insulin for30min, or0.5mmol/L AICAR for30min with or without preincubation with1μmol/L Compound C for30min, the expressions of p-AMPK (Thr172), AMPK, p-ACC (Ser79), ACC, p-AS160, and AS160were detected by Western blot analysis and the ratios of p-AMPK/AMPK, p-ACC/ACC, and p-AS160/AS160were calculated. Results:(1) APS stimulated glucose uptake in time-and concentration-dependent manners;(2) Compound C reduced APS-stimulated glucose uptake in L6myotubes, but STO-609did not affect the stimulatory effect of APS on glucose uptake in L6myotubes;(3) Significant increases in the p-AS160expression and the p-AS160/AS160ratio were observed in APS-treated L6myotubes;(4) The sites of AS160-4P plasmid were mutated correctly, i.e., the four Akt phosphorylation sites (Ser318, Ser588, Thr642, and Ser751) had been mutated to alanine;(5) The expression of Myc-Tag could be determined in L6myoblasts transfected with AS160-WT or AS160-4P plasmids, and the expression of AS160in L6myoblasts transfected with AS160-WT or AS160-4P plasmids was significantly higher than that in L6myoblasts transfected with empty pCAGGS vector;(6) L6myoblasts overexpressing AS160-4P exhibited a robust impairment in APS-stimulated glucose uptake relative to myoblasts expressing AS160-WT;(7) Preincubation with Compound C abrogated the enhanced phosphorylation of AMPK, ACC, and AS160by APS in L6myotubes.Conclusions:(1) APS stimulated glucose uptake through the activation of AMPK in L6myotubes;(2) APS increased glucose uptake through the enhanced phosphorylation of AS160in L6myotubes;(3) Phosphorylation of AS160by APS was AMPK-dependent. |
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