Transgenerational inheritance of increased fat depot size, stem cell reprogramming, and hepatic steatosis elicited by prenatal exposure to the obesogen tributyltin in mice

Abstract

Background: We have previously shown that exposure to tributyltin (TBT) modulates critical steps of adipogenesis through RXR/PPARγ and that prenatal TBT exposure predisposes multipotent mesenchymal stem cells (MSCs) to become adipocytes by epigenetic imprinting into the memory of the MSC compartment.

Objective: We tested whether the effects of prenatal TBT exposure were heritable in F2 and F3 generations.

Methods: We exposed C57BL/6J female mice (F0) to DMSO vehicle, the pharmaceutical obesogen rosiglitazone (ROSI), or TBT (5.42, 54.2, or 542 nM) throughout pregnancy via the drinking water. F1 offspring were bred to yield F2, and F2 mice were bred to produce F3. F1 animals were exposed in utero and F2 mice were potentially exposed as germ cells in the F1, but F3 animals were never exposed to the chemicals. We analyzed the effects of these exposures on fat depot weights, adipocyte number, adipocyte size, MSC programming, hepatic lipid accumulation, and hepatic gene expression in all three generations.

Discussion: Prenatal TBT exposure increased most white adipose tissue (WAT) depot weights, adipocyte size, and adipocyte number, and reprogrammed MSCs toward the adipocyte lineage at the expense of bone in all three generations. Prenatal TBT exposure led to hepatic lipid accumulation and up-regulated hepatic expression of genes involved in lipid storage/transport, lipogenesis, and lipolysis in all three subsequent generations.

Conclusions: Prenatal TBT exposure produced transgenerational effects on fat depots and induced a phenotype resembling nonalcoholic fatty liver disease through at least the F3 generation. These results show that early-life obesogen exposure can have lasting effects.

Figure 1

Transgenerational effect of DMSO (vehicle), ROSI, or TBT (5.42, 54.2, or 542 nM) on adipose depots (epididymal WAT, perirenal WAT, interscapular WAT, and interscapular BAT) from F1, F2, and F3 male mice. (A) Adipose tissue weights are represented as the percentage of total body weight. (B) Relative adipocyte size in epididymal, perirenal, and subscapular WAT (expressed as the number of pixels per selected area, with measurements for 100 cells/animal), and lipid accumulation in subscapular BAT (expressed as a percentage of the area covered by lipid vesicles). (C) Number of adipocytes per fat depot as assayed by total DNA quantitation. All data are expressed as the mean ± SEM of 9–18 animals per exposure group. *p < 0.05, **p < 0.01, and ^#p < 0.001 compared with DMSO vehicle by one-way ANOVA with Dunnett’s post hoc test.

Figure 2

Transgenerational effects of DMSO (vehicle), ROSI, or TBT (5.42, 54.2, or 542 nM) on the gene expression profile of MSCs from F1, F2, and F3 male mice. The relative mRNA levels of specific transcripts for adipogenic (A) or osteogenic (B) differentiation were assayed by QPCR in undifferentiated MSCs, with the expression of each target gene normalized to β-actin. Data are expressed as mean fold change ± SEM of three biological replicates assayed in duplicate. *p < 0.05, **p < 0.01, and ^#p < 0.001 compared with DMSO vehicle by unpaired t-test.

Figure 3

Transgenerational effects of DMSO (vehicle), ROSI, or TBT (5.42, 54.2, or 542 nM) on hepatic lipid accumulation in F1, F2, and F3 male (A) and female (B) mice. Histological sections of frozen livers were stained with Oil Red O and hematoxylin; at least five animals per exposure group were analyzed, and representative photomicrographs are shown. Bars = 50 μm.

Figure 4

Transgenerational effects of DMSO (vehicle), ROSI, or TBT (5.42, 54.2, or 542 nM) on markers of hepatic lipid metabolism in F1, F2, and F3 male (A) and female (B) mice. Relative mRNA levels of PPARγ2, SREBP1c, GyK, and FASN (lipogenic markers); PPARα and ACOX (lipolytic markers); and Fsp27 and FATP (lipid droplet markers) were evaluated in total RNA from mouse liver. Data are expressed as mean fold change ± SEM of three biological replicates assayed in duplicate. *p < 0.05, **p < 0.01, and ^#p < 0.001 compared with DMSO vehicle by unpaired t-test.