Sex-specific role of high-fat diet and stress on behavior, energy metabolism, and the ventromedial hypothalamus

Abstract

Background: Scientific evidence highlights the influence of biological sex on the relationship between stress and metabolic dysfunctions. However, there is limited understanding of how diet and stress concurrently contribute to metabolic dysregulation in both males and females. Our study aimed to investigate the combined effects of high-fat diet (HFD) induced obesity and repeated stress on fear-related behaviors, metabolic, immune, and hypothalamic outcomes in male and female mice.

Methods: To investigate this, we used a highly reliable rodent behavioral model that faithfully recapitulates key aspects of post-traumatic stress disorder (PTSD)-like fear. We subjected mice to footshock stressor followed by a weekly singular footshock stressor or no stressor for 14 weeks while on either an HFD or chow diet. At weeks 10 and 14 we conducted glucose tolerance and insulin sensitivity measurements. Additionally, we placed the mice in metabolic chambers to perform indirect calorimetric measurements. Finally, we collected brain and peripheral tissues for cellular analysis.

Results: We observed that HFD-induced obesity disrupted fear memory extinction, increased glucose intolerance, and affected energy expenditure specifically in male mice. Conversely, female mice on HFD exhibited reduced respiratory exchange ratio (RER), and a significant defect in glucose tolerance only when subjected to repeated stress. Furthermore, the combination of repeated stress and HFD led to sex-specific alterations in proinflammatory markers and hematopoietic stem cells across various peripheral metabolic tissues. Single-nuclei RNA sequencing (snRNAseq) analysis of the ventromedial hypothalamus (VMH) revealed microglial activation in female mice on HFD, while male mice on HFD exhibited astrocytic activation under repeated stress.

Conclusions: Overall, our findings provide insights into complex interplay between repeated stress, high-fat diet regimen, and their cumulative effects on health, including their potential contribution to the development of PTSD-like stress and metabolic dysfunctions, emphasizing the need for further research to fully understand these interconnected pathways and their implications for health.

Fig. 1

A high-fat diet inhibits fear extinction in male mice. (A) Experimental timeline used to study the effect of diet and repeated stress on mice. (B) Percent freezing on day 2 of SEFL (n = 20/19; two-tailed unpaired t test, t = 1.913; p = 0.0635). (C) Shock reactivity measured as velocity (cm/s) on day 2 (n = 20/19; two-tailed unpaired t test, t = 1.875; p = 0.0687). (D) Percent freezing on day 3 of SEFL (n = 20/19; two-tailed unpaired t test, t = 1.814; p = 0.3916). (E) Percent freezing of HFD-fed male mice during 14 weeks of RS/NRS treatment (n = 4,5; two-way ANOVA, F_1,98 =69.18; ****p < 0.0001; post hoc comparison, n = 5/4; Sidak’s multiple comparisons test, p > 0.99). (F) The freezing percentage of HFD-fed male mice during the freezing test conducted after 14 weeks (n = 5/4, two-tailed paired t test; t = 2.518, p = 0.08). (G) Percent freezing of HFD female mice during 14 weeks of RS/NRS treatment (n = 5; two-way ANOVA, F_1,112 =157.1; ****p < 0.0001) (post hoc comparison, n = 5; Sidak’s multiple comparisons test, **p < 0.01). (H) Percent freezing in HFD females in the freezing test after 14 weeks (n = 5, two-tailed paired t test; t = 3.514, *p < 0.05)

