Sex-specific metabolic responses to high-fat diet in mice with NOX4 deficiency

Abstract

Reactive oxygen species (ROS) are critical mediators of cellular signaling that regulate metabolic homeostasis, including lipid uptake, synthesis, and storage. NADPH oxidase 4 (NOX4), a significant enzymatic source of ROS, has been identified as a redox-sensitive regulator of glucose and lipid metabolism. However, its contribution to sex-specific metabolic regulation remains poorly defined. This study compared how NOX4 knock-out (NOX4 KO) shifted systemic and tissue-specific metabolic phenotypes between male and female mice fed with a high-fat diet (HFD) for 20-weeks. We observed that male NOX4 mice on HFD exhibited reduced adiposity, diminished liver lipid accumulation, and improved glucose and insulin tolerance compared to male WT mice on HFD. In contrast, female NOX4 KO mice developed increased adiposity and lipid accumulation in peripheral adipose depots, accompanied by impaired glucose tolerance. Gene expression profiling in skeletal muscle and liver revealed distinct, sex-specific patterns of changes in genes related to lipid uptake, synthesis, and storage, possibly implicating differential activation of PPAR signaling pathways supportive of in vivo data. These findings identify NOX4 as a central regulator of sexually dimorphic lipid metabolism, acting through redox-sensitive transcriptional networks to shape divergent metabolic responses to HFD.

Graphical abstract

Fig. 1

Weight gain and lipid deposition. (A) Percent change in body mass (%) recorded weekly over 20 weeks, relative to baseline. Males, left, blue; Females, right, pink. (B) Absolute body masses (g) at the start (0 months) and end (5 months) of the study. Males, left, blue; Females, right, pink. (C) Monthly absolute lean mass and (D) fat mass (g), measured by NMR. Males, left, blue; Females, right, pink. (E) Absolute masses of subcutaneous adipose tissue (iWAT, inguinal depot, g) and visceral adipose tissue (pgWAT, perigonadal depot, g) at sacrifice. Males, left, blue; Females, right, pink. Values represent means ± S.E.M. Statistical analyses: In panels (A), (C), and (D), significance was determined using a repeated measures full factorial mixed model for each sex separately (fixed effects: time, diet, genotype; random effects: mouse, cohort; covariance structure: unequal variances), followed by Tukey's multiple comparison test. More statistical details provided in Methods. In panels (B) and (E), significance was assessed using a full factorial 2-way ANOVA by sex, followed by Tukey's multiple comparisons test. Statistical significance was set at p < 0.05. Symbols: “∮” indicates a diet effect in WT (WT chow vs. WT HFD); “∯” indicates a diet effect in KO (KO chow vs. KO HFD); “†” indicates a genotype effect on chow diet (KO chow vs. WT chow); “‡” indicates a genotype effect in HFD (WT HFD vs. KO HFD). Sample sizes: n = 8–10 (females), n = 12–17 (males).

Fig. 2

Glucose handling and circulating lipids. (A) Fasted levels of triglycerides in plasma. Males, left, blue; Females, right, pink. (B) Fasted levels of non-esterified fatty acids (NEFA) in plasma. Males, left, blue; Females, right, pink. (C) Monthly measurements of glucose tolerance test (GTT) area-under-the-curve (gAUC) values. Males, left, blue; Females, right, pink. (D) Monthly measurements of insulin tolerance test (ITT) AUC (iAUC) values. Males, left, blue; Females, right, pink. Values represent means ± S.E.M. Statistical analyses: In panels (A) and (B), significance was assessed using a full factorial 2-way ANOVA by sex, followed by Tukey's multiple comparisons test. Further statistical details are available in Methods. Sample sizes: n = 8–10 (females), n = 12–17 (males). In panels (C) and (D), significance was determined using a repeated measures full factorial mixed model for each sex separately (fixed effects: time, diet, genotype; random effects: mouse, cohort; covariance structure: unequal variances), followed by Tukey's multiple comparison test. Sample sizes: n = 6–10 (females), n = 5–11 (males). Statistical significance was set at p < 0.05. Symbols: “∮” indicates a diet effect in WT (WT chow vs. WT HFD); “∯” indicates a diet effect in KO (KO chow vs. KO HFD); “†” indicates a genotype effect on chow diet (KO chow vs. WT chow); “‡” indicates a genotype effect in HFD (WT HFD vs. KO HFD).

