1), L6 myoblasts were incubated in the presence and absence of ATM inhibitors and subjected to a DCFDA ROS assay. was inhibited, and prevented the pyrogallol-induced decrease in insulin signaling and insulin-stimulated glucose transport. Taken together, the data suggest that GLUT1 plays a role in regulation of ROS and could contribute to maintenance of insulin action in the presence of ROS. reductase in the electron transport chain [21]. Pyrogallol generates superoxide by nonenzymatic reaction with oxygen [22]. Finally, a kinetic fluorescence reading was taken. A portion of the plates contained no cells or non-loaded cells as negative controls. Insulin action experiments L6 myoblasts transiently transfected with FLAG-GLUT1 constructs were serum starved in HBS for 2?h and then treated plus or minus 100?nM insulin and 50?/4M pyrogallol, a ROS generator, for 1?h. Subsequently, the cells were washed C188-9 twice with phosphate buffered saline (PBS) then subjected to either a Western blot analysis or glucose transport assay (both described below). Western blot analysis L6 myoblasts transiently transfected with FLAG-GLUT1 constructs and incubated as explained above were subjected to Western blot analysis as described previously [14]. Transport assays for cultured myoblasts L6 myoblasts, treated as explained in insulin action experiments above, were exposed to 2-deoxy-glucose (2DG) transport media (0.5?/4Ci/ml 3H-labeled 2DG, 10?/4M 2DG, dissolved in glucose-free HBS) for 10?min. An ice-cold 0.9% saline solution was used to wash the cells. The cells were then incubated with lysis buffer (0.2% SDS and 0.2?N NaOH) for 30?min and processed for scintillation counting as described previously [14]. A BCA assay was performed using the samples to determine protein content. To account for non-specific 2DG uptake, a subgroup of the cells was treated in the presence of 10?/4M cytochalasin B. Cytochalasin B prevents transport by GLUTs [23]. Statistical analysis Univariate one-way analysis of variance (ANOVA) with a significance determined as em p /em ? ?0.05 was used to analyze the data. The software used was SPSS Statistics 21.0 (IBM, Armonk, NY, USA). Results Inhibition of GLUT1 elevates ROS levels The oxidative state of the cell is maintained by a balance between antioxidant and pro-oxidant levels. In cultured muscle cells, GLUT1 and GLUT4 transport glucose [23] and DHA [24], both of which can aid in antioxidant defense [11,13]. To determine the effects of GLUT1 inhibition on ROS levels, L6 myoblasts that had been loaded with DCFDA, a fluorescent ROS probe, were incubated in press plus and minus GLUT1 inhibitors, and then oxidative stress was induced by antimycin A C188-9 (Ant-A) or pyrogallol (PG). GLUT1 inhibition with 80?/4M fasentin increased both Ant-A- and PG-induced ROS levels (30?40%, em p /em ? ?0.05) (Fig. 1A and B). GLUT1 inhibition with 100?/4M phloretin raised Ant-A-induced ROS by 20% and PG-induced ROS levels by 2-fold ( em p /em ? ?0.05) (Fig. 1C and D). These data suggest that GLUT1 plays a role in the rules of ROS levels. Open in a separate windowpane Fig.?1 Inhibition of GLUT1 increases ROS levels.?L6 myoblasts were subjected to a DCFDA ROS assay in the presence or absence of (A) GLUT1 inhibitor, 80?/4M fasentin with induction of ROS by 100?/4M antimycin A (AntA), (B) 80?/4M fasentin with 50?/4M pyrogallol (PG), a superoxide generator, (C) GLUT1 inhibitor, 100?/4M phloretin, with induction of ROS by 100?/4M AntA, (D) 100?/4M phloretin with 50?/4M PG, and (E) 100?/4M indinavir, which inhibits GLUT4, with induction of ROS by Ant-A or (F) by PG. ( em n /em ?=?6/group. Variations are statistically significant for all time PLCB4 points after time 0, em C188-9 p /em ? ?0.05. Subpanels E and F do not display significant variations, except 1F at 2?min, em p /em ? ?0.05). To determine whether GLUT4 plays a role in rules of ROS in muscle mass cells, DCFDA loaded L6 myoblasts were incubated in the presence or absence of 100?M indinavir, which inhibits GLUT4 but not GLUT1, and then oxidative stress was induced by Ant-A or PG. From 4?min post-induction and beyond, indinavir treated muscle tissue displayed no significant switch in Ant-A or PG-induced ROS levels from control muscle mass cells (Fig. 1E and F). Therefore, in muscle mass cells, GLUT4 does not play a role in rules of ROS levels. Together, the data suggest that GLUT1 takes on a more prominent part in rules C188-9 of ROS.