Loss of the alternative calcineurin variant CnAβ1 enhances brown adipocyte differentiation and drives metabolic overactivation through FoxO1 activation

  1. Elísabet Bello-Arroyo
  2. Blanca Rubio
  3. Alfonso Mora
  4. Marina López-Olañeta
  5. Jesús M. Gómez-Salinero
  6. Laura Ramos
  7. Carlos G. Sánchez-Cabezudo
  8. Emilio Camafeita
  9. Lorena Cusso
  10. Manuel Desco
  11. Nolwenn Joffin
  12. Johanne Le Coq
  13. Jasminka Boskovic
  14. Kathryn A. McGurk
  15. James S. Ware
  16. Paul J. R. Barton
  17. Jesús Vázquez
  18. Philipp E. Scherer
  19. Guadalupe Sabio
  20. María Victoria Gómez-Gaviro
  21. Enrique Lara-Pezzi

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

The alternative calcineurin A variant CnAβ1 has a unique C-terminal domain that provides it with distinct subcellular localization and mechanism of action different from other calcineurin isoforms. Here, we used mice lacking CnAβ1’s C-terminal domain (CnAβ1 Δi12 ) to show that the absence of this specific isoform strongly reprograms metabolism. CnAβ1 Δi12 mice on a high-fat diet showed reduced body weight, white adipose tissue (WAT) mass, and circulating triglycerides, together with enhanced insulin sensitivity. In brown adipose tissue (BAT), CnAβ1 deficiency increased mitochondrial content and upregulated fatty acid oxidation and thermogenic proteins, improving cold resistance. Conversely, under starvation, CnAβ1 Δi12 mice experienced rapid fat depletion and hypothermia. Importantly, BAT-specific FoxO1 knockout in CnAβ1 Δi12 mice reduced catabolism-related gene expression and partially reversed the metabolic phenotypes, increasing body weight and WAT mass. Our findings reveal a relevant role for CnAβ1 in orchestrating BAT metabolism, highlighting its potential as a therapeutic target for obesity and metabolic syndrome.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Manuscript number: RC-2025-03031

    Corresponding author(s): Lara-Pezzi, Enrique and Gómez-Gaviro, María Victoria

    1. General Statements [optional]

    Dear Editors,

    Following the review of our article entitled "Loss of the alternative calcineurin variant CnAβ1 enhances brown adipocyte differentiation and drives metabolic overactivation through FoxO1 activation", we propose below a number of experiments to be performed in order to address the issues raised by the reviewers.

    While we acknowledge the limitations of the full CnAβ1 knockout mouse and we unfortunately lack a tissue-specific knockout mouse, we believe that the proposed new experiments together with the (abundant) existing information in the paper will help clarify the concerns raised by the reviewers.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    *The current study examines the metabolic phenotype of mice lack the calcineurin variant CnAb1 (CnAb1KO). On a high fat diet, CnAb1KO mice gain less weight compared to WT controls, which is accompanied by improvements in obesity-related metabolic dysfunction, such as glucose/insulin intolerance and hyperlipidemia. The authors attribute most of the observed phenotypes to enhanced brown fat function, notably fatty acid catabolism and the thermogenic capacity. Mechanistically, the authors propose that CnAb1KO increases FoxO1 transcriptional activity, as a result of reduced mTOR/Akt signaling, which in turn mediates the hyper-catabolism of BAT in CnAb1KO mice. *

    Major comments: *

    *Q1. The main issue of the study is it's not hypothesis driven. Based on high fat diet-induced metabolic phenotype of the whole body CnAb1KO mice, the authors put together a mechanism focusing on potential roles of CnAb1 in BAT functions that affect systemic metabolic homeostasis. However, the rationales to establish this link were based largely on correlative results and at times incorrect data interpretation (for instance, using the expression of Myf5 and Pax7 as markers for brown adipocyte differentiation). The sequential event from CnAb1 loss of function to reduced mTOR signaling and increased FoxO1 activity (or conversely, how CnAb1 increases mTOR signaling to reduce FoxO1 activity) has not been mechanistically characterized. There are also no studies to explain how FoxO1 is involved in brown fat differentiation and hyper-catabolism of BAT downstream of the CnAb1-mTOR pathway. In addition, the UCP-1 FoxO1KO experiment in Fig. 6 fails to provide strong evidence to support the claim. Thus, there are many gaps between the observed phenotype and the proposed mechanism. *

    A1. We thank the reviewer for the insightful comments. We agree with the reviewer that, historically, this project did not originally focus on the BAT. Instead, we arrived at the BAT after ruling out other possibilities to explain the reduced body weight observed in these animals, together with the reduced body temperature after starvation, which was our first observation. While the BAT involvement was not our first hypothesis a priori, we do not agree that this would invalidate or reduce the interest of our work. While our initial evidence may have been correlative at first, the FoxO1 BAT-specific knockout experiments and the AAV/Ucp1-Cre CnAβ1 expression restoration experiments prove that the BAT is indeed involved in the phenotype observed in CnAβ1Δi12 (KO) mice. It is likely that other organs may be also involved (since the phenotype is not fully prevented by the BAT-specific approaches) but the BAT is definitely involved.

    To further substantiate the involvement of the BAT in the improved metabolic phenotype observed in CnAβ1Δi12 mice, we propose to perform BAT transplantation, monitoring body weight over 8 weeks following transplantation. If successful, BAT transplantation from CnAβ1Δi12 mice into WT mice should improve their metabolic response to high-fat diet (HFD), thereby reinforcing the role of the BAT in these mice.

            In addition, we propose to measure the __*levels of so-called batokines*__ FGF21, VEGFA, IL6, and also of 12,13-diHOME in BAT and serum from 12-week-old chow and HFD mice.

        With regards to Pax7 and Myf5, while we agree that these are common precursors to other lineages (skeletal muscle), we show in Fig. S1E additional differentiation markers such as Cox2 and Cpt1b. __*The 5 markers assessed showed an increase in *____*CnAβ1Δi12 mice, pointing towards a cell-autonomous effect of the absence of CnAβ1 on the BAT*__. Nevertheless, to further substantiate the accelerated differentiation of brown preadipocytes in the absence of CnAβ1, we propose to __*measure the expression of additional BAT markers*__ (although they are not exclusive of BAT), such as Ucp1, Prdm16, PPARγ, and AdipoQ in brown preadipocytes isolated from 6–8-week-old mice.

        With regards to the activation of mTOR (specifically mTORC2) by CnAβ1, we published this in previous papers from our group: Gómez-Salinero et al (Cell Chem Biol, 2016), Felkin et al (Circulation, 2011), Lara-Pezzi et al (J Cell Biol 2007), Padrón-Barthe et al (J Am Coll Cardiol 2018). The mechanism involves the interaction between CnAβ1 and mTORC2 in cellular membranes. Knockdown of CnAβ1 results in mTORC2 mislocalisation and Akt inhibition. In addition, we show in Fig. 6C in this paper that PTEN inhibition reduces the improved differentiation of BAT adipocytes from CnAβ1Δi12 mice, further involving the Akt pathway in the observed phenotype. Furthermore, Fig. 6 shows a significant increase in body weight and BAT weight in BAT-specific FoxO1 knockout CnAβ1Δi12 mice, together with a significant decrease in different Pnpla1, Irf4, and Bcat2 expression. While we agree that the reversal of the phenotype is only partial, the effect of knocking out FoxO1 in the BAT of CnAβ1Δi12 mice is both statistically significant and biologically relevant. We would be happy to provide additional information at the Editors’ request. In addition, we propose to carry out __BAT preadipocyte differentiation experiments comparing cells isolated from CnAβ1Δi12 mice to those isolated from CnAβ1Δi12 mice with BAT-specific FoxO1 knockout__.

    Q2. A second issue is that most of the phenotypes can be explained by the difference in weight gain. With the available data, it's difficult to pinpoint the tissue origin(s) mediating the weight gain/loss phenotype. The authors would first need to generate a BAT-CnAb1KO mouse line to convincingly show a main role for BAT CnAb1 in systemic metabolic homeostasis. There are also many problems with data presentations/interpretations of the metabolic phenotyping studies. For example, Fig. 1A shows that CnAb1KO mice are about 5 g lighter than controls. However, Fig. 1G indicates a 10 g difference in fat mass. The EM images in Fig. 3B are of poor quality, which seems to suggest that HFD fed CnAb1KO mice have the highest mitochondrial density. Lastly, in Fig. 4C/D, the authors interpret the reduced FFA and glycerol levels in CnAb1KO after b3-agonist injection as increased fatty acid burning by BAT, which is incorrect. If anything, the reduced glycerol release in the KO mice would suggest a reduction in lipolysis. However, the most likely explanation is that WT mice have more fat mass and as such, more fat hydrolysis.

    A2. While we agree with the reviewer that some of the features may be explained by reduced body weight gain (reduced WAT weight, for instance), many other changes showed by CnAβ1Δi12 mice cannot be explained by reduced body weight gain alone, including higher expression of differentiation markers in BAT, higher number of mitochondria in BAT, or improved cold-tolerance, among others. Therefore, we respectfully disagree with the reviewer’s opinion.

    Unfortunately, we do not have a tissue-specific CnAβ1 knockout mouse and we cannot commit to having one in the short term. While we acknowledge the limitations of using a full knockout mouse, we provided several pieces of evidence that the BAT is involved in the observed phenotype, as pointed out in the discussion: 1) Placing CnAβ1Δi12 mice in thermoneutral conditions mitigated the weight loss. 2) Reintroducing CnAβ1 in BAT with a CnAβ1-overexpressing virus partially prevented the weight loss. 3) Minimal changes in mitochondrial gene expression were observed in skeletal muscle and liver, suggesting that the phenotype is primarily driven by alterations in BAT. 4) BAT adipocytes from CnAβ1Δi12 mice differentiated more effectively than those from wild type mice, suggesting a cell-autonomous effect. While a direct effect of CnAβ1 on WAT cannot be entirely ruled out, our results strongly suggest that loss of CnAβ1 in BAT is a major contributor to the observed metabolic changes.

            With regards to Fig. 1E, this is an estimation of fat weight from __MRI__ images. We agree with the reviewer that this is obviously wrong and we will __revise this quantification__. We propose to __add measurements of subcutaneous WAT__, which we also have, to further support the difference observed in eWAT.

        With regards to Fig. 3B, we agree that some of the individual figures may have been poorly chosen, but the graph in Fig. 3C (which quantifies the electron microscopy pictures) clearly shows that the reduction in mitochondria in WT mice as a result of HFD feeding is prevented in CnAβ1Δi12 mice. Fig. 3C does not show an increase in mitochondria with HFD, as implied by the reviewer based on Fig. 3B. We propose to __provide adequate panels for Fig. 3B that better reflect the averages shown in Fig. 3C__.

        Regarding Fig. 4C and D, we thank the reviewer for this correction, which we agree with. We still believe that the BAT of CnAβ1Δi12 mice is burning fat more effectively than that of WT mice, but we agree that these experiments are not the proof of this claim. We will__ move or remove panels C and D from Fig. 4__ and focus this figure on thermogenic capacity.

        To assess systemic lipolysis, we will __measure in vivo serum levels of NEFA__ (non-esterified fatty acids) __and glycerol__ in 12-week-old mice fed a HFD. Additionally, to evaluate BAT lipolytic activation, we will perform __BAT explant and *ex vivo* experiments__ to determine the lipolysis rate. This should provide valuable information supporting the role of the BAT in the observed phenotype in CnAβ1Δi12 mice.

    *Q3. The authors should take a fresh, unbiased look at existing data, form a testable hypothesis and design a series of new experiments (including new tissue-specific KO mice) to assess the function of CnAb1 in BAT or other tissues responsible for the metabolic phenotype. If BAT is indeed involved, the authors need to mechanistically determine the role of CnAb1 in brown adipocyte differentiation vs BAT function and explain why the ratio of CnAb1/CnAb2 ratio matters in this context, as this is the basis for the entire study. A revision addressing main issues of the manuscript will not likely to be completed in a typical revision time (e.g. 3 months). *

    A3. As explained above, unfortunately we do not have tissue-specific CnAβ1 knockout mice. If the Editors consider that this is essential for resubmission of a revised article, we are afraid that we cannot comply. This said, we believe that our manuscript contains relevant data about metabolic regulation by the CnAβ1 calcineurin isoform that are new and relevant to the field.

            Our data provide clear evidence that the BAT is indeed involved in the phenotype observed in CnAβ1Δi12 mice, as explained in our previous answers above. It may not be the *only* tissue involved, but it is most definitely involved. The BAT transplant experiments will add further evidence of this.

        We already show evidence of the role of CnAβ1 (or rather, its absence) in the differentiation of BAT pre-adipocytes (Fig. S1E and Fig. 6C) and we will __provide additional evidence through the proposed new experiments__. Similarly, we provide evidence of the role of CnAβ1 in BAT weight, transcriptional profile, lipid content, and number of mitochondria. Also here, we believe that __the proposed experiments will reinforce this aspect of the paper__.

    Reviewer #1 (Significance (Required)):

    *Q4. The thermogenic capacity of brown and beige adipocytes has shown promise as a means to reduce fat burden to treat obesity and related metabolic diseases. Identification of brown/beige adipocyte promoting mechanisms may provide druggable targets for therapeutic development. As such, the topic and findings of the current study would be of interest to researchers in the metabolism and drug development fields. The weakness of the study is that it's descriptive and the authors jump to conclusions without strong supporting evidence. Most of the metabolic phenotypes associated with CnAb1KO mice are likely secondary to the weight difference. The rationale to focus on BAT is not well justified. A well-thought-out approach would be needed to identify the tissue origins mediating the metabolic phenotypes of CnAb1KO mice and to dissect the underlying mechanisms. *

    *Reviewer's field of expertise: adipose tissue biology, systemic metabolic regulation, immunometabolism *

    A4. We agree with the reviewer about the potential relevance of our findings. The shortcomings pointed out in this comment have been addressed above. Overall, we thank the reviewer for their thorough review of our ms.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    *The manuscript entitled « Loss of the alternative calcineurin variant CnAβ1 enhances brown adipocyte differentiation and drives metabolic overactivation through FoxO1 activation » by Dr Lara-Pezzi and colleagues describes the role of the calcium/calmodulin dependent serine/threonine phosphatase catalytic subunit calcineurin variant CnAß1 in brown adipose tissue physiology and function. Through the use of global CnAß1 KO mice, the authors show that these mice are resistant to diet-induced obesity, have increased thermogenesis due to increased mitochondrial activity, decreased body weight, improved glucose homeostasis, increased fatty acid oxidation. The authors also demonstrate that these effect are mostly mediated through improved brown adipose tissue (BAT) function, through increased Foxo1 activation in BAT. Genetic deletion of Foxo1 in BAT resulted in increased body weight and impaired mitochondrial gene expression. In addition, the authors also correlate their findings to potential CNAß1 polymorphism from the UK biobank associated to improved metabolic traits in humans (blood glucose mainly). *

    Although interesting, the conclusion are not always supported by the data. The manuscript requires additional experiments to further consolidate their claims.

    *Q1. It should be mentioned that all experiments are performed in global CnAβ1 KO mice. Thus, it is difficult to assess the cell-autonomous role if this protein in BAT function (even if an AAV9 driving CnAβ1 expression is used; or if other tissues have been studied). This should be discussed at least as a limitation of the study, except if floxed mice are available. *

    A1. We thank the reviewer for the positive comments about our work.

    Unfortunately, we do not have a tissue-specific CnAβ1 knockout mouse. However, we believe we provide abundant evidence of the involvement of the BAT in the phenotype observed in CnAβ1Δi12 mice, including the following: 1) Placing CnAβ1Δi12 mice in thermoneutral conditions mitigated the weight loss. 2) Reintroducing CnAβ1 in BAT with a CnAβ1-overexpressing virus partially prevented the weight loss. 3) Minimal changes in mitochondrial gene expression were observed in skeletal muscle and liver, suggesting that the phenotype is primarily driven by alterations in BAT. 4) BAT adipocytes from CnAβ1Δi12 mice differentiated more effectively than those from wild type mice, suggesting a cell-autonomous effect. While a direct effect of CnAβ1 on WAT cannot be entirely ruled out, our results strongly suggest that loss of CnAβ1 in BAT is a major contributor to the observed metabolic changes.

    This said, we fully agree with the reviewer to acknowledge in the discussion the limitation of using a full knockout mouse for this study.

    Q2. Is there good antibodies for CnAβ1? The protein levels of the protein should be shown in, at least, adipose tissues of WT and KO mice under chow and HFD.

    A2. There is no good antibody against CnAβ1. The main reason is that the C-ter domain of this isoform is not very immunogenic. We did try to generate an antibody, but we got no immune response against the unique C-ter domain. We do have an old antibody generated against CnAβ1 years ago. We propose to try to perform WB and immunohistochemistry in WT and ____CnAβ1Δi12 mice. However, we need to be clear that we cannot make any commitments towards these results, since the antibody may not work. In any case, we believe that the RT-PCR results, which clearly discriminate both isoforms, are very clear.

