Author response:
The following is the authors’ response to the original reviews.
We sincerely thank the reviewers for their thoughtful and constructive comments. We fully agree that when two independent variables (genotype and age) are being evaluated, the statistical analysis must appropriately account for both factors and their potential interaction. We appreciate the reviewers’ guidance in strengthening the statistical rigor of our study.
In response to this concern, we have carefully reanalyzed the relevant datasets using two-way ANOVA to properly assess the effects of genotype, age, and their interaction. The manuscript, figures, and figure legends have been revised accordingly. Specifically:
Figure 1:
The quantification of p16 expression in Fig. 1F has been reanalyzed using two-way ANOVA. The figure has been replotted, and the corresponding legend has been updated to reflect the revised statistical approach.
Figure 2:
The quantification of AUC in Fig. 2F has been reanalyzed using two-way ANOVA. The figure and legend have been updated accordingly.
Figure 3:
The quantification of F4/80 in Fig. 3C and 3D has been reanalyzed using two-way ANOVA. The figures and corresponding legends have been revised to reflect this updated analysis.
Public Reviews:
Reviewer #1 (Public review):
Sebag et al. addressed the role of ADH5 in BAT in the development of aging and metabolic disarrangements associated with it. This is a follow-up study after the authors' demonstration of the role of BAT ADH5 in glucose homeostasis, obesity, and cold tolerance. By ablating ADH5 specifically in brown adipocytes or pharmacologically modulating ADH5 through activation of its transcription factor, the authors conclude that preservation of BAT function is crucial for healthy aging and ADH5 is causally involved in this process. The topic is appealing given the rise in the aging population and the unclear role of BAT function in this process. Overall, the study uses several techniques, is easy to follow, and addresses several physiological and molecular manifestations of aging. However, the study lacks an appropriate statistical analysis, which severely affects the conclusions of the work. Therefore, interpretation of the findings is limited and must be done with caution.
We sincerely thank the reviewer for their thoughtful and constructive comments. We fully agree that when two independent variables (genotype and age) are being evaluated, the statistical analysis must appropriately account for both factors and their potential interaction. We appreciate the reviewers’ guidance in strengthening the statistical rigor of our study.
Reviewer #2 (Public review):
Weaknesses:
(1) Sex needs to be considered as a biological variable, at a minimum in the reporting of the phenotypes observed in this manuscript, but also potentially by further experimentation. The only mention of sex I could find is that the authors reported the general protein SNO status in BAT is increased with age in male C57Bl/6J mice. Is this also true in female mice?
We thank the reviewer for this insightful comment. In response, we examined whether aging affects Hsf1 and Adh5 transcript levels in wild-type female mice (3 months vs. 19 months). Our analysis did not reveal significant age-associated changes in the expression of either gene. These results have now been incorporated into the revised manuscript and are presented in Figure 4A.
(2) It would be helpful to know the extent of ADH5 loss in the adipose tissue of knockout mice, either by mRNA or by immunoblotting for ADH5. It could also be helpful to know if ADH5 is deleted from the inguinal adipose tissue of these mice, especially since they seem to accumulate fat mass as they age (Figure 2B).
We thank the reviewer for this suggestion. Indeed, we have previously measured ADH5 expression in both brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT). These data were published in Cell Reports (PMID: 3478865).
(3) For Figure 4D, it's not clear how these BAT samples were treated with HSF1A - was this done in vivo or ex vivo?
We thank the reviewer for their thoughtful comment. We have now provided additional methodological details in the revised manuscript. In Figure 4D (current Figure 4E), BAT was collected from wild-type mice and cultured ex vivo as explants. The BAT explants were treated for 24 hours with HSF1A (an HSF1 activator; 20 µM). Following treatment, mRNA levels of the indicated genes were measured by RT-qPCR.
(4) I didn't understand what was on the y-axis in Figure 5A, nor how it was measured.
We apologize for not making these critical points clearer in the initial submission. Figure 5A shows the release profiles of HSF1A from collagen gels with nanoclay (Collagen–NC–HSF1A) and without nanoclay (Collagen–HSF1A), determined using an established standard curve method (Hu et al., PMID: 33225042).
The concentration of HSF1A was quantified by UV–Vis spectroscopy. Briefly, a standard curve for HSF1A was generated by measuring the UV–Vis spectra of HSF1A at known concentrations (1.25, 2.5, 5, 10, and 20 µM) prepared in phosphate-buffered saline (PBS). Collagen gels with or without nanoclay were then fabricated to evaluate the release profile. At predetermined time points (1, 5, 9, 14, and 21 days), the PBS supernatant from each sample was collected and analyzed by UV–Vis spectroscopy. The amount of released HSF1A was calculated using the previously established standard curves. A brief description has now been included in the figure legend.
