https://orcid.org/0000-0003-2638-3954
Figures
Abstract
Excessive consumption of high-fat diets is linked to peripheral insulin resistance, neuroinflammation, and obesity. While dietary methionine restriction improves peripheral metabolic health, its effectiveness in attenuating central insulin resistance and neuroinflammation, especially in a sex-dependent manner, remains unclear. This study investigates whether methionine restriction can mitigate high-fat diet-induced alterations in insulin resistance and neuroinflammation in male and female mice and explores the role of endogenous fibroblast growth factor 21 (FGF21) in mediating these effects. We utilized wild-type and Fgf21 knockout (Fgf21-/-) mice to assess the impact of methionine restriction on body composition, insulin sensitivity, central insulin signaling, and neuroinflammation. methionine restriction reduced body weight and adiposity in males, regardless of diet or genotype. In females, methionine restriction reduced weight gain under high-fat diet conditions in both genotypes but had limited effects on female adiposity. Methionine restriction improved insulin sensitivity in WT mice, but this effect was absent in Fgf21-/- mice, highlighting the importance of FGF21. Likewise, methionine restriction enhanced hepatic insulin signaling in WT males, but not Fgf21-/- males. In contrast, methionine restriction had minimal impact on insulin signaling in the liver or brain of female mice. Methionine restriction decreased neuroinflammatory gene expression in the hippocampus of males following the high-fat diet, a process dependent on FGF21. These findings demonstrate that methionine restriction confers sex-specific protection against high-fat diet-induced metabolic disturbances, with FGF21 playing a critical role in both peripheral and central insulin sensitivity, particularly in males. Future studies should further elucidate the molecular mechanisms underlying the sex-specific effects of methionine restriction and the role of FGF21 in mediating these responses.
Citation: Lail H, Lawrence R, Pinheiro F, Price E, Wanders D (2026) Methionine restriction provides sex-specific protection against high-fat diet-induced adiposity, peripheral insulin resistance, and neuroinflammation in FGF21-dependent and independent manners in mice. PLoS One 21(3): e0343503. https://doi.org/10.1371/journal.pone.0343503
Editor: Víctor Sánchez-Margalet, Virgen Macarena University Hospital, School of Medicine, University of Seville, SPAIN
Received: March 26, 2025; Accepted: February 5, 2026; Published: March 10, 2026
Copyright: © 2026 Lail et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: Funding for this study was provided by the Center for Neuroinflammation and Cardiometabolic Diseases Seed Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Excessive dietary fat intake is strongly linked to the development of chronic diseases including obesity [1] and type 2 diabetes [2]. Consumption of a high-fat diet (HFD) induces widespread alterations in both peripheral and central systems [3–6], notably impairing insulin sensitivity [7–13]. In peripheral tissues, HFDs disrupt insulin signaling pathways, leading to reduced glucose uptake, dysregulated glucose metabolism, and elevated circulating glucose concentrations [14]. These systemic effects are closely linked to the development of low-grade chronic inflammation, which is characterized by elevated pro-inflammatory cytokines and immune cell activation [15]. Importantly, this inflammatory state is not confined to peripheral tissues; it extends to the central nervous system, where HFDs contribute to neuroinflammation and impair neuronal insulin signaling [16,17]. However, the metabolic and central consequences of high dietary fat intake reported in the literature vary considerably, depending on multiple factors including the duration of HFD exposure [18], the animal model used [19], and the age [20,21], and sex [22,23] of the animals. These variables can significantly influence the magnitude and nature of peripheral and central insulin resistance, as well as inflammatory responses. Therefore, research examining the effects of HFDs and potential dietary interventions must consider these factors, with attention to sex as a biological variable, to capture the full spectrum of metabolic and neurobiological outcomes.
Dietary methionine restriction (MR) confers numerous metabolic benefits, including reduced adiposity, enhanced lipid and glucose metabolism, reduced inflammation, increased energy expenditure, and extended lifespan [24–29]. These effects occur despite hyperphagia and increased energy intake [30]. In experimental animal models, MR reduces dietary methionine content by 80% and eliminates the nonessential amino acid cysteine [30]. MR increases peripheral insulin sensitivity in several mouse models of obesity by enhancing protein kinase B (Akt) phosphorylation [27,31–34]. Centrally, MR increases the expression of insulin signaling genes and reduces neuroinflammation and oxidative stress in the hippocampus of male mice [35]. This evidence, at least indirectly, suggests that MR may be used as a strategy to improve insulin sensitivity in the brain; however to date, this has not been directly tested.
