Dietary sulfur amino acid restriction elicits a cold-like transcriptional response in inguinal but not epididymal white adipose tissue of male mice
- Functional Genomics and Metabolism Unit, Department for Biochemistry and Molecular Biology, University of Southern Denmark, Denmark
- Novo Nordisk Foundation Center for Adipocyte Signaling, University of Southern Denmark, Denmark
Figures
Figure 1 with 1 supplement
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 https://www.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). Sample sizes for experiment 1 are 5 animals/group and 5–7 animals/group for Experiment 2. Error bars are SEM.
Figure 1—figure supplement 1
Physiological and metabolic effects of methionine restriction (MetR) and cold exposure (CE).
(A) ANCOVA analysis of average nighttime EE on days 6 and 11 over average nighttime locomotor activity. (B) Cumulative food intake (kcal) over the entire study period. (C) Cumulative water intake (mL) over the entire study period. (D) Cumulative locomotor activity (beam breaks) over the entire study period. (E) Average daily locomotor activity over all RT (22°C) or CE (4°C) experimental days. (F) Average light and dark phase RER at days 4–6 (RT) and days 9–11 (CE). (G) Relative organ weights for liver, iWAT, gWAT, iBAT. Statistics were done within tissues. (H) Serum β-hydroxybutyrate (βOHB), Non-esterified fatty acids (NEFA), and IL-6 levels. EE was analyzed by ANCOVA using average locomotor activity as a covariate (via https://www.mmpc.org/); p-values were Bonferroni-corrected. Average daily locomotor activity and average RER were analyzed via two-way ANOVA with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Serum parameters and relative organ weights were analyzed using one-way ANOVA with Tukey’s post hoc test. Different letters indicate statistically significant differences (p < 0.05). Sample sizes for Experiment 1 are 5 animals/group and 5–7 animals/group for Experiment 2. Error bars are SEM.
Figure 2 with 1 supplement
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, and 7 (iBAT); 7, 7, 7, and 7 (iWAT); and 4, 7, 6, and 7 (eWAT; see Materials and methods).
Figure 2—figure supplement 1
The transcriptional responses to cold exposure (CE) and MetR-induced thermogenesis are tissue-specific.
(A) PCA plot of all RNA-seq samples colored by tissue and shaped by experimental group. (B) Number of DEGs (adjusted P-value <0.05, |FC|>1.5) in each tissue across the indicated comparisons, split into up- (red) and down- (blue) regulated genes. (C–D) UpSet plots showing overlap of DEGs across tissues for each contrast: (C) MetR_CE vs MetR_RT, (D) MetR_CE vs Ctrl_CE. 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, and 7 (iBAT); 7, 7, 7, and 7 (iWAT); and 4, 7, 6, and 7 (eWAT; see Materials and methods).
Figure 3 with 1 supplement
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 gray dots (see Materials and 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.
Figure 3—figure supplement 1
Cold exposure (CE) drives gene induction while methionine restriction (MetR) and CE cooperatively repress genes in the liver.
(A) Volcano plots showing DEGs for MetR_CE vs MetR_RT (left) and MetR_CE vs Ctrl_CE (right). Numbers indicate significantly up- and downregulated genes (adjusted P-value <0.05, |FC|>1.5). (B) Scatter plot comparing log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs significant in either condition. Colored dots indicate DEGs specific to each contrast or MetR_CE. n denotes DEGs shown. (C) Schematic highlighting gene expression profiles. Arrows indicate significant regulation for induced (up; red) or repressed (down; blue) genes. Non-significant regulation is depicted as gray dots (see Materials and methods). (D) Regulation classification of DEGs found in Ctrl_CE vs Ctrl_RT (left) or MetR_RT vs Ctrl_RT (right), but not MetR_CE vs Ctrl_RT. (E) Heatmap of Top 30 genes induced under Ctrl_CE. (F) Heatmap of Top 30 genes induced under MetR_RT. (G) Heatmap of Top 30 genes with negative interaction. (H) Heatmap of Top 30 genes repressed under Ctrl_CE. (I) Heatmap of Top 30 genes repressed under MetR_RT. (J) Heatmap of Top 30 genes with positive interaction.
