Figure 3. Activity of PLD1-WT and PLD1-mutants under glucose deprivation. A. HEK-293 cells were transfected with the indicated plasmids of vector, PLD1-WT, S558A, S505Q or S505E. At 40 h after transfection, cells were incubated in glucose free-DMEM with or without 25 mM glucose for 2 h. PLD activity in each group was then measured by the formation of [3H]PBtOH. The expression levels of PLD1 were analyzed by western blotting. Quantitative data represent the means of four independent experiments. *, p,0.01 versus vector- transfected cells with glucose; #, p,0.01 versus vector-transfected cells without glucose. B. HEK-293 cells were transfected with siRNAs directed against PLD. At 24 h post-transfection, cells were transfected with siRNA-resistant forms of the indicated PLD1 [45]. After 24 h, cells were treated with 25 mM of 2-DOG for 10 min and the formation of [3H]PBtOH was then quantified as previously described. Re-expression levels of PLD1 were analyzed by western blotting. Quantitative data represent the means of three independent experiments. *, p,0.01 versus vector-transfected cells without 2-DOG; #, p,0.01 versus vector- transfected cells with 2-DOG. doi:10.1371/journal.pone.0009600.g003 PLD1 Regulation by AMPK PLD1-WT showed an approximately 1.9-fold increase in 14C- glucose uptake under glucose deprivation conditions (Columns 2 and 5), while PLD1-S505Q showed only a 1.2-fold increase in 14C-glucose uptake under the same conditions (Columns 3 and 6). The changes in 14C-glucose uptake in response to glucose deprivation showed a similar pattern to that of PLD1 activity. These results indicate that the Ser-505 residue of PLD1 is required for 14C-glucose uptake in response to glucose deprivation. Next, we measured the 14C-glucose uptake in response to AICAR (used as an AMPK activator) and AMPK-DN to determine the effects of AMPK on glucose uptake. As shown in Figure 5B, the 14C-glucose uptake was increased by about 1.9-fold by AICAR, but was significantly reduced by AMPK-DN (Columns 2 and 3). To examine the effects of PLD and ERK on glucose uptake, we treated the cells with 1-BtOH (a PLD inhibitor) and PD98059 (an ERK inhibitor). The 14C-glucose uptake was attenuated in response to treatment with 1-BtOH as well as PD98059. Furthermore, the increase in 14C-glucose uptake regulated by AICAR was correlated with that of the 14C-glucose uptake induced by glucose deprivation (Columns 2 and 7). The increase in 14C-glucose uptake induced by glucose deprivation was decreased by AMPK-DN, 1-BtOH and PD98059 (Columns 8, 9 and 11). Taken together, these results imply that AMPK-PLD1-induced ERK activation is necessary for glucose uptake in differentiated C2C12 cells. To further define the role of ERK activation in glucose uptake, we investigated the amount of cell surface glucose transport (GLUT) 4 and the 14C-glucose uptake in differentiated L6 GLUT4-myc myotube cells. The levels of cell surface GLUT4 were enhanced by about 1.5-fold by glucose deprivation when compared to the presence of glucose. AICAR increased the amount of cell surface GLUT4 by approximately 1.8-fold. However, the levels of cell surface GLUT4 induced by both glucose deprivation and AICAR were attenuated by the presence of PD98059 (Figure 5C). As shown in Figure 5D, glucose deprivation increased the 14C-glucose uptake by approximately 1.6-fold in the absence of PD98059 when compared to the presence of 25 mM glucose (Columns 1 and 2). AICAR enhanced the 14C-glucose uptake by about 1.5-fold under the same conditions (Columns 1 and 3). However, the 14C-glucose uptake stimulated by both glucose deprivation and AICAR were attenuated by the presence of PD98059. Taken together, these results suggest that the stimulation of ERK is required for glucose uptake in differentiated L6 GLUT4-myc myotube cells. Discussion Although it is known that extracellular stressors such as AICAR increase glucose uptake through the AMPK pathway in muscle cells, the downstream aspects of AMPK signaling are largely unknown. In the present study, we found that PLD1 is a novel signaling effector of AMPK. Several lines of evidence support the notion that PLD1-mediated AMPK activation is required for ERK activation and glucose uptake in muscle cells (Figure 5E). For example, AMPK stimulates PLD1 through phosphorylation as demonstrated by a reduction of PLD1 activity in AMPK-DN adenovirus-infected cells (Figure 1). Moreover, mutagenesis and in vitro and in vivo phosphorylation assays demonstrated that AMPK phosphorylates PLD1 on the Ser-505 (Figure 2). ERK activation was found to be dependent on energy status, but not hyperosmolar stresses (Figure 4). Finally, ERK activation by AMPK-mediated PLD1 was found to be essential for 14C-glucose uptake in differentiated C2C12 and L6 GLUT4-myc myotube cells (Figure 5). Taken together, these results suggest that PLD1 is a downstream effector of AMPK in a signaling pathway related to PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9600