Fig. 3

Muscle mass, muscle triglycerides, and mRNA gene expression. (A) Wet weight of skeletal muscles at sacrifice. Sample sizes: n = 12–17 (males, top, blue), n = 8–10 (females, bottom, pink). (B) Amount of intramuscular triglycerides in gastrocnemius (GC) skeletal muscle normalized to total protein content. n = 5–11. Males, top, blue; Females, bottom, pink. (C) mRNA expression of Nox4 in the GC. n = 8–11. (D) mRNA expression of genes related to insulin signaling and lipid metabolism. (E) mRNA expression of genes related lipid storage and hypoxia. (F) mRNA expression of genes related to redox homeostasis. Gene expression in skeletal muscle was normalized to the mRNA expression of Rpl14a (n = 4–6). Values represent means ± S.E.M. Statistical analyses: Significance was assessed using a full-factorial 2-way ANOVA by sex, followed by Tukey's multiple comparisons test or simple main effects as appropriate. More statistical details provided in Methods. Please see Supplementary Fig. S6 for ANOVA tables. Statistical significance was set at p < 0.05. Symbols: “∮” indicates a diet effect in WT (WT chow vs. WT HFD); “∯” indicates a diet effect in KO (KO chow vs. KO HFD); “†” indicates a genotype effect on chow diet (KO chow vs. WT chow); “‡” indicates a genotype effect in HFD (WT HFD vs. KO HFD).

Fig. 4

Liver mass, fat accumulation, histology, and mRNA gene expression. (A) Liver wet weight at sacrifice. Males, left, blue; Females, right, pink. (B) Amount of neutral lipids in the liver (mg) normalized to total liver mass. Males, left, blue; Females, right, pink. (C) Representative H&E histological micrograph of livers at sacrifice (n = 3–5), 10×-magnification, scale bar measures 100 μm. Males, top, blue lines; Females, bottom, pink lines. (D) mRNA expression of genes related to lipid uptake and transport. (E) mRNA expression of genes related to lipogenesis, gluconeogenesis and insulin signaling. (F) mRNA expression of genes related to fatty acid activation or oxidation. (G) mRNA expression of transcription factors related to lipid storage and hypoxia. (H) mRNA expression of genes related to lipid storage, lipolysis, and lipid droplet dynamics. (I) mRNA expression of genes involved with redox signaling and homeostasis. Gene expression in the liver was normalized to the mRNA expression of Tbp (n = 4–6). Values represent means ± S.E.M. Statistical analyses: Significance was assessed using a full factorial 2-way ANOVA by sex, followed by Tukey's multiple comparisons test or comparison of the simple main effects as appropriate. More statistical details provided in Methods. Statistical significance was set at p < 0.05. Symbols: “∮” indicates a diet effect in WT (WT chow vs. WT HFD); “∯” indicates a diet effect in KO (KO chow vs. KO HFD); “†” indicates a genotype effect on chow diet (KO chow vs. WT chow); “‡” indicates a genotype effect in HFD (WT HFD vs. KO HFD).

(A) Mass of food (g) consumed over 20 weeks by cage and averaged by number of mice per cage. (B) Conversion of total food consumed by diet-specific energy content. (C) Sex-specific changes in estimated energy efficiency in mice NOX4 KOs on HFD calculated by total body mass gained per individual mouse (g)/total energy consumed per cage averaged by number of mice per cage during the 20 weeks of chow diet or HFD.

(A) Pearson correlation coefficient matrix. Δ refers to the difference from baseline (8 weeks of age) to after 20 weeks of chow diet or HFD. BM = body mass. FG = fasting glucose. GTT = glucose tolerance test area-under-the-curve values. ITT = insulin tolerance test area-under-the-curve values. Fat and lean refer specifically to the difference in total adiposity or lean mass measured via NMR. (B) Partialed correlations. Values on the left/bottom correspond to the partial correlations which adjusts for the effect of all variables (bolded cells and text indicates p < 0.05), the values on the top/right correspond to the p-values for the partial correlations (orange text signifies the p-value is < 0.05). Results confirm significant relationship between liver mass, glucose tolerance, and metrics related to lipid handling (iWAT and pgWAT mass, plasma triglycerides and NEFA), but do not reflect as significant of relationships between the GC and iWAT and pgWAT mass, plasma triglycerides and NEFA. n = 65–70.

Hepatic (A) and gastrocnemius (B) expression of genes related to adipogenesis, the import and transport of lipids, and proteins secreted from the liver known to regulate lipid accumulation and adipogenesis. Dietary and genotypic conditions were pooled by sex to examine the main effect of sex. Numbers above bars represent fold change of all females relative to all males. All genes shown demonstrated a significant main effect of sex (p < 0.05). Full factorial 3-way ANOVA. Sample sizes: n = 22 (males), n = 23 (females).

Expression of Nox4 in the liver relative to WT male on chow diet normalized to Tbp. n = 69.

Hepatic gene expression in heatmap form (Heatmapper.ca). Red color indicates increased expression, blue indicates decreased expression, while white color is the averaged expression across samples. Hierarchical clustering was used with Spearman Rank Correlation settings. Sample sizes: n = 22 (males), n = 23 (females).