Figure 4 with 1 supplement
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. (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.
Figure 4—figure supplement 1
CE dominates gene induction in iBAT with limited contribution by MetR feeding.
(A) Volcano plots showing DEGs for MetR_CE vs MetR_RT (left) and MetR_CE vs Ctrl_CE (right). Numbers indicate significantly up- and downregulated genes (adjusted p-value <0.05, |FC|>1.5). (B) Scatter plot comparing log₂FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs significant in either condition. Colored dots indicate DEGs specific to each contrast or MetR_CE. n denominates DEGs shown. (C) Regulation classification of DEGs found in Ctrl_CE vs Ctrl_RT (left) or MetR_RT vs Ctrl_RT (right), but not MetR_CE vs Ctrl_RT. (D) Heatmap of Top 30 genes induced under Ctrl_CE. (E) Heatmap of Top 30 genes induced under MetR_RT. (F) Heatmap of Top 30 genes with negative interaction. (G) Heatmap of Top 30 genes repressed under Ctrl_CE. (H) Heatmap of Top 30 genes repressed under MetR_RT. (I) Heatmap of Top 30 genes with positive interaction.
Figure 5 with 1 supplement
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. (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.
Figure 5—figure supplement 1
Additive and synergistic gene regulation by MetR and CE in iWAT.
(A) Volcano plots showing DEGs for MetR_CE vs MetR_RT (left) and MetR_CE vs Ctrl_CE (right). Numbers indicate significantly up- and downregulated genes (adjusted p-value <0.05, |FC|>1.5). (B) Scatter plot comparing log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs significant in either condition. Colored dots indicate DEGs specific to each contrast or MetR_CE. n denominates DEGs shown. (C) Regulation classification of DEGs found in Ctrl_CE vs Ctrl_RT (left) or MetR_RT vs Ctrl_RT (right), but not MetR_CE vs Ctrl_RT. (D) Heatmap of Top 30 genes induced under Ctrl_CE. (E) Heatmap of Top 30 genes induced under MetR_RT. (F) Heatmap of Top 30 genes with negative interaction. (G) Heatmap of Top 30 genes repressed under Ctrl_CE. (H) Heatmap of Top 30 genes repressed under MetR_RT. (I) Heatmap of Top 30 genes with positive interaction.
Figure 6 with 1 supplement
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. (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.
Figure 6—figure supplement 1
Limited yet codependent and antagonistic gene regulation by MetR and CE in eWAT.
(A) Volcano plots showing DEGs for MetR_CE vs MetR_RT (left) and MetR_CE vs Ctrl_CE (right). Numbers indicate significantly up- and downregulated genes (adjusted p-value <0.05, |FC|>1.5). (B) Scatter plot comparing log2FC values in Ctrl_CE vs Ctrl_RT and MetR_RT vs Ctrl_RT for DEGs significant in either condition. Colored dots indicate DEGs specific to each contrast or MetR_CE. n denominates DEGs shown. (C) Regulation classification of DEGs found in Ctrl_CE vs Ctrl_RT (left) or MetR_RT vs Ctrl_RT (right), but not MetR_CE vs Ctrl_RT. (D) Heatmap of Top 30 genes induced under Ctrl_CE. (E) Heatmap of Top 30 genes induced under MetR_RT. (F) Heatmap of Top 30 genes with negative interaction. (G) Heatmap of Top 30 genes repressed under Ctrl_CE. (H) Heatmap of Top 30 genes repressed under MetR_RT. (I) Heatmap of Top 30 genes with positive interaction.
Additional files
-
Source data 1
List of all differentially expressed genes.
- https://cdn.elifesciences.org/articles/108825/elife-108825-data1-v1.zip
-
Source data 2
List of all Gene set enrichment analyses.
- https://cdn.elifesciences.org/articles/108825/elife-108825-data2-v1.zip
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108825/elife-108825-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Dietary sulfur amino acid restriction elicits a cold-like transcriptional response in inguinal but not epididymal white adipose tissue of male mice
eLife 14:RP108825.
https://doi.org/10.7554/eLife.108825.3
