Elsevier

Cellular Signalling

Volume 20, Issue 8, August 2008, Pages 1555-1563
Cellular Signalling

ATM protein kinase mediates full activation of Akt and regulates glucose transporter 4 translocation by insulin in muscle cells

https://doi.org/10.1016/j.cellsig.2008.04.011Get rights and content

Abstract

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. Patients with A-T also have high incidences of type 2 diabetes mellitus. The gene mutated in this disease, ATM (A-T, mutated), encodes a protein kinase. Previous studies have demonstrated that cytoplasmic ATM is an insulin-responsive protein and a major upstream activator of Akt following insulin treatment. To further investigate the function of ATM in insulin signal transduction, insulin resistance was induced in rats by feeding them a high-fat diet. Muscle tissue of rats with insulin resistance had both dramatically reduced ATM levels and substantially decreased Akt phosphorylation at Ser473 in comparison to that of regular chow-fed controls. The decreased ATM expression suggests that ATM is involved in the development of insulin resistance through down-regulation of Akt activity. The role of ATM in activation of Akt was further confirmed in mouse embryonic fibroblast (MEF) A29 (ATM+/+) and A38 (ATM−/−) cells. In addition, insulin-mediated Akt phosphorylation in mouse L6 muscle cells was greatly reduced by KU-55933, a specific inhibitor of ATM. A 2-deoxyglucose incorporation assay showed that this inhibitor also caused a significant reduction in insulin-mediated glucose uptake in L6 cells. An immunofluorescence experiment demonstrated that in L6 cells transfected with wild-type (WT) ATM, insulin caused a dramatic increase of the cell surface glucose transporter 4 (GLUT4), while in cells transfected with kinase-dead (KD) ATM, translocation of GLUT4 to the cell surface in response to insulin was markedly inhibited.

Introduction

Ataxia-telangiectasia (A-T) is a monogenic, autosomal recessive disorder. A-T was initially noticed in children who appeared to have an unsteady gait (ataxia) that reflects cerebellar degeneration. Other symptoms of A-T include oculocutaneous telangiectasias, cancer predisposition, premature aging, growth retardation, and variable immune deficiencies [1], [2]. In addition, A-T patients are known to have higher incidences of type 2 diabetes mellitus and exhibit both insulin resistance and glucose intolerance, two typical symptoms of type 2 diabetes [3], [4], [5], [6].

In 1970, Schalch et al. [3] reported that 10 out of 17 A-T patients developed type 2 diabetes. Although only a subset of patients with A-T has been found to have type 2 diabetes mellitus, it should be noted that A-T patients usually die before the third decade of their life. Since type 2 diabetes usually develops at a later stage (> 40 years old) of a patient's life, the percentage of A-T patients who were found to develop type 2 diabetes mellitus may have been significantly underestimated [6].

A-T disease is caused by the lack or inactivation of the ATM protein. This protein is a 370-kDa protein kinase encoded by ATM, the gene mutated in A-T. The ATM protein is a member of a family of proteins related to phosphatidylinositol 3-kinase (PI 3-kinase). ATM was previously reported primarily as a nuclear protein in proliferating cells [7], [8], and it was thought to function mainly in controlling cell cycle progression after DNA damage. In response to ionizing radiation and DNA double strand breaks, ATM was shown to phosphorylate p53 [9], [10], Brca1 [11], Chk2 [12], and a number of other substrates.

However, many of the growth abnormalities associated with the A-T disease, including insulin resistance and glucose intolerance, cannot be explained by defective DNA damage responses in the nuclei of A-T cells. Several recent lines of evidence indicate that ATM is also present in the cytoplasm and is associated with vesicular structures in proliferating cells [13], [14], [15], [16]. Moreover, ATM was found to bind to β-adaptin, a cytoplasmic protein involved in vesicle or protein transport processes [14]. In certain postmitotic cells, it was even demonstrated that ATM is predominantly cytoplasmic [17], [18], [19].