Fibroblast growth factor 21 (FGF21) is an endogenous stress-response hormone produced primarily by the liver, and it plays a central role in mediating many of the beneficial effects of dietary MR, including in insulin sensitivity [28] and those related to the brain and cognition [36]. FGF21 exerts pleiotropic effects on metabolism by enhancing glucose uptake, promoting lipid oxidation, and modulating energy expenditure [37,38]. Exogenous FGF21 administration protects against HFD-induced body weight gain [39], adiposity [40], insulin resistance [41], and systemic inflammation [42]. Beyond its peripheral actions, FGF21 has been shown to influence the brain directly, supporting neuronal function, synaptic plasticity, and protection against neuroinflammation [43,44].
Despite this evidence, significant gaps remain in our understanding of how MR and FGF21 affect the brain under conditions of HFD, particularly in females. While MR has been shown to improve peripheral and central metabolic outcomes in male mice, the efficacy of MR in mitigating HFD-induced neuroinflammation and central insulin resistance in females remains unclear. Similarly, the role of FGF21 in regulating central insulin signaling and neuroprotection in females has not been fully elucidated. To address these gaps, this study investigates whether MR can attenuate HFD-induced peripheral and central insulin resistance and neuroinflammation in both male and female mice. Furthermore, it examines the contribution of endogenous FGF21 in mediating these protective effects, with the goal of clarifying sex-specific differences in the metabolic and neuroprotective responses to dietary methionine restriction.
Methods
Animals and diets
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (Approval Code: A21030, Approval Date: 22 January 2021) at Georgia State University, ensuring compliance with the National Research Council guidelines, Animal Welfare Act, and Public Health Service Policy on the humane care and use of laboratory animals. Breeding pairs of male and female wild-type (WT, Jax#000664) and Fgf21 knockout (Fgf21-/-, Jax#033846) mice purchased from the Jackson Laboratory (Bar Harbor, ME). Experimental groups were researcher-generated using genotype-specific littermates until the specified group sizes were attained. At five to six weeks of age, mice were randomly assigned to either normal-fat (NFD, 3.815 kcal/g; 14.5% energy from protein, 67% energy from carbohydrate, 18.5% energy from fat) or high-fat (HFD, 5.45 kcal/g; 14.7% energy from protein, 25.7% energy from carbohydrate, 59.6% energy from fat) versions of control (0.86%) or methionine restriction (MR, 0.172%) diets for five weeks (n = 3–11, diet x sex x genotype). Due to the large number of experimental groups, a cohort-based approach was implemented to ensure feasibility and manageability. Each mouse participated in all aspects of the study, and the average cohort size was 16 mice. Diets were purchased from Dyets Inc. (Bethlehem, PA) as extruded pellets. Diet information is found in Table 1. All animals were group-housed under standard temperature conditions and humidity, followed a 12-hour light/dark schedule, and were provided food and water ad libitum. Body weights were recorded weekly.
Insulin tolerance test
After four weeks on diets, mice underwent an insulin tolerance test (ITT). Mice were fasted in new cages for four hours prior to ITTs. Fasting blood glucose was measured via tail nick using a glucometer (OneTouch Ultra2) followed by administration of insulin (0.6 U/kg body weight) via intraperitoneal (I.P.) injection. Human insulin was purchased from Sigma; catalog # I2643. Blood glucose concentrations were measured every 15 minutes for 1 hour following insulin administration.
Sacrifice.
After five weeks on diets, mice were sacrificed by decapitation following carbon dioxide-induced overdose. Fifteen minutes before sacrifice mice were administered insulin (0.6 U/kg body weight; Sigma catalog # I2643) or vehicle (saline) via I.P. injection. Trunk blood was collected and clotted for 30 minutes at room temperature, and serum was isolated after centrifugation at 5,000 RPM for 15 minutes at 4°C (accuSpin Micro 17R, Fisher Scientific). Brains were carefully removed and dissected in ice-cold phosphate-buffered saline. The hypothalamus and hippocampus were snap-frozen in liquid nitrogen and stored at −80°C. Additional tissues, including the liver, gonadal white adipose tissue (gWAT), and retroperitoneal white adipose tissue (rpWAT) were collected and stored at −80°C.
