Molecular Mechanisms of the Diabetogenic Effects of Arsenic: Inhibition of Insulin Signaling by Arsenite and Methylarsonous Acid

Background Increased prevalences of diabetes mellitus have been reported among individuals chronically exposed to inorganic arsenic (iAs). However, the mechanisms underlying the diabetogenic effects of iAs have not been characterized. We have previously shown that trivalent metabolites of iAs, arsenite (iAsIII) and methylarsonous acid (MAsIII) inhibit insulin-stimulated glucose uptake (ISGU) in 3T3-L1 adipocytes by suppressing the insulin-dependent phosphorylation of protein kinase B (PKB/Akt). Objectives Our goal was to identify the molecular mechanisms responsible for the suppression of PKB/Akt phosphorylation by iAsIII and MAsIII. Methods The effects of iAsIII and MAsIII on components of the insulin-activated signal transduction pathway that regulate PKB/Akt phosphorylation were examined in 3T3-L1 adipocytes. Results Subtoxic concentrations of iAsIII or MAsIII had little or no effect on the activity of phosphatidylinositol 3-kinase (PI-3K), which synthesizes phosphatidylinositol-3,4,5-triphosphate (PIP3), or on phosphorylation of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a PIP3 phosphatase. Neither iAsIII nor MAsIII interfered with the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1) located downstream from PI-3K. However, PDK-1 activity was inhibited by both iAsIII and MAsIII. Consistent with these findings, PDK-1-catalyzed phosphorylation of PKB/Akt(Thr308) and PKB/Akt activity were suppressed in exposed cells. In addition, PKB/Akt(Ser473) phosphorylation, which is catalyzed by a putative PDK-2, was also suppressed. Notably, expression of constitutively active PKB/Akt restored the normal ISGU pattern in adipocytes treated with either iAsIII or MAsIII. Conclusions These results suggest that inhibition of the PDK-1/PKB/Akt-mediated transduction step is the key mechanism for the inhibition of ISGU in adipocytes exposed to iAsIII or MAsIII, and possibly for impaired glucose tolerance associated with human exposures to iAs.


Research
Arsenic (As) is a naturally occurring toxic metalloid and a potent human carcinogen [International Agency for Research on Cancer (IARC) 1987]. The cancer-promoting effects of environmental exposures to inorganic arsenic (iAs) have been examined by epidemiologic studies and in laboratory experiments. Much less attention has been paid to the adverse effects of iAs that do not involve malignancies. Epidemiologic evidence suggests that type 2 (noninsulin dependent) diabetes mellitus may be one of the most common noncancerous diseases associated with chronic exposures to iAs. Increased prevalences of type 2 diabetes or symptoms consistent with this disease have been associated with the consumption of drinking water containing high levels of iAs (Chen et al. 1995;Lai et al. 1994;Rahman et al. 1998Rahman et al. , 1999Tseng et al. 2000Tseng et al. , 2002Wang et al. 2003) or with chronic exposures to iAs in occupational settings (Jensen and Hansen 1998;Rahman and Axelson 1995;Rahman et al. 1996). Although not all epidemiologic studies support the association between iAs exposure and diabetes (Navas-Acien et al. 2006), the existing evidence provides sufficient basis for investigation of the diabetogenic effects of iAs.
Type 2 diabetes is characterized by disruptions in whole-body glucose homeostasis due to insulin resistance and impaired glucose utilization by peripheral tissues, including skeletal muscle and adipose tissue. The insulin-dependent activation of glucose uptake in these tissues is one of the key mechanisms that regulates glucose homeostasis. The insulin-activated signal transduction mechanism that stimulates glucose uptake by adipocytes has been extensively studied. It includes the autophosphorylation of the β-subunit of the insulin receptor (IRβ) upon binding of insulin to the α-subunit of the receptor (IRα), the subsequent tyrosine phosphorylation of insulin receptor substrate 1 or 2 (IRS-1 or -2), and the binding of a phosphorylated IRS (p-IRS) to the regulatory (p85) subunit of the class IA phosphatidylinositol 3-kinase (PI-3K) that leads to the activation of its catalytic (p110) subunit. The activated PI-3K catalyzes the phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) at the plasma membrane to phosphatidylinositol-3,4,5-triphosphate (PIP 3 ) (Farese 2001;Ruderman et al. 1990;White and Kahan 1994). PIP 3 facilitates 3-phosphoinositide-dependent kinase-1/2 (PDK-1/2) dependent phosphorylation/activation of protein kinase B (PKB/Akt) and two atypical enzymes of the protein kinase C (PKC) family, PKCλ and ζ (Chou et al. 1998;Le Good et al. 1998;Standaert et al. 1997). The phosphorylation of PKB/Akt results in the translocation of intracellular vesicles containing glucose transporter-4 (GLUT4) from the perinuclear region to the plasma membrane and in the stimulation of glucose uptake (Kohn et al. 1996a;Tanti et al. 1997). In addition to phosphorylated PKB/Akt (p-PKB/Akt), phosphorylated PKCλ (p-PKCλ), and PKCζ (p-PKCζ) are thought to participate in the stimulation of GLUT4 translocation in response to insulin signaling (Elmendorf and Pessin 1999;Ruderman et al. 1990). The mechanism by which p-PKB/Akt, p-PKCλ and ζ induce the translocation and fusion of GLUT4-containing vesicles with the plasma membrane, as well as the degree to which each of these kinases participates in this event, are unclear. Recent studies have indicated that the PI-3K-dependent rearrangement of actin filaments ) and activation of the microtubule-associated motor protein kinesin (Imamura et al. 2003) contribute to the translocation of GLUT4 to the plasma membrane. The disruption of cytoskeletal components may represent a potential mechanism by which As exposure inhibits insulinstimulated glucose uptake (ISGU). Notably, As has been shown to bind to actin and tubulin in human lymphoblastoid cells (Menzel et al. 1999) and to inhibit the cytoskeletal protein synthesis in Swiss 3T3 mouse cells (Li and Chou 1992).
