Inhibition of insulin-dependent glucose uptake by trivalent arsenicals: possible mechanism of arsenic-induced diabetes

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Abstract

Chronic exposures to inorganic arsenic (iAs) have been associated with increased incidence of noninsulin (type-2)-dependent diabetes mellitus. Although mechanisms by which iAs induces diabetes have not been identified, the clinical symptoms of the disease indicate that iAs or its metabolites interfere with insulin-stimulated signal transduction pathway or with critical steps in glucose metabolism. We have examined effects of iAs and methylated arsenicals that contain trivalent or pentavalent arsenic on glucose uptake by 3T3-L1 adipocytes. Treatment with inorganic and methylated pentavalent arsenicals (up to 1 mM) had little or no effect on either basal or insulin-stimulated glucose uptake. In contrast, trivalent arsenicals, arsenite (iAsIII), methylarsine oxide (MAsIIIO), and iododimethylarsine (DMAsIIIO) inhibited insulin-stimulated glucose uptake in a concentration-dependent manner. Subtoxic concentrations of iAsIII (20 μM), MAsIIIO (1 μM), or DMAsIIII (2 μM) decreased insulin-stimulated glucose uptake by 35–45%. Basal glucose uptake was significantly inhibited only by cytotoxic concentrations of iAsIII or MAsIIIO. Examination of the components of the insulin-stimulated signal transduction pathway showed that all trivalent arsenicals suppressed expression and possibly phosphorylation of protein kinase B (PKB/Akt). The concentration of an insulin-responsive glucose transporter (GLUT4) was significantly lower in the membrane region of 3T3-L1 adipocytes treated with trivalent arsenicals as compared with untreated cells. These results suggest that trivalent arsenicals inhibit insulin-stimulated glucose uptake by interfering with the PKB/Akt-dependent mobilization of GLUT4 transporters in adipocytes. This mechanism may be, in part, responsible for the development of type-2 diabetes in individuals chronically exposed to iAs.

Introduction

Arsenic is a toxic metalloid that occurs in the environment in a variety of chemical forms. Inorganic arsenic (iAs) is classified as human carcinogen (IARC, 1987). Drinking water containing high levels of iAs and industrial pollution are major sources of exposure to iAs for populations worldwide. Numerous epidemiological studies have associated chronic exposure to iAs with increased prevalences of skin cancer and cancers of internal organs Bates et al., 1992, Bates et al., 1995, Chen et al., 1985, Chen et al., 1992, Chiang et al., 1988, Chiou et al., 1995, Guo et al., 1995, Hopenhayn-Rich et al., 1996, Hopenhayn-Rich et al., 1998, Lewis et al., 1999, Smith et al., 1992, Smith et al., 1998, Tseng et al., 1968, Tsuda et al., 1995, Wu et al., 1989. Although the primary emphasis in research of the effects of chronic exposure to iAs has commonly focused on its carcinogenic potency, epidemiological studies demonstrate that iAs exerts other adverse effects that do not involve the induction of cancer. For example, blackfoot disease, an occlusive disease of the peripheral vasculature, has been reported in arsenic endemic areas of Taiwan (Tseng, 1989). Among other noncancerous disorders associated with chronic exposure to iAs are peripheral vascular, cardiovascular and cerebrovascular diseases Chen et al., 1995, Chen et al., 1996, Chiou et al., 1997, Engel et al., 1994, Thomas and Goyer, 1995, Tseng et al., 1995, Tseng et al., 1997, hypertension (Chen et al., 1995), goiter (Chang et al., 1991), hepatomegaly (Santra et al., 1999), respiratory system dysfunctions (Mazumder et al., 2000), and diseases of the peripheral and central nervous systems Bencko et al., 1977, Chisolm and Thomas, 1983, Masahiko and Hideyasu, 1973. In 1994, Lai and associates found a significant dose–response relationship between cumulative exposure to iAs and prevalence of diabetes mellitus in Taiwanese residents exposed to iAs from drinking water (Lai et al., 1994). The association between chronic exposure to iAs and diabetes mellitus was later confirmed by several epidemiological studies from Bangladesh Rahman et al., 1998, Rahman et al., 1999 and Taiwan (Tseng et al., 2000). In arseniasis endemic areas of Taiwan, the overall incidence of diabetes has reached 27.4 cases per 1000 person years (Tseng et al., 2000). Importantly, an increased incidence of diabetes mellitus as a cause of death has also been found among workers exposed to arsenic trioxide in copper smelter and glass producing plants in Sweden Rahman and Axelson, 1995, Rahman et al., 1996. In addition, elevated concentrations of glycosylated hemoglobin, an indicator of high blood glucose levels, were reported in several groups of Danish workers occupationally exposed to arsenic, including taxidermists, wood workers, and workers impregnating wood (Jensen and Hansen, 1998). Thus, chronic exposure to iAs represents a risk factor for diabetes mellitus in both environmental and occupational settings. Mechanisms by which iAs induces cancer, diabetes mellitus, or other noncancerous diseases are now under investigation. In general, the nature and extent of the biological effects of arsenic strongly depends on its chemical form, especially on its oxidation state. Arsenic species that contain trivalent arsenic (AsIII) have profound affinities toward SH-groups in peptides and proteins. Interactions between trivalent arsenicals and biologically active thiols are thought to underlie most of the adverse effects of arsenic. Trivalent iAs, and especially methylated trivalent arsenicals, methylarsonous acid (MAsIII), and dimethylarsonous acid (DMAsIII), that are products of the metabolism of iAs in humans are potent cytotoxins, genotoxins, and enzyme inhibitors (Thomas et al., 2001). In addition, trivalent iAs and methylated arsenicals interfere with major signal transduction pathways in human cells Drobná et al., 2003, Simeonova and Luster, 2000.