Enzyme-linked immunosorbent assay.
Serum FGF21 concentrations were measured using an enzyme-linked immunosorbent assay (ELISA), according to the solid phase sandwich method (Cat# MF2100; R&D Systems, Minneapolis, MN) following the manufacturer’s protocol. The plate was read at 450 nm using a spectrophotometer (Synergy HT, BioTek).
RNA isolation, reverse transcription, and quantitative real-time PCR.
Quantitative polymerase chain reaction (qPCR, LightCycler 96) was used to evaluate gene expression. Briefly, total RNA was extracted from the liver and hippocampus tissue using the QIAzol reagent method (Cat#79306) following the manufacturer’s protocol, reverse transcribed (Promega RT-System, C-1000 Touch Thermal cycler), and diluted to 3.33 ng/L. qPCR was used to measure gene expression of Fgf21 in the liver and (Tnfa, Il6, Il1b, Il23, Sirt1) and other genes (Nfkb, Il10, Cat, Nox2, Bdnf, Trkb, Synpo, Psd95, Nrgn, Reln, Creb, Irs1) related to health status in the hippocampus. The housekeeping gene cyclophilin (Ppia) was used to normalize target gene expression (S1 Table).
Protein isolation and immunoblot analysis
Western blot was used for quantifying changes in protein expression. Western blots followed standard protocols as previously described [45]. In short, protein was extracted from the liver and brain tissues via incubation and homogenization in radioimmunoprecipitation assay lysis buffer containing a protease inhibitor cocktail (Halt P&P cocktail, Thermo Scientific) and phosphatase inhibitors (50 mM sodium fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate decahydrate, 10 mM 2,3-bisphosphoglyceric acid). Total protein concentration was determined using a detergent-compatible protein assay and a microplate reader (Synergy HT, BioTek). Proteins were then separated by 10% SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 5% non-fat dry milk for 1 hour followed by incubation with the primary antibody at 4°C overnight. Membranes were washed and incubated with secondary antibodies at room temperature for 1 hour before imaging. β-actin or total lane protein (Sigma-Aldrich Cat#T54801) was used to normalize all results for statistical analyses. Protein expression was visualized using the Bio-Rad ChemiDoc Imaging System, and band densities were quantified using Image Lab (Bio-Rad Inc) (S2 Table).
Statistical analysis
Statistical analyses were conducted using Excel (Version 16.95.1 2025) and GraphPad Prism 10.0 (GraphPad Software, San Diego, CA). One-way ANOVA with Tukey’s or Sidak’s multiple comparisons tests were used for statistical analysis of within genotype and within sex comparisons. Two-way ANOVA with Tukey’s multiple comparisons test were used for statistical analysis of between genotype but within sex comparisons. Two-way ANOVA with mixed effects analysis were used for ITTs. All data are expressed as the standard error of the mean (SEM). Shapiro-Wilks test was used for normality detection. The ROUT 1% test was used to identify statistical outliers which were excluded from analyses. A value of (p < 0.05) was considered statistically significant and results approaching significance were reported as relevant scientific trends.
Results
Body weight and composition
Initial body weights were different between diets in males and genotypes in females due to slight differences in age at the beginning of the study (Fig 1). Therefore, the percent change in body weight was reported. Given their young age at the start of the study, all groups gained weight during the five-week feeding regimen, with mice fed MR versions of their diets gaining significantly less weight (Fig 1). MR decreased the percent increase in body weight and reduced the final body weight of males regardless of diet or genotype (Fig 1B, 1C). MR reduced percent body weight gain in HFD female mice of both genotypes (Fig 1E) and reduced the final body weight of HFD WT females, while final body weight in HFD Fgf21-/- females showed a near significant reduction (p = 0.07) (Fig 1F).
Male (A-C), Female (D-F). Wild-type (WT), FGF21 knockout (Fgf21-/-), normal-fat diet (NFD), high-fat diet (HFD), or methionine restriction diet (MR). n = 3–11. Two-way ANOVA for statistical significance with Sidak’s multiple comparisons test. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****). Bar indicates significance between genotypes.
