ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

Stanley Andrisse,Larry D Spears,Rikki M Koehler,Rona M Robinson-Hill,Gaytri D Patel,Andrea M Webber,Jonathan S Fisher,James K Ching,Joseph E Chen,Imju Jeong,Josep V Planas

doi:10.1371/journal.pone.0066027

Abstract

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.

Highlights

Impaired insulin-stimulated glucose transport by glucose transporter 4 (GLUT4) is a well-documented contributor to the reduced glucose clearance found in subjects with type 2 diabetes mellitus (T2DM) [1,2,3,4]
ataxia telangiectasia mutated (ATM) Inhibition Diminishes Basal Glucose Transport Glucose transport in skeletal muscle is predominantly mediated by GLUT1 and GLUT4, with GLUT1 being responsible for physiologically-relevant portion of basal glucose transport [33,34,35]
To follow up on the results described above, we determined the effects of ATM inhibition on glucose and dehydroascorbic acid (DHA) transport, GLUT1 localization, and GLUT1 trafficking

Summary

Results

ATM Inhibition Diminishes Basal Glucose Transport Glucose transport in skeletal muscle is predominantly mediated by GLUT1 and GLUT4, with GLUT1 being responsible for physiologically-relevant portion of basal glucose transport [33,34,35]. To determine whether ATM inhibition decreases the phosphorylation of GLUT1-S490, wild-type and GLUT1-S490A L6 myoblasts incubated in DMSO or 6 mM CP were harvested, immunoprecipitated with antibodies against FLAG, and subjected to Western Blot analysis. ATM mutant soleus and extensor digitorum longus (EDL) muscles exhibited diminished DHA transport compared to wild type control muscles (55% and 65%, respectively; p,0.05, Fig. 4A–B) suggesting that ATM plays a role in DHA transport in skeletal muscle. Wild type (WT) and ATM mutant (ATM 2/2) mouse muscles were excised and subjected to a glucose transport assay in the presence of indinavir, a GLUT4 inhibitor. ATM mutant soleus muscles displayed reduced glucose transport (20%; p,0.05, Fig. 5A), which, given the inhibition of GLUT4 by indinavir, is attributable to GLUT1-mediated transport. Soleus muscles exposed to the ATM activator, DXR, exhibited an increase in glucose transport (42%; p,0.05, Fig. 5F). Differences in basal transport in ATM-deficient muscles do not appear to be caused by changes in GLUT1 or GLUT4 abundance

Introduction

Discussion