Dietary sulfur amino acid restriction elicits a cold-like transcriptional response in inguinal but not epididymal white adipose tissue of male mice
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eLife Assessment
Ruppert et al. investigated how activation of thermogenesis by cold exposure (CE) and methionine restriction (MetR) impacts health and leads to weight loss in mice. The authors provided valuable datasets showing that the responses to MR and CE are tissue-specific, while MR and CE affect beige adipose similarly. Although the study is descriptive, the data analyses are solid, with well-supported conclusions drawn from the findings.
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Abstract
Abstract
Introduction
About 1 billion people are living with obesity worldwide. GLP-1-based drugs have massively transformed care, but long-term consequences are unclear in part due to reductions in energy expenditure with ongoing use. Diet-induced thermogenesis (DIT) and cold exposure (CE) raise EE via brown adipose tissue (BAT) activation and beiging of white adipose tissue (WAT). Methionine restriction (MetR) is a candidate DIT trigger, but its EE effect has not been benchmarked against CE, nor have their tissue-level interactions been defined.
Objective & Methods
In a 2×2 design (Control vs. MetR; room temperature, RT: 22 °C vs. CE: 4 °C for 24 h), we used male C57BL/6N mice to benchmark MetR-induced thermogenesis against CE and mapped how diet and temperature interact across tissues. Bulk RNA-seq profiled liver, iBAT, iWAT, and eWAT. Differential expression was modeled with main effects and a diet×temperature interaction; KEGG GSEA assessed pathways.
Results
MetR increased EE at RT and shifted fuel use toward lipid oxidation, supporting MetR as a bona fide DIT factor. CE elevated EE across diets and blunted diet differences. Transcriptomic responses were tissue-specific: in liver, CE dominated gene induction while MetR and CE cooperatively repressed genes. The combination enriched glucagon/AMPK-linked and core metabolic pathways. In iBAT, CE dominated thermogenic and lipid-oxidation programs with minimal MetR contribution. In iWAT, MetR and CE acted largely additively with high concordance, enhancing fatty-acid degradation, PPAR signaling, thermogenesis, and TCA cycle pathways. In eWAT, robust co-dependent differential expression emerged only with MetR+CE, yet pathway-level enrichment was limited.
Conclusion
MetR is a genuine DIT stimulus that remodels metabolism in a tissue-specific manner. Our study provides a tissue-resolved transcriptomic resource that benchmarks diet-induced (MetR) against cold-induced thermogenesis and maps their interactions across liver, iBAT, iWAT, and eWAT.
Article activity feed
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eLife Assessment
Ruppert et al. investigated how activation of thermogenesis by cold exposure (CE) and methionine restriction (MetR) impacts health and leads to weight loss in mice. The authors provided valuable datasets showing that the responses to MR and CE are tissue-specific, while MR and CE affect beige adipose similarly. Although the study is descriptive, the data analyses are solid, with well-supported conclusions drawn from the findings.
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Reviewer #1 (Public review):
Summary:
Activation of thermogenesis by cold exposure and dietary protein restriction are two lifestyle changes that impact health in humans and lead to weight loss in model organisms - here, in mice. How these affect liver and adipose tissues has not been thoroughly investigated side by side. In mice, the authors show that the responses to methionine restriction and cold exposure are tissue-specific, while the effects on beige adipose are somewhat similar.
Strengths:
The strength of the work is the comparative approach, using transcriptomics and bioinformatic analyses to investigate the tissue-specific impact. The work was performed in mouse models and is state-of-the-art. This represents an important resource for researchers in the field of protein restriction and thermogenesis.
Weaknesses:
The findings …
Reviewer #1 (Public review):
Summary:
Activation of thermogenesis by cold exposure and dietary protein restriction are two lifestyle changes that impact health in humans and lead to weight loss in model organisms - here, in mice. How these affect liver and adipose tissues has not been thoroughly investigated side by side. In mice, the authors show that the responses to methionine restriction and cold exposure are tissue-specific, while the effects on beige adipose are somewhat similar.
