Paraquat-induced Oxidative Stress Represses Phosphatidylinositol 3-Kinase Activities Leading to Impaired Glucose Uptake in 3T3-L1 Adipocytes

Abstract

Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquatinduced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3-OH kinase (PI 3-kinase) p85 regulatory subunit or p110␣ catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulindependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110␣ and p110␤ that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110␣ mutated at Cys 90 in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes. Reactive oxygen species (ROS)2 are generated in organisms during metabolic reactions that use oxygen. Increased ROS production causes oxidative stress, which is frequently associated with various disorders such as hypertension, inflammation, and diabetes (1, 2). For example, it is well known that in many diabetic patients and animal models of diabetes, increased generation of ROS and the onset of diabetes are closely associated with oxidative stress (2). Antioxidants such as ␣-lipoic acid and vitamins C and E have been shown to improve insulin sensitivity in diabetic models, further evidence that oxidative stress is associated with insulin resistance (3-5).In general, the intracellular insulin signal is initiated by insulin binding to its receptor (IR), resulting in activation of the intrinsic tyrosine kinase of the receptor. This leads to the recruitment and tyrosine phosphorylation of intracellular insulin receptor substrates (IRS) 1-4 (6). Phosphorylated IRS proteins bind signaling molecules possessing the Src homology 2 domain such as the class Ia PI 3-kinases that are heterodimers of a p85 regulatory subunit and a p110 catalytic subunit. Three isoforms of p110 catalytic subunit, ␣, ␤, and ␦, have been identified. ...

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