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, 24 (3), 526-39

Prenatal Exposure to the Environmental Obesogen Tributyltin Predisposes Multipotent Stem Cells to Become Adipocytes

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Prenatal Exposure to the Environmental Obesogen Tributyltin Predisposes Multipotent Stem Cells to Become Adipocytes

Séverine Kirchner et al. Mol Endocrinol.

Abstract

The environmental obesogen hypothesis proposes that pre- and postnatal exposure to environmental chemicals contributes to adipogenesis and the development of obesity. Tributyltin (TBT) is an agonist of both retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARgamma). Activation of these receptors can elevate adipose mass in adult mice exposed to the chemical in utero. Here we show that TBT sensitizes human and mouse multipotent stromal stem cells derived from white adipose tissue [adipose-derived stromal stem cells (ADSCs)] to undergo adipogenesis. In vitro exposure to TBT, or the PPARgamma activator rosiglitazone increases adipogenesis, cellular lipid content, and expression of adipogenic genes. The adipogenic effects of TBT and rosiglitazone were blocked by the addition of PPARgamma antagonists, suggesting that activation of PPARgamma mediates the effect of both compounds on adipogenesis. ADSCs from mice exposed to TBT in utero showed increased adipogenic capacity and reduced osteogenic capacity with enhanced lipid accumulation in response to adipogenic induction. ADSCs retrieved from animals exposed to TBT in utero showed increased expression of PPARgamma target genes such as the early adipogenic differentiation gene marker fatty acid-binding protein 4 and hypomethylation of the promoter/enhancer region of the fatty acid-binding protein 4 locus. Hence, TBT alters the stem cell compartment by sensitizing multipotent stromal stem cells to differentiate into adipocytes, an effect that could likely increase adipose mass over time.