The mechanisms by which exposure to iAs may induce impaired glucose tolerance have not been systematically studied. Data on the effects of As on glucose homeostasis have been generated almost exclusively in studies that examined the metabolism of nutrients under severe stress induced by chemical or physical stimuli. Results of in vitro studies have consistently shown significant increases in basal (insulin-independent) glucose uptake by various types of cells or dissected tissues exposed to cytotoxic concentrations of a trivalent iAs, arsenite (iAs III ), or an aromatic derivative of As III , phenylarsine oxide (PAO) (Bazuine et al. 2003(Bazuine et al. , 2004Brazy et al. 1980;McDowell et al. 1997;Pasternak et al. 1991;Short 1965;Sviderskaya et al. 1996;Widnell et al. 1990). Consistent with these findings, some in vivo studies have reported moderate or severe hypoglycemia in animals chronically exposed to toxic, often lethal, concentrations of iAs III or arsenate (iAs V ), in drinking water (Hughes and Thompson 1996;Pal and Chatterjee 2004a, 2004b. Only limited information is available on the effects of arsenicals on glucose metabolism at low concentrations that are compatible with environmental or occupational exposures. Micromolar concentrations of PAO have been shown to inhibit basal or ISGU by cultured cells (Liebl et al. 1992(Liebl et al. , 1995 and by intact skeletal muscle (Henriksen and Holloszy 1990;Sowell et al. 1988). PAO did not interfere with the insulin-dependent phosphorylation of IRβ and did not interact directly with glucose transporters (Frost and Lane 1985;Frost et al. 1987). The effects of physiologically relevant arsenicals on insulinstimulated glucose metabolism have only recently been examined in this laboratory (Walton et al. 2004). We have shown that iAs III and the products of iAs methylation in humans, methylarsonous acid (MAs III ), and dimethylarsinous acid (DMAs III ) (Thomas et al. 2001), inhibit ISGU by 3T3-L1 adipocytes at concentrations that do not affect cell viability. Exposures to these arsenicals did not prevent IRβ and IRS phosphorylation or formation of the PI-3K-p-IRS complex. However, both iAs III and MAs III inhibited the insulin-dependent phosphorylation of PKB/ Akt that mediates ISGU in adipocytes. In contrast, DMAs III did not inhibit PKB/Akt phosphorylation, suggesting that this metabolite of iAs inhibits ISGU by a PKB/Akt-independent mechanism.
In the present study we examined the molecular mechanisms of ISGU inhibition by iAs III and MAs III , focusing mainly on the components of the insulin-activated signal transduction pathway that regulate PKB/Akt phosphorylation in adipocytes. Results of this work show that iAs III and MAs III inhibit PDK-1 activity, thus suppressing PDK-1catalyzed phosphorylation of PKB/Akt and p-PKB/Akt-mediated translocation of GLUT4 transporters to the plasma membrane. Notably, MAs III was an order of magnitude more potent than iAs III as an inhibitor of the PDK-1/PKB/Akt signal transduction step and of glucose uptake in insulin-stimulated adipocytes. Thus, the formation of MAs III in the methylation pathway for iAs may play a critical role in determining the extent of the diabetogenic effects associated with chronic exposures to iAs.

