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

Abstract

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 (iAs(III)) and methylarsonous acid (MAs(III)) 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 iAs(III) and MAs(III).

Methods: The effects of iAs(III) and MAs(III) on components of the insulin-activated signal transduction pathway that regulate PKB/Akt phosphorylation were examined in 3T3-L1 adipocytes.

Results: Subtoxic concentrations of iAs(III) or MAs(III) had little or no effect on the activity of phosphatidylinositol 3-kinase (PI-3K), which synthesizes phosphatidylinositol-3,4,5-triphosphate (PIP(3)), or on phosphorylation of PTEN (phosphatase and tensin homolog deleted on chromosome ten), a PIP(3) phosphatase. Neither iAs(III) nor MAs(III) 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 iAs(III) and MAs(III). 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 iAs(III) or MAs(III).

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 iAs(III) or MAs(III), and possibly for impaired glucose tolerance associated with human exposures to iAs.

Figure 1

iAs^III and MAs^III inhibit ISGU by adipocytes at concentrations that do not compromise cell viability. 3T3-L1 adipocytes were treated with iAs^III (A) or MAs^III (B) for 4 hr. [¹⁴C]-2-Deoxyglucose uptake was assessed in treated and untreated (control) cells after a 10-min activation with insulin. Cell viability was measured by MTT assay. Each value represents the mean ± SD; n = 3–5 experiments. *Statistically significant differences (p < 0.05) between treated and control cells. The concentrations of 50 μM iAs^III and 2 μM MAs^III (indicated by arrows) were used in all subsequent experiments.

Figure 2

ISGU in adipocytes exposed to subtoxic concentrations of iAs^III and MAs^III is independent of caspase-3 activation. (A) Caspase-3 activity was measured in 3T3-L1 adipocytes treated for 4 hr with 50 μM iAs^III or 2 μM MAs^III and in untreated (control) cells in the presence or absence of 75 μM AC-DEVD-CHO, a caspase-3 inhibitor. Adipocytes treated with 500 μM H₂O₂ for 4 hr were used as positive controls. (B) Basal and insulin-stimulated uptake of [¹⁴C]-2-deoxyglucose was measured in adipocytes exposed for 4 hr to 50 μM iAs^III or 2 μM MAs^III in the presence or absence of 75 μM AC-DEVD-CHO. Each value represents the mean ± SD; n = 3–5 experiments. *Statistically significant differences (p < 0.05) between treated and untreated (control) cells.

Figure 3

Four-hour exposures to subtoxic concentrations of iAs^III or MAs^III do not increase DNA fragmentation in cultured adipocytes. DNA fragmentation was measured by TUNEL in 3T3-L1 adipocytes treated with 50 μM iAs^III or 2 μM MAs^III for 4, 24, 48, and 72 hr. Untreated adipocytes were used as controls. Color images show green fluorescein signal of fragmented DNA in apoptotic cells. Gray-scale images illustrate the corresponding cell morphology. Representative fields of two independent experiments are shown. Bars = 40 μm.

Figure 4

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.

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.

Figure 6

Exposures to subtoxic concentrations of iAs^III or MAs^III do not affect insulin signal mediators that regulate PIP₃ 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.

Figure 8

Exposures to subtoxic concentrations of iAs^III or MAs^III inhibit the phosphorylation and PKB/Akt. (A) Immunoblot analysis of total PKB/Akt and Ser473- or Thr308-phosphorylated PKB/Akt in insulin-activated 3T3-L1 adipocytes treated for 4 hr with 50 μM iAs^III or 2 μM MAs^III and in insulin-activated control (untreated) cells. Representative blots of three independent experiments are shown. (B) The activity of PKB/Akt immunoprecipitated from insulin-activated control and treated adipocytes. Each value represents the mean ± SD; n = 4–5 experiments. *Statistically significant differences (p < 0.05) between treated and control cells.

Figure 9

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 [¹⁴C]-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.