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. 2023 Apr 25;42(4):112390.
doi: 10.1016/j.celrep.2023.112390. Epub 2023 Apr 12.

Estradiol cycling drives female obesogenic adipocyte hyperplasia

Rocío Del M Saavedra-Peña  1 , Natalia Taylor  1 , Clare Flannery  2 , Matthew S Rodeheffer  3
Affiliations

Estradiol cycling drives female obesogenic adipocyte hyperplasia

Rocío Del M Saavedra-Peña et al. Cell Rep. .

Abstract

White adipose tissue (WAT) distribution is sex dependent. Adipocyte hyperplasia contributes to WAT distribution in mice driven by cues in the tissue microenvironment, with females displaying hyperplasia in subcutaneous and visceral WAT, while males and ovariectomized females have visceral WAT (VWAT)-specific hyperplasia. However, the mechanism underlying sex-specific hyperplasia remains elusive. Here, transcriptome analysis in female mice shows that high-fat diet (HFD) induces estrogen signaling in adipocyte precursor cells (APCs). Analysis of APCs throughout the estrous cycle demonstrates increased proliferation only when proestrus (high estrogen) coincides with the onset of HFD feeding. We further show that estrogen receptor α (ERα) is required for this proliferation and that estradiol treatment at the onset of HFD feeding is sufficient to drive it. This estrous influence on APC proliferation leads to increased obesity driven by adipocyte hyperplasia. These data indicate that estrogen drives ERα-dependent obesogenic adipocyte hyperplasia in females, exacerbating obesity and contributing to the differential fat distribution between the sexes.