HFD feeding increased the gWAT and rpWAT of males in both genotypes compared to NFD. MR reduced the adiposity of males in both genotypes independent of the diet (Fig 2A, 2B). Neither HFD feeding nor MR altered female gWAT weight (Fig 2D). However, the HFD increased WT female rpWAT weight compared to NFD, and MR did not attenuate HFD-induced increases in rpWAT weight in this group (Fig 2E). Overall, Fgf21-/- females had lower adiposity compared to WT females independent of the diet (Fig 2D, 2E). In males, the HFD did not affect liver weight; however, MR reduced liver weight in both genotypes (Fig 2C). Overall, Fgf21-/- males had larger livers compared to WT males regardless of the diet. In females, neither diet nor genotype altered liver weight (Fig 2F).
Gonadal white adipose tissue (gWAT; A&D), retroperitoneal white adipose tissue (rpWAT; B&E) and liver (C&F) weights of male (A-C) and female (D-F) wild-type (WT) and FGF21 knockout (Fgf21-/-) mice fed a normal-fat diet (NFD), high-fat diet (HFD), or methionine restriction diet (MR) for 5 weeks. n = 3–11. Two-way ANOVA for statistical significance with Sidak’s multiple comparisons test. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). Bar indicates significance between genotypes.
Hepatic FGF21
HFD feeding did not affect hepatic Fgf21 gene expression or circulating FGF21 concentrations in either sex (Fig 3). MR increased hepatic Fgf21 gene expression in both NFD and HFD WT males (Fig 3A). Although not statistically significant, MR approximately doubled the serum concentrations of FGF21 in WT males on the NFD by 2.4-fold and led to a significant 5.3-fold increase in FGF21 in HFD males (Fig 3B). Similarly, Fgf21 gene expression was increased by MR in NFD (5.8-fold) WT females, but only the HFD reached statistical significance (Fig 3C). Serum FGF21, on the other hand, was increased by MR feeding in both NFD and HFD WT females by 3.5-fold and 6.8-fold, respectively (Fig 3D). Serum FGF21 was undetectable in Fgf21-/- mice.
Hepatic Fgf21 gene expression (n = 8–10; A&C) and FGF21 serum concentrations (n = 4–6; B&D) in male (A&B) and female (C&D) wild-type (WT) and FGF21 knockout (Fgf21-/-) mice fed a normal-fat diet (NFD), high-fat diet (HFD), or methionine restriction diet (MR), Not detected (ND). One-way ANOVA for statistical significance with Tukey’s multiple comparisons test. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).
Insulin tolerance
We aimed to examine whether FGF21 was essential for any MR-related improvements in both sexes. Due to small sample sizes for females during the ITT, male and female ITT data were combined to determine the effect of MR on peripheral insulin sensitivity. Consumption of an HFD was sufficient to increase fasting blood glucose concentrations and to impair insulin response during ITT of WT mice. Methionine restriction normalized fasting blood glucose concentrations and restored insulin sensitivity in HFD-fed WT mice (Fig 4A-4C). Meanwhile, Fgf21-/- mice displayed elevated fasting blood glucose concentrations and failed to respond to insulin administration, indicative of insulin resistance in this group (Fig 4D-4F). While MR completely normalized the blood glucose concentrations of HFD WT mice, MR did not affect the blood glucose concentrations or insulin response of Fgf21-/- mice.
Fasting blood glucose (A&D), ITT (B&E), and area under the curve (AUC; C&F) of wild-type (WT) and FGF21 knockout (Fgf21-/-) mice fed a normal-fat diet (NFD), high-fat diet (HFD), or methionine restriction diet (MR). Sexes are combined. n = 2–13 (WT = NFD 8M:0F, NFD-MR 8M:0F, HFD 7M:1F, HFD-MR 6M:2F, Fgf21-/- = NFD 3M:0F, NFD-MR 2M:0F, HFD 4M:9F, HFD-MR 4M:7F. One-way ANOVA with Tukey’s multiple comparisons or two-way ANOVA with mixed effects analysis. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. p < 0.05 (*), p < 0.0001 (****).
Hepatic insulin signaling
To assess the short-term effects of HFD and MR feeding on insulin sensitivity in specific tissues, insulin or vehicle was administered via I.P. injection 15 minutes before sacrifice. Phosphorylation of protein kinase B (Akt) was measured as a marker of insulin signaling using western blot analysis. Insulin increased Akt phosphorylation by 3.4-fold in NFD WT males and by 2.5-fold in NFD MR males, but neither reached statistical significance (Fig 5A). WT males on the HFD failed to respond to the insulin treatment, and MR ameliorated this effect (Fig 5B). In comparison, insulin failed to increase Akt phosphorylation in Fgf21-/- males and MR was unable to restore insulin signaling in these mice regardless of the diet (Fig 6). Together, these data suggest that mice lacking FGF21 have impaired hepatic insulin signaling and that the ability of MR to increase insulin signaling in males is dependent upon FGF21.
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Total lane protein for NFD and HFD (C&D). Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 4–5. Fold change from control. Two-way ANOVA for statistical significance with Sidak’s multiple comparisons test. Shapiro-Wilks for normality detection. Data are presented as mean ± SEM. p < 0.001 (***). Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to total lane protein for loading control.
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Total lane protein for NFD and HFD (C&D). Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 3–4. Fold change from control. Two-way ANOVA for statistical significance with Sidak’s multiple comparisons test. Shapiro-Wilks for normality detection. Data are presented as mean ± SEM. Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to total lane protein for loading control.
In NFD WT females, insulin increased Akt phosphorylation 9.4-fold (Fig 7A). Interestingly, MR significantly increased basal Akt phosphorylation (Fig 7A). The HFD impaired insulin response in WT females, and MR did not improve this response but again appeared to elevate basal Akt phosphorylation, though this did not reach statistical significance (Fig 7B). There was no significant effect of insulin, HFD feeding, or MR on hepatic Akt phosphorylation, in Fgf21-/- females suggesting that FGF21 is also important for hepatic insulin action in females (Fig 8).
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Total lane protein for NFD and HFD (C&D). Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 3–5. Fold change from control. Two-way ANOVA for statistical significance with Sidak’s multiple comparisons test. Shapiro-Wilks for normality detection. Data are presented as mean ± SEM. p < 0.05 (*), p < 0.0001 (****). Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to total lane protein for loading control.
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups and total lane protein (C). Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 1–5. Fold change from control. Two-way ANOVA for statistical significance with Sidak’s multiple comparisons test. Shapiro-Wilks for normality detection. Data are presented as mean ± SEM. Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to total lane protein for loading control.
Hypothalamic insulin signaling
Contrary to our expectations, insulin decreased Akt phosphorylation in the hypothalamus of WT NFD and Fgf21-/- NFD males. This effect was lost in male mice fed the MR diet in both genotypes (Fig 9A, 10A). In male mice fed the HFD, neither insulin nor MR affected Akt phosphorylation independent of genotype (Fig 9B, 10B). Insulin did not alter Akt phosphorylation in the hypothalamus of females regardless of diet or genotype (Figs 11, 12).
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 4–5. Fold change from control. Two-way ANOVA for statistical significance. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. p < 0.001 (***). Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to beta-actin for loading control.
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 3–5. Fold change from control. Two-way ANOVA for statistical significance. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. p < 0.05 (*). Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to beta-actin for loading control.
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 3–5. Fold change from control. Two-way ANOVA for statistical significance. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to beta-actin for loading control.
Quantified band densities for normal-fat diet (NFD; A) and high-fat diet (HFD; B) groups. Protein kinase B (Akt), methionine restriction diet (MR), saline (s), insulin (i). n = 1–5. Fold change from control. Two-way ANOVA for statistical significance. Shapiro-Wilks for normality detection. ROUT 1% for statistical outliers. Data are presented as mean ± SEM. Vertical bands in western blot images denote grouping, not blot splicing. Bands were normalized to beta-actin for loading control.
Hippocampal insulin signaling
Insulin did not affect Akt or glycogen synthase-3 kinase (GSK-3) phosphorylation in the hippocampus of male and female mice regardless of diet or genotype (S3-S6 Fig).
Hippocampal neuroinflammation
HFD feeding increased inflammatory gene expression in the hippocampus of HFD WT males. Although these effects are somewhat inconsistent, there are clear upward trends in the gene expression of cytokines Tnfa, Il1b, and Il23. MR trended to reduce Tnfa and Il23 inflammatory gene expression in the hippocampus of WT HFD males (p = 0.06, p = 0.09), respectively. There were no changes in neuroinflammatory gene expression in the hippocampus of females. There were no changes in Sirt1 gene expression in either sex (S7 Fig).
