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. 2013 Feb;121(2):237-43.
doi: 10.1289/ehp.1205731. Epub 2012 Dec 5.

Association between arsenic suppression of adipogenesis and induction of CHOP10 via the endoplasmic reticulum stress response

Yongyong Hou  1 , Peng Xue, Courtney G Woods, Xia Wang, Jingqi Fu, Kathy Yarborough, Weidong Qu, Qiang Zhang, Melvin E Andersen, Jingbo Pi
Affiliations

Association between arsenic suppression of adipogenesis and induction of CHOP10 via the endoplasmic reticulum stress response

Yongyong Hou et al. Environ Health Perspect. 2013 Feb.

Abstract

Background: There is growing evidence that chronic exposure to inorganic arsenic (iAs) is associated with an increased prevalence of type 2 diabetes (T2D). However, the mechanisms for the diabetogenic effect of iAs are still largely unknown. White adipose tissue (WAT) actively stores and releases energy and maintains lipid and glucose homeostasis.

Objective: We sought to determine the mechanisms of arsenic suppression of adipogenesis.

Methods: The effects and associated mechanisms of iAs and its major metabolites on adipogenesis were determined in 3T3-L1 preadipocytes, mouse adipose-derived stromal-vascular fraction cells (ADSVFCs), and human adipose tissue-derived stem cells (ADSCs).

Results: Exposure of 3T3-L1 preadipocytes to noncytotoxic levels of arsenic, including inorganic arsenite (iAs3+, ≤ 5 μM), inorganic arsenate (≤ 20 μM), trivalent monomethylated arsenic (MMA3+, ≤ 1 μM), and trivalent dimethylated arsenic (DMA3+, ≤ 2 μM) decreased adipogenic hormone-induced adipogenesis in a concentration-dependent manner. In addition, iAs3+, MMA3+, and DMA3+ exhibited a strong inhibitory effect on adipogenesis in primary cultured mouse ADSVFCs and human ADSCs. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by arsenic occurred in the early stage of terminal adipogenic differentiation and was highly correlated with the induction of C/EBP homologous protein (CHOP10), an endoplasmic reticulum (ER) stress response protein. Induction of CHOP10 by arsenic is associated with reduced DNA-binding activity of CCAAT/enhancer-binding protein β (C/EBPβ), which regulates the transcription of peroxisome proliferator-activated receptor γ and C/EBPα.

Conclusions: Low-level iAs and MMA3+ trigger the ER stress response and up-regulate CHOP10, which inhibits C/EBPβ transcriptional activity, thus suppressing adipogenesis. Arsenic-induced dysfunctional adipogenesis may be associated with a reduced capacity of WAT to store lipids and with insulin resistance.

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Conflict of interest statement

This research was supported in part by National Institutes of Health grant ES016005 to J.P. M.E.A. received funding from the DOW Chemical Company and Unilever. The other authors declare they have no actual or potential competing financial interests.

