Medial preoptic area FoxO1 controls metabolic adaptation in a sexually dimorphic manner

Abstract

The medial preoptic area (MPOA) of the hypothalamus is essential for metabolic adaptation to environmental challenges, though the molecular mechanisms underlying this process remain poorly understood. Here, we investigate the role of Forkhead transcription factor O1 (FoxO1), a key mediator of stress adaptation, in MPOA-dependent metabolic responses to temperature and nutritional changes. Our findings reveal sex-specific responses to both nutritional and temperature challenges. In female mice, but not males, a high-fat diet (HFD) challenge decreased FoxO1 expression in the MPOA. Specific deletion of FoxO1 in MPOA neurons (FoxO1-KO^MPOA) had no effect on body weight under normal chow-fed conditions but protected females from HFD-induced obesity (DIO). These protected females exhibited increased lean mass, decreased fat mass, enhanced thermogenesis, increased energy expenditure, and reduced food intake under HFD conditions. They also showed enhanced cold-induced heat production at 6°C, though this effect vanished at thermoneutrality (30°C). The protection against DIO was abolished by ovariectomy (OVX) and was not restored by 17β-estradiol supplementation, suggesting an estrogen-independent mechanism. Conversely, constitutive activation of FoxO1 in MPOA neurons (FoxO1-CA^MPOA) increased DIO susceptibility in both sexes. Together, these findings demonstrate that FoxO1^MPOA plays a crucial role in coordinating metabolic adaptation to nutritional and temperature challenges specifically in female mice.

Keywords: FoxO1; MPOA; sex difference.

Figure 1.. FoxO1^MPOA sexual dimorphism responds to a High-Fat Diet (HFD) challenge.

(A) FoxO1 mRNA expression in MPOA after 2-week chow or HFD feeding in male and female C57BL/6J Mice. (B-C) Representative images (B) and quantification (C) of FoxO1-GFP fluorescence in the MPOA of FoxO1-YFP mice fed either a chow diet, 3-day HFD, or 7-day HFD. Representative images showing the four nuclei of the Medial Preoptic Area (MPOA): Medial Part of Medial Preoptic Nucleus (MPOM), Lateral Part of Medial Preoptic Nucleus (MPOL), Ventral Medial Preoptic Nucleus (VMPO), and Ventrolateral Preoptic Nucleus (VLPO). Results are expressed as means ± SEM. In panels A and C, significance levels are indicated as #P < 0.05 and ##P < 0.01 for two-way ANOVA analysis, and *P < 0.05 and **P < 0.01 for subsequent post hoc Sidak tests.

Figure 2.. Selective deletion of FOXO1 in the MPOA during adulthood increases fat mass and is associated with higher energy expenditure and food intake in females.

(A) Schematic of the experimental strategy using AAV-CMV-Cre-GFP virus to selectively delete FoxO1 in FoxO1^flox/flox mice (FoxO1-KO^MPOA, 8 weeks). FoxO1^flox/flox mice receiving AAV-CMV-GFP virus injections served as controls. (B) Body weight percentage compared to initial weight at two weeks post-surgery. Female control and FoxO1-KO^MPOA mice received virus injections at eight weeks of age and were fed a chow diet for 25 weeks (n = 8/7). (C-D) Body composition percentage relative to body weight (C) and tissue index percentage relative to body weight (D) in female mice 25 weeks after virus injection (n = 8/7). (E-I) Energy expenditure (E, n = 7/8), light/dark/24-hour average energy expenditure (F, n = 7/8), ANCOVA analysis of daily total energy expenditure using body weight as a covariate (G, n = 7/8), cumulative food intake (H, n = 6/8), and light/dark/24-hour food intake (I, n = 6/8) in female control and FoxO1-KO^MPOA mice. Indirect calorimetry was performed on a separate cohort of mice from those used for chronic body weight recording. These mice had comparable body weight and lean mass at the time of study, 8 weeks after virus injection. Data in panel G were analyzed using ANCOVA with daily total energy expenditure as the dependent variable, genotype as the fixed variable, and body weight as the covariate. Results are displayed as means ± SEM. (E and H) #P < 0.05, ####P < 0.0001 in the two-way ANOVA analysis. (C and D) *P < 0.05, ****P < 0.0001 in the two-way ANOVA analysis followed by post hoc Sidak tests.

Figure 3.. FoxO1-KO^MPOA enhances cold-induced heat production in females.

(A) Temperature scheme used for the exposure experiment. (B-E) Energy expenditure measured before, during, and after six-hour exposure to various temperatures: cold (6°C, B), room temperature (22°C, C), thermoneutrality (30°C, D), and warm (37°C, E, n = 7/8). Mice had comparable body weight and lean mass during the indirect calorimetry study, conducted 8 weeks after virus injection. (F) Average energy expenditure during temperature exposures (n = 7/8). (G-H) Energy expenditure (G) and average energy expenditure during light/dark/24 hours (H) in female control mice after 48-hour adaptation to cold, room temperature, and thermoneutrality (n = 5/7/6). Mice had comparable body weight and lean mass during the indirect calorimetry study, conducted 8 weeks after virus injection. (I-J) Energy expenditure (I) and average energy expenditure during light/dark/24 hours (J) in female FoxO1-KO^MPOA mice after 48-hour adaptation to cold, room temperature, and thermoneutrality (n = 5/8/6). (K) Energy expenditure changes in mice after 48-hour exposure to 22°C or 6°C, compared to thermoneutral conditions at 30°C. Changes calculated from area under the curve from G and I, representing cold-induced heat production (n = 7/8 and 5/5). Results are shown as means ± SEM. (B-F, and K) #P < 0.05, ##p < 0.01, ###P < 0.001, ####p < 0.0001 in two-way ANOVA. (F, H, J, and K) *p < 0.05, **p < 0.01, ***P < 0.001, ****p < 0.0001 in two-way ANOVA followed by post hoc Sidak tests.

