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

Hou Y, Xue P, Woods CG, Wang X, Fu J, Yarborough K, Qu W, Zhang Q, Andersen ME, Pi J - Environ. Health Perspect. (2012)

Bottom Line: 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).In addition, iAs3+, MMA3+, and DMA3+ exhibited a strong inhibitory effect on adipogenesis in primary cultured mouse ADSVFCs and human ADSCs.Arsenic-induced dysfunctional adipogenesis may be associated with a reduced capacity of WAT to store lipids and with insulin resistance.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709, USA.

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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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.
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f3: 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.

Mentions: iAs3+ inhibits C/EBPβ transactivation during adipogenesis. C/EBPβ and C/EBPδ are transiently expressed and play a role at the early stage of adipogenic differentiation by sensing adipogenic stimuli and initiating expression of PPARγ and C/EBPα (Rosen and MacDougald 2006). Upon exposure to adipogenic signals, such as DMI or DMIRI cocktail, C/EBPβ and C/EBPδ are rapidly expressed and transcriptionally regulate the expression of PPARγ and C/EBPα, whereas C/EBPε and CHOP10 serve as negative regulators of PPARγ transcription (Batchvarova et al. 1995; Clarke et al. 1997; Darlington et al. 1998). As C/EBPβ acquires its DNA-binding activity, it becomes localized to centromeres and results in a characteristic “punctuated” pattern in immunofluorescence staining, which is a well-accepted measure of C/EBPβ–DNA-binding activity (Tang and Lane 2000). As shown in Figure 3A,B, iAs3+ did not reduce the mRNA and/or protein expression of C/EBPβ and C/EBPδ in the early stage (≤ 48 hr) of adipogenesis. However, the “punctuated” nuclear accumulation of C/EBPβ was reduced by iAs3+ after a 16-hr DMI treatment (Figure 3C,D), suggesting that iAs3+ directly suppresses centromere accumulation of C/EBPβ and/or interferes with the acquisition of C/EBPβ–DNA-binding activity. In addition, C/EBP–luciferase reporter assay (Figure 3E) and the mRNA expression of C/EBP-target gene Pparγ2 (Figure 3A) revealed that iAs3+ treatment significantly attenuates the transcriptional activity of C/EBPs after ≥ 12 hr of DMI treatment. It appears that the reduced transcriptional activity of C/EBPs caused by iAs3+ treatment results from a reduced DNA-binding activity of the C/EBPs, in particular C/EBPβ. Concomitant with the DNA-binding activity of C/EBPβ measured by immunostaining and C/EBP-reporter assay, the expression of PPARγ and C/EBPα in iAs3+-treated cells was markedly decreased at a later stage of adipogenesis, particularly after 24 hr of DMI treatment (Figure 3A,B).


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

Hou Y, Xue P, Woods CG, Wang X, Fu J, Yarborough K, Qu W, Zhang Q, Andersen ME, Pi J - Environ. Health Perspect. (2012)

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.
© Copyright Policy - public-domain
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569692&req=5

f3: 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.
Mentions: iAs3+ inhibits C/EBPβ transactivation during adipogenesis. C/EBPβ and C/EBPδ are transiently expressed and play a role at the early stage of adipogenic differentiation by sensing adipogenic stimuli and initiating expression of PPARγ and C/EBPα (Rosen and MacDougald 2006). Upon exposure to adipogenic signals, such as DMI or DMIRI cocktail, C/EBPβ and C/EBPδ are rapidly expressed and transcriptionally regulate the expression of PPARγ and C/EBPα, whereas C/EBPε and CHOP10 serve as negative regulators of PPARγ transcription (Batchvarova et al. 1995; Clarke et al. 1997; Darlington et al. 1998). As C/EBPβ acquires its DNA-binding activity, it becomes localized to centromeres and results in a characteristic “punctuated” pattern in immunofluorescence staining, which is a well-accepted measure of C/EBPβ–DNA-binding activity (Tang and Lane 2000). As shown in Figure 3A,B, iAs3+ did not reduce the mRNA and/or protein expression of C/EBPβ and C/EBPδ in the early stage (≤ 48 hr) of adipogenesis. However, the “punctuated” nuclear accumulation of C/EBPβ was reduced by iAs3+ after a 16-hr DMI treatment (Figure 3C,D), suggesting that iAs3+ directly suppresses centromere accumulation of C/EBPβ and/or interferes with the acquisition of C/EBPβ–DNA-binding activity. In addition, C/EBP–luciferase reporter assay (Figure 3E) and the mRNA expression of C/EBP-target gene Pparγ2 (Figure 3A) revealed that iAs3+ treatment significantly attenuates the transcriptional activity of C/EBPs after ≥ 12 hr of DMI treatment. It appears that the reduced transcriptional activity of C/EBPs caused by iAs3+ treatment results from a reduced DNA-binding activity of the C/EBPs, in particular C/EBPβ. Concomitant with the DNA-binding activity of C/EBPβ measured by immunostaining and C/EBP-reporter assay, the expression of PPARγ and C/EBPα in iAs3+-treated cells was markedly decreased at a later stage of adipogenesis, particularly after 24 hr of DMI treatment (Figure 3A,B).

Bottom Line: 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).In addition, iAs3+, MMA3+, and DMA3+ exhibited a strong inhibitory effect on adipogenesis in primary cultured mouse ADSVFCs and human ADSCs.Arsenic-induced dysfunctional adipogenesis may be associated with a reduced capacity of WAT to store lipids and with insulin resistance.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709, USA.

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.

Show MeSH
Related in: MedlinePlus