Early life stress exacerbates obesity in adult female mice via mineralocorticoid receptor-dependent increases in adipocyte triglyceride and glycerol content

Abstract

Previously, we have shown that Maternal Separation and Early Weaning (MSEW) exacerbates high fat diet (HF)-induced visceral obesity in female offspring compared to normally reared female mice. Stress hormones such as glucocorticoids and mineralocorticoids are critical mediators in the process of fat expansion, and both can activate the mineralocorticoid receptor (MR) in the adipocyte. Therefore, this study aimed to, comprehend the specific effects of MSEW on adipose tissue basic homeostatic function, and investigate whether female MSEW mice show an exacerbated obesogenic response mediated by MR. Gonadal white adipose tissue (gWAT), a type of visceral fat, was collected to assess lipidomics, transcriptomics, and in vitro lipolysis assay. Obese female MSEW mice showed increased adiposity, elevated 44:2/FA 18:2 + NH4 lipid class and reduced mitochondrial DNA density compared to obese control counterparts. In addition, single-cell RNA sequencing in isolated pre- and mature adipocytes showed a ~9-fold downregulation of aquaglycerolporin 3 (Aqp3), a channel responsible for glycerol efflux in adipocytes. Obese MSEW mice showed high levels of circulating aldosterone and gWAT-derived corticosterone compared to controls. Further, the MR blocker spironolactone (Spiro, 100 mg/kg/day, 2 weeks) normalized the elevated intracellular glycerol levels, the greater in vitro lipolysis response, and the number of large size adipocytes in MSEW mice compared to the controls. Our data suggests that MR plays a role promoting adipocyte hypertrophy in female MSEW mice by preventing lipolysis via glycerol release in favor of triglyceride formation and storage.

Keywords: Adiposity; Early life stress; Lipidomics; Single cell RNA-seq; Spironolactone.

Figure 1.. MSEW increases triglyceride content and whitening of gWAT in HF-fed female mice:

(A) preadipocyte gating strategy example and flow cytometry results showing the percentage of preadipocytes and macrophages in gWAT and scWAT stromal vascular fractions (B) gWAT mtDNA, n=8/group (C) scWAT primary cell culture preadipocyte (T0, day 0 of confluence) gene expression (E) volcano plot of gWAT lipidomic analysis and (F) bar graph of lipidomic analysis. Data expressed as mean ± SEM. An unpaired Student’s t-test was used to determine significance between the means of control and MSEW. *p MSEW<0.05 vs control. BW, Body Weight; MSEW, Maternal Separation and Early Weaning; gWAT, gonadal white adipose tissue; scWAT, subcutaneous white adipose tissue; HF, high fat diet; mtDNA; mitochondrial DNA; gDNA, genomic DNA; Cebpα/β, CCAAT/enhancer-binding protein alpha/beta; PparƔ(1+2), Peroxisome Proliferator Activated Receptor Gamma(1+2); Fabp4, Fatty Acid Binding Protein 4.

Figure 2.. Preadipocyte and Adipocyte transcriptome of MSEW mice evidence metabolic growth:

(A) volcano plot of gWAT preadipocyte single cell RNAseq analysis, (B) volcano plot of gWAT mature adipocyte, (C) heatmap of pre- and mature adipocyte adipogenic genes, (D) dietary excess enrichment plot, (E) preadipocyte increased biological processes, and (F) mature adipocyte increased biological processes. Data expressed as mean ± SEM. *p MSEW<0.05 vs control. MSEW, Maternal Separation and Early Weaning; gWAT, gonadal white adipose tissue; HF, high fat diet; Adrb3, Beta 3 Adrenergic Receptor; Aqp3/7/9, Aquaglycerolporin 3/7/9; Cebpα/β, CCAAT/enhancer-binding protein alpha/beta; Fabp3/4, Fatty Acid Binding Protein 3/4; GK, Glycerol Kinase; Lipe, Hormone Sensitive Lipase; Lpl, Lipoprotein Lipase; Nr3c1/2, Nuclear Receptor Class 1 (Mineralocorticoid Receptor) 2 (Glucocorticoid Receptor); Plin1, Perilipin-1; Pnpla2, Adipose triglyceride lipase; Pparα/Ɣ(1+2), Peroxisome Proliferator Activated Receptor Alpha/Gamma(1+2); Srebp 1/ 2, Sterol Response Element Binding Protein 1/ 2.

