Molecular analysis of insulin action using high throughput genetic screens
The insulin-stimulated uptake of glucose by muscle and adipose is vital for the maintenance of glucose homeostasis in the body. This uptake primarily occurs through the action of the insulin-regulatable glucose transporter GLUT4, which is rapidly translocated to the plasma membrane in response to the insulin signal. However, in the insulin resistant state, insulin is unable to effect a normal biological response in its target tissues, characterized by decreased glucose uptake as a result of defects in insulin signaling and attenuated GLUT4 translocation. Elucidating the molecular causes underlying insulin resistance is important for the development of therapeutics for this disease. Identifying the components involved in the propagation or regulation of insulin signaling is an important step in understanding insulin resistance. To date, the upstream components of the signaling pathway are well established, demonstrating the importance of the insulin receptor, IRS proteins and PI3K/Akt signaling axis in this process. As a result much work has focused on defects at the point of IRS in the development of insulin resistance. However, it has recently been suggested that defects associated with insulin resistance occur independently of IRS. Therefore, identifying the sites that this dysfunction occurs at is of great interest in understanding this disease. The purpose of this study is to discover novel proteins involved in the regulation of the insulin signaling and GLUT4 translocation. A GLUT4-overexpressing HeLa cell line was developed and optimized for use in high throughput screening for regulators of insulin stimulated GLUT4 translocation. Insulin stimulation caused GLUT4 translocation to the plasma membrane, and the activation of the PI3K/Akt signaling pathway in these cells. Using this cell line, I performed an siRNA screen of kinase and DUB libraries which identified a number of novel targets that may play a role in the regulation of insulin stimulated GLUT4 translocation. In conclusion, this assay can be used to identify novel regulators of insulin stimulated GLUT4 translocation, which may potentially represent targets for drug development in the treatment of insulin resistance
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