In skeletal muscle cells, GLUT1 is responsible for a large portion of basal uptake of glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would decrease GLUT1-mediated transport would cause increased reactive oxygen species (ROS) levels in L6 myoblasts, while conditions that would increase GLUT1-mediated transport would result in decreased ROS levels. We found that the GLUT1 inhibitors fasentin and phloretin increased the ROS levels induced by antimycin A and the superoxide generator pyrogallol. However, indinavir, which inhibits GLUT4 but not GLUT1, had no effect on ROS levels. Ataxia telangiectasia mutated (ATM) inhibitors and activators, previously shown to inhibit and augment GLUT1-mediated transport, increased and decreased ROS levels, respectively. Mutation of an ATM target site on GLUT1 (GLUT1-S490A) increased ROS levels and prevented the ROS-lowering effect of the ATM activator doxorubicin. In contrast, expression of GLUT1-S490D lowered ROS levels during challenge with pyrogallol, prevented an increase in ROS when ATM 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.
ObjectiveThe glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1.Research Design and MethodsL6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity.ResultsIn myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL.ConclusionsThese results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction.
The GLUT1 PDZ‐binding motif that plays a role in GLUT1 localization contains a reported phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study is to determine whether ATM and/or modification of GLUT1 S490 affect GLUT1 localization, trafficking, and transport activity. Mouse extensor digitorum longus (EDL) muscles were excised from wild type and ATM −/− mice and then subjected to a DHA transport assay. ATM inhibition by KU55933 or CP466722 or ATM dysfunction (ATM−/−) in EDL resulted in reduced DHA transport (~30–65%, p<0.05). Also, L6 myoblasts were transfected with FLAG tagged GLUT1 (FLAG‐GLUT1) or FLAG‐GLUT1 S490A and S490D site mutants. ATM inhibition significantly diminished cell surface FLAG‐GLUT1 (~20–80%, p<0.05) and glucose transport in L6 myotubes (~50%, p<0.05) with a concomitant reduction in GLUT1 externalization (~20%, p<0.05). The S490D mutation increased cell surface GLUT1 (~35%, p<0.05) as well as GLUT1‐mediated glucose transport (~2‐fold, p<0.05), while S490A had opposite effects. These results provide evidence that ATM and GLUT1 S490 mutations affect cell surface GLUT1 and GLUT1‐mediated transport. Supported by USPHS award 1R15DK091904.
Elevated reactive oxygen species (ROS) levels are associated with metabolic abnormalities such as insulin resistance. Glucose transporter 1 (GLUT1) transports glucose and dehydroascorbic acid, both of which play roles in antioxidant defense. We hypothesized that conditions that would increase GLUT1 activity would result in decreased ROS levels and conditions that would decrease GLUT1 activity would increase ROS levels. . L6 myoblasts loaded with H2‐DCFDA, a fluorescent ROS probe, were treated under various conditions to alter GLUT1 activity, and fluorescence readings were taken under basal conditions or after increases in ROS were induced by exposure to pyrogallol or antimycin A. GLUT1 inhibitors, fasentin and phloretin, increased induced ROS levels. Ataxia telangiectasia mutated inhibitors and activators, previously shown to inhibit and augment GLUT1 activity, increased and decreased basal and induced ROS levels, respectively. GLUT1‐S490A, previously shown to decrease GLUT1 activity, enhanced basal and induced ROS levels. GLUT1‐S490D, previously shown to increase GLUT1‐mediated transport, lowered basal ROS levels. Thus, the data suggest that GLUT1 plays a role in regulation of ROS levels. Grant Funding Source: Supported by: William Townsend Porter Predoctoral Fellowship from the American Physiological Society
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