Figure 3.. MSEW increased stress hormones in female mice fed a HF:

(A) Serum aldosterone and (B) gWAT media explant-derived aldosterone (C) Serum corticosterone (D) gWAT media explant-derived corticosterone Steroidogenic gene profile using RNAseq read counts in (E) Preadipocytes and (F) Mature Adipocytes. Data expressed as mean ± SEM. An unpaired Student’s t-test was used to determine significance between the means of control and MSEW. *p MSEW<0.05 vs control. MSEW, Maternal Separation and Early Weaning; gWAT, gonadal white adipose tissue; HF, high fat diet. Cyp11β2, Aldosterone Synthase; Cyp21a1, 21-hydroxylase; Cyp11β1, 11-beta-hydroxylase; Cyp11a1, Cholesterol side-chain cleavage enzyme; Hsd3β1, 3 beta-hydroxysteroid dehydrogenase; Hsd11β1, 11β-hydroxysteroid dehydrogenase type 1; Hsd11β2, 11β-hydroxysteroid dehydrogenase type 2.

Figure 4.. MSEW mice, and not Controls, lose fat mass in response to MR Blockade:

(A) trajectory of adiposity from 16W of HF to 22W with Spironolactone treatment from 20W to 22W, n=21 Control, n=21 MSEW; (B) fat mass loss with 2 weeks of either Vehicle or Spironolactone treatment, (C) histogram of gWAT vehicle adipocyte area (D) histogram of gWAT Spironolactone treated adipocyte area (E) representative 100um images of gWAT adipocytes, (F) MSEW/Control log plot of adipocyte area frequency for Vehicle and Spironolactone treated groups. Data expressed as mean ± SEM. Two-way ANOVA was used to determine the effect and interaction of Spironolactone treatment on control and MSEW followed by a Tukey’s post hoc test. Histograms were analyzed for normalcy and significant shifts using a nonparametric t test Komogorov-Smirnov for distributions. Statistical outliers were identified and removed using the ROUT statistical analysis with Q=1%. *p<0.05 vs Control, #p<0.05 vs Vehicle. MSEW, Maternal Separation and Early Weaning; gWAT, gonadal white adipose tissue; Spiro, Spironolactone; VEH, Vehicle; HF, high fat diet.

Figure 5.. Spiro treatment reduces intracellular glycerol content in gWAT of MSEW female mice:

(A) gene profile mRNA expression (B) basal intracellular glycerol (C) basal media explant glycerol (D) stimulated media explant glycerol (E) plasma glycerol n=8 Control/group. (G) AQP3 protein expression (G) PPARα protein expression (H) PPARα, AQP3, and HSP90 western blot images, Data expressed as mean ± SEM. Two-way ANOVA was used to determine the effect and interaction of Spironolactone treatment on control and MSEW followed by a Tukey’s post hoc test. *p <0.05 vs Control, #p<0.05 vs Vehicle, &p<0.05 vs basal. MSEW, Maternal Separation and Early Weaning; gWAT, gonadal white adipose tissue; HF, high fat diet; Adrb3, Beta 3 Adrenergic Receptor; Aqp3, Aquaglycerolporin 3; Cebpα/β, CCAAT/enhancer-binding protein alpha/beta; Nr3c1/2, Nuclear Receptor Class 1 (MR, Mineralocorticoid Receptor) 2 (GR, Glucocorticoid Receptor); Pparα/Ɣ(1+2)/ Ɣ2, Peroxisome Proliferator Activated Receptor Alpha/Gamma.