Figures

Figure 1
Figure 1
Noncytotoxic levels of iAs suppress adipogenesis in 3T3-L1 preadipocytes. (A) Cytotoxicity of iAs3+ and iAs5+. Cells were exposed to iAs3+ and iAs5+ in DMEM with 10% FBS for 48 hr, followed by immediate cell viability measurements (n = 5–6). (B) iAs concentration-dependently inhibits adipogenesis. Cells were differentiated 1 day after 100% confluence (designated as day 0) by replacing growth medium with differentiation medium containing DMI (1 μM dexamethasone, 0.5 mM 3-isobutylmethylxanthine, and 1 μg/mL insulin) in DMEM with 10% FBS for 2 days, followed by an additional 6 days of culture in DMEM with 10% FBS and 1 μg/mL insulin; iAs was added during days 1 and 2 of differentiation; lipid vesicles were stained using ORO. (C) Suppression of iAs3+ on adipogenesis occurs in the early stage of DMI-induced differentiation. During adipogenesis, iAs3+ was added at the indicated periods, and ORO staining was performed on day 8. (D,F) Cytotoxicity of methylated arsenicals. Cells were exposed to the arsenicals in DMEM with 10% FBS for 48 hr (n = 5–6). (E,G) Trivalent, but not pentavalent, methylated arsenicals caused a concentration-dependent inhibition of adipogenesis. MMA3+, DMA3+, MMA5+, and DMA5+ were added during days 1 and 2 of differentiation; ORO staining was performed on day 8. Time (days), day of differentiation. *p < 0.05, arsenic-treated vs. vehicle-treated cells.
Figure 2
Figure 2
iAs3+, MMA3+, and DMA3+ suppress adipogenesis in mouse ADSVFCs and human ADSCs. Abbreviations: Pre, preadipocytes; Cont, control. Mouse ADSVFCs (A,B) isolated from C57BL/6J mice and human ADSCs (C,D) were cultured to 95% confluence and differentiated for 5 days using the DMIRI protocol. iAs3+ (5 μM), MMA3+ (1 μM for mouse ADSVFCs; 0.2 μM for human ADSCs), and DMA3+ (2 μM) were added during days 1 and 2 of differentiation. (A,C) Photomicrographs of stained cells. After differentiation, cells were stained with ORO to visualize lipid accumulation (20×). (B,D) mRNA expression of PPARγ (n = 3). *p < 0.05, preadipocytes vs. control cells. #p < 0.05, arsenic-treated vs. control cells.
Figure 3
Figure 3
Inhibitory effect of iAs3+ on the transcriptional activity of C/EBPβ and expression of PPARγ and C/EBPs during adipogenesis in 3T3-L1 preadipocytes. Abbreviations: C/EBPβ (LAP), C/EBPβ isoform liver-enriched activator protein; C/EBPβ (LIP), C/EBPβ isoform liver-enriched inhibitory protein; Control, cells were differentiated using the DMI protocol for the indicated time; iAs3+, cells were treated with iAs3+ (5 μM) during DMI treatment; vehicle, cells were maintained in growth medium without DMI. (A) mRNA expression of Cebps and Pparγ at the same differentiation time (n = 3). (B) Effects of DMI treatment on protein expression of PPARγ and C/EBPs during adipogenesis. Two isoforms of C/EBPα (42 kDa and 30 kDa) are shown on the blot. (C) Representative images of immunostaining of nuclear C/EBPβ after 16-hr DMI treatment. (D) Quantification of fluorescence intensity of nuclear C/EBPβ shown in (C) (n = 4–5). (E) Activity of C/EBP-luciferase reporter following DMI treatment in control and iAs3+-treated vs. control cells.
Figure 4
Figure 4
iAs3+ activates UPR in 3T3-L1 preadipocytes. Abbreviations: BFA, brefeldin A; TG, thapsigargin; TUN, tunicamycin. (A) Six hours of treatment with iAs3+ concentration-dependently enhanced mRNA expression of Atf4 and Chop10 (n = 3). (B) Time-course of mRNA expression of Atf4 and Chop10 after exposure to 5 μM iAs3+ (n = 3). (C,D) Concentration–response (C) and time course (D) of protein expression of eIF2α, p-eIF2α, ATF4, and CHOP10 in response to iAs3+ treatment. Cells were treated with iAs3+ for 6 hr (C) or 5 μM iAs3+ for the indicated time (D) (n = 3). (E) ER stressors suppress adipogenesis in 3T3-L1 cells. Cells were treated with the stressors at the indicated concentrations during the first 2 days of DMI-induced adipogenesis, followed by ORO staining. *p < 0.05, iAs3+-treated vs. vehicle-treated cells.
Figure 5
Figure 5
Effects of arsenicals on the DMI-induced reduction of CHOP10 in the early stage of adipogenesis in 3T3-L1 preadipocytes. (A) Effects of various arsenicals on the expression of CHOP10 protein in the early stage of adipogenesis. Cells were treated 1 day after confluence with DMI in the absence (control) or presence of arsenicals. Vehicle, growth medium. (B–D) Quantification of the protein expression of CHOP10 in response to iAs3+, iAs5+, or MMA3+ exposure in the early stage of adipogenesis (n = 3). *p < 0.05, compared with control cells at the same time. #p < 0.05, compared with vehicle-treated control cells.
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