Materials and Methods
Cell culture and treatment. We obtained 3T3-L1 preadipocytes from Y. Patel (University of North Carolina, Greensboro, North Carolina). Myr-PKB/Akt-3T3-L1 preadipocytes expressing constitutively active PKB/Akt lacking the pleckstrin homology (PH) domain were provided by S. Summers (University of Colorado at Boulder, Boulder, Colorado). Addition of the src myristoylation sequence promotes constitutive membrane association and activation of PKB/Akt (Kohn et al. 1996a(Kohn et al. , 1996b. A2myr-PKB/Akt-3T3-L1 adipocytes, which express PKB/Akt containing a nonfunctional src myristoylation domain, and 3T3-L1 adipocytes containing the empty expression vector were also provided by S. Summers. All cell lines were cultured in Dulbecco's modified Eagle medium (Gibco, Grand Island, NY) with high glucose, 10% fetal bovine serum (HyClone, Logan, UT), penicillin, and streptomycin (Sigma Chemical Co., St. Louis, MO). Cells were cultured at 37°C in a humidified incubator in a 90% air and 10% CO 2 atmosphere. To induce differentiation, postconfluent cells were treated with a mixture of 0.5 mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 1 µg/mL insulin (all from Sigma Chemical Co.) for 48 hr and cultured in insulin-containing medium for an additional 48 hr (Paul et al. 2003). All experiments were performed between days 9 and 12 postinduction, when more than 90% of cells were fully differentiated. Differentiated adipocytes were treated with iAs III (sodium salt, Sigma Chemical Co.) or methylarsine oxide (provided by W. Cullen, University of British Columbia, Vancouver, Canada). Identity and purity of methylarsine oxide was confirmed by 1 H-NMR and mass spectrometry. In aqueous solutions, methylarsine oxide is hydrolyzed to form MAs III (Petrick et al. 2001). Fresh stock solutions of iAs III and MAs III in sterile phosphatebuffered saline (PBS) were prepared before each experiment to minimize the oxidation of iAs III to iAs V or MAs III to methylarsonic acid (MAs V ). Adipocytes were incubated with arsenicals or vehicle in a cell culture incubator for 4 hr.
Glucose uptake assay. The glucose uptake assay followed the previously described procedures (Paul et al. 2003). Briefly, adipocytes were serum starved in the presence or absence of arsenicals for 4 hr, washed with Krebs-Ringer phosphate (KRP) buffer, and treated with 1 µM insulin at 37°C for 10 min. Insulin-activated cells were incubated for 10 min with 200 µM 2-[1-14 C]-deoxy-D-glucose (0.1 µCi/well) (NEN Life Science Products, Inc., Boston, MA). To measure basal (insulin-independent) glucose uptake, we incubated cells with radiolabeled glucose without pretreatment with insulin. After the incubation, cells were washed twice with PBS (0°C), and lysed in a solution of 0.5 N NaOH and 10% SDS. Radioactivity in cell lysates was measured, using a Wallac 1409 liquid scintillation counter (Wallac, Turku, Finland).

Evaluation of cytotoxic and apoptotic effects of arsenicals.
To determine cell viability, we used the MTT assay, which measures the conversion of 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) to purple formazan by mitochondrial dehydrogenases of viable cells (Carmichael et al. 1987), as previously described (Walton et al. 2004). Caspase-3 activity was examined in an assay mixture containing cell lysate and aminomethylcoumarin (AMC)-derived substrate, Z-DEVD-AMC (Molecular Probes, Carlsbad, CA). Cleavage of Z-DEVD-AMC by caspase-3 yields a blue-fluorescent product (excitation/emission wavelength = 342/ 441 nm) that was quantified by an HTS 7000 Bio Assay Reader (Perkin-Elmer, Norwalk, CT). We examined DNA fragmentation in adipocytes using TUNEL (terminal deoxynucleotidyltransferase-mediated nick end labeling). For this assay, adipocytes were cultured on glass coverslips coated with poly-L-lysine (Sigma Chemical Co.) and treated with arsenicals. Cells were then fixed in 4% bufferedparaformaldehyde and permeabilized in a solution of 0.1% Triton X-100 and 0.1% sodium citrate (Sigma Chemical Co.). DNA strand breaks were enzymatically labeled on 3´-OH termini with fluorescein-linked nucleotides, using the In Situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN). Nuclei of both normal and apoptotic cells were stained with 100 nM 4´,6diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma Chemical Co.). Labeled cells were visualized using a Nikon Microphot FXA fluorescent microscope (Nikon, Tokyo, Japan).
