Ataxia Telangiectasia Mutated in Glucose Transport

Ataxia Telangiectasia Mutated in Glucose Transport A role for ataxia telangiectasia mutated in insulin-independent stimulation of glucose transport Abstract Literature reports suggest that ataxia telangiectasia mutated (ATM) can activate the AMP-activated protein kinase (AMPK), a protein that can stimulate glucose transport in skeletal muscle. We hypothesized that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, would increase glucose transport in mouse extensor digitorum longus (EDL) muscles in an ATM-dependent manner. AICAR-stimulated glucose transport was prevented by the ATM inhibitor KU-55933 and in ATM-deficient (ATM-/-) muscle despite normal stimulation of AMPK phosphorylation. S231 of TBC1D1 matches the sequence motif of ATM substrates, and phosphorylation of this site is known to inhibit TBC1D1 and lead to increased glucose transport. Accordingly, we assessed TBC1D1 phosphorylation and found that AICAR-stimulated phosphorylation of TBC1D1 at S231did not occurin ATM-/- muscle. However, activation of ATM without activation of AMPK was insufficient to increase TBC1D1 phosphorylation.The data suggest that ATM plays a role in AICAR stimulated glucose transport downstream of AMPK. Keywords: AMP-activated protein kinase; ataxia telangiectasia mutated; TBC1D1; AICAR; glucose transport; skeletal muscle Introduction The serine-threonine kinase ataxia telangiectasia mutated (ATM) appears to play a role in glucose homeostasis. For example, recent genome-wide association studies have found that genetic variations near the ATM gene are related to glycemic responses to metformin [1, 2], a commonly-prescribed drug for blood glucose control. While the mechanism for metformin’s effect on blood glucose levels is under debate [3-6], it is known that metformin acutely stimulates glucose transport into skeletal muscle concomitant with activation of the AMP-activated protein kinase (AMPK) [7]. Activation of AMPK is sufficient to stimulate insulin-independent glucose transport into skeletal muscle [8, 9]. Intriguingly, ATM dependence has been reported for activation of AMPK in response to DNA damage or insulin-like growth factor 1 in HeLa cells and fibroblasts, exposure of lung cancer cells to ionizing radiation,exposure of lymphoblaststo H 2 O 2 , or treatment of HeLa cells and mouse embryonic fibroblasts with the adenosine analog AICAR [10-14]. Despite these suggestive data on the role of ATM upstream of AMPK, the potential role of ATM in AMPK-dependent stimulation of glucose transport has not previously been investigated in skeletal muscle, the predominant whole-body storage depot for glucose. Accordingly, the purpose of this study was to test the hypothesis that glucose uptake stimulated by the AMPK activator AICAR would be dependent on ATM in skeletal muscle. Methods Materials Antibodies against TBC1D1, AMPK, phosphorylated AMPKÃŽ ± T172 (P-AMPK), and phosphorylated ATM S1981 (P-ATM) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies aga inst phosphorylated TBC1D1 (P-TBC1D1) S237 (S231 in mouse) were purchased from EMD Millipore Corporation (Billerica, MA, USA). Antibodies against tubulin and ATM were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Pierce Biotechnology (Rockford, IL, USA). The ATM inhibitor KU-55933 was a generous gift from Dr. Graeme Smith (KuDOSPhramaceuticals, Cambridge, UK). The AMPK inhibitor Compound C was provided by Merck & Co., Inc. (Rahway, NJ, USA). Doxorubicin was purchased from Sigma-Aldrich Corporation. Radiolabeled 2-deoxyglucose and mannitol were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA).