Thermogenic Adipose ADH5 Counteracts Age-related Metabolic Decline

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.

Reviewer #2 (Public review):

Summary:

This study investigates the role of the enzyme Alcohol Dehydrogenase 5 (ADH5) in brown adipose tissue (BAT) during aging. BAT is crucial for thermogenesis and energy balance, but its function and mass diminish with age, contributing to metabolic dysfunction and age-related diseases. ADH5, also known as S-nitrosoglutathione reductase, regulates nitric oxide (NO) signaling by damaging S-nitrosylation modifications from proteins. The authors show that aging in mice leads to increased protein S-nitrosylation but reduced ADH5 expression in BAT, resulting in impaired metabolic and cognitive functions. Deletion of ADH5 in BAT accelerates tissue senescence and systemic metabolic decline.

Mechanisticaremoving lly, aging suppresses ADH5 via downregulation of heat shock factor 1 (HSF1), a master regulator of protein homeostasis. Importantly, pharmacologically boosting HSF1 improves BAT function and mitigates both metabolic and cognitive declines in aged mice. The findings highlight a critical HSF1-ADH5 pathway in BAT that protects against aging-related dysfunction, suggesting that targeting this pathway may offer new therapeutic strategies for improving metabolic health and cognition during aging.

Strengths:

This research provides insight into the interplay between redox biology, proteostasis, and metabolic decline in aging. By identifying a specific enzyme that controls SNO status in BAT and further developing a therapy to target ADH5 in BAT to prevent age-related decline, the authors have identified a putative mechanism to combat age-related decline in BAT function.

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? For all of the ADH5 knockout mouse data, are these also male mice? Do female ADH5 knockout mice have a consistent phenotype, or are the sex differences?

(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).

(3) For Figure 4D, the ChiP, it would be better to show the IgG control pulldowns. Also, there's an unexpected thing where all the values for the Adh5 flox mice are exactly the same - how is this possible? Finally, it's not clear how these BAT samples were treated with HSF1A - was this done in vivo or ex vivo?

(4) I didn't understand what was on the y-axis in Figure 5A, nor how it was measured. I assume it's HSF1A, and maybe it's the part in the methods with the Metabolomic Analysis, but this wasn't clear. It would also help if release from the NC-Vehicle formulation could be included as a negative control.

(5) What happens to BAT protein S-nitrosylation in HSF1A-treated mice?

(6) Figure 1B: What is the age of the positive (ADH5BKO) and negative (Adh5 fl) mice?

(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?

(8) Figure 3B looks a bit odd since 7 of the 12 total mice seem to have an IL-beat level of exactly 5. I was a bit unclear about why arbitrary units were used for IL-1β levels since it says an ELISA was used to quantify IL-1β; however, in the methods the authors describe a Bio-Rad Laboratories Bio-plex Pro Mouse Cytokine 23-Plex approach, which I don't think is an ELISA. 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?

(9) Figure 2C and 2D: I don't really understand why the Heat or VO2 need to be expressed as fold changes. Can't these just be expressed with absolute units? It's also confusing why the heat fold change is 1.0 in the light and the dark for the floxed animal. I bet this is because the knockout is normalized to the floxed animal for light and then normalized again for the dark period, but since both are on the same graph, readers could be confused into thinking there is no difference in the heat production or VO2 between light and dark, which would be surprising. This could all just be solved if absolute units were used.

Author response:

Reviewer #1 (Public review):

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 greatly appreciate the reviewer’s encouragement. Our team is fully committed to maintaining clarity and rigor in the design, execution, and reporting of this study. We are grateful to the reviewers for bringing these issues to our attention. We also acknowledge and are working on that several statistical analyses could be reperformed to better emphasize our focus on the genetic effect of ADH5 deletion in mice of the same age.

Reviewer #2 (Public review):

Strengths:

This research provides insight into the interplay between redox biology, proteostasis, and metabolic decline in aging. By identifying a specific enzyme that controls SNO status in BAT and further developing a therapy to target ADH5 in BAT to prevent age-related decline, the authors have identified a putative mechanism to combat age-related decline in BAT function.

We greatly appreciate the reviewer’s encouragement.

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.

We thank the reviewer for the insightful remark, and we agree with the reviewer that sex needs to be considered as a biological variable. We will assess ADH5 expression in aged female mice.

(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 the comment/suggestion. Indeed, we have measured the ADH5 expression in both brown adipose tissue (BAT) and inguinal adipose tissue (iWAT). We regret that we did not include our results in the first submission and will provide these results in the revised manuscript.

(3) For Figure 4D, the ChiP, it would be better to show the IgG control pulldowns. Finally, 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 and will provide detailed information in the revised manuscript.

(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 first submission. In the revised manuscript we will include, in detail, the logistics of the experiments in the materials and methods section, figure annotation and figure legends.

(5) What happens to BAT protein S-nitrosylation in HSF1A-treated mice?

We thank the reviewer for the insightful remark, and we will measure general protein Snitrosylation status in the BAT of HSF1A-treated mice.

(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 will provide 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 will provide detailed information 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 will provide detailed information in the revised manuscript.

(9) Figure 2C and 2D: I don't really understand why the Heat or VO2 need to be expressed as fold changes. Can't these just be expressed with absolute units?

We thank the reviewer for the insightful comment. We will present these results as suggested in the revised manuscript.