Immunofluorescent analysis of GLUT4. Adipocyte cultures on glass cover slips were treated with arsenicals, activated with insulin, and incubated with D-glucose (Sigma Chemical Co.). After fixation with 4% bufferedparaformaldehyde, adipocytes were rinsed with ice-cold PBS and incubated with poly-L-lysine (0.5 mg/mL) for 1 min. Cells were then treated
Speciation analysis of As. We analyzed arsenicals in cell cultures exposed to iAs III or MAs III using hydride generation atomic absorption spectrometry (HG-AAS) as previously described (Devesa et al. 2004). Cells and culture medium were analyzed separately for each treatment. Arsines were generated at pH 1, cold-trapped, separated by their boiling points, and analyzed, using a Perkin-Elmer model 5100 PC atomic absorption spectrometer (Perkin-Elmer). Under these conditions, arsines were generated from both As III and As V species. Thus, total iAs (iAs = iAs III + iAs V ), total methylarsenic (MAs = MAs III + MAs V ), and total dimethylarsenic (DMAs = DMAs III + DMAs V ) species were determined. We confirmed the identities of arsenicals in spectral peaks using aliquots of samples spiked with standards. Calibration curves for each of the arsenicals (0.5, 2.5, 10, 20, 80 ng As) were generated to quantify results of the analyses.
Protein kinase activity assays. We measured PI-3K, PKB/Akt, and PDK-1 activities in cell lysates from adipocytes treated with arsenicals and activated with insulin after immunoprecipitation with specific antibodies bound to protein G agarose beads (Santa Cruz Biotechnology). For a single assay we used the immunoprecipitate from cells cultured in one 10-cm plate. The assay conditions were as follows: PI-3K assay. PI-3K was immunoprecipitated from control (untreated) adipocytes or from adipocytes exposed to arsenicals with an anti-phosphotyrosine (PY20) antibody. PI-3K immunoprecipitated from insulin-activated adipocytes pretreated with 1 nM wortmannin (a specific inhibitor of PI-3K) was used as a negative control. The enzyme activity was measured in a 50-µL assay mixture containing the immunoprecipitated PI-3K, 20 mM HEPES (pH 7.4), 50 mM MgCl 2 , 200 µM adenosine, 40 µM adenosine 5´-triphosphate (ATP) (all from Sigma Chemical Co.), 20 µCi [γ-32 P]-ATP (NEN Life Science Products, Inc.), and L-α-phosphatidylinositol (PI) (Avanti Polar Lipids Inc., Alabaster, AL) as a substrate (Augustine et al. 1991). The reaction was stopped by 1 N HCl. A 30-min incubation at 37°C of the assay mixture containing PI-3K from control cells resulted in the formation of radiolabeled phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2 ). To simplify the analysis, a 10-min incubation that yielded only PIP was used  throughout this study. Radiolabeled phospholipids were extracted in chloroform:methanol (CHCl 3 :CH 3 OH) (1:1). The organic phase was washed with CH 3 OH:HCl (1:1), evaporated under nitrogen, and the residue was dissolved in CHCl 3 :CH 3 OH (2:1). The extract was separated by thin-layer chromatography (TLC) on glass silica plates pretreated with 1% potassium oxalate, using an n-propanol:2 N acetic acid (65:35) solvent system (Augustine et al. 1991). The distribution of radioactivity on TLC plates was evaluated, using a computerized Fuji FLA-2000 imaging system (Fujifilm, Stamford, CT). The following standards were used to confirm the identity of separated phospholipids: L-α-phosphatidylinositol, L-α-phosphatidylinositol-4-phosphate, L-αphosphatidylinositol-4,5-bisphosphate (Avanti Polar Lipids Inc.). Standards were visualized on developed TLC plates by treatment with 50% sulfuric acid at 100°C for 1 hr.
Statistical analysis. All experiments were replicated to ensure the reproducibility of results. Representative findings are shown.
Results of the cell viability, glucose uptake, and protein kinase activity assays were evaluated by analysis of variance with Tukey multiple comparison posttest using a GraphPad Instat statistical software package (GraphPad Software, San Diego, CA). Differences among means with p < 0.05 were considered statistically significant.