Figure 4.. FoxO1-KO^MPOA prevents diet-induced obesity in females.

(A-C) Body weight (A), body composition as percentage of total weight (B), and tissue weight (C) of female control and FoxO1-KO^MPOA mice after 10 weeks of high-fat diet (HFD) feeding (n = 9/8). HFD feeding began two weeks after virus injection when mice were 10 weeks old. (D-G) Representative Hematoxylin and Eosin (H&E) staining and cell size quantification in inguinal white adipose tissue (iWAT, D and E) and gonadal white adipose tissue (gWAT, F and G) fat pads after 10 weeks of HFD feeding (n = 3/3). Results are presented as means ± SEM. (A-C, E, and G) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by two-way ANOVA with post hoc Sidak tests.

Figure 5.. FoxO1-KO^MPOA reduces food intake and stimulates energy expenditure during metabolic adaptation to a high-fat diet challenge in female mice.

(A-B) Cumulative food intake (A) and feeding efficiency (B) of female control and FoxO1-KO^MPOA mice. Feeding efficiency represents the ratio of body weight change to cumulative food intake (n = 9/8). (C-D) Cumulative food intake (C) and total food intake during light/dark/24-hour periods (D) of female control and FoxO1-KO^MPOA mice following acute adaptation to a high-fat diet challenge (n = 5/6). (E-F) Energy expenditure (E) and average energy expenditure during light/dark/24-hour periods (F) of female control and FoxO1-KO^MPOA mice following acute adaptation to a high-fat diet challenge (n = 5/5). (G) Representative image of brown adipose tissue (BAT) with H&E staining, taken 10 weeks after high-fat diet challenge. (H-J) mRNA levels of genes in BAT (H), iWAT (I), and gWAT (J) 10 weeks after high-fat diet challenge. Measured genes include estrogen receptors (ESR1 and ESR2), thermogenic genes (UCP1, Dio2, Cidea, PRDM16), lipolytic gene (ATGL), adipogenic gene (PPARγ), and fatty acid sensor and transporter (CD36, n = 6/6). (K-L) Circulating levels of 17β-estradiol (K, n = 6/8) and relative mRNA levels of LH and FSH to housekeeping gene GAPDH in the pituitary (L, n = 8/8) 10 weeks after high-fat diet challenge. Results are shown as means ± SEM. (A-B, D, and F) *P < 0.05, **P < 0.01 in two-way ANOVA analysis followed by post hoc Sidak tests. (H-K) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in unpaired t tests.

Figure 6.. FoxO1-CA^MPOA promotes diet-induced obesity in both sexes.

(A) Schematic of the experimental strategy using AAV-CMV-Cre virus to selectively overexpress the constitutively active form of FoxO1AAA/GFP in Rosa26-LSL-FoxO1AAA mice (FoxO1-CA^MPOA, 8 weeks). Rosa26-LSL-FoxO1AAA mice receiving AAV-CMV-GFP virus injections served as controls. (B) GFP immunoreactivity was observed in a Rosa26-LSL-FoxO1AAA mouse after AAV-CMV-Cre injection into the MPOA, but not in a Rosa26-LSL-FoxO1AAA mouse without virus injection. (C-H) Body weight (C and F), cumulative food intake (D and G), and feeding efficiency (E and H) were measured in female and male control and FoxO1-CA^MPOA mice. Feeding efficiency was calculated as the ratio of body weight change to cumulative food intake. Mice were fed a high-fat diet for 8 weeks, beginning two weeks after virus injection at 10 weeks of age (females: n = 10/9; males: n = 12/10). Results are shown as means ± SEM. (C-H) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 in two-way ANOVA analysis followed by post hoc Sidak tests.

Figure 7.. The gender-specific anti-DIO effects of FoxO1-KO^MPOA require ovarian hormones, though estrogens alone are not sufficient.

(A-K) Analysis of body weight, body weight gain, body composition, tissue index (as percentage of total weight), cumulative food intake, and feeding efficiency in two groups: ovariectomized female control and FoxO1-KO^MPOA mice (OVX, n = 7/8, A-E), and OVX female mice with subcutaneous 17β-estradiol pellet implants (OVX+E, 0.025 mg/pellet, 60-day maximum release, n = 7/7, F-J). All mice received a high-fat diet for 28–56 days, beginning four days after virus injection and either OVX or OVX+E pellet implantation at 8 weeks of age. Gas: gastrocnemius muscle; TA: tibialis anterior muscle. (L) Comparison of body weight gain between female FoxO1-KO^MPOA mice and control mice after 4 weeks of HFD feeding under naïve, OVX, or OVX+E conditions (n = 9/8/7). Results are shown as means ± SEM. (C, D, and I) *P < 0.05, **P < 0.01 in two-way ANOVA followed by post hoc Sidak tests. (L) *P < 0.05, ***P < 0.001 in one-way ANOVA followed by post hoc Dunnett tests.