Fig. 2

HFD induces weight gain and an increase in blood glucose levels in a sex-specific manner. (A) Percent weight gain over 14 weeks in HFD-fed mice (n = 4/5, two-way ANOVA; F _3,225 =37.12; ****p < 0.0001) (Post hoc comparison, n = 4/5, Tukey’s multiple comparisons test, p < 0.05 from week 7). (B) Percent weight gain over 14 weeks plotted for chow-fed mice (n = 5; two-way ANOVA, F _3,240 =5.698, ***p < 0.001) (Post hoc comparison, n = 5, Tukey’s multiple comparisons test, p < 0.05 in week 10 only). (C) Week 10 plasma glucose levels during the GTT test after 4 h of fasting in HFD mice (n = 5, two-way ANOVA, F _3,90=33.23, ****p < 0.0001) (post hoc comparison, n = 5, Tukey’s multiple comparisons test, p < 0.05). AUC graph from the 10-week GTT test in HFD mice (N = 5, one-way ANOVA, F _3,15 =1.579, ***p < 0.001) (post hoc comparison, N = 5, Tukey’s multiple comparisons test, p < 0.05) (D) Week 14 plasma glucose levels during the GTT test in HFD mice (n = 5, two-way ANOVA, F _3,90=14.91, ****p < 0.0001) (post hoc comparison, N = 5, Tukey’s multiple comparisons test, p < 0.05). AUC graph from the 14-week GTT test in HFD mice (n = 5, one-way ANOVA; F _3,15=0.8293, *p < 0.05) (post hoc comparison, n = 5; Tukey’s multiple comparison test; p < 0.05). (E) Week 10 plasma glucose levels during the GTT test in chow-fed mice (n = 5, two-way ANOVA, F _3,96=12.54, ****p < 0.0001) (post hoc comparison, n = 4/5, Tukey’s multiple comparisons tests, p < 0.05). AUC graph from the 10-week GTT test in chow-fed mice (n = 5, one-way ANOVA; F _3,16=0.0884, *p < 0.05) (post hoc comparison, n = 4/5, Tukey’s multiple comparisons test, p < 0.05). (F) Week 14 plasma glucose levels during the GTT test in chow-fed mice (n = 5, two-way ANOVA, F _3,96=6.201, ***p < 0.001) (post hoc comparison, n = 4/5, Tukey’s multiple comparisons tests, p = 0.08). AUC graph from the 14-week GTT test in chow-fed mice (n = 5, one-way ANOVA; F _3,65=0.075; p = 0.59) (*Compares No repeated Shock F vs. No repeated Shock M, #Compares No repeated shock F to Repeated Shock M, +Compares Repeated shock F to No repeated Shock M, %Compares Repeated shock F to Repeated Shock M)

Fig. 3

A HFD and repeated reminder shocks led to sex-specific metabolic dysregulation. (A) Energy expenditure (kCal/hr) of HFD-fed mice over 60 h in the metabolic chamber (n = 4,5; one-way ANOVA, p < 0.05) (post hoc comparison, n = 4,5; Tukey’s multiple comparisons test, *p < 0.05). (B) Energy expenditure (kCal/hr) of chow-fed mice over 60 h in the metabolic chamber (n = 5; one-way ANOVA, p > 0.05). (C) Respiratory exchange ratio (RER) of HFD-fed mice aged more than 60 h in the metabolic chamber (n = 4/5; one-way ANOVA, p < 0.05) (post hoc comparisons, n = 4,5; Tukey’s multiple comparisons test, *p < 0.05). (D) RERs of chow-fed mice aged more than 60 h in the metabolic chamber (n = 5; one-way ANOVA, p > 0.05). (E) Pedestrian locomotion in HFD-fed mice over 60 h in the metabolic chamber (n = 5; one-way ANOVA, p < 0.05) (post hoc comparisons, n = 4,5; Tukey’s multiple comparison test, *p < 0.05). (F) Pedestrian locomotion in chow-fed mice over 60 h in the metabolic chamber (n = 5; one-way ANOVA, p < 0.05) (post hoc comparison, n = 4,5, Tukey’s multiple comparisons test, *p < 0.05)

Fig. 4

A HFD did not affect acute stress-induced fear behaviors but did cause sex-specific metabolic alterations. (A) Experimental design of the comprehensive 10-week study conducted to investigate the role of diet and acute stress. (B) Weight gain percentages plotted through 10 weeks of the HFD/chow diet regimen (n = 10, two-way ANOVA, F_3,528 = 188.3, ****p < 0.0001) (Post hoc comparison, n = 10, Tukey’s multiple comparisons test, p < 0.05 from week 2) (C) Percent fat mass in HFD/chow-fed mice (n = 10; two-way ANOVA, F_1,48 = 17.69; ***p < 0.001) (post hoc comparison, n = 10, Tukey’s multiple comparisons test, **p < 0.01). (D) Percent freezing in HFD/chow-fed stressed and no stressed groups (n = 5, one-way ANOVA, F_7,44 = 1.388, ****p < 0.0001) (post hoc comparison, n = 5, Tukey’s multiple comparison test, Chow-NS-F v. Chow-S-F, Chow-NS-M v. Chow-S-M, HFD-NS-F v. HFD-S-F, HFD-NS-M v. HFD-S-M; ****p < 0.0001). (E) Plasma glucose levels from the GTT performed at week 10 in HFD-NS/S female and male mice (n = 5, two-way ANOVA, F_3,168 = 18.53, ****p < 0.0001). AUC graph for the week 10 GTT test in HFD/chow-fed female mice (n = 5, one-way ANOVA; F_3,16 = 0.1454, p = 0.07). (F) Plasma glucose levels from the GTT performed at week 10 in Chow–NS/S female and male mice (n = 5, two-way ANOVA, F_3,120 = 9.057, ****p < 0.0001). AUC graph for the week 10 GTT test in chow-fed mice (n = 5, one-way ANOVA, F_3,16 = 1.206, p = 0.41). (G) Energy expenditure (EE) (kCal/hr) in HFD-fed S and NS mice (n = 5; one-way ANOVA, p > 0.05). (H) Energy expenditure (EE) (kCal/hr) in chow-fed mice under either the S or NS conditions (n = 5; one-way ANOVA, p > 0.05)

