Physiological and metabolic effects of methionine restriction (MetR) and cold exposure (CE).

(A) Schematic depicting 11 day experimental setup and dietary composition for mouse experiment 1 (n = 5 animals/group). (B) Energy expenditure (EE) over the entire experiment duration. (C) ANCOVA analysis of average nighttime EE on days 6 and 11 over body weight. (D) Bodyweight and body weight loss (%). (E) Respiratory exchange ratio (RER) over the entire experiment duration. (F) Average daily food and water intake over all RT (22 °C) or CE (4 °C) experimental days. (G) Schematic depicting 8 day experimental setup for mouse experiment 2 (n = 7 animals/group). Dietary compositions for Ctrl and MetR diets are the same as in mouse experiment 1. Groups are denominated as Ctrl_RT, Ctrl_CE, MetR_RT and MetR_CE. (H) Bodyweight and body weight loss (%). (I) Absolute organ weights for Liver, inguinal WAT, gonadal WAT, interscapular BAT. Statistics were done within tissues. (J) Blood glucose, serum triglycerides, and FGF21 levels. EE was analyzed by ANCOVA using body weight as a covariate (via mmpc.org); p-values were Bonferroni-corrected. Bodyweight, average food and water intake were analyzed via two-way ANOVA with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Body Weight loss, serum parameters and absolute organ weights were analyzed using one-way ANOVA with Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05). Samples sizes for experiment 1 are 5 animals/group and 5-7 animals/group for Experiment 2.

The transcriptional responses to Cold exposure (CE) and MetR-induced thermogenesis are tissue-specific.

(A) Tissue-specific PCA plots for Liver, iBAT, iWAT, and eWAT. (B) Number of differentially expressed genes (DEGs), split into induced (red) and repressed (blue) genes, per contrast (adjusted p-value < 0.05, |FC| > 1.5). (C-F) UpSet plots showing overlap of DEGs across tissues for each contrast: (C) Ctrl_CE vs Ctrl_RT, (D) MetR_RT vs Ctrl_RT, (E) MetR_CE vs Ctrl_RT, and (F) Interaction. Set size bars indicate the total number of DEGs per tissue; intersection bars indicate the number of shared DEGs between tissues. Dots and bars in orange represent tissue-specific DEGs. In the order of Ctrl_RT, Ctrl_CE, MetR_RT and MetR_CE the following number of samples were included in the transcriptomic analysis: 7, 7, 7 and 7 (Liver), 7, 6, 7 an 7 (iBAT), 7, 7, 7, and 7 (iWAT) and 4, 7 6, and 7 (eWAT) (see methods).

Cold exposure (CE) drives gene induction while methionine restriction (MetR) and CE cooperatively repress genes in the liver.

(A) Volcano plots showing DEGs for each contrast (Ctrl_CE vs Ctrl_RT, MetR_RT vs Ctrl_RT, MetR_CE vs Ctrl_RT, and the diet × temperature interaction). Numbers indicate significantly up- and downregulated genes (adjusted p-value < 0.05, |FC| > 1.5). (B) Upset plot showing the overlap of DEGs across contrasts. Intersection bars indicate the number of shared DEGs between contrasts. Dots in orange represent contrast-specific DEGs. (C) Scatter plot of log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs identified in MetR_CE. n denominates DEGs shown. (D) Schematic highlighting gene expression profiles. Arrows indicate significant regulation for induced (up; red) or repressed (down; blue) genes. Non-significant regulation is depicted as grey dots (see methods). (E) Classification of MetR_CE DEGs based on their mode of regulation mode. (F) GSEA of the top 20 positively enriched pathways in MetR_CE vs Ctrl_RT. Dot size represents gene ratio, color denotes contrast, and significance is indicated by filled circles (adjusted p-value < 0.05). (G) Heatmap of Top 30 induced genes under MetR_CE. (H) GSEA for the top 14 negatively enriched pathways in the MetR_CE vs Ctrl_RT comparison. Dot size reflects gene ratio, colors indicate contrast group, and significance is shown by filled circles (adjusted p-value < 0.05). (I) Heatmap of Top 30 repressed genes under MetR_CE.

