(S)-[6]-Gingerol enhances glucose uptake in L6 myotubes by activation of AMPK in response to [Ca 2+ ] i

– PURPOSE . The aim of this study was to investigate the mechanism of ( S )-[6]-gingerol in promoting glucose uptake in L6 skeletal muscle cells. METHODS . The effect of ( S )-[6]-gingerol on glucose uptake in L6 myotubes was examined using 2-[1,2- 3 H]-deoxy-D-glucose. Intracellular Ca 2+ concentration was measured using Fluo-4. Phosphorylation of AMPK α was determined by Western blotting analysis. RESULTS . ( S )-[6]-Gingerol time-dependently enhanced glucose uptake in L6 myotubes. ( S )-[6]-Gingerol elevated intracellular Ca 2+ concentration and subsequently induced a dose- and time-dependent enhancement of threonine172 phosphorylated AMPK α in L6 myotubes via modulation by Ca 2+ /calmodulin-dependent protein kinase kinase . CONCLUSION. The results indicated that ( S )-[6]-gingerol increased glucose uptake in L6 skeletal muscle cells by activating AMPK. ( S )-[6]-gingerol, a major component of Zingiber officinale, may have potential for development as an antidiabetic agent. _______________________________________________________________________________________


INTRODUCTION
High calorie intake and low physical activity has led to a dramatic increase in the incidence of type 2 diabetes and in particular hyperglycemia and its complications over the past few decades. A number of intracellular signalling pathways are associated with regulation of blood glucose and may be targets of drug action. AMP-activated protein kinase (AMPK) has a key role in regulating energy fuel, as demonstrated at both the cellular and whole body level (1,2). AMPK is a heterotrimeric complex comprised of one catalytic subunit (α) and two regulatory subunits (β and γ). Activation of AMPK occurs by phosphorylation at threonine172 (Thr172) on the loop of the catalytic domain of the α-subunit (3,4). Currently, two major upstream kinases have been identified, the LKB1/STRAD/MO25 complex, which maintains the basal level of phosphorylation of AMPKα Thr172 , and the Ca 2+ /calmodulin-dependent protein kinase kinase (CAMKK), which is triggered by increased intracellular Ca 2+ concentration (5)(6)(7).
In skeletal muscle AMPK activation in response to metabolic stress leads to a switch of cellular metabolism from anabolic to catabolic states. Studies with AMP-mimetic compound 5aminoimidazole -4 -carboxamide -1 -β -Dribofuranoside (AICAR) showed that acute activation of AMPK increased glucose uptake by promoting glucose transporter (GLUT4) translocation to the plasma membrane as well as facilitated fatty acid influx and β-oxidation (8)(9)(10). Repetitive AMPK activation results in upregulation of numerous genes and proteins involved in energy metabolism.
Cell culture L6 myoblasts were maintained in α-minimal essential medium with 10% fetal bovine serum (FBS) at 37°C in an atmosphere of 5% CO2. When myoblasts grew to confluence, cells were allowed to fuse into multinucleated myotubes in α-MEM containing 2% heat-treated newborn calf serum (CS).
Glucose uptake assay L6 myoblasts were seeded in 48-well plates at a density of 4×10 4 cells/cm 2 and allowed to fuse into myotubes as described above. The assays were performed when over 70% myotubes had formed. On the day of testing, the cells were washed twice with phosphate-buffered saline solution (PBS), and starved in serum-free α-MEM in 0.5% (w/v) bovine serum albumin containing treatment samples for 5 hours, followed by a quick wash with Krebs-Ringer-phosphate-Hepes buffer (KRPH, 20 mM Hepes, 5 mM KH2PO4, 1 mM MgSO4, 136 mM NaCl, 4.7 mM KCl, 1 mM CaCl2, pH 7.4).
Test samples were incubated for the indicated time periods. Where inhibitors were used, cells were pre-treated for 30 minutes before adding treatment compounds. The cells were then incubated in KRPH and 2-deoxy-glucose uptake was measured over a 5 minute period (100 µM 2-deoxy-Dglucose with 2-[1,2-3 H]-deoxy-D-glucose 0.3 µCi/well) at room temperature (20-25°C). The uptake was terminated by 5 quick washes with icecold PBS. The plates were air-dried for 15 minutes and cells were lysed in 0.05 N NaOH solution. The radioactivity of 2-[1,2-3 H]-deoxy-D-glucose in the cell lysate was determined in a scintillation counter. Intracellular Ca 2+ concentration L6 myoblasts were seeded in 96-well black clear bottom plates and differentiated into myotubes as described above. The intracellular Ca 2+ concentration was measured using Fluo-4 NW Calcium Assay Kit following the manufacturer's instruction. Briefly, on the day of experiment, the cells were washed twice with PBS, then 100 µl of Fluo-4 NW solution was added quickly to each well and incubated at 37°C for 30 minutes and left at room temperature for a further 30 minutes. Addition of treatment compounds was followed by immediate measurement of the fluorescence intensity of Fluo-4 at ex 485 nm / em 520 nm by a real-time Novostar plate reader over 120 second time periods.
Western blot analysis L6 myotubes were treated with (S)-[6]-gingerol for the indicated time periods. Where inhibitors were used, cells were pre-treated for 30 minutes before adding treatment compounds. Then the cells were washed twice with PBS, and lysed with RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktail. The protein content of cell lysates was determined by Micro BCA™ protein assay kit. Thirty micrograms of protein was resolved on 4-12% SDS-PAGE, and then transferred to nitrocellulose membrane. The membrane was blocked with 5% BSA/TBST for 1 hour, incubated with primary antibody overnight at 4°C, then probed with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5000, Cell Signaling) for 1 hour. Primary antibodies used were monoclonal rabbit antiphospho-AMPKα (Thr172) (1:2500, Cell Signaling), monoclonal rabbit anti-AMPKα (1:2500, Cell Signaling), polyclonal rabbit antiphospho-acetyl-CoA carboxylase (Ser79) (1:2500, Cell Signaling), and monoclonal rabbit anti-acetyl-CoA carboxylase (1:2500, Cell Signaling). Protein bands were detected with LumiGLO ® and Phototope ® -HRP detection reagent. The intensity of bands was determined using the ImageJ image processing program.