Results
Our previous work has shown that trivalent arsenicals inhibit ISGU by 3T3-L1 adipocytes. However, the possible association between the inhibition of ISGU and a general loss of cell functions due to the cytotoxicity of arsenicals has not been thoroughly examined. In this study we examined ISGU and cell viability in adipocytes exposed for 4 hr to iAs III or MAs III at a wide range of concentrations. Consistent with our previous report (Walton et al. 2004), stimulation of 3T3-L1 adipocytes with insulin increased glucose uptake by 9-to 11-fold over basal levels (data not shown). ISGU was significantly inhibited by concentrations as low as 5 µM iAs III and 0.5 µM MAs III ( Figure 1A, B). In contrast, cell viability decreased only when concentrations of iAs III and MAs III exceeded 1 mM and 5 µM, respectively. Gross abnormalities in adipocyte morphology were absent at all concentrations tested, although minor cell detachment did occur at higher concentrations (≥ 200 µM iAs III and ≥ 10 µM MAs III ). The estimated IC 50 (concentration that results in the inhibition of ISGU by 50%) values for the inhibition of ISGU were 25 µM for iAs III and 4 µM for MAs III . In comparison, the LC 50 (concentration that results in a decrease of cell viability by 50%) values characterizing the cytotoxic effects were 11 mM for iAs III and 15 µM for MAs III . Thus, the inhibition of ISGU by iAs III and MAs III at or below IC 50 values was not due to impaired adipocyte viability. However, both iAs III and MAs III can induce cell apoptosis (Lau et al. 2004;McCollum et al. 2005;Namgung and Xia 2001). At early stages, apoptotic processes may affect cell functions without having

Inhibition of insulin signal by inorganic and methyl arsenic
Environmental Health Perspectives • VOLUME 115 | NUMBER 5 | May 2007 immediate effects on cell viability. We examined apoptotic markers in adipocytes exposed for 4 hr to 50 µM iAs III and 2 µM MAs III , the concentrations that effectively inhibit ISGU, but are far below the minimal cytotoxic concentrations. Under these exposure conditions, both iAs III and MAs III significantly increased the activity of caspase-3, an early marker of apoptosis ( Figure 2A). Adipocytes treated with 500 µM H 2 O 2 were used as positive controls for this experiment. Pretreatment with 75 µM Ac-Asp-Glu-Val-Asp-CHO (AC-DEVD-CHO), a cell-permeable caspase-3 inhibitor, prevented caspase-3 activation by both arsenicals and by hydrogen peroxide. However, pretreatment with AC-DEVD-CHO did not prevent the decrease in ISGU in cells treated with either iAs III or MAs III ( Figure 2B), suggesting that the inhibition of ISGU was independent of processes associated with early stages of apoptosis. TUNEL was used to determine the degree of DNA fragmentation in adipocytes exposed to 50 µM iAs III or 2 µM MAs III (Figure 3). Adipocyte nuclei were stained with DAPI to determine the total number of cells (data not shown). The average apoptotic index (percentage of TUNEL-positive cells) was about 16% for control adipocytes and did not change after a 4-hr exposure to either iAs III or MAs III . However, the apoptotic index increased considerably after longer exposure times, reaching an average of 32% for iAs III and 39% for MAs III after 24 hr and more than 90% after 72-hr exposure to either arsenical. These data suggest that 4-hr exposures to 50 µM iAs III or 2 µM MAs III did not compromise cell viability or integrity. In addition, neither 50 µM iAs III nor 2 µM MAs III induced p38 MAPK phosphorylation during the 4-hr exposure (data not shown). Therefore, the inhibition of ISGU is not associated with stress and is likely due to specific effects of these arsenicals on mediators of insulin signaling or on the cellular components involved in glucose transport. Based on these results, 4-hr exposures to 50 µM iAs III and 2 µM MAs III were used in further experiments to examine the effects of iAs III or MAs III on components of the insulin-activated signal transduction pathway in 3T3-L1 adipocytes. The effects of iAs III or MAs III on mediators of insulin signaling would ultimately depend on the intracellular concentrations and metabolic conversion of these arsenicals. We examined the distribution of As species in adipocytes after a 4-hr exposure to iAs III or MAs III , using HG-AAS. Cells exposed to 50 µM iAs III retained about 3 times more As than cells exposed to 2 µM MAs III (Figure 4). Retained As represented 2.5 and 16% of the total As in cultures exposed to iAs III or MAs III , respectively. Only iAs and MAs species were detected in adipocyte cultures exposed to iAs III and MAs III , respectively, indicating that no methylation conversion took place during the 4-hr exposures. These findings are consistent with previous reports that found adipocytes to be inefficient methylators of iAs (Walton et al. 2004).
The translocation of GLUT4 from the perinuclear compartment to the plasma membrane is a prerequisite for glucose uptake in adipocytes stimulated with insulin. We used immunofluorescent staining in this study to examine the association of GLUT4 with the plasma membranes of insulin-stimulated 3T3-L1 adipocytes treated with 50 µM iAs III or 2 µM MAs III for 4 hr and from control (untreated) cells that were or were not stimulated with insulin ( Figure 5). Stimulation with insulin dramatically increased the GLUT4-specific fluorescent signal in plasma membrane lawns of control cells. GLUT4 signals in plasma membrane lawns isolated from insulin-stimulated cells treated with either iAs III or MAs III were noticeably weaker compared with control insulin-stimulated cells, suggesting that both arsenicals interfered with the translocation of GLUT4 in response to insulin stimulation.  . Retention of As species in 3T3-L1 adipocytes exposed to iAs III or MAs III . As species retained in 3T3-L1 adipocytes exposed for 4 hr to 50 µM iAs III or 2 µM MAs III were analyzed by HG-AAS. Note: The HG-AAS technique used in this study cannot distinguish between As III and As V species. Each value represents the mean ± SD; n = 3 experiments.