Additional genes related to neuroinflammation, oxidative stress, neurogenesis, and neuroplasticity that could have been altered by either the HFD and/or MR were examined. Neither the HFD nor MR had any effect on inflammation or oxidative stress (Nfkb, Il10, Cat, Nox2) in the hippocampus in either sex or genotype. There were also no differences in the expression of genes related to neurogenesis or neuroplasticity (Bdnf, Trkb, Synpo, Psd95, Nrgn, Reln, Creb, Irs1) regardless of diet, sex, or genotype (S3 Table).
Antioxidant activity
To determine how MR may be attenuating HFD-induced neuroinflammation in WT males, protein expression of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) and one of its downstream antioxidant products, heme oxygenase 1 (HO-1) were measured in the hippocampus. Despite MR reducing neuroinflammatory gene expression in the hippocampus of WT males on the HFD, MR did not alter the protein expression of NRF2 or HO-1 in the hippocampus of males or females (S8 Fig).
Discussion
This study investigated whether MR could mitigate HFD-induced insulin resistance and neuroinflammation in male and female mice and explored the role of endogenous FGF21 in mediating these effects. Methionine restriction significantly reduced gWAT and rpWAT weights in HFD-fed male WT and Fgf21-/- mice (Fig 2A, 2B), consistent with previous findings from our lab showing that FGF21 is not essential for MR’s ability to reduce body weight or adiposity in males under normal-fat diet conditions [28]. In contrast, MR had no significant effect on gWAT or RPWAT weight in either genotype among females (Fig 2E). Factors such as the timing of MR initiation may influence these outcomes. MR reduces adiposity only in male mice when the diet is introduced in young animals (aged eight weeks) [46]. However, when the diet is introduced in adulthood (four months), MR produces comparable reductions in adiposity in both sexes [46].
Methionine restriction also reduced liver weights in both WT and Fgf21-/- male mice. Interestingly, Fgf21-/- males exhibited consistently larger livers across all diet conditions (Fig 2C), aligning with previous reports showing that global FGF21 deficiency leads to hepatic enlargement due to suppressed expression of lipolytic genes and increased expression of genes promoting fatty acid synthesis [47–49]. In contrast, liver weights in female mice remained consistent regardless of genotype or diet (Fig 2F). Thus, while MR effectively prevented HFD-induced liver enlargement in males, it had no significant effect in females.
We found that short-term HFD consumption impairs peripheral insulin sensitivity in young mice, an effect counteracted by MR in an FGF21-dependent manner. These findings are consistent with earlier work demonstrating that FGF21 is essential for MR’s insulin-sensitizing effects [28]. To further investigate the mechanism behind MR’s benefits, hepatic insulin signaling was assessed. Although previous studies examined MR’s effects on hepatic insulin signaling, this is the first to do so under HFD conditions in both sexes while evaluating FGF21 dependence [33]. MR increases hepatic insulin sensitivity in WT males, restoring HFD-induced reductions in liver Akt phosphorylation (Figs 5, 7). However, MR did not restore Akt phosphorylation in females. Previous studies have similarly reported MR-induced increases in hepatic Akt phosphorylation in male mice [33,50]. Notably, MR failed to increase hepatic insulin signaling in both male and female Fgf21 ⁻ / ⁻ mice (Figs 6, 8), indicating that MR’s protective effects are both FGF21-dependent and sex-specific.
This study also examined sex-dependent effects of HFD on central insulin resistance and whether MR confers protection, with a focus on the role of endogenous FGF21. The hypothalamus, a key regulator of energy balance [51,52], responds to insulin through activation of the insulin receptor and subsequent phosphorylation of Akt [53,54], a pathway disrupted by HFD consumption [9]. Unexpectedly, intraperitoneal insulin administration decreased Akt phosphorylation in the hypothalamus of both WT and Fgf21-/- NFD-fed males (Figs 9, 10). In contrast, neither MR nor insulin administration altered hypothalamic Akt phosphorylation in HFD-fed males or in females fed the NFD or HFD (Figs 11, 12). One possible explanation for the absence of an insulin-induced increase in Akt phosphorylation is that 15 minutes post-injection may have been insufficient for insulin to cross the blood-brain barrier. Intranasal delivery, which reaches the brain in primates in approximately 13 minutes [55] and has been used effectively in mice [56,57], may represent a more suitable approach for future studies on central insulin signaling.
