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-[(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.


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.


Introduction
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. Skeletal muscle, 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-lation at the Thr172 residue of AMPKα, which is mediated by one or more upstream kinases. In addition, AMPK is allosterically activated by AMP [6,7] . Once activated, AMPK switches on energy-generating pathways such as glucose transport. This finding has led to the concept that AMPK is a potential therapeutic drug target in situations of insulin resistance. Biguanides and thiazolidinediones, two classic antidiabetic drugs, have been used to treat T2DM patients due (at least in part) to their abilities to activate AMPK [8] .
Activation of AMPK has been involved in the regulation of glucose uptake [9] ; however, the signaling molecules connecting these events are still unknown. Akt substrate of 160 kDa (AS160; also known as TBC1D4) has been thought to be a key molecule linking the insulin and AMPK signaling pathways to glucose transporter 4 (GLUT4) translocation and subsequent glucose uptake in skeletal muscle [10] . AS160 has a Rab GTPase-activating protein (GAP) domain that plays a critical role in regulating vesicle formation, vesicle movement, and membrane fusion [11] . Phosphorylation of AS160 is suspected to exhibit impairments in its Rab GAP activity, which permits target Rabs to return to their active GTP-bound forms and initiate GLUT4 exocytotic trafficking [12,13] . AS160 phosphorylation is reduced in T2DM patients, which is accompanied by impaired GLUT4 translocation [14] .
Many natural products have been used in traditional medicine to treat a variety of human diseases. Astragalus membranaceus Bunge (Huang Qi) is a popular traditional Chinese herb that has a long history of medicinal use in both Chinese and Western herbal medicine [15] . Astragalus polysaccharide (APS) was extracted from Astragalus membranaceus Bunge and identified as its main active component. Our previous studies have shown that APS has the ability to restore glucose homeostasis through the stimulation of glucose uptake in skeletal muscle cells [16] . To detail the mechanisms of this process, we examined the effects of APS on the AMPK signaling pathway in L6 myotubes. We hypothesized that APS might be capable of stimulating glucose uptake through the activation of AMPK and the phosphorylation of AS160 in L6 myotubes.

Cell culture and differentiation
The L6 myoblast cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Following the manufacturer's protocol for cell culture and differentiation, L6 myoblasts were cultured in HG-DMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere with 5% CO 2 at 37 °C.
For differentiation, cells were allowed to reach confluence, and the medium was switched to HG-DMEM containing 2% (v/v) FBS. During the subsequent 7-d period, the medium was changed every other day. All treatments in this study were initiated after differentiation had been successfully completed. Cells from passages 3-15 were used in the experiments, and undifferentiated cells were not allowed to grow to more than 60%-70% confluence.

2-Deoxy-[ 3 H]-D-glucose uptake assay
Glucose uptake was measured using the 2-deoxy-[ 3 H]-D-glucose method [17] with modifications as previously described [18] . Briefly, the assay was initiated by replacing the HG-DMEM with a glucose-free medium containing 50 μmol/L 2-deoxy-[ 3 H]-D-glucose (370 Bq/nmol). After incubation at 37 °C for 15 min, the cells were rinsed 3 times with ice-cold phosphatebuffered saline (PBS) and digested in 0.2 mol/L NaOH at 60 °C for 1 h. Liquid scintillation counting of the samples was performed to assess the amount of labeled glucose taken up by the cells. The net uptake of 2-deoxy-[ 3 H]-D-glucose was expressed in pmol·min -1 ·mg -1 (protein)±SEM.
AMPK activity assay AMPK activity was measured using the HMRSAMSGL-HLVKRR (SAMS) peptide as previously described [19] . Briefly, 200 μg of protein from each sample was prepared in triplicate and mixed with 500 μL of IP buffer (lysis buffer plus 1 mmol/L dithiothreitol). AMPK was immunoprecipitated by an incubation with an anti-AMPKα antibody prebound to protein A/G-agarose (Santa Cruz Biotechnology) at 4 °C for 2 h. The beads were collected after centrifugation at 14 000×g for 1 min and washed once with IP buffer and twice with www.chinaphar.com Liu J et al Acta Pharmacologica Sinica npg 10×reaction buffer (400 mmol/L HEPES, pH 7.4, 50 mmol/L MgCl 2 , 800 mmol/L NaCl, and 1 mmol/L dithiothreitol). Following the washes, 50 μL of a reaction mixture containing 5 μL of reaction buffer, 10 μL of ATP working stock (0.1 μL of 100 mmol/L ATP, 1 μL of [ 32 P]ATP, and 8.9 μL of H 2 O), 10 μL of SAMS peptide (1 g/L), and 25 μL of H 2 O was incubated with the beads for 10 min at 37 °C. The supernatants were spotted onto P81 Whatman filter papers, and the free [ 32 P]ATP was subsequently removed by washing the filter papers with 1% phosphoric acid 4-5 times. After the washes, the filter papers were quickly dried, and the radioactivity was measured using a liquid scintillation counter.