CE dominates gene induction in iBAT with limited contribution by MetR feeding

(A) Volcano plots showing DEGs for each contrast (Ctrl_CE vs Ctrl_RT, MetR_RT vs Ctrl_RT, MetR_CE vs Ctrl_RT, and the diet × temperature interaction). Numbers indicate significantly up- and downregulated genes (adjusted p-value < 0.05, |FC| > 1.5). (B) Upset plot showing the overlap of DEGs across contrasts. Intersection bars indicate the number of shared DEGs between contrasts. Dots in orange represent contrast-specific DEGs. (C) Scatter plot of log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs identified in MetR_CE. n denominates DEGs shown. (D) Classification of MetR_CE DEGs based on their mode of regulation mode. (E) GSEA of the top 10 positively enriched pathways in MetR_CE vs Ctrl_RT. Dot size represents gene ratio, color denotes contrast, and significance is indicated by filled circles (adjusted p-value < 0.05). (F) Heatmap of Top 30 induced genes under MetR_CE. (G) GSEA for the top 20 negatively enriched pathways in the MetR_CE vs Ctrl_RT comparison. Dot size reflects gene ratio, colors indicate contrast group, and significance is shown by filled circles (adjusted p-value < 0.05). (H) Heatmap of Top 30 repressed genes under MetR_CE.

Additive and synergistic gene regulation by MetR and CE in iWAT

(A) Volcano plots showing DEGs for each contrast (Ctrl_CE vs Ctrl_RT, MetR_RT vs Ctrl_RT, MetR_CE vs Ctrl_RT, and the diet × temperature interaction). Numbers indicate significantly up- and downregulated genes (adjusted p-value < 0.05, |FC| > 1.5). (B) Upset plot showing the overlap of DEGs across contrasts. Intersection bars indicate the number of shared DEGs between contrasts. Dots in orange represent contrast-specific DEGs. (C) Scatter plot of log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs identified in MetR_CE. n denominates DEGs shown. (D) Classification of MetR_CE DEGs based on their mode of regulation mode. (E) GSEA of the top 20 positively enriched pathways in MetR_CE vs Ctrl_RT. Dot size represents gene ratio, color denotes contrast, and significance is indicated by filled circles (adjusted p-value < 0.05). (F) Heatmap of Top 30 induced genes under MetR_CE. (G) GSEA for the top 20 negatively enriched pathways in the MetR_CE vs Ctrl_RT comparison. Dot size reflects gene ratio, colors indicate contrast group, and significance is shown by filled circles (adjusted p-value < 0.05). (H) Heatmap of Top 30 repressed genes under MetR_CE.

Limited yet codependent and antagonistic gene regulation by MetR and CE in eWAT

(A) Volcano plots showing DEGs for each contrast (Ctrl_CE vs Ctrl_RT, MetR_RT vs Ctrl_RT, MetR_CE vs Ctrl_RT, and the diet × temperature interaction). Numbers indicate significantly up- and downregulated genes (adjusted p-value < 0.05, |FC| > 1.5). (B) Upset plot showing the overlap of DEGs across contrasts. Intersection bars indicate the number of shared DEGs between contrasts. Dots in orange represent contrast-specific DEGs. (C) Scatter plot of log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs identified in MetR_CE. n denominates DEGs shown. (D) Classification of MetR_CE DEGs based on their mode of regulation mode. (E) GSEA of the top 10 positively enriched pathways in MetR_CE vs Ctrl_RT. Dot size represents gene ratio, color denotes contrast, and significance is indicated by filled circles (adjusted p-value < 0.05). (F) Heatmap of Top 30 induced genes under MetR_CE. (G) GSEA for the top 20 negatively enriched pathways in the MetR_CE vs Ctrl_RT comparison. Dot size reflects gene ratio, colors indicate contrast group, and significance is shown by filled circles (adjusted p-value < 0.05). (H) Heatmap of Top 30 repressed genes under MetR_CE.