As species in cells (ng As/well)
iAs III + iAs V MAs III + MAs V Figure 5. Exposures to subtoxic concentrations of iAs III or MAs III inhibit GLUT4 association with the plasma membrane of insulin-activated adipocytes. Immunofluorescent images of GLUT4 in plasma membrane lawns isolated from control (untreated) 3T3-L1 adipocytes before (A) and after activation (B) with insulin and from insulin-activated adipocytes treated for 4 hr with 50 µM iAs III (C) or 2 µM MAs III (D). Adipocytes were fixed and sonicated to prepare plasma membrane lawns. GLUT4 was labeled with an anti-GLUT4 antibody and visualized with a fluorescent secondary antibody. Representative fields of two independent experiments are shown. Bars = 10 µm.
The impaired ISGU in adipocytes exposed to trivalent arsenicals has previously been linked to the inhibition of components of the insulin signal transduction pathway located downstream of IRS1/2, but upstream of PKB/Akt (Walton et al. 2004). PI-3K is located downstream of IRS. The binding of p-IRS to the regulatory (p85) subunit of PI-3K in response to insulin stimulates the PI-3K-catalyzed production of PIP 3 from PIP 2 . In this study, the association of p-IRS with PI-3K was examined in insulin-stimulated adipocytes exposed for 4 hr to 50 µM iAs III or 2 µM MAs III . Neither iAs III nor MAs III affected the amount of PI-3K (p85), immunoprecipitated with an anti-phosphotyrosine (PY20) antibody, which reacts with phosphorylated tyrosine residues of IRS in the insulin-activated PI-3K complex ( Figure 6A). PI-3K activity was measured in adipocytes exposed for 4 hr to 50 or 100 µM iAs III or to 2 or 5 µM MAs III . Exposures to iAs III had no effect on PI-3K activity. A relatively small decrease in PI-3K activity was detected in cells exposed to 2 µM MAs III ; however, no changes were found in cells exposed to 5 µM MAs III (data not shown). Effects of MAs III on PI-3K activity were further analyzed in an in vitro assay mixture containing PI-3K immunoprecipitated from control insulin-stimulated adipocytes. Addition of MAs III into this mixture at concentrations up to 50 µM did not inhibit PI-3K activity (data not shown). PTEN, a PIP 3 phosphatase, is involved in the regulation of PIP 3 levels in adipocytes. PTEN activity is regulated by a casein kinase 2-catalyzed phosphorylation on its C-terminal noncatalytic regulatory domain, which includes Ser380 (Torres and Pulido 2001). Neither 50 µM iAs III nor 2 µM MAs III altered the levels of total PTEN or pPTEN (Ser380) ( Figure 6A). No changes in the of ratio of phosphorylated pPTEN (Ser380) to total PTEN were found in insulin-stimulated adipocytes exposed to either iAs III or MAs III ( Figure 6B).
Phosphorylation on Ser241 is required for optimal activity of PDK-1, a downstream effector of PI-3K (Casamayor et al. 1999). Figure 7A shows that exposures to 50 µM iAs III or 2 µM MAs III had no significant effects on the level of Ser241-phosphorylated PDK-1 in insulin-stimulated adipocytes. However, PDK-1 activity was significantly lower in cells exposed to either iAs III or MAs III , 47% and 57% of that in control cells, respectively ( Figure 7B).
In the insulin-activated signal transduction pathway, PKB/Akt is the downstream effector of PDK-1. The activation of PKB/Akt in response to insulin stimulation includes the phosphorylation of Ser473 and Thr308 residues (Toker and Newton 2000). Our previous work demonstrated that exposures to iAs III or MAs III inhibit PKB/Akt phosphorylation on Ser473 (Walton et al. 2004), which is thought to be catalyzed by a putative Ser-kinase, PDK-2 (Toker and Newton 2000). PDK-1 is responsible for Thr308 phosphorylation, which is required for maximal PKB/Akt activity (Scheid et al. 2002). Immunoblot analysis carried out in this study showed that 4-hr exposures to 50 µM iAs III or 2 µM MAs III inhibited the insulin-dependent phosphorylation of PKB/Akt on both Ser473 and Thr308 residues ( Figure 8A). PKB/Akt activity in

Inhibition of insulin signal by inorganic and methyl arsenic
Environmental Health Perspectives • VOLUME 115 | NUMBER 5 | May 2007 Figure 6. Exposures to subtoxic concentrations of iAs III or MAs III do not affect insulin signal mediators that regulate PIP 3 levels in insulin-activated adipocytes. (A) Immunoblot analyses of the activated PI-3K, total PTEN, and phosphorylated PTEN (Ser380) in control 3T3-L1 adipocytes before or after activation with insulin and in insulin-activated adipocytes treated for 4 hr with 50 µM iAs III or 2 µM MAs III . Activated PI-3K was immunoprecipitated from control and exposed cells with an anti-phospho-Tyr (PY20) antibody and immunoblotted with an antibody against the regulatory (p85) subunit. Representative blots of three independent experiments are shown. (B) The ratio of phosphorylated PTEN (Ser380) to total PTEN expressed as a percent of the ratio found in control adipocytes before activation with insulin. Each value represents the mean ± SD; n = 3 experiments.  