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. 2017 Feb 24;7:43269.
doi: 10.1038/srep43269.

High-Fat Diet Induces Unexpected Fatal Uterine Infections in Mice With aP2-Cre-mediated Deletion of Estrogen Receptor Alpha

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Free PMC article

High-Fat Diet Induces Unexpected Fatal Uterine Infections in Mice With aP2-Cre-mediated Deletion of Estrogen Receptor Alpha

Zsofia Ban et al. Sci Rep. .
Free PMC article

Abstract

Estrogen receptor alpha (ERα) is a major regulator of metabolic processes in obesity. In this study we aimed to define the relevance of adipose tissue ERα during high-fat diet (HFD)-induced obesity using female aP2-Cre-/+/ERαfl/fl mice (atERαKO). HFD did not affect body weight or glucose metabolism in atERαKO- compared to control mice. Surprisingly, HFD feeding markedly increased mortality in atERαKO mice associated with a destructive bacterial infection of the uterus driven by commensal microbes, an alteration likely explaining the absence of a metabolic phenotype in HFD-fed atERαKO mice. In order to identify a mechanism of the exaggerated uterine infection in HFD-fed atERαKO mice, a marked reduction of uterine M2-macrophages was detected, a cell type relevant for anti-microbial defence. In parallel, atERαKO mice exhibited elevated circulating estradiol (E2) acting on E2-responsive tissue/cells such as macrophages. Accompanying cell culture experiments showed that despite E2 co-administration stearic acid (C18:0), a fatty acid elevated in plasma from HFD-fed atERαKO mice, blocks M2-polarization, a process known to be enhanced by E2. In this study we demonstrate an unexpected phenotype in HFD-fed atERαKO involving severe uterine bacterial infections likely resulting from a previously unknown negative interference between dietary FAs and ERα-signaling during anti-microbial defence.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Metabolic phenotyping of female atERαKO mice.
(A) Body-weight of atERαKO- compared to wt-mice fed a control (CD) or a high-fat diet (HFD) at the age of 15 weeks. (n = 5 (CD) and 17–19 (HFD)). (B) Body weight development during HFD-feeding, showing no differences between genotypes. (C,D) Intraperitoneal glucose- (C) and insulin- (D) tolerance test (n = 9–10). No differences occurred in energy expenditure (E) and in locomotor activity (F) between the genotypes, while atERαKO mice showed an increase in food intake (G) (n = 10). (H) Gene expression analyses in white adipose tissue of wt and atERαKO mice (n = 5–8). (I) Survival curves of atERαKO- and wt-mice on CD and HFD. Onset of HFD-feeding at 42 days of age is indicated in the graph. n.s. = non-significant (P > 0.05, 2-way-ANOVA (Bonferroni-posttest) and 2-way-ANOVA with repeated measures (Bonferroni-posttest)). *P < 0.05 vs. wt (two-tailed t-test).
Figure 2
Figure 2. Characterization of uterine phenotype.
(A,B) Representative macro- and microscopic images of uteri of atERαKO vs. wt-mice fed a CD or HFD. (C) Quantification of the grade of inflammation (n = 9–17). Legend: 0 = no inflammation; 1 = beginning inflammation; 2 = visible inflammation; 3 = severe inflammation (D) Representative Mac3- and Ly6G-stainings. (E) Quantification of macrophages (Mac3) and neutrophils (Ly6G) in uteri of atERαKO mice (n = 9–17). Legend: 0 = no immune cells; 1 = few immune cells; 2 = high quantity of immune cells; 3 = very high quantity of immune cells (F) Relative mRNA-expression of CD47 and PAI-1 normalized to Ly6G mRNA-expression in uteri, comparing atERαKO mice on CD or HFD (n = 4–7). Relative Nos2 and Arg1 mRNA expression in uteri (G) and white adipose tissue (H) of atERαKO mice (n = 7–8). *P < 0.05, **P < 0.01 two-tailed t-test or 2-way-ANOVA (Bonferroni-posttest).
Figure 3
Figure 3. ERα-signaling and lipid profile of atERαKO mice.
(A) Relative ERα mRNA-expression in indicated tissues (n = 5). (B) 17β-estradiol levels in plasma of female atERαKO- and wt-mice (n = 5–10). (C) Relative mRNA expression of ERα-target genes in hypothalamus (Wnt4), uterus (Ltf) and spleen (CTSD) in CD or HFD-fed atERαKO- and wt-mice (WT) (n = 4–8). (D) Measurement of plasma concentration of fatty acids in HFD-fed atERαKO mice vs. CD-fed (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. atERαKO CD; #P < 0.05, ###P < 0.001 vs. WT; one-way ANOVA, 2-way-ANOVA or unpaired t-test.
Figure 4
Figure 4. Impact of stearic acid on macrophage polarization (and function).
Characterization of M1- and M2-markers on differentiated THP-1 cells after stimulation with estradiol (E2)+BSA, E2+C18:0, E2+LPS and E2+LPS+C18:0. (A) Relative mRNA-expression of CCR7 (M1) and CD206 (M2) over E2+LPS stimulated cells. (B) Representative images of CD209 (M2-marker) staining of E2+BSA or E2+C18:0 stimulated THP-1 cells. (C) Representative FACS-dot plots of CD11b and CD209-staining. First column represents unstained cells in forward (FSC) and side scattered blots (SSC); second column shows staining of CD11b- (y-axis) and 7AAD- (x-axis) staining. The third column is analogous to the second one for CD209-staining. Average MFI of viable (7AAD negative) CD11b positive cells (left upper quarter of the blots) and MFI of viable CD209 cells are indicated. (D) Relative mean-fluorescence-intensity of cd11-positive cells and (E) relative mean-fluorescence-intensity of cd209-positive cells (x-fold induction over E2/LPS-stimulated cells). (F) Representative images of phagocytotic analysis of E2+BSA or E2+C18:0 stimulated THP-1 cells. **P < 0.01, ***P < 0.001 vs. E2+BSA; #P < 0.05, ###P < 0.001 vs. E2+LPS (n = 3; one-way-ANOVA).
Figure 5
Figure 5. Stearic acid modulation of ERα transcriptional activity.
(A) Relative mRNA expression of IL4-receptor after stimulation with E2 and C18:0 in primary murine bone marrow-derived macrophages (n = 7–9 per condition). (B) Inhibition of ligand-dependent activation of ERE by C18:0 in THP-1 cells (n = 3–8 per condition). (C) Inhibition of E2-induced ERE-activity by C18:0 in cells transiently expressing mutated C447A (left bars) or wt (right bars) ERα. (D) Experimental settings of HPLC/MS lipid analysis from protein (ERα) precipitates (E) Relative concentration of main fatty acids bound to ERα per 1 mg protein. (inlay: IP- and transfection-controls by Western-Blot (cropped) of transfected HeLa cells with pSG5 and hERα-pSG5 plasmids; full-length blot was provided as supplemental data). #P < 0.05, ##P < 0.01, ###P < 0.001 vs. E2+BSA; **P < 0.01, ***P < 0.001 vs. vehicle; (one-way-ANOVA or unpaired t-test).

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