PLD1 Regulation by AMPK Figure 5. Glucose uptake in differentiated C2C12 and L6 GLUT4-myc myotube cells. A. C2C12 cells were transfected with PLD siRNAs. At 24 after transfection, cells were transfected with the indicated siRNA-resistant PLD1 plasmids for vector, PLD1-WT or PLD1-S505Q [45]. After 24 h, cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h. The expression levels of PLD1 were then analyzed by western blotting. The amounts of 14C-glucose uptake and [3H]PBtOH formation were analyzed as described in the materials and methods. The quantitative data shown represent the means of three independent experiments. *, #, p,0.05 versus vector-transfected cells with glucose; ", 1, p,0.05 versus vector- transfected cells without glucose. B. After C2C12 cells were differentiated according to the procedures described in the materials and methods, cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h. Cells were then treated with the indicated exogenous agents (AICAR [1 mM], 1-BtOH [0.4%], t-BtOH [0.4%], PD98059 [20 mM]) for 30 min. For the case of infection of AMPK-DN adenovirus, the differentiated C2C12 cells were incubated with the virus for 48 h. Cells were then incubated in glucose free-DMEM with or without 25 mM glucose for 2 h and were treated with 1 mM of AICAR for 15 min. The uptake of 14C-glucose was measured and quantified. The quantitative data shown represent the means of four independent experiments. *, p,0.05 versus vehicle treatment in the presence of glucose; #, p,0.05 versus vehicle treatment in the absence of glucose. C. L6 GLUT4-myc myoblast cells were differentiated according to the procedures described in the materials and methods. The differentiated L6 GLUT4-myc myotube cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h or pre-treated with 1 mM AICAR for 30 min. After treatment with 20 mM of PD98059 for 30 min, the amount of cell surface GLUT4 was quantified as described in the materials and methods. This experiment was performed as three independent assays. *, p,0.01 versus vehicle treatment in the presence of 25 mM glucose; #, p,0.01 versus PD98059 treatment in the presence of 25 mM glucose. D. The differentiated L6 GLUT4-myc myotube cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h or pre-treated with 1 mM AICAR for 30 min. After treatment with 20 mM of PD98059 for 30 min, the uptake of 14C- glucose was measured and quantified. Quantitative data represent the means of three independent experiments. *, p,0.01 versus vehicle treatment in the presence of 25 mM glucose; #, p,0.01 versus PD98059 treatment in the presence of 25 mM glucose. E. A hypothetical model. This diagram shows a hypothetical model of extracellular stress-mediated glucose uptake. Glucose uptake is induced by the AMPK-activated signaling pathway. In this pathway, AMPK increases PLD1 activity through phosphorylation and promotes glucose uptake by triggering subsequent ERK activation in muscle cells. doi:10.1371/journal.pone.0009600.g005 However, further studies are necessary to characterize the specificity of the signaling pathways. In this study, we demonstrated that ERK stimulated by AMPK- induced PLD1 activation regulates the translocation of GLUT4 in L6 GLUT4-myc myotube cells. Several groups have reported that ERK activation is required for increased glucose uptake through the cell surface translocation of GLUT4 by AICAR [23], [39]. PLD1 isoform is involved in the translocation of GLUT4 and PLD1-induced PA generation is also required for the trafficking mechanisms of GLUT4 during membrane fusion and docking [26]. Based on these previous reports and the results of the present study, we suggest that the AMPK-mediated PLD1 pathway increases glucose uptake through regulation of the translocation machinery of GLUT4. However, the mechanism by which ERK regulates the translocation machinery of GLUT4 is still not clear. In conclusion, we suggest that AMPK-mediated PLD1 activity is essential for glucose uptake via ERK activation in differentiated C2C12 and L6 GLUT4-myc myotube cells. PLD1 and its phosphorylation may serve as important points in the regulation of energy exhaust caused by both glucose deprivation and extracellular stresses. Accordingly, we suggest that the physiological interaction between AMPK and PLD1 may provide clues to solving problems in metabolic syndromes such as diabetes. The AMPK- mediated PLD1 pathway may provide attractive target molecules for prospective pharmacological studies or genetic manipulation. However, further studies are necessary to understand the relationship between this signaling cascade and related diseases. Materials and Methods Materials An enhanced chemiluminescence kit (ECL system) and glutathione Sepharose 4B were purchased from Amersham Pharmacia Biotech. Protease inhibitor cocktail, 2-deoxyglucose (2-DOG), mannitol and o-phenylenediamine dihydrochloride were obtained from Sigma. Polyclonal anti-pAMPK antibody (Thr-172), anti-pERK (Thr-202/Tyr-204) antibody and anti-ERK antibody were purchased from Cell Signaling. AMPK, anti- AMPK antibody and polyclonal anti-pACC antibody (Ser-79) were obtained from Upstate Biotechnology, Inc. Anti-actin antibody was obtained from ICN Pharmaceuticals. b-octylgluco- pyranoside and PD98059 were purchased from Calbiochem. Horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgA, IgM and IgG were obtained from Kirkegaard & Perry Laboratories. [c-32P]ATP, [3H]myristic acid and [32P]or- thophosphate were purchased from Perkin-Elmer Life Sciences. C6-phosphatidic acid (PA) was obtained from Avanti Lipids Inc. 2- deoxy[14C-glucose] was acquired from ARC. Immobilized protein A was purchased from Pierce. Glucose free-Dulbecco’s modified Eagle’s medium (DMEM), phosphate free-DMEM and a-MEM were obtained from Life Technologies, Inc. Polyclonal antibody for the recognition of PLD1 and PLD2 was generated as previously described [40]. Purification of Recombinant PLD from sf9 Cells Hexa-histidine (His6)-tagged PLD was expressed in sf9 cells and then purified by Chelating Sepharose-affinity column chromatog- raphy, which was performed as previously described [40]. Cell Culture, Differentiation and Transfection HEK-293 cells and C2C12 cells were obtained from American Type Culture Collection (ATCC) and were maintained according to the standard ATCC protocol. HEK-293 cells and C2C12 cells were supplemented with 10% (v/v) fetal bovine serum (FBS) at 37uC in a humidified, CO2-controlled (5%) incubator. L6 GLUT4-myc myoblast cells were grown in a-MEM containing 10% fetal bovine serum, 50 U of penicillin/ml and 50 mg of streptomycin/ml and then maintained in a 5% CO2 humidified atmosphere at 37uC. To induce myogenic differentiation, C2C12 cells were grown to 70–90% confluency and then shifted from growth medium (DMEM + 10% FBS) to differentiation medium (DMEM + 2% horse serum). To induce differentiation of L6 GLUT4-myc myoblast cells, cells were diluted to a concentration of 46104/ml with a-MEM medium containing 2% FBS and then seeded in a 24 well plate. The plate was then incubated in a 5% CO2 humidified atmosphere at 37uC and the medium was changed every other day. After 7 days, L6 GLUT4-myc myoblast cells had differentiated into myotubes. HEK-293 cells were transfected using Lipofectamine and C2C12 cells were transfected with Lipofectamine 2000 to induce transient expression of the indicated plasmids and siRNAs. siRNA Sequences The small interfering RNAs (siRNA) for PLD1, PLD2 or controls (Luciferase) were purchased from Dharmacon Research, PLoS ONE | www.plosone.org 10 March 2010 | Volume 5 | Issue 3 | e9600