Figures

Figure 1
Figure 1
Experimental design and characterization of mADSCs. A, Study of the effect of prenatal TBT exposure. The effect of fetal and neonatal exposure to TBT was observed in C57BL/6J mice exposed to the chemical in utero. Pregnant females were dosed by gavage feeding with 0.1 mg/kg TBT, 1 mg/kg ROSI, or 0.5% CMC vehicle at E16.5. At 8 wk of age, mice were killed to isolate ADSCs from n = 5 pooled WAT. Subsequent treatment with nuclear receptor agonists is described in Figs. 2 and 5. B, Gene expression profile of mADSCs for multilineage progenitors and surface markers. The presence or absence of surface markers or transcripts specific to adipogenic, osteogenic chondrogenic lineages was detected by real-time RT-PCR in undifferentiated mADSCs, with the expression of each target gene normalized to β-actin. A gene was considered not expressed in the cell preparation if its mean Ct value exceeded 38. The symbol (+/−) was assigned to genes the expression of which was low (mean Ct value exceeding 32), but consistently detectable. C, Multilineage assays. ADSCs were differentiated into adipose (A), bone (B), and cartilage (C) throughout the experiments (up to passage 6). The expression level of specific lineage markers was assayed by real-time PCR as previously described. Adipogenic (LEP, ADIPQ), osteogenic (OST), and chondrogenic (ACAN) markers that were not significantly expressed in undifferentiated cells, were detectable in induced mADSCs. Data were expressed as average fold change in expression ± sem in differentiated relative to undifferentiated ADSCs. Asterisks represent significant differences (***, P < 0.001). ACAN, Aggrecan; OST, osteocalcin.
Figure 2
Figure 2
In vitro effect of TBT exposure on ADSCs adipogenic and proliferative capacities. A, Adipogenesis was induced in mADSCs by the addition of an adipogenic cocktail for 14 d in the absence (DMSO) or presence of nuclear receptor agonists 50 nm TTNPB (RAR, negative control), 50 nm AGN (RXR), 500 nm ROSI (PPARγ), or two doses of TBT (5 and 50 nm) (n = 3 wells per treatment). Undifferentiated cells were kept in basic MSCs expansion media, able to prevent differentiation, as a negative control (untreated cells). Lipid accumulation was stained by Oil Red O and quantified with Image J software. The number of cells with lipid droplets was also visually counted. B, Proliferative capacities of mADSCs were assayed by CyQuant cell counting of culture in regular expansion media, with 400 cells as a starting quantity. Media was supplemented with DMSO, adipogenic cocktail, 50 nm TTNPB, 50 nm AGN, 10, 100, or 500 nm ROSI, or 5, 50, or 100 nm TBT (n = 3 wells per treatment and time point, n = 3 pictures per well). C, Adipogenic capacities of hADSCs were performed as described for hADSCs. Mouse cells were isolated as described in Fig. 1. All data were expressed as average fold change in n = 9 replicates ± sem (n = 3 wells per treatment in triplicates) relative to vehicle (DMSO) controls. Asterisks show significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). D, Proliferative capacities of hADSCs harvested from mice not prenatally exposed to RXR and/or PPARγ ligands.
Figure 3
Figure 3
In vitro effect of TBT exposure on adipogenic markers in differentiated ADSCs. Gene expression profile of adipogenesis-induced hADSCs (panels A and B) and mADSCs (panels C and D) was assayed by real-time RT-PCR (1. Early adipogenesis markers: Pref-1, Fapb4, and PPARγ2 (panels A and C); 2. Late adipogenesis markers: ADIPOQ and LEP, panels B and D). Expression was normalized to β-actin and expressed as average fold change in expression ± sem (n = 3 wells per treatment in triplicates) relative to vehicle (DMSO) controls.
Figure 4
Figure 4
Effect of the PPARγ2 agonist ROSI, and the PPARγ2 antagonist T0070907 on the TBT-induced increase of the adipogenic abilities of mADSCs in vitro. Mouse cells were isolated as described in Fig. 1. All pictures are representative of n = 9 replicates ± sem (n = 3 wells per treatment in triplicate) relative to vehicle (DMSO) controls. Asterisks show significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Lipid accumulation was stained by Oil Red O and quantified with Image J software. A, Adipogenesis was induced in mADSCs by the addition of an adipogenic cocktail for 14 d with 100 nm ROSI (upper row) or 50 nm TBT (lower row) in the presence of DMSO, 10, 100, or 1000 nm T0070907 (PPARγ antagonist). B, Adipogenesis was induced in mADSCs by the addition of an adipogenic cocktail with 0 or 100 nm T0070907 for 14 d in the presence of DMSO, 100 nm TBT, or 1000 nm ROSI. C, Adipogenesis was induced in mADSCs by the addition of an adipogenic cocktail for 14 d with three doses of ROSI (0, 10, and 100 nm) in combination with three doses of TBT (0, 10, 50 nm). Data were expressed as average fold change in n = 9 replicates ± sem (n = 3 wells per treatment in triplicate) relative to vehicle (DMSO) controls.
Figure 5
Figure 5
Effect of TBT prenatal exposure on adipogenic capacities of mADSCs. TBT and ROSI exposure in utero was performed as described in Fig. 1. mADSCs harvested from exposed animals was additionally treated in vitro with 50 nm TBT, 500 nm ROSI [PPARγ agonist]), or a DMSO vehicle control, in the presence of an adipogenic induction cocktail (n = 3 wells per treatment in triplicates) for 14 d. Undifferentiated cells were kept in basic MSCs expansion media, able to prevent differentiation, as a negative control (untreated cells). A, Lipid accumulation was stained by Oil Red O and quantified by Image J software. Mice in utero treatments are presented from left to right (CMC, TBT, and ROSI). Additional in vitro TBT and ROSI treatments of ADSCs are presented from the bottom up. B, Gene expression profile of adipogenesis-induced mADSCs was assayed by real-time RT-PCR as previously described (early adipogenesis markers: Pref-1 and Fapb4; late adipogenesis marker: LEP), using cells treated with cocktail only (+DMSO vehicle control) as a reference group. Asterisks show significant effects of in utero (black bars) and in vitro treatments.
Figure 6
Figure 6
Effect of TBT prenatal exposure on osteogenic capacities of mADSCs. TBT and ROSI exposure in utero was performed as described in Fig. 1. mADSCs harvested from exposed animals were additionally treated in vitro with 50 nm TBT or a DMSO vehicle control, in the presence of an osteogenic induction cocktail (n = 3 wells per treatment in triplicate) for 21 d. Undifferentiated cells were kept in basic MSC expansion media (which did not allow differentiation), as a negative control (untreated cells). A, Calcium accumulation was stained by Alizarin Red S and quantified by Image J software. Mice in utero treatments are presented from left to right (CMC, TBT, and ROSI). Additional in vitro TBT treatment of ADSCs are presented on lower rows, with calcium accumulation (Ost, left panel) and lipid accumulation (Adi, right panel), performed as previously described. B, Gene expression profile of osteogenesis-induced mADSCs was assayed by real-time RT-PCR as previously described (adipogenesis markers: Fapb4; osteogenesis marker: OPN), using cells treated with cocktail only (+DMSO vehicle control) as a reference group. Asterisks show significant effects of in utero (black bars) and in vitro treatments. Ost, Osteocalcin; Adi, adipocyte.
Figure 7
Figure 7
Effect of prenatal TBT exposure on the gene expression profile of undifferentiated mADSCs. TBT exposure in utero was performed as described in Fig. 1. A, Undifferentiated mADSCs were cultured, and gene expression levels of marcrophage surface and adipogenic markers were assayed by real-time RT-PCR with the expression of each target gene normalized to β-actin. Data were expressed as average fold change in expression ± sem (n = 3 per treatment in triplicates) relative to CMC controls. B, Flow cytometry. Undifferentiated mADSCs were cultured, and the number of CD68- and Fabp4-postive cells were counted. The upper panel is a frequency histogram displaying the Fabp4 relative fluorescence plotted against the total number or events (maximum number of cells) from untreated mADSCs harvested from animals in utero exposed to CMC (red) or TBT (green). The two lower panels are the density plots for the same population of cells, with the x- and y-axis representing the CD68 and Fabp4 fluorescence, respectively, and the cell count height on a density gradient. Particle counts are shown by dot density. The Fabp4-positive but CD68-negative cells, thought to be preadipocytes, appear in the boxed upper left quadrant of the histogram.
Figure 8
Figure 8
Effect of prenatal TBT exposure on the methylation status of Fapb4 promoter/enhancer region. A, Fapb4 and PPARγ2 promoter/enhancer regions. Schematic representation of the CpG islands, which spans −1500 to +100 with respect to the transcription initiation site (right angle arrow at +1) and restriction map (arrows). The closed box represents the location of Exon1, and the vertical bars represent the location of CpG sites. Horizontal bars represent the fragments (Met1, Met2, and Met3) spanning the restriction sites and the uncut control amplified by real-time RT-PCR. B, Occurrence of methylation in Fapb4 and PPARγ2 promoter/enhancer regions. Genomic DNA extracted from undifferentiated mADSCs harvested from TBT- or CMC-exposed mice was digested by the methylation-sensitive enzyme AciI. Uncut fragments corresponding to potentially methylated were detected by real-time RT-PCR with the expression of each target normalized to a region without restriction sites (uncut). Data are presented as previously described.

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