Pubertal exposure to high fat diet causes mouse strain-dependent alterations in mammary gland development and estrogen responsiveness

Abstract

Objective: Increased adolescent obesity rates in the United States are a significant public health concern. Obesity or increased adiposity during puberty in girls, an important period of breast development and a window of exposure sensitivity, may influence breast development and cancer risk. The purpose of this study was to investigate the impact of a high fat diet (HFD) on mammary gland development in obesity-susceptible C57BL/6 and obesity-resistant BALB/c mice.

Design: Pubertal or adult C57BL/6 and BALB/c mice were fed an HFD or control diet (CD) from 3 to 7 weeks of age or from 10 to 14 weeks of age, respectively. The effects of HFD diet on body weight, adiposity, mammary gland development, and mammary gland response to estrogen were evaluated.

Results: Pubertal C57BL/6 mice fed the HFD had a significant increase in body weight and adiposity, and this was accompanied by stunted mammary duct elongation and reduced mammary epithelial cell proliferation. Ovariectomy and estrogen (17-β-estradiol, E) treatment of pubertal HFD-fed C57BL/6 mice showed decreased mammary gland stimulation by E. Amphiregulin, a downstream mediator of pubertal E action, was reduced in mammary glands of HFD-fed C57BL/6 mice. Weight loss and reduced adiposity initiated by switching C57BL/6 mice from HFD to CD restored ductal elongation. Pubertal BALB/c mice fed the HFD did not exhibit a significant increase in body weight or adiposity; HFD caused increased mammary epithelial cell proliferation and had no effect on response to E. HFD had no effect on body weight or the mammary glands of adult mice.

Conclusions: HFD during puberty had a profound strain-specific effect on murine mammary gland development. Obesity and increased adiposity were associated with reduced responsiveness to estrogen and stunted ductal growth. Importantly, the effect of diet and adiposity on the mammary gland was specific to the pubertal period of development.

Figure 1. Mammary duct epithelium of peripubertal C57BL/6 mice fed HFD has reduced outgrowth and terminal end bud formation

A. Whole mounts of representative mammary glands from peripubertal C57BL/6 mice fed CD or HFD for 3 or 4 weeks. Scale bar, 300 μm. B. Mammary duct length and TEB number in C57BL/6 mice fed CD or HFD for 4 weeks (n = 15). *, p < 0.001 or #, p < 0.05 when compared to CD fed C57BL/6 mice. C. Percentage of PCNA-positive epithelial cells in ducts, duct ends and TEB of C57BL/6 mice during estrus and fed CD or HFD (n = 3). #, p < 0.05 when compared to CD fed C57BL/6 mice.

Figure 2. Effect of HFD on mammary duct epithelium development in pubertal BALB/c mice

A. Representative whole mounts of mammary glands from peripubertal BALB/c mice fed CD or HFD for 4 weeks (n = 7). Whole mounts from BALB/c mice fed CD or HFD for 2 or 3 weeks are not shown because no distinct differences were observed. Scale bar, 300 μm. B. Mammary duct length and TEB number in BALB/c mice fed CD or HFD for 4 weeks (n = 14). C. Percentage of PCNA-positive epithelial cells in ducts, duct ends and TEB of BALB/c mice during estrus and fed CD or HFD (n = 3). *, p < 0.05 when compared to CD fed BALB/c mice.

Figure 3. Effect of HFD on mammary gland morphology in adult BALB/c and C57BL/6 mice

Adult BALB/c and C57BL/6 mice were fed CD or HFD from 13 to 17 weeks of age. A. Mammary gland whole mounts. Scale bar, 300 μm. B. Mammary gland weights. *, p < 0.05 HFD fed compared to CD fed BALB/c mice.

Figure 4. Effect of HFD on leptin, ERα and amphiregulin mRNA levels in peripubertal C57BL/6 and BALB/c mice

Peripubertal C57BL/6 and BALB/c mice were fed CD or HFD for 4 weeks, after which total RNA was isolated from mammary glands. Leptin, ERα and amphiregulin mRNA levels were determined as described in the Materials & Methods. Data are expressed as fold change in HFD-fed mice relative to mice fed CD. Values are mean ± SEM (n = 3). *, p < 0.005 when compared to CD-fed C57BL/6 mice. #, p < 0.05 when compared to CD-fed BALB/c mice.

Figure 5. HFD modifies mammary gland response to E in ovariectomized C57BL/6 mice treated with estrogen (E)

Peripubertal C57BL/6 or BALB/c mice were fed CD or HFD for 4 weeks (7 weeks of age), after which they were ovariectomized and maintained on respective diets for 3 additional weeks to allow for TEB regression. The mice were then treated with saline (control) or E for 5 days as described in Materials & Methods. Representative mammary gland whole mounts from (A) C57BL/6 mice or (B) BALB/c mice. Scale bar, 100 μm.

Figure 6. HFD-fed C57BL/6 mice have reduced amphiregulin protein expression

Representative merged images of mammary gland Areg (green) immunofluorescent staining in mammary glands of peripubertal C57BL/6 mice fed CD or HFD for 4 weeks. Nuclei were counterstained with DAPI. Scale bar, 25 μm.

Figure 7. Effect of switching C57BL/6 mice fed HFD to CD on adipocyte size and mammary gland development

A. Representative H&E staining of mammary glands from BALB/c and C57BL/6 mice fed CD or HFD for 4 weeks; note significant increase in adipocyte size in C57BL/7 glands in HFD group. Scale bar, 60 μm. B. Representative H&E staining and mammary gland whole mounts from peripubertal C57BL/6 mice fed HFD for 4 weeks, then switched to CD or maintained on HFD for an additional 6 weeks; note decrease in adipocyte size and increase in duct length in C57BL/6 glands in group switched to CD. C. Representative H&E staining of mammary glands from CD- or HFD-fed OVX-C57BL/6 mice treated with saline (control) or estrogen. Note that E treatment for 5 days had no discernible effect on adipocyte size. H&E images scale bar, 60 μm. Whole mount images scale bar, 300 μm.