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Cited by 161Arsenic contamination and mitigationHeavy Metal Exposure and ToxicityMetabolism, Diabetes, and Cancer

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

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Paul, David S., Harmon, Anne W., Devesa, Vicenta, Thomas, David J., Stýblo, Miroslav·2007·Environmental Health Perspectives
DOI 10.1289/ehp.9867PMC PMC1867998
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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 (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.

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Figure 1 iAsIII and MAsIII inhibit ISGU by adipocytes at concentrations that do not compromise cell viability. 3T3-L1 adipocytes were treated with iAsIII (A) or MAsIII (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 iAsIII and 2 μM MAsIII (indicated by arrows) were used in all subsequent experiments.
Figure 1 iAsIII and MAsIII inhibit ISGU by adipocytes at concentrations that do not compromise cell viability. 3T3-L1 adipocytes were treated with iAsIII (A) or MAsIII (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 iAsIII and 2 μM MAsIII (indicated by arrows) were used in all subsequent experiments.CC0 · PubMed Central
Figure 2 ISGU in adipocytes exposed to subtoxic concentrations of iAsIII and MAsIII is independent of caspase-3 activation. (A) Caspase-3 activity was measured in 3T3-L1 adipocytes treated for 4 hr with 50 μM iAsIII or 2 μM MAsIII 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 iAsIII or 2 μM MAsIII 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 2 ISGU in adipocytes exposed to subtoxic concentrations of iAsIII and MAsIII is independent of caspase-3 activation. (A) Caspase-3 activity was measured in 3T3-L1 adipocytes treated for 4 hr with 50 μM iAsIII or 2 μM MAsIII 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 iAsIII or 2 μM MAsIII 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.CC0 · PubMed Central
Figure 3 Four-hour exposures to subtoxic concentrations of iAsIII or MAsIII do not increase DNA fragmentation in cultured adipocytes. DNA fragmentation was measured by TUNEL in 3T3-L1 adipocytes treated with 50 μM iAsIII or 2 μM MAsIII 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 3 Four-hour exposures to subtoxic concentrations of iAsIII or MAsIII do not increase DNA fragmentation in cultured adipocytes. DNA fragmentation was measured by TUNEL in 3T3-L1 adipocytes treated with 50 μM iAsIII or 2 μM MAsIII 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.CC0 · PubMed Central
Figure 4 Retention of As species in 3T3-L1 adipocytes exposed to iAsIII or MAsIII. As species retained in 3T3-L1 adipocytes exposed for 4 hr to 50 μM iAsIII or 2 μM MAsIII were analyzed by HG-AAS. Note: The HG-AAS technique used in this study cannot distinguish between AsIII and AsV species. Each value represents the mean ± SD; n = 3 experiments.
Figure 4 Retention of As species in 3T3-L1 adipocytes exposed to iAsIII or MAsIII. As species retained in 3T3-L1 adipocytes exposed for 4 hr to 50 μM iAsIII or 2 μM MAsIII were analyzed by HG-AAS. Note: The HG-AAS technique used in this study cannot distinguish between AsIII and AsV species. Each value represents the mean ± SD; n = 3 experiments.CC0 · PubMed Central
Figure 5 Exposures to subtoxic concentrations of iAsIII or MAsIII 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 iAsIII (C) or 2 μM MAsIII (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 5 Exposures to subtoxic concentrations of iAsIII or MAsIII 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 iAsIII (C) or 2 μM MAsIII (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.CC0 · PubMed Central
Figure 6 Exposures to subtoxic concentrations of iAsIII or MAsIII 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 iAsIII or 2 μM MAsIII. 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 6 Exposures to subtoxic concentrations of iAsIII or MAsIII 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 iAsIII or 2 μM MAsIII. 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.CC0 · PubMed Central
Figure 7 Exposures to subtoxic concentrations of AsIII or MAsIII 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 iAsIII or 2 μM MAsIII 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 7 Exposures to subtoxic concentrations of AsIII or MAsIII 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 iAsIII or 2 μM MAsIII 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.CC0 · PubMed Central
Figure 8 Exposures to subtoxic concentrations of iAsIII or MAsIII 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 iAsIII or 2 μM MAsIII 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 8 Exposures to subtoxic concentrations of iAsIII or MAsIII 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 iAsIII or 2 μM MAsIII 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.CC0 · PubMed Central
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 iAsIII or MAsIII. Basal and insulin-stimulated [14C]-2-deoxyglucose uptake by 3T3-L1 adipocytes treated for 4 hr with 50 μM iAsIII or 2 μM MAsIII 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 9 Constitutive activation of protein kinase B (PKB/Akt) prevents the inhibition of insulin-stimulated glucose uptake in adipocytes treated with subtoxic concentrations of iAsIII or MAsIII. Basal and insulin-stimulated [14C]-2-deoxyglucose uptake by 3T3-L1 adipocytes treated for 4 hr with 50 μM iAsIII or 2 μM MAsIII 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.CC0 · PubMed Central
Figure 10 The molecular mechanism underlying the inhibition of insulin-stimulated glucose uptake by adipocytes exposed to iAsIII or MAsIII. The inhibition of PDK-1 and putative PDK-2 activities by iAsIII and MAsIII results in suppression of the downstream signaling steps, including PKB/Akt phosphorylation and GLUT4 translocation to the plasma membrane.
Figure 10 The molecular mechanism underlying the inhibition of insulin-stimulated glucose uptake by adipocytes exposed to iAsIII or MAsIII. The inhibition of PDK-1 and putative PDK-2 activities by iAsIII and MAsIII results in suppression of the downstream signaling steps, including PKB/Akt phosphorylation and GLUT4 translocation to the plasma membrane.CC0 · PubMed Central