The function of cytoplasmic ATM in insulin signal transduction has recently started to emerge. ATM was shown to be an insulin-responsive protein that controls protein translation through its phosphorylation of a cytoplasmic, translational regulatory protein, 4E-BP1 [16]. The functional significance of ATM in insulin signaling has been further verified by a recent finding showing that the ATM protein kinase mediates the full activation of Akt/PKB activity by stimulating its phosphorylation at Ser473 following insulin treatment [20].

Insulin initiates several signal transduction pathways in the cytoplasm of the cell. One of the most important pathways activated by insulin is the PI 3-kinase pathway. Akt is a major component of the PI 3-kinase signaling pathway and is known to participate in multiple physiological processes. In response to insulin, Akt not only stimulates protein translation by controlling the activity of several protein translation initiation factors [21], [22], but also controls the glucose uptake process by regulating insulin-mediated GLUT4 translocation [23], [24].

Although the cause of type 2 diabetes mellitus is still unclear, it is known that insulin resistance is closely related to the development of the disease. Defective glucose uptake in muscle and adipose tissues plays a major role in causing the insulin resistance and glucose intolerance symptoms associated with type 2 diabetes [25], [26]. The rate-limiting step in glucose uptake is glucose transport mediated by GLUT4, which is mainly present in muscle and adipose tissue. In response to insulin, GLUT4 translocates from the cytoplasm to the cell membrane and mediates the transport of glucose. Zisman et al. [26] reported that mice carrying a muscle-specific deletion of the GLUT4 gene developed severe insulin resistance and glucose intolerance. A study using adipose-specific GLUT4 knockout mouse models also showed that these mice developed insulin resistance and glucose intolerance [25]. These results demonstrate that GLUT4 has an essential role in the maintenance of normal glucose homeostasis.

In this study, we induced insulin resistance in rats by feeding them a high-fat diet and measured the expression of the ATM protein and the phosphorylation of Akt in their skeletal muscle tissue. The functional link between ATM and Akt was further examined in MEF A29 (ATM+/+) and A38 (ATM−/−) cells. In addition, the effect of ATM on Akt phosphorylation following insulin treatment in L6 muscle cells was studied using a specific inhibitor of ATM. We also conducted experiments to see if there is a functional connection between the ATM protein kinase and the translocation of GLUT4 in response to insulin in L6 cells.

Section snippets

Materials

The antibody against β-tubulin was from Sigma. The anti-c-myc antibody was from Santa Cruz. The Cy3-conjugated goat–anti-mouse antibody was from Jackson ImmunoResearch Laboratories. The antibodies against phospho-Ser473 and phospho-Thr308 of Akt, as well as the antibodies against the different Akt isoforms were from Cell Signaling Technology. The antibodies against total Akt, phospho-c-Jun, and total c-Jun were from Santa Cruz Biotechnology. The antibodies against phospho-Tyr612 of insulin

Results

To determine whether the ATM protein is involved in the development of insulin resistance in type 2 diabetes mellitus, we induced insulin resistance in rats by feeding them a high-fat diet. This is an established method and is based on previous studies performed in many other laboratories [28], [29], [30], [31]. Control rats were given standard rodent chow. Insulin resistance was determined by the QUICKI method. The QUICKI method is a mathematical model that has been found to correlate well

Discussion

A commonly used animal model of insulin resistance involves feeding lean rodents a high-fat diet which results in obesity and insulin resistance [28], [29], [30], [31]. In the case of the rat model, substantial increases in fasting insulin levels are usually seen in the high-fat-fed group when compared to a chow-fed control group, with varying responses in fasting glucose levels [28], [31]. In order to eliminate the effects of other diabetes-prone genes on our results, we chose to use this

Acknowledgements

We are grateful to Dr. Amira Klip (The Hospital for Sick Children, Toronto) for her kindness in providing us with the GLUT4myc plasmid and L6 muscle cells, as well as detailed experimental protocols for our GLUT4-related experiments. We are also grateful to Dr. Graeme Smith (KuDos Pharmaceuticals) for providing us with the ATM inhibitor KU-55933. We would also like to thank Dr. Doug Martin and Dr. Keith Miskimins (University of South Dakota) for their critical reading of this manuscript and Dr.

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