Adenine nucleotide extraction and measurement
The ATP, ADP, and AMP contents were measured by highperformance liquid chromatography (HPLC, Dionex Corporation, Sunnyvale, CA, USA) as previously described [20] with some modifications. Briefly, after treatment with different reagents, the cells were rinsed with PBS and digested with trypsin. Total cell numbers were counted before the cells were collected by centrifugation at 1000×g for 3 min. The cell pellets were resuspended in 150 μL of ice-cold perchloric acid (4% v/v) and incubated on ice for 30 min. After incubation, the cell lysates were neutralized with 2 mol/L KOH and centrifuged at 13 000×g at 4 °C for 10 min to remove the precipitated KClO 4 . After passage through a 0.22-μm filter, the supernatants were aliquoted and stored at -80 °C.
The adenine nucleotide contents were determined using HPLC with a chromatography column (Phenomenex Luna C18 100A, 250×4.60 mm, 5 μm). Mobile phase A was phosphate buffer (50 mmol/L potassium dihydrogen orthophosphate, 50 mmol/L dipotassium hydrogen orthophosphate, pH 7.0, and 1 mmol/L EDTA), and mobile phase B was methyl hydrate. The adenine nucleotides were assayed spectrophotometrically at 259 nm and eluted after 15 min of isocratic elution at a flow rate of 1.0 mL/min. The HPLC peaks were identified and quantified by comparing the retention times and integrated peak areas with known external standards.

Plasmid DNA extraction and transient transfection
The cDNAs encoding AS160 were kindly provided by Dr Laurie J GOODYEAR (Joslin Diabetes Center, Boston, MA, USA) after we had obtained permission from the Kazusa DNA Research Institute (Kisarazu, Chiba, Japan). After signing a Material Transfer Agreement (MTA) with Dr Jun-ichi MIYAZAKI (Osaka University Medical School, Osaka, Japan), we obtained the pCAGGS vector from Dr Laurie J GOOD-YEAR. The wild-type AS160 (AS160-WT) DNA and 4P mutant AS160 (AS160-4P) DNA constructs have been previously characterized [21] . The accuracy of the DNA sequences was confirmed using the high-throughput DNA sequencing service provided by Invitrogen. Plasmid DNAs were amplified in

Statistical analysis
Data are expressed as the mean±SEM. Differences were determined by an analysis of variance (ANOVA) followed by Tukey's test. A level of P<0.05 was considered to be statistically significant.