STATISTICS
All data are presented as mean ± SEM of three independent experiments. Results were analysed using one way analysis of variance (ANOVA) followed by the Newman-Keuls post hoc test. Differences were considered significant when P values were less than 0.05 (P < 0.05).
The involvement of AMPK and phosphorylated AMPKα Thr172 (p-AMPKα Thr172 ) in (S)-[6]-gingerol stimulated glucose uptake were examined after (S)- [6]-gingerol (150 µM) treatment by Western blot analysis. P-AMPKα Thr172 was found to be elevated rapidly and reached a peak of 5.5-fold over basal level at 10 minutes, then declined gradually and was maintained at 3.5-fold thereafter (Figure 2A). Consistent with the increase of p-AMPKα Thr172 , phosphorylated acetyl-CoA carboxylase (p-ACC Serine79 ), one of the downstream targets of AMPK, was elevated maximally within 5 min and was maintained thereafter ( Figure 2B).

(S)-[6]-Gingerol -induced AMPK phosphorylation was mediated by CaMKK in L6 myotubes
The Ca 2+ signal is involved in a broad range of skeletal muscle activities. (S)-[6]-Gingerol increased intracellular Ca 2+ concentration in a dosedependent manner within 1 minute in L6 myotubes ( Figure 3). The increase appeared more gradual than the rapid increase seen with carbachol as a control, which indicated that (S)-[6]-gingerolinduced intracellular Ca 2+ rise occurred through a mechanism distinct from that with carbachol. P-AMPKα Thr172 increased significantly in L6 myotubes treated with the calcium ionophore A23187 (1 µM). AMPK activator AICAR (1 mM) also increased the AMPKα Thr172 phosphorylation level, and the activation by AICAR was blocked by the AMPK inhibitor Compound C, which was consistent with a previous study (15). We next investigated the role of Ca 2+ and CaMKK activation in (S)-[6]-gingerol-stimulated glucose uptake. Pretreatment of L6 myotubes with the intracellular Ca 2+ chelator BAPTA-AM abolished the stimulation of glucose uptake by (S)-[6]-gingerol. Glucose uptake was also abolished by pretreatment with the CaMKK inhibitor STO609 ( Figure 5). The significant increment of glucose uptake by AICAR was diminished by adding its inhibitor Compound C. The calcium ionophore A23187 increased glucose uptake slightly (1.27fold), and it was decreased by adding calcium chelator BAPTA-AM.

Which AMPKα isoform is involved in glucose uptake?
To determine which AMPKα isoform was dominant in mediating (S)-[6]-gingerol stimulated glucose uptake in L6 myotube, AMPKα1 or AMPKα2 was selectively knocked down by transfecting their corresponding siRNAs. The transfection efficiency was tested using a FAMconjugated MISSION ® Universal Negative Control, and achieved 80.43% knockdown of target gene using GAPDH positive control (data not shown but available as Supplementary information).
A significant reduction in protein expression was found to occur to a similar extent in both AMPKα1 and AMPKα2 knockdown L6 myotubes ( Figure 6A and 6B).  The (S)-[6]-gingerol stimulated glucose uptake was diminished in AMPKα1 and AMPKα1/α2 knockdown L6 myotubes, whilst the increment of glucose uptake by (S)-[6]-gingerol was not affected in AMPKα2 knockdown cells ( Figure 6C). This indicated that AMPKα1 was the dominant isoform involved in (S)- [6]-gingerol stimulated glucose uptake in L6 skeletal muscle cells.