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Metadata

Authors
Paul, D. S., Harmon, A. W., Devesa, V., Thomas, D. J., & Stýblo, M.
Affiliations
  • University of North Carolina at Chapel Hill (US)
  • Department of Nutrition and
  • Environmental Protection Agency (US)
Corresponding author
Stýblo, Miroslav
ORCID
Venue
Environmental Health Perspectives
Publisher
American Chemical Society (ACS)
Published
2007-01-29
Volume
115
Issue
5
Pages
734-742
ISSN
  • 0091-6765 (ISSN-L)
  • 1552-9924
DOI
10.1289/ehp.9867
PMC
PMC1867998
OpenAlex
W1987873876
Cited by
161
References
75
Citations by year
2026: 3 · 2025: 2 · 2024: 6 · 2023: 11 · 2022: 8 · 2021: 11 · 2020: 5 · 2019: 8 · 2018: 5 · 2017: 15 · 2016: 9 · 2015: 12 · 2014: 9 · 2013: 11 · 2012: 13
FWCI
5.54
Type
journal article
Language
en
Open access
Licence
Locations
  • Environmental Health Perspectives (publishedVersion, public-domain)
  • Europe PMC (PubMed Central) (publishedVersion, public-domain)
  • PubMed Central (publishedVersion, public-domain)
  • PubMed (publishedVersion)
Funders
  • U.S. Environmental Protection Agency
  • National Institutes of Health (DK56350)
  • National Institute of Diabetes and Digestive and Kidney Diseases (T32DK007686, 5 T32 DK07686, DK56350, P30DK056350)
Topics
  • Physical Sciences › Environmental Science › Environmental Chemistry › Arsenic contamination and mitigation
  • Physical Sciences › Environmental Science › Health, Toxicology and Mutagenesis › Heavy Metal Exposure and Toxicity
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
Keywords
inorganic arsenic, arsenite, diabetogenic effect, insulin signaling, insulin-stimulated glucose uptake, adipocytes, 3T3-L1 cells, Akt/protein kinase B, PDK1, phosphatidylinositol 3-kinase, PTEN, phosphatidylinositol 3,4,5-trisphosphate, chronic arsenic exposure, diabetes mellitus, arsenic metabolites
MeSH terms
Animals, Arsenicals, Diabetes Mellitus, Glucose, Insulin, Microscopy, Fluorescence, Phosphorylation, Immunoblotting, Signal Transduction, 3T3 Cells, Protein Serine-Threonine Kinases, Adipocytes, Arsenites, Phosphatidylinositol 3-Kinases, Proto-Oncogene Proteins c-akt, PTEN Phosphohydrolase, Glucose Transporter Type 4, Mice, 3-Phosphoinositide-Dependent Protein Kinases

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