Results
APS stimulated glucose uptake in time-and concentrationdependent manners L6 myotubes were treated with 400 μg/mL APS for 6 to 48 h. As shown in Figure 1A, APS exposure increased glucose uptake in a time-dependent manner (P<0.05). Furthermore, the increased glucose uptake mediated by APS was concentration-dependent. APS increased glucose uptake when administered at a concentration of 100 μg/mL and induced maximal glucose uptake at 400 μg/mL ( Figure 1B, P<0.05). Because 400 μg/mL APS resulted in an optimal increase of glucose uptake at 24 h, we used this concentration of APS along with a 24-h incubation period for all subsequent experiments.
APS stimulated the activation of AMPK and the phosphorylation of AS160 The signaling pathways triggered by APS were explored by Western blot analysis. APS increased the phosphorylation of AMPK-Thr172 (Figure 2A, P<0.05), which indicated an elevation in AMPK activity. Meanwhile, AMPK activation by APS was further confirmed by increased phosphorylation of ACC-Ser79, a direct substrate of AMPK (Figure 2A, P<0.05). In As shown in Figure 2A, an increase in AS160 phos phorylation was also observed in APS-treated L6 myotubes (P<0.05). In comparison, the phosphorylation of AS160, but not that of AMPK and ACC, was considerably increased in insulintreated cells (P<0.05). AICAR, a well-known AMPK activator, robustly stimulated the phosphorylation of AS160 as well as that of AMPK and ACC (P<0.05).
Role of CaMKKβ and LKB1 in APS-induced AMPK activation Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ) and LKB1 are two upstream AMPK kinases (AMP-KKs). To evaluate whether either of these kinases is involved in the activation of AMPK by APS, we investigated the effect of STO-609, a selective inhibitor for CaMKKβ, on the activation of AMPK by APS in L6 myotubes. Exposure to ionomycin, a Ca 2+ ionophore, stimulated the phosphorylation of AMPK and ACC in L6 myotubes, and the stimulatory effect was abrogated by STO-609 ( Figure 3A, P<0.05). However, incubation with STO-609 did not abolish the enhanced phosphorylation of AMPK and ACC by APS in L6 myotubes ( Figure 3A, P>0.05), which suggested that CaMKKβ might not be the major AMPK kinase in APS-induced AMPK activation. To explore the role that LKB1 plays in the activation of AMPK by APS, HeLa cells lacking LKB1 were used ( Figure 3B). APS caused a significant increase in the phosphorylation of AMPK and ACC in HeLa cells ( Figure 3C, P<0.05), indicating that LKB1 might not be the major AMPK kinase involved in APS-induced AMPK activation. Notably, incubation of HeLa cells with STO-609 abrogated the activation of AMPK by APS, ionomycin, and AICAR ( Figure 3C, P<0.05).

APS increased the cellular AMP:ATP ratio
The above results demonstrate that alternate pathways leading to APS-induced AMPK activation may exist. Increases in AMP levels can induce AMPK activation [22] . To investigate the possibility that AMP participates in APS-induced AMPK activation, we measured the adenine nucleotide levels in L6 myotubes subjected to different AMPK-activating treatments. As shown in Figure 4 and Supplementary Table 1, incubation with APS caused marked increases in the levels of AMP and the calculated AMP:ATP ratio compared with untreated control cells (P<0.05). Similar increases were also observed in the treatment of L6 myotubes with rosiglitazone (P<0.05). Metformin increased the cellular AMP levels (P<0.05), but no difference in the calculated AMP:ATP ratio was observed compared with untreated control cells (P>0.05).
Increase in glucose uptake by APS was AMPK-dependent AMPK has been previously demonstrated to increase glucose uptake in skeletal muscle in an insulin-independent manner [4] . To explore the possibility that AMPK is involved in APS-stimulated glucose uptake, we examined the role of Compound C, a selective AMPK inhibitor, on glucose uptake mediated by APS in L6 myotubes. As shown in Figure 5A, Compound C did not affect insulin-stimulated glucose uptake (P>0.05). However, Compound C reduced APS-stimulated glucose uptake (P<0.05), indicating that AMPK played a role in the effect of APS on glucose uptake. A similar attenuation was observed following incubation with AICAR (P<0.05).
Because incubation with the CaMKKβ inhibitor STO-609 did not abolish the enhanced phosphorylation of AMPK by APS in L6 myotubes ( Figure 3A), we reasoned that APS-stimulated glucose uptake should not be inhibited by this inhibitor. Indeed, STO-609 did not significantly inhibit APS-stimulated glucose uptake in L6 myotubes ( Figure 5B, P>0.05). Similar effects were also observed in the treatment of L6 myotubes Overexpression of AS160 in transfected L6 myoblasts The expression of recombinant AS160-WT and AS160-4P iso-forms was accomplished using our gene transfer approach. Lysates from transfected cells were immunoblotted with antibodies against the Myc epitope tag and the carboxyl terminus of rat AS160 (amino acids 1178-1189) ( Figure 5C). Notably, the AS160 antibody detected endogenous rat AS160 (rAS160, lower band) and exogenous Myc-tagged human AS160 (hAS160, upper band). Both bands were used in densitometry quantifications. On the basis of the histogram in Figure 5C, approximately equal amounts of the wild-type and mutant forms of Myc-tagged human AS160 were expressed, and the transfected cells expressed approximately 7 times more Myctagged human AS160 than endogenous rat AS160 (P<0.01). In contrast, there were no significant changes in the phosphorylation of ACC and AMPK in L6 myoblasts transfected with empty pCAGGS vector, AS160-WT, or AS160-4P ( Figure 5D, P>0.05).