DISCUSSION
The main finding of this study is that (S)-[6]gingerol increased glucose uptake in L6 skeletal muscle cells, and that the stimulation involves AMPKα activation. Though mounting evidence has suggested that ginger and its major chemical components were effective in alleviating hyperglycaemia and dyslipidaemia, the mechanisms underlying these actions remained largely unclear (16). To our knowledge this is the first study to reveal the role of AMPK in (S)-[6]gingerol stimulated glucose uptake. It has been well established that AMPK plays an important role in mediating energy homeostasis. Recent evidence showed that AMPK activation facilitated acute glucose uptake by triggering glucose transporter GLUT4 trafficking via phosphorylation of TBC1D1, a downstream protein shared in the insulin signalling transduction pathway (17,18). AMPK activation also up-regulated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a transcriptional cofactor regulating mitochondrial biogenesis and GLUT4 expression (19)(20)(21)(22). Therefore AMPK has been considered a potential target for developing therapeutic agents to treat type 2 diabetes (23). In support of a role of AMPK in (S)-[6]gingerol stimulated glucose uptake we showed that the downstream target of AMPK, acetyl-CoA carboxylase (ACC), was also phosphorylated in (S)-[6]-gingerol treated L6 myotubes. ACC plays a key role in de novo fatty acid biosynthesis by catalysing carboxylation of acetyl-CoA to form malonyl-CoA. Accumulation of malonyl-CoA inhibits carnitine palmitoyltransferase 1 (CPT1) to transfer long-chain fatty acyl-CoA from cytosol into mitochondria for further oxidation (24). A decrease of malonyl-CoA was reported to diminish insulin resistance in fat-fed rats (25). Phosphorylation of Ser 79 in ACC will subsequently inactivate the enzyme, leading to a switch of the cellular metabolism from energy storage to expenditure.
The glucose transporter GLUT4 is the principle isoform responsible for glucose clearance in peripheral tissues. Though the dynamic nature of GLUT4 has been extensively studied in the past few decades, the role of Ca 2+ in mediating GLUT4 trafficking is not completely understood (30). Recent studies supported the Ca 2+ requirement for GLUT4 trafficking along the cortical actin filaments and fusion into the plasma membrane (31)(32)(33). Our previous study showed that (S)-[8]gingerol, a more potent homologue of (S)-[6]gingerol, enhanced glucose uptake either in the presence or absence of insulin by promoting GLUT4 translocation and fusion into plasma membrane (14). In this study the intracellular Ca 2+ chelator BAPTA-AM completely abolished the effect of (S)- [6]-gingerol on glucose uptake in L6 myotubes, suggesting a pivotal role of Ca 2+ in GLUT4 dynamics. The same result was observed in 3T3-L1 adipocytes in a previous study (34).
AMPK is highly expressed in skeletal muscle tissue (35). The two isoforms of the catalytic subunit AMPKα1 and AMPKα2 are encoded by distinct genes (36). It has been suggested that AMPKα1 and AMPKα2 have different physiological roles in mediating energy homeostasis. AMPKα2 tends to be more sensitive to cellular AMP variation (37). The results from isoform knockout rodent models demonstrated that AMPKα2 knockout mice were resistant to AICAR stimulated glucose uptake and presented insulin resistance (38,39). However, it was found that in obese subjects, the basal AMPKα1 activity was reduced significantly compared to lean control, whilst AMPKα2 activity remained at the same level (40). In AMPKα1 knockout mice, low intensity contraction-stimulated glucose uptake in skeletal muscle was markedly decreased, but this was not the case in AMPKα2 knockout mice. A recent study showed that caffeine increased AMPKα1 activity and glucose uptake in rat epitrochlearis muscle without affecting energy status (41). In the present study, it was found that the (S)- [6]-gingerol stimulated increase of glucose uptake was completely abolished in AMPKα1 knockdown L6 myotubes, which indicated that (S)-[6]-gingerol increased glucose uptake preferentially via activation of the AMPKα1, rather than the AMPKα2 isoform.

CONCLUSION
The present study showed a significant and rapid increase of glucose uptake in (S)-[6]-gingerol treated L6 myotubes. This action of (S)-[6]gingerol was associated with an elevation of cytosolic Ca 2+ concentration and enhancement of levels of phosphorylated AMPKα Thr172 , preferentially through the AMPKα1 isoform. Our data supports (S)-[6]-gingerol, the major pungent component of ginger (Zingiber officinale), as a candidate potential hypoglycaemic agent at least in part through its effectiveness in promoting glucose uptake in skeletal muscle.