Strengths:
The strength of the work is the comparative approach, using transcriptomics and bioinformatic analyses to investigate the tissue-specific impact. The work was performed in mouse models and is state-of-the-art. This represents an important resource for researchers in the field of protein restriction and thermogenesis.
Weaknesses:
The findings are descriptive, and the conclusions remain associative. The work is limited to mouse physiology, and the human implications have not been investigated yet.
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Reviewer #2 (Public review):
Summary:
This study provides a library of RNA sequencing analysis from brown fat, liver, and white fat of mice treated with two stressors - cold challenge and methionine restriction - alone and in combination (interaction between diet and temperature). They characterize the physiologic response of the mice to the stressors, including effects on weight, food intake, and metabolism. This paper provides evidence that while both stressors increase energy expenditure, there are complex tissue-specific responses in gene expression, with additive, synergistic, and antagonistic responses seen in different tissues.
Strengths:
The study design and implementation are solid and well-controlled. Their writing is clear and concise. The authors do an admirable job of distilling the complex transcriptome data into …
Reviewer #2 (Public review):
Summary:
This study provides a library of RNA sequencing analysis from brown fat, liver, and white fat of mice treated with two stressors - cold challenge and methionine restriction - alone and in combination (interaction between diet and temperature). They characterize the physiologic response of the mice to the stressors, including effects on weight, food intake, and metabolism. This paper provides evidence that while both stressors increase energy expenditure, there are complex tissue-specific responses in gene expression, with additive, synergistic, and antagonistic responses seen in different tissues.
Strengths:
The study design and implementation are solid and well-controlled. Their writing is clear and concise. The authors do an admirable job of distilling the complex transcriptome data into digestible information for presentation in the paper. Most importantly, they do not overreach in their interpretation of their genomic data, keeping their conclusions appropriately tied to the data presented. The discussion is well thought out and addresses some interesting points raised by their results.
Weaknesses:
The major weakness of the paper is the almost complete reliance on RNA sequencing data, but it is presented as a transcriptomic resource.
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Reviewer #3 (Public review):
Summary:
Ruppert et al. present a well-designed 2×2 factorial study directly comparing methionine restriction (MetR) and cold exposure (CE) across liver, iBAT, iWAT, and eWAT, integrating physiology with tissue-resolved RNA-seq. This approach allows a rigorous assessment of where dietary and environmental stimuli act additively, synergistically, or antagonistically. Physiologically, MetR progressively increases energy expenditure (EE) at 22{degree sign}C and lowers RER, indicating a lipid utilization bias. By contrast, a 24-hour 4 {degree sign}C challenge elevates EE across all groups and eliminates MetR-Ctrl differences. Notably, changes in food intake and activity do not explain the MetR effect at room temperature.
Strengths:
The data convincingly support the central claim: MetR enhances EE and shifts fuel …
Reviewer #3 (Public review):
Summary:
Ruppert et al. present a well-designed 2×2 factorial study directly comparing methionine restriction (MetR) and cold exposure (CE) across liver, iBAT, iWAT, and eWAT, integrating physiology with tissue-resolved RNA-seq. This approach allows a rigorous assessment of where dietary and environmental stimuli act additively, synergistically, or antagonistically. Physiologically, MetR progressively increases energy expenditure (EE) at 22{degree sign}C and lowers RER, indicating a lipid utilization bias. By contrast, a 24-hour 4 {degree sign}C challenge elevates EE across all groups and eliminates MetR-Ctrl differences. Notably, changes in food intake and activity do not explain the MetR effect at room temperature.
Strengths:
The data convincingly support the central claim: MetR enhances EE and shifts fuel preference to lipids at thermoneutrality, while CE drives robust EE increases regardless of diet and attenuates MetR-driven differences. Transcriptomic analysis reveals tissue-specific responses, with additive signatures in iWAT and CE-dominant effects in iBAT. The inclusion of explicit diet×temperature interaction modeling and GSEA provides a valuable transcriptomic resource for the field.
Weaknesses:
Limitations include the short intervention windows (7 d MetR, 24 h CE), use of male-only cohorts, and reliance on transcriptomics without complementary proteomic, metabolomic, or functional validation. Greater mechanistic depth, especially at the level of WAT thermogenic function, would strengthen the conclusions.
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