We further investigated whether MR exerts sex-specific effects on neuroinflammation and oxidative stress in the brain and whether these depend on FGF21. Our comprehensive analysis of gene and protein expression related to inflammatory and antioxidant pathways revealed no significant impact of diet or genotype on markers of inflammation or oxidative stress in either the hypothalamus or hippocampus (see supplementary information). These findings contrast with previous studies reporting that dietary MR reduces oxidative stress and inflammation in the hippocampus and cortex of male db/db mice [58]. Notably, when the FGF21 receptor (FGFR1) was selectively knocked down in the brain via intracerebroventricular injection of an adeno-associated virus, the anti-inflammatory benefits of MR were diminished [58]. This suggests a key role for FGF21 in mediating MR’s neuroprotective effects. Another study found that MR actually increased markers of neuroinflammation in the hippocampus of female mice in a tauopathy model, even though cognitive outcomes such as short-term memory, habituation, and motor control improved; notably, males were not assessed in this context [59].
Similarly to MR, protein-restricted diets improve metabolic health by increasing FGF21, which coordinates shifts in macronutrient preference, increases thermogenesis, and promotes insulin sensitivity [60–63]. Importantly, these responses are not due to a limitation of a single amino acid but rather reflect a generalized sensing of protein restriction, triggering integrated stress responses in the liver that upregulate FGF21 and modify systemic metabolism [64–66]. Moreover, FGF21-dependent responses to dietary protein restriction are sexually dimorphic, as males and females exhibit distinct peripheral metabolic adaptations, with females generally showing reduced sensitivity to the effects of protein restriction [67–70].
Our study has several limitations. First, we used a global FGF21 knockout model to assess whether FGF21 mediates the effects of methionine restriction on central insulin sensitivity and neuroinflammation. While this approach provides an important foundation for understanding FGF21’s role, it does not distinguish between the roles of circulating FGF21 (primarily liver-derived) and centrally produced FGF21. Future studies employing tissue specific and inducible FGF21 knockout models will be essential to delineate the relative contributions of peripheral versus central FGF21 and to minimize potential confounding factors of lifelong FGF21 deficiency. A second limitation is the relatively short duration of HFD exposure. Although our goal was to assess the early effects of HFD consumption in juvenile animals, the exposure period may have been too short to induce measurable changes in markers of neuroinflammation or oxidative stress in the brain. Third, we did not perform a glucose tolerance test (GTT), which limits our ability to assess glucose handling in response to a glycemic challenge. While the ITT provides insight into whole-body insulin action by evaluating the glycemic response to a defined insulin dose, GTTs reflect both β-cell function and insulin sensitivity. Therefore, the absence of GTT data limits our ability to fully characterize glucose tolerance and pancreatic β-cell contributions to the observed metabolic phenotype. An additional limitation is the absence of comprehensive morphometric measurements of peripheral organs. This limitation is particularly relevant in the context of a global, non-inducible knockout model, where metabolic phenotypes may reflect developmental adaptations in addition to, or instead of, alterations in metabolic regulation. Finally, the absence of littermate controls is a key limitation, as it introduces potential confounding factors related to genetic background, in utero environment, and early postnatal experiences. Future experiments should prioritize the use of littermate controls to improve the rigor and interpretability of findings.
Conclusion
In conclusion, the present study demonstrated that even short-term HFD feeding can negatively affect body weight, adiposity, and peripheral insulin sensitivity of young mice. MR is more effective at preventing these alterations in males compared to females, and this protection occurs in both FGF21-dependent and independent manners. While MR improved peripheral insulin tolerance and hepatic insulin signaling in male mice, it did not alter hypothalamic or hippocampal insulin signaling in males or females. The sex-specific effects of MR highlight the importance of well-powered preclinical studies that include female subjects.
Supporting information
S1 Fig. Western blot loading controls for hypothalamus of male mice.
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S2 Fig. Western blot loading controls for hypothalamus of female mice.
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S3 Fig. Hippocampal insulin signaling in wild-type male mice.
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S4 Fig. Hippocampal insulin signaling in Fgf21-/- male mice.
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S5 Fig. Hippocampal insulin signaling in wild-type female mice.
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S6 Fig. Hippocampal insulin signaling in Fgf21-/- female mice.
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S7 Fig. Neuro inflammatory gene expression in hippocampus.
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S8 Fig. Antioxidant protein expression in hippocampus.
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(PNG)
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