Chemico-Biological Interactions 182 (2009) 199–203 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Dual action of phenylarsine oxide on the glucose transport activity of GLUT1 Jordan Scott, Adeleye Opejin, Andrew Tidball, Nathan Stehouwer, Janelle Rekman, Larry L. Louters ∗ Department of Chemistry and Biochemistry, Calvin College, Grand Rapids, MI 49546, USA article info Article history: Received 1 June 2009 Received in revised form 24 July 2009 Accepted 9 August 2009 Available online 15 August 2009 Keywords: Phenylarsine oxide GLUT1 Arsenical toxicity Glucose uptake L929 fibroblast cells abstract An early event in the toxic effects of organic arsenic compounds, such as phenylarsine oxide (PAO), is an inhibition of glucose uptake. Glucose uptake involving the glucose transporter, GLUT4 is inhibited by PAO indicating an importance of vicinal sulfhydryls in insulin-stimulated glucose uptake. However, the data on effects of PAO on GLUT1 are conflicting. This study investigated the effects of PAO on glucose uptake in L929 fibroblast cells, cells, which express only GLUT1. The data presented here reveal a dual effect of PAO. At low concentrations or short exposure times PAO stimulated glucose uptake reaching a peak activation of about 400% at 3 M. At higher concentrations (40 M), PAO clearly inhibited glucose uptake. At intermediate concentrations (10 M), PAO had no effect under basal conditions but completely inhibited activation of glucose uptake by glucose deprivation and partially inhibited methylene blue- stimulated glucose uptake. PAO increased the specific binding of cytochalasin B to GLUT1 suggesting a direct interaction with the transporter. These data are most consistent with PAO interacting with multiple proteins that regulate the activity of this transporter, one of which may be GLUT1 itself. The identity of these proteins will require further investigation. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Phenylarsine oxide (PAO) is a trivalent arsenic compound that forms cyclic thioarsenite complexes with vicinal sulfhydryls. It has been extensively used as a tool for detecting and blocking thiol dependent biological processes. In particular PAO has been used to probe membrane receptor or transport mediated events such as glucose uptake. It has been demonstrated that PAO inhibits insulin-stimulated glucose uptake in adipocytes [1–5] and hepa- tocytes [6], as well as inhibiting both insulin-stimulated glucose and amino acid uptake in muscle [7–9]. This effect may be a func- tion of inhibition of GLUT4 translocation [2] and/or stimulation of GLUT4 degradation [10]. PAO also inhibits binding of insulin to its receptor [11] as well as the feedback inhibitory effects of insulin and IGF-1 on insulin release [12]. The effects of PAO on glucose uptake under basal or non-insulin- stimulating conditions are less clear. PAO has been reported to inhibit basal glucose uptake in kidney cells [13,14], adipocytes [1] and endothelial cells [15]. Indeed, the inhibition of glucose uptake appears to be a very early event in the pathogenesis of organic arsenical toxicity [13,14]. Notably, however, some investigators report that PAO either has no effect under basal conditions [2,8], Abbreviations: PAO, phenylarsine oxide; DMSA, meso-2,3-dimercaptosuccinic acid; MB, methylene blue. ∗ Corresponding author. Tel.: +1 616 526 6493; fax: +1 616 526 8551. E-mail address: lout@calvin.edu (L.L. Louters). or that it activates glucose uptake [7,16]. These conflicting data can be attributed, in part, to differential effects of PAO on GLUT4 ver- sus GLUT1. PAO has been shown to inhibit cytochalasin B binding in insulin-sensitive adipoctyes suggesting that it directly binds to GLUT4 [17]. A trivalent arsenical affinity column complexes with GLUT4, but not with GLUT1 [18]. Also, PAO is reported to have no effect on transport in erythrocytes or vesicles, which contain only GLUT1 [19,20]. However, there is also evidence that PAO does affect the transport activity of GLUT1 as well as GLUT4. It has been previously reported that PAO can inhibit the acute activation of glu- cose uptake in L929 fibroblast cells, cells that contain only GLUT1 [21]. The activation of glucose uptake by either methylene blue, a redox dye, or by glucose deprivation was completely blocked by PAO [22,23]. The effects of PAO at basal conditions in these studies were mixed, reporting both an inhibition [22] and an activation of glucose uptake [23]. The purpose of this study was to make a careful systematic study of the effects of PAO on GLUT1 mediated glucose uptake, under both basal and activating conditions in order to clarify the effects of PAO and the potential importance of vicinal thiols in the transport activity of GLUT1. 2. Materials and methods 2.1. Reagents Phenylarsine oxide (PAO), meso-2,3-dimercaptosuccinic acid (DMSA), 2-deoxy-d-glucose-[1,2- 3 H], d-mannitol-1- 14 C and 0009-2797/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2009.08.008