Fig. 5

Repeated reminder shocks and a high-fat diet (HFD) induced distinctive alterations in peripheral myeloid lineage cells and inflammatory markers, exhibiting a sexually dimorphic pattern. (A) Quantification of the bone marrow population of MPP2 progenitors in HFD-fed mice (n = 9; two-way ANOVA, F1,14 = 290.1; ****p < 0.0001) (post hoc comparison, n = 5; Tukey’s multiple comparison test, *p < 0.05 and ****p < 0.0001). (B) Quantification of the MPP3 progenitor population in the bone marrow of HFD-fed mice (n = 9; two-way ANOVA; F1,14 = 34.48; ****p < 0.0001) (post hoc comparison, n = 5; Tukey’s multiple comparison test, p = 0.08 and ***p < 0.001). (C) Quantification of granulocyte monocyte progenitor (GMP) cells in HFD-fed mice (n = 9; two-way ANOVA, F1,14 = 42.52; ****p < 0.0001) (post hoc comparison, N = 4/5; Tukey’s multiple comparison test, **p < 0.01). (D) Quantification of monocyte-dendritic cell progenitors (MDPs) in the bone marrow of HFD-fed mice (n = 9; two-way ANOVA, F1,14 = 6.017, *p < 0.05) (post hoc comparison, n = 5; Tukey’s multiple comparison test, *p < 0.05). (E) Quantification of neutrophils in the blood of HFD-fed mice (n = 9; two-way ANOVA, p > 0.05). (F) Quantification of Ly6Chi monocytes in the blood of HFD-fed mice (n = 9; two-way ANOVA, p > 0.05). (G) Quantification of neutrophils in the gonadal white adipose tissue (gWAT) of HFD-fed mice (n = 9; two-way ANOVA, F1,14 = 24.65, ***p < 0.001) (post hoc comparison, n = 5; Tukey’s multiple comparison test, *p < 0.05 and **p < 0.01). (H) Quantification of macrophages in the gWAT of HFD-fed mice (n = 9; two-way ANOVA; F1,14 = 6.296; *p < 0.05) (post hoc comparison; n = 4/5; Tukey’s multiple comparison test; *p < 0.05). (I) Quantification of aortic neutrophils from HFD-fed mice (n = 9; two-way ANOVA; F1,14 = 59.59; ****p < 0.0001) (post hoc comparison; n = 4/5; Tukey’s multiple comparison test; ***p < 0.001). (J) Quantification of macrophages in the aortas of HFD-fed mice (n = 4/5; Tukey’s multiple comparison test, p > 0.05). K. Quantification of neutrophils in the hearts of HFD-fed mice (n = 9; two-way ANOVA, p > 0.05). L. Quantification of macrophages in the hearts of HFD-fed mice (n = 9; two-way ANOVA; F1,14 = 32.04; ****p < 0.0001) (post hoc comparison; n = 4/5; Tukey’s multiple comparison test; *p < 0.05 and **p < 0.01)

Fig. 6

Repeated reminder shock and a high-fat diet (HFD) induced differential changes in the ventromedial hypothalamus (VMH) of male and female mice. (A) Illustration outlining the snRNA-seq procedure utilizing the 10X Genomics platform workflow, which involves isolating nuclei from the VMH of male and female mice fed a HFD (n = 5 pooled). (B) UMAP plot displaying single cells from this study, color coded by cell type, with cell types identified based on the expression of canonical marker genes. (C) UMAP plot of single cells from the VMH cohort, color coded by cell type and segregated by sample. (D) Heatmap depicting the relative fractions of each cell type in each sample. (E) Heatmap illustrating the regulon activity of the indicated transcription factors, indicating the intensity of gene regulation in specific cell types from the VMH of males and females. (F) UMAP plot of the indicated cell types from the VMH cohort separated by sample; adjacent violin plots displaying the expression levels of specific genes are shown. G, H & I. Volcano plot illustrating differentially expressed genes (DEGs) with fold changes plotted against p values; female vs. male in microglia (G), male vs. female in astrocytes (H), and female vs. male in oligodendrocytes (I). Violin plots highlighting the highly upregulated genes. J, K & L. Pathway analysis of DEGs showing neuroinflammation (J), astrocyte activation (K), and microglial activation (L). M. Interactome representation based on DEGs from male and female mice indicating the strength of interaction between specific cell types. N. Heatmap exhibiting the indicated ligand-cell interactions in male and female mice