Figure 7. Exposures to subtoxic concentrations of As III or MAs III do not affect the phosphorylation of PDK-1 but inhibit PDK-1 activity in adipocytes. (A) Immunoblot analysis of Ser241-phosphorylated PDK-1 in insulin-activated 3T3-L1 adipocytes treated for 4 hr with 50 µM iAs III or 2 µM MAs III and in control (untreated) insulin-activated adipocytes. Representative blot of three independent experiments is shown. (B) The kinase activity of PDK-1 immunoprecipitated from insulin-activated control or treated 3T3-L1 adipocytes. Each value represents the mean ± SD; n = 4-5 experiments. *Statistically significant differences (p < 0.05) between treated and control cells. *Statistically significant differences (p < 0.05) between treated and control cells. insulin-stimulated adipocytes exposed to iAs III and MAs III was 47 and 28% of that in control insulin-activated cells, respectively ( Figure 8B). To further evaluate the role of the PDK-1/PKB/Akt signal transduction step as a target for trivalent arsenicals in the insulinactivated signal transduction pathway, we examined the effects of iAs III or MAs III on ISGU by adipocytes expressing constitutively active myr-PKB/Akt. Adipocytes expressing an inactive A2myr-PKB/Akt mutant or empty expression vector were used as negative controls. Consistent with the constitutive activation of PKB/Akt, glucose uptake by adipocytes expressing myr-PKB/Akt was elevated even in the absence of insulin stimulation ( Figure 9). Four-hour exposures to 50 µM iAs III or 2 µM MAs III had no effect on ISGU by myr-PKB/Akt expressing cells. In contrast, both arsenicals inhibited ISGU in cells expressing the inactive A2myr-PKB/Akt mutant or the empty expression vector.

Discussion
Previous studies have shown that As III -containing species may affect glucose uptake by cultured cells or dissected tissues by two independent mechanisms that strictly depend on the concentration of As III . Highly-toxic concentrations of As III stimulate glucose uptake in the absence of insulin (Bazuine et al. 2003(Bazuine et al. , 2004Brazy et al. 1980;McDowell et al. 1997;Pasternak et al. 1991;Short 1965;Sviderskaya et al. 1996;Widnell et al. 1990) through a mechanism that involves activation of p38 MAPK-mediated stress signaling and PI-3K-dependent phosphorylation of PKB/Akt (Souza et al. 2001). In our experiments, exposure of 3T3-L1 adipocytes to 50 µM iAs III and 2 µM MAs III for 4 hr did not activate p38 MAPK, thus providing further evidence of the subtoxic nature of our exposure conditions. Treatments with toxic concentrations of arsenicals are not comparable to environmental or occupational exposures to iAs that do not typically induce acute stress or tissue damage. In contrast, subtoxic concentrations of As III inhibit ISGU (Henriksen and Holloszy 1990;Liebl et al. 1992Liebl et al. , 1995Sowell et al. 1988) in a manner consistent with impaired glucose tolerance reported among individuals chronically exposed to relatively low concentrations of iAs. We have shown that inhibition of ISGU in adipocytes exposed to subtoxic concentrations of trivalent metabolites of iAs, iAs III , or MAs III is associated with the suppression of PKB/Akt phosphorylation (Walton et al. 2004). Because neither iAs III nor MAs III interfered with insulin signaling upstream of PI-3K (Walton et al. 2004), the present work focused on the signal transduction steps immediately preceding PKB/Akt phosphorylation, specifically, on the enzymatic system controlling PIP 3 levels in insulin-activated cells and on PDK-1.
The formation of PIP 3 catalyzed by the insulin-activated PI-3K-IRS complex is an essential step in ISGU by adipocytes. PIP 3 is required for PDK-1-catalyzed phosphorylation of PKB/Akt on Thr308 (Casamayor et al. 1999). PIP 3 is thought to interact directly with the PH-domain of PDK-1 and PKB/Akt, activating PDK-1 or facilitating Thr308 phosphorylation of PKB/Akt. Other studies suggest that PIP 3 promotes the phosphorylation of PKB/Akt on Ser473 by a putative PDK-2, thereby priming PKB/Akt for PDK-1-catalyzed phosphorylation of Thr308 (Toker and Newton 2000). PIP 3 concentration in the membrane region of cells is subjected to strict regulation involving PI-3K and specific lipid phosphatases, including PTEN (Maehama and Dixon 1998) and SHIP2 (Src homology 2-containing inositol 5´-phosphatase 2) (Wada et al. 2001). Both PTEN, a D-3 lipid phosphatase, and SHIP2, a D-5 lipid phosphatase, are expressed in adipocytes. However, a recent report suggests that only PTEN is capable of suppressing insulin signaling in 3T3-L1 adipocytes (Tang et al. 2005). PTEN is phosphorylated on Ser380 and Thr382/383 by casein kinase 2 (Torres and Pulido 2001). The phosphorylated PTEN (p-PTEN) is less susceptible to degradation by the proteosome but is less active. Inhibition of Ser380 phosphorylation increases PTEN activity, but destabilizes the enzyme (Georgescu et al. 1999;Tolkacheva and Chan 2000). Factors that interfere with PI-3K activation in response to insulin or inhibit PTEN phosphorylation may decrease PIP 3 levels in adipocytes and, ultimately, prevent PDK-1/2-catalyzed phosphorylation of PKB/Akt.