Keywords: CP: Metabolism; adipocyte precursors; adipogenesis; estradiol; estrogen receptor alpha; estrogen signaling; estrous cycle; female obesity; high-fat diet; obesity.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Estrogen pathways are upregulated in female APCs at the onset of HFD
(A) Schematic representation of RNA sequencing experiment in female mice. (B) Venn diagram of HFD-induced DEGs in female VWAT and SWAT. (C) Volcano plot of HFD-induced DEGs in female VWAT. Top 3 genes with lowest p values are colored in red (upregulated) or blue (downregulated). (D) Top positive upstream regulators on an HFD in VWAT as predicted by Ingenuity Pathway Analysis. Gene predictions are in uppercase. (E) Volcano plot of HFD-induced DEGs in female SWAT. Top 2 genes with lowest p values are colored in red (upregulated) or blue (downregulated). (F) Top positive upstream regulators on an HFD in SWAT as predicted by Ingenuity Pathway Analysis. Gene predictions are in uppercase. (G) Top canonical pathways with a positive Z score induced by HFD as predicted by IPA. n = 5 samples per group, 3 mice pooled for each sample. VWAT, perigonadal fat; SWAT, inguinal subcutaneous fat; SD, standard diet; HFD, high-fat diet; DEGs, differentially expressed genes. See also Figure S1.
Figure 2.
Figure 2.. Estrous cycling affects obesogenic APC proliferation
(A) Schematic of circulating estradiol (E2) levels in female mice during the 4 stages of the estrous cycle (adapted from McLean et al.). Grouping of females based on transition into proestrus happening early (days 0 to 2) or late (days 3 to 7) of a 1-week-long HFD feeding. (B) Representative images of vaginal smears stained with crystal violet from female mice during the 4 stages of the estrous cycle. Nucleated epithelial cells are highlighted by purple arrows, cornified squamous epithelial cells by black arrows, and leukocytes by green arrows. Scale bar is 100 μm, images taken at 20×. (C) Representative flow cytometry dot plots to measure BrdU incorporation into APCs. Briefly, APCs are lineage negative (CD45−, CD31−) and positive for CD34, CD29, and Sca-1. BrdU incorporation is measured to assess proliferation. (D) Representative BrdU histograms from APCs in the different groups including BrdU FMO. (E) APC proliferation from females after 1 week of SD or HFD feeding. (F) CD45+ cell proliferation from females after 1 week of SD or HFD feeding. n = 7–11 mice per group. Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test for (E) and (F). Error bars represent mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ****p < 0.0001. APCs, adipocyte precursor cells; VWAT, perigonadal fat; SWAT, inguinal subcutaneous fat; E2, 17β-estradiol; SD, standard diet; HFD, high-fat diet; BrdU, bromodeoxyuridine; FMO, fluorescence minus one.
Figure 3.
Figure 3.. Estradiol drives female APC proliferation in an ERα-dependent manner
(A) Food intake (cage average) during vehicle or E2 treatment (days 0–2 of an HFD) in female mice (n = 14–19 mice per group). (B) Representative BrdU histograms in APCs from estradiol treatment experiment including BrdU FMO. (C) APC proliferation after 1 week of HFD feeding in vehicle or E2 treated female mice. (D) Expression of estrogen receptors in female APCs from RNA-seq data (n = 5 samples per group, 3 mice pooled per sample). (E) Schematic of APC transplantation assay into female SWAT. (F) Representative BrdU histograms in APCs from APC-ERαKO transplant experiment including BrdU FMO. (G) APC proliferation of transplanted ERα-KO and endogenous APCs after 1 week of HFD feeding in females (n = 4 mice per group). Statistical significance was determined by one-way ANOVA with Šidák’s tests for (A) and unpaired t tests for (C) and (F). Error bars represent mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01. APCs, adipocyte precursor cells; VWAT, perigonadal fat; SWAT, inguinal subcutaneous fat; BrdU, bromodeoxyuridine; E2, 17β-estradiol; Vh, vehicle; SD, standard diet; HFD, high-fat diet; BrdU, bromodeoxyuridine; Endog, endogenous; FMO, fluorescence minus one. See also Figure S2.
Figure 4.
Figure 4.. Timing of HFD determines hyperplasia in female mice
(A) Schematic of BrdU pulse-chase assay in combination with estrous scoring. Briefly, BrdU was given during the first week of diet along with estrous scoring and then chased for 12 weeks to allow APCs to differentiate into adipocytes. (B) Representative images of immunofluorescence staining in SWAT to quantify BrdU incorporation in adipocyte nuclei. Tissue is stained for caveolin-1 to visualize adipocyte plasma membranes, DAPI to visualize nuclei, and BrdU. Adipocyte nuclei are indicated with arrowheads (yellow indicates BrdU+ and white indicates BrdU−) and are identified by their location inside the adipocyte plasma membrane. Scale bar is 25 μm. (C) Representative images of WAT from females fed an SD or HFD for 12 weeks stained with caveolin-1, DAPI, and BrdU. Examples of adipocyte nuclei (white arrows) and BrdU+ adipocyte nuclei (yellow arrows) are shown. 30–35 images were taken per tissue to ensure at least 200 adipocyte nuclei were counted. Scale bar is 100 μm, images taken at 40×. (D) Adipocyte hyperplasia analysis from female SWAT and VWAT after 12 weeks of HFD feeding. (E) Total fat mass during 12 weeks of SD or HFD feeding in female mice. (F) SWAT and VWAT weight after 12 weeks of SD or HFD feeding. (G and H) Histogram of VWAT (G) and SWAT (H) adipocyte size from females after 12 weeks of feeding. n = 6–10 mice per group. Statistical significance was determined by unpaired t test for (D), ordinary one-way ANOVA with Tukey’s test for (F), and ordinary two-way ANOVA with Tukey’s test for (E), (G), and (H). Error bars represent mean ± SEM. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. APCs, adipocyte precursor cells; VWAT, perigonadal fat; SWAT, inguinal subcutaneous fat; SD, standard diet; HFD, high-fat diet; BrdU, bromodeoxyuridine. See also Figures S3 and S4 and Table S1.
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