(6) Figure 1B: What is the age of the positive (ADH5BKO) and negative (Adh5 fl) mice?
We regret that we did not describe our results clearly in the first submission and have included detailed information in the revised manuscript.
(7) Figure 1F: Can you clarify what I'm looking at in the P16ink4a panels? The red staining? Is the blue staining DAPI? This is also a problem in Figures 3C, 3D and 5G, and 5I. Figure 4B looks great - maybe this could be used as an example?
We regret that we did not present results clearly in the first submission and have provided detailed information in these figures in the revised manuscript.
(8) Figure 3B looks a bit odd. Can the approach to measuring IL-1β be clarified, and could the authors explain why they can't show units of mass for IL-1β levels?
We have provided information in the revised manuscript.
(9) What are the levels of nitric oxide synthase in the BAT of the aging model? Since protein S-nitrosylation is regulated by a balance of both, the attribution of greater protein S-nitrosylation to ADH5 is incomplete without determining nitric oxide synthase.
We thank the reviewer for this thoughtful comment. In response, we have now included the analysis of iNOS transcript expression levels in the revised manuscript. These data are presented in Figure 1C.
Recommendations for the authors:
Reviewer #2 (Recommendations for the authors):
(2) Presentation of metabolomics is not appropriate. The authors described, using color coding, the metabolites up- or downregulated in the experimental design. However, the current approach does not allow the reader to detect sample size, magnitude of changes, variability of the data, p-values, etc. This approach does not follow the standard practices of scientific rigor and should be modified. Metabolomic data could be uploaded as supplementary data, or a table with all necessary information to allow a full interpretation of the data should be provided.
We have now provided the the metabolimic data in a table format as Figure 3I.
(6) What are the levels of nitric oxide synthase in the BAT of the aging model? Since protein S-nitrosylation is regulated by a balance of both, the attribution of greater protein S-nitrosylation to ADH5 is incomplete without determining nitric oxide synthase.
We thank the reviewer for their thoughtful comment. We have now included iNOS transcript levels expression level in the revised manuscript (Figure 1C).
Minor Comments:
(1) The conclusion of the abstract is somewhat vague. I suggest the authors rewrite it to better recapitulate what was found in the study.
We thank the reviewers for this helpful suggestion. In response, we have revised the Abstract to improve the specificity and clarity of our conclusions.
(2) In the introduction, the authors mention that an increased level of mitochondrial ROS activates UCP1. Given that the evidence for this statement is circumstantial and not supported by the current state-of-the-art (PMID: 28710335), where it is accepted that UCP1 activation diminishes ROS production, I suggest that the authors tone down this statement or at least acknowledge conflicting findings and interpretations.
We thank the reviewer’s insight, we have included this important notion in the introduction.
(3) Figure 2H - It is unclear what this figure (and statistical analysis) represents. Please, improve the description of the experiment and how the data were plotted to reach such a conclusion.
We regret that we did not present results clearly in the first submission. The trend lines show the relationship between body weight and time on rotarod. The P value is the comparison of the slope of the line between Adh5 BKO mice and Adh5 fl/fl mice. The data implicate that the heavier the BKO mouse, the less time spent on the rotarod.
(4) Figure 2M - The unit of LV thickness is missing. Please, provide it. In addition, I am missing the other cardiac parameters obtained from the echocardiogram.
We have included this information in Figure 2M in the revised manuscript.
(5) Figure 2G - I believe force is not the right unit for the grip strength test. Please, revise accordingly.
We regret that we did not describe our results clearly in the first submission. We have corrected this unit in the revised figure.
(6) Figure 3H - What is the unit when reporting mitochondrial area?
We regret that we did not describe our results clearly in the first submission. We have added this information in the revised figure.
(7) Is HFS1 also downregulated in iWAT?
We thank the reviewer for this thoughtful comment. In response, we measured Hsf1 expression in iWAT from young and aged wild-type male mice. Our analysis did not reveal any significant age-associated changes in Hsf1 expression in iWAT. These results have now been included in the revised manuscript (Figure 4C).
(8) Can the authors explain how HFS1 expression increases upon HSF1 activation? I understand ADH5 is controlled by HSF1, but what would control HSF1 itself? Off targets?
We thank the reviewer for this insightful comment. At present, we do not have direct mechanistic evidence to definitively support this notion, and we cannot exclude the possibility of off-target effects of HSF1A. However, previous studies have reported that the HSF1 promoter contains heat shock elements (HSEs) in humans and HSE-like domains in mice. Based on this, we speculate that activated HSF1 may enhance its own transcription through an autoregulatory or positive feedback mechanism.