Non-insulin-dependent (type-2) diabetes is the prevalent form of diabetes mellitus found in populations chronically exposed to iAs from the environment (Tseng et al., 2002). Type-2 diabetes is characterized by insulin resistance of internal organs and peripheral tissues that results in impaired glucose utilization, and consequently, in abnormally high blood glucose levels between and especially after meals. The symptoms of the disease as described in individuals chronically exposed to iAs suggest that iAs or its metabolites interfere with insulin signaling or with the metabolism of glucose at the cellular level. Trivalent arsenicals are relatively potent inhibitors of several enzymes involved in glucose metabolism, including α-ketoglutarate dehydrogenase, succinyl-CoA synthase, and pyruvate dehydrogenase Boquist et al., 1988, Petrick et al., 2001. In addition, phenylarsine oxide (PAO), an organic derivative of AsIII, has been shown to inhibit basal or insulin-stimulated glucose uptake by adipocytes Douen and Jones, 1988, Douen et al., 1988, Frost et al., 1987, canine kidney (MDCK) cells Liebl et al., 1992, Liebl et al., 1995, and by intact skeletal muscle Henriksen and Hollotszy, 1990, Sowell et al., 1988. However, PAO is a synthetic compound with chemical and metabolic characteristics that may be different from those of iAs and of its known metabolites. Effects of physiologically relevant arsenicals on insulin-stimulated glucose uptake and insulin-activated signal transduction have never been systematically examined.

The present work describes effects of tri- and pentavalent arsenicals that are consistent with known metabolites of iAs on insulin-dependent glucose uptake by murine 3T3-L1 adipocytes. The results of this work show that trivalent arsenicals, particularly MAsIII and DMAsIII derivatives, are potent inhibitors of insulin-dependent glucose uptake by 3T3-L1 cells. Examination of insulin-activated signal transduction pathway in exposed cells suggests that trivalent arsenicals inhibit expression or activation (phosphorylation) of protein kinase B (PKB/Akt), an essential component of this pathway. Thus, the inhibition of insulin-dependent signal transduction at the PKB/Akt level may be at least in part responsible for type-2 diabetic symptoms as reported in individuals chronically exposed to iAs.

Section snippets

Arsenicals

Sodium arsenate (iAsV) and sodium arsenite (iAsIII) were purchased from Sigma (St. Louis, MO). Monosodium methylarsonate (MAsV) and dimethylarsinic acid (DMAsV) were obtained from Chem Service (West Chester, PA). Methylated trivalent arsenicals, methylarsine oxide (MAsIIIO), and iododimethylarsine (DMAsIIII) were synthesized by Dr. William R. Cullen (University of British Columbia, Vancouver, Canada) using previously described methods Cullen et al., 1984, Styblo et al., 1997. Identity and

Metabolism of [73As]iAsIII in differentiated 3T3-L1 adipocytes

To examine the capacity of 3T3-L1 cells to metabolize arsenicals, we exposed differentiated adipocytes to 0.1 μM [73As]iAsIII (100 pmol iAsIII/ml) for 48 h and analyzed the production and distribution of radioactive metabolites in the culture (Fig. 1). At the end of the incubation period, about 21% of 73As was associated with cells. Cells contained iAs as well as products of its methylation, MAs and DMAs. Methylated metabolites accounted for about 6% of the total 73As in the culture. No

Discussion

The activation of glucose transport across the cell membrane in response to insulin is a key mechanism in the regulation of glucose homeostasis. This mechanism facilitates the utilization of glucose in tissues and activates its subsequent metabolism, including glycogen synthesis and breakdown of glucose for energy production. The failure of insulin to activate glucose uptake in cases of insulin resistance represents the mechanistic basis for the development of type-2 diabetes. The signal

Acknowledgements

The authors thank Dr. David J. Thomas (U.S. EPA) for helpful discussions and Professor William Cullen and his colleagues at the University of British Columbia in Vancouver for provision of custom-synthesized methylated trivalent arsenicals that made this study possible. This work was supported by grants ES09941, ES11496, and CNRC-DK56350 from NIH. Some of the preliminary data from this study were presented at the BITREL 2002 Symposium, October 28 to November 2, 2002, Japan.

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