This review reflects comments and contributions from Femi Arogundade & Pablo Ranea-Robles. Review synthesized by Pabloe Raneo-Robles.

The study investigates the distinct effects of exercise training (voluntary running) and cold exposure on inguinal white adipose tissue (iWAT) in mice, a fat depot that is metabolically active in response to nutritional and environmental challenges. The authors hypothesized that there would be relevant differences in the molecular adaptations to these interventions, since only transplantation of adipose tissue from trained animals exerts a positive effect on glucose tolerance. This has been demonstrated before by the same group, which has a large expertise on these types of experiments. Results reveal that while both interventions induce phenotypic changes in iWAT, exercise training enhances its endocrine function, positively impacting systemic glucose metabolism, whereas cold exposure primarily promotes thermogenesis but does not improve glucose tolerance. The findings emphasize the distinct roles of iWAT in metabolic health and highlight specific proteins, such as Rilpl2 and Rab GTPases, as potential exercise-induced regulators of glucose homeostasis. The paper is well-structured, presenting comprehensive data and analyses, with clear limitations and future research directions. We have provided several comments to improve the clarity of the paper, stated below.

Major comments:

• Once one goes through the methods, it is revealed that some of the datasets were generated (and reported) for another manuscript, for example the exercise-induced proteome, or the spatial transcriptomics, as I understood the reported methods. In general, it is not clear in the manuscript which datasets are novel for this study, which could generate confusion in the reader.

• The spatial transcriptomic images would require a more detailed explanation to make it easier to the reader. In my opinion, it is not clear only with what is shown, that the transcript of interest is more expressed in beige cells. Perhaps a higher quality image with some shapes to guide the reader would help

• The strength of the conclusions lies in the clear differentiation of the effects of exercise training and cold exposure on iWAT and their respective systemic impacts. The identification of specific proteins correlated with fasting glucose levels, such as Rilpl2 and Rab GTPases, adds depth to the findings. However, further mechanistic insights into how these proteins influence glucose metabolism would enhance the conclusions.

• Some limitations are well-stated, including the focus on male mice and the potential for lower molecular weight proteins in the exercise-induced secretome. However, there could be alternative explanations for the observed phenotype after iWAT transplantation, for example, the putative role of different metabolites coming from iWAT is not discussed in this manuscript.

• The conclusions of this study are based on findings in mice. It's important to emphasize that translating these results to humans requires additional research. There is no mention on how these pathways could be involved in human response to exercise in the discussion.

Minor comments:

• It is not clear on Fig. 2E whether the 20x images correspond to the same images shown at 4x. The addition of an inset showing the amplified region would be more informative for the reader.

• In the description of control sedentary mice, authors could specify whether they were also exposed to a wheel (locked of course) in the cage, to control for that variable.

• Since this study compares exercise vs cold at different levels, it would be interesting to see the amount of extracellular vesicles produced in iWAT in response to cold.

Comments on reporting:

• When describing the proteomic analysis, specify which statistical tests were used for determining significance.

Suggestions for future studies:

• Explore possible alternatives for mediators of this phenotype, for example metabolomics of iWAT

• Conduct in-depth mechanistic studies to elucidate how specific proteins identified in this research, such as Rilpl2 and Myosin Va, influence glucose metabolism and the endocrine function of iWAT.

• Explore the role of adipocyte-derived extracellular vesicles (Ad-EVs) in mediating the effects of exercise training on glucose metabolism and inter-organ communication. Investigate the content and function of these vesicles in more detail.

• Investigate the temporal aspects of exercise training and cold exposure effects. How do these adaptations change over time, and are there long-term effects that differ from the short-term responses observed in this study?

• Examine the systemic effects of iWAT adaptations on other metabolic tissues and organs. Investigate how iWAT communicates with liver, muscle, and pancreas in response to exercise training and cold exposure.

• Consider conducting translational studies to determine if the findings in mice hold true in humans. Do exercise-induced adaptations in human subcutaneous adipose tissue have similar effects on glucose metabolism?

• Composition of those extracellular vesicles secreted from iWAT after exercise

• Administration of those ecV from sedentary and exercise iWAT

Competing interests

The author declares that they have no competing interests.