Molecular mechanisms of the diabetogenic effects of arsenic: inhibition of insulin signaling by arsenite and methylarsonous acid.

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. 1998, 1999; Tseng et al. 2000, 2002; Wang 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 (PIP2) at the plasma membrane to phosphatidylinositol-3,4,5-triphosphate (PIP3) (Farese 2001; Ruderman et al. 1990; White and Kahan 1994). PIP3 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 (Patel et al. 2003) 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 insulin-stimulated 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 (iAsIII), or an aromatic derivative of AsIII, phenylarsine oxide (PAO) (Bazuine et al. 2003, 2004; Brazy 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 iAsIII or arsenate (iAsV), in drinking water (Hughes and Thompson 1996; Pal and Chatterjee 2004a, 2004b, 2005). 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, 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 insulin-stimulated glucose metabolism have only recently been examined in this laboratory (Walton et al. 2004). We have shown that iAsIII and the products of iAs methylation in humans, methylarsonous acid (MAsIII), and dimethylarsinous acid (DMAsIII) (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 iAsIII and MAsIII inhibited the insulin-dependent phosphorylation of PKB/Akt that mediates ISGU in adipocytes. In contrast, DMAsIII 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 iAsIII and MAsIII, 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 iAsIII and MAsIII inhibit PDK-1 activity, thus suppressing PDK-1–catalyzed phosphorylation of PKB/Akt and p-PKB/Akt–mediated translocation of GLUT4 transporters to the plasma membrane. Notably, MAsIII was an order of magnitude more potent than iAsIII as an inhibitor of the PDK-1/PKB/Akt signal transduction step and of glucose uptake in insulin-stimulated adipocytes. Thus, the formation of MAsIII 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.