In this study, iAs III and MAs III inhibited PDK-1/2 catalyzed phosphorylation of PKB/Akt on Thr308 and Ser473 but had little or no effect on PI-3K activity or PTEN phosphorylation. In addition, neither iAs III nor MAs III affected PDK-1(Ser241) phosphorylation, which is essential for PDK-1 activity. These results suggest that iAs III and MAs III inhibit PDK-1 activity through direct interactions with the enzyme. Sulfhydryl groups of vicinal or closely spaced cysteines are typical high-affinity targets for trivalent arsenicals in protein structures (Altamirano et al. 1989;Carlson et al. 1978;Chakraborti et al. 1992;Delnomdedieu et al. 1993;Li et al. 2001;Lopez et al. 1990). Two such closely spaced cysteines (Cys21 and Cys23) are present in the N-terminus of both mouse and human PDK-1 (Alessi et al. 1997;Dong et al. 1999). Thus, it is plausible that binding of iAs III and MAs III to Cys21 and Cys23 is the proximate cause of PDK-1 inhibition by these arsenicals. However, unlike MAs III , which can form a  . Constitutive activation of protein kinase B (PKB/Akt) prevents the inhibition of insulin-stimulated glucose uptake in adipocytes treated with subtoxic concentrations of iAs III or MAs III . Basal and insulin-stimulated [ 14 C]-2-deoxyglucose uptake by 3T3-L1 adipocytes treated for 4 hr with 50 µM iAs III or 2 µM MAs III and by untreated adipocytes expressing a constitutively active myr-PKB/Akt) (black bar, an inactive PKB/Akt mutant (A2myr-PKB/Akt) (blue bar), or an empty expression vector (white bar). Each value represents the mean ± SD; n = 4-5 experiments. *Statistically significant differences (p < 0.05) between treated and control cells.  Figure 10. The molecular mechanism underlying the inhibition of insulin-stimulated glucose uptake by adipocytes exposed to iAs III or MAs III . The inhibition of PDK-1 and putative PDK-2 activities by iAs III and MAs III results in suppression of the downstream signaling steps, including PKB/Akt phosphorylation and GLUT4 translocation to the plasma membrane. stable cyclic structure with two thiols, iAs III may require three coordination bonds to form a stable enzyme-inhibitor complex. A lower affinity for binding to Cys21 and Cys23 may explain why iAs III is a weaker inhibitor of PDK-1 than MAs III . In addition, the difference in potencies of iAs III and MAs III to inhibit PDK-1 activity and ISGU may be in part due to differences in the uptake and/or retention of these arsenicals by adipocytes. Our data suggest that MAs III was retained by 3T3-L1 adipocytes more efficiently than iAs III . These findings are consistent with the results of previous studies in other cell types (Dopp et al. 2004;Drobna et al. 2005). Importantly, our data show that the expression of constitutively active myrPKB/Akt prevents the inhibition of ISGU by either iAs III or MAs III . These data provide further evidence that the inhibition of ISGU by 3T3-L1 adipocytes exposed to iAs III and MAs III is due to the inhibition of the PDK-1-catalyzed activation of PKB/Akt and that neither iAs III nor MAs III disrupts signal transduction steps downstream from PDK-1/PKB/Akt, or events associated with GLUT4 translocation to the plasma membrane.
In summary, subtoxic concentrations of iAs III and MAs III inhibit ISGU by 3T3-L1 adipocytes through a mechanism that involves the inhibition of PDK-1 activity and of PDK-1/2-catalyzed phosphorylation of PKB/Akt ( Figure 10). The inhibition of ISGU by iAs III and MAs III , trivalent metabolites of iAs, is consistent with impaired glucose tolerance reported in individuals chronically exposed to iAs from the environment. In addition, the concentrations of iAs III and MAs III that inhibit ISGU by cultured adipocytes (as low as 5 and 0.5 µM, respectively) appear to be compatible with this type of exposure. Thus, taken together, this work provides a mechanistic basis for the diabetogenic effects of chronic environmental and occupational exposures to iAs.