APS increased glucose uptake through AS160
In skeletal muscle, the phosphorylation and deactivation of AS160 are involved in the regulation of AMPK on glucose uptake, which is able to facilitate the translocation of GLUT4 vesicles to the plasma membrane by a relief of inhibition mechanism [23,24] . In L6 myotubes, APS increased glucose uptake ( Figure 1) and AS160 phosphorylation (Figure 2A). Hence, we further attempted to determine whether APSstimulated glucose uptake was mediated by AS160. AS160 is phosphorylated at several sites, and Ser318, Ser588, Thr642, and Ser751 are considered to be the most important sites for AS160-mediated regulation of glucose uptake [21] . Therefore, we expressed AS160-WT and AS160-4P (containing 4 mutations, ie, S318A, S588A, T642A, and S751A) constructs in L6 myoblasts by transient transfection. As shown in Figure 5E, L6 myoblasts overexpressing AS160-4P exhibited a robust impairment in APS-stimulated glucose uptake relative to myoblasts expressing AS160-WT (P<0.05). Similar attenuations were observed following incubation with insulin or AICAR (P<0.05). In accordance with these results, AS160 is a key molecule in APS-stimulated glucose uptake.
Phosphorylation of AS160 by APS was AMPK-dependent AS160 has been reported to be an AMPK substrate in vitro, and the phosphorylation of AS160 is regulated by AMPK in the isolated extensor digitorum longus muscle [24] . To determine whether AMPK is involved in APS-stimulated AS160 phosphorylation, L6 myotubes were pretreated with Compound C, a selective AMPK inhibitor, prior to Western blot analysis. As indicated in Figure 6, Compound C attenuated the APS-and AICAR-mediated increase in AS160 phosphorylation as well as phosphorylation of AMPK-Thr172 and ACC-Ser79 (P<0.05). However, insulin-stimulated AS160 phosphorylation was not inhibited by Compound C (P>0.05). These results suggest that APS-stimulated AS160 phosphorylation is AMPK-dependent.

Discussion
First, we show that APS stimulates glucose uptake in L6 myotubes through AMPK, which has been previously reported in our experiments using animal models of T2DM to test this hypothesis [16] . These results highlight the importance of AMPK as a promising antidiabetic drug target. Second, 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] . Finally, we provide evidence that APS strongly increases AS160 phosphorylation, which mediates APS-stimulated glucose uptake in L6 myotubes. These results suggest that AS160 participates in the hypoglycemic effect of APS. AMPK is believed to play a central role in the energy homeostasis system, especially in reports concerning the pathophysiology and therapy of T2DM. Following activation by exercise or pharmacological agents, AMPK could reverse the abnormal energy processes associated with T2DM [8] . These observations suggest that it is possible to treat T2DM patients with AMPK activators. In this study, we determined that APS activated AMPK in L6 myotubes and exhibited some important beneficial metabolic effects in response to AMPK activation, including the phosphorylation of ACC and AS160.
There are two major signaling pathways that participate in glucose transport in skeletal muscle. One is the insulin signaling pathway, and the other is the AMPK signaling pathway [26] . Whether crosstalk exists between these two signaling pathways is still unclear. In this study, we showed that APS-stimulated glucose uptake was dependent on AMPK in L6 myotubes. In our previous study, we determined that the insulin signaling pathway was activated after treatment with APS [27] . These data suggest that both signaling pathways could be activated by APS. However, we could not conclude that intercommunication was occurring between these two signaling pathways in the presence of APS. Therefore, further studies are required to elucidate how these two signaling pathways collectively contribute to the hypoglycemic effect of APS.  The name "AMP-activated protein kinase" comes from a study in which the activity of this kinase was shown to be regulated by AMP [28] . AMP augments the activity of AMPK in two possible ways. One mechanism is the induction of a conformational change in the γ subunit following binding to AMP, which can be activated by allosteric regulation [29] . The other mechanism is the inhibition of AMPK dephosphoryla-tion by protein phosphatases [25,30] . In cell-free systems, protein phosphatase-2A and protein phosphatase-2C (PP2A/PP2C) dephosphorylate AMPK at Thr172 after binding to the threonine residue of AMPK, and AMP could inhibit this reaction by preventing this binding, which could increase the activity of AMPK [25,30,31] . Therefore, AMP activates AMPK by causing allosteric activation of AMPK as well as by making AMPK a Here, we showed that APS increased the cellular AMP levels and the AMP:ATP ratio in L6 myotubes, which may provide a mechanistic explanation for the activation of AMPK by APS.
Other possible mechanisms involved in APS-induced AMPK activation were tested using HeLa cells (lacking LKB1) and the CaMKKβ inhibitor STO-609. We observed that the activation of AMPK in HeLa cells by APS was blocked by pretreatment with STO-609. However, we showed that STO-609 did not block the activation of AMPK by APS in the L6 myotubes that express LKB1 and CaMKKβ. These findings suggest that APSinduced AMPK activation can be mediated by either LKB1 or CaMKKβ. Additionally, we discovered that AMPK was activated in HeLa cells by AICAR, an adenosine analogue, and that this activation was abolished by STO-609. As discussed above, AMP, LKB1, and CaMKKβ may serve as upstream activators that mediate APS-induced AMPK activation.
In adult skeletal muscle, insulin, the AMPK activator AICAR, and contraction stimulate AS160 phosphorylation concomitant with glucose uptake [23,32] . AS160 phosphorylation is induced by different signaling molecules due to various stimuli. In skeletal muscle, Akt mediates insulin-stimulated AS160 phosphorylation; AMPK induces AICAR-stimulated AS160 phosphorylation; and AMPK and to a lesser extent Akt regulate contraction-stimulated AS160 phosphorylation [24,32] . Our present study demonstrated that APS could stimulate AS160 phosphorylation and that AMPK contributed to APS-stimulated AS160 phosphorylation. Whether Akt mediated this APS-stimulated AS160 phosphorylation was unresolved in this study. Therefore, in the future, it will be important to elucidate the role of Akt in APS-stimulated AS160 phosphorylation and glucose uptake.
To summarize, we demonstrate for the first time that APS enhances AS160 phosphorylation to stimulate glucose uptake in an AMPK-dependent manner, which supports the development of APS as a promising and potent candidate for the treatment of T2DM.