    *Q3. A general comment is that most of the conclusions are drawn from qRT-PCR data. It lacks functional experiments that may reinforce the conclusion. For example, did the authors measure mitochondrial function in BAT of WT and KO mice using different substrate (fatty acids, glucose, ...)? *

    A3. We thank the reviewer for this suggestion and we therefore propose to include in the revised paper measurements of mitochondrial activity with different substrates in WT and ____CnAβ1Δi12 mice.

    *Q4. Lack of validation of the mouse model used (CnAβ1 expression in BAT upon AAV9 over expression confirmed? What about the other tissues?). *

    A4. We showed in Fig. 5E the increase in CnAβ1 expression in the BAT of Ucp1-Cre mice infected with the floxed AAV-CnAβ1 virus. We propose to include similar expression analyses in other tissues.

    Reviewer #2 (Significance (Required)):

    Q5. This is a novel study addressing the role of CnAβ1 in energy homeostasis, more specifically in BAT function. This study reports for the first time the role of CnAβ1 in energy homeostasis, with new mechanistic insights related to the crosstalk between CnAβ1 and Foxo1.

    The authors have previously described the role of this protein in cardiac function. There are not a lot of publications describing the function of this protein, thus this study may be interested for the community working on diabetes/obesity/cardio-metabolic field.

    *Limitations : see below (lack of functional data, ...). *

    A5. We thank the reviewer for these comments, with which we agree.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    4. Description of analyses that authors prefer not to carry out

    As much as we would like to have a tissue-specific CnAβ1 knockout mouse, the reality is that we do not have it. In any case, we believe that our paper provides a considerable amount of data that is relevant to the field.

    We remain open to incorporating the suggested experiments, or others, should they be considered necessary to further strengthen the manuscript.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    The manuscript entitled « Loss of the alternative calcineurin variant CnAβ1 enhances brown adipocyte differentiation and drives metabolic overactivation through FoxO1 activation » by Dr Lara-Pezzi and colleagues describes the role of the calcium/calmodulin dependent serine/threonine phosphatase catalytic subunit calcineurin variant CnAß1 in brown adipose tissue physiology and function. Through the use of global CnAß1 KO mice, the authors show that these mice are resistant to diet-induced obesity, have increased thermogenesis due to increased mitochondrial activity, decreased body weight, improved glucose homeostasis, increased fatty acid oxidation. The authors also demonstrate that these effect are mostly mediated through improved brown adipose tissue (BAT) function, through increased Foxo1 activation in BAT. Genetic deletion of Foxo1 in BAT resulted in increased body weight and impaired mitochondrial gene expression. In addition, the authors also correlate their findings to potential CNAß1 polymorphism from the UK biobank associated to improved metabolic traits in humans (blood glucose mainly).

    Although interesting, the conclusion are not always supported by the data. The manuscript requires additional experiments to further consolidate their claims.

    It should be mentioned that all experiments are performed in global CnAβ1 KO mice. Thus, it is difficult to assess the cell-autonomous role if this protein in BAT function (even if an AAV9 driving CnAβ1 expression is used; or if other tissues have been studied). This should be discussed at least as a limitation of the study, except if floxed mice are available. Is there good antibodies for CnAβ1? The protein levels of the protein should be shown in, at least, adipose tissues of WT and KO mice under chow and HFD.

    A general comment is that most of the conclusions are drawn from qRT-PCR data. It lacks functional experiments that may reinforce the conclusion. For example, did the authors measure mitochondrial function in BAT of WT and KO mice using different substrate (fatty acids, glucose, ...) ? Lack of validation of the mouse model used (CnAβ1 expression in BAT upon AAV9 over expression confirmed? What about the other tissues?).

    Significance

    This is a novel study addressing the role of CnAβ1 in energy homeostasis, more specifically in BAT function. This study reports for the first time the role of CnAβ1 in energy homeostasis, with new mechanistic insights related to the crosstalk between CnAβ1 and Foxo1.

    The authors have previously described the role of this protein in cardiac function. There are not a lot of publications describing the function of this protein, thus this study may be interested for the community working on diabetes/obesity/cardio-metabolic field.

    Limitations: see below (lack of functional data, ...).

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The current study examines the metabolic phenotype of mice lack the calcineurin variant CnAb1 (CnAb1KO). On a high fat diet, CnAb1KO mice gain less weight compared to WT controls, which is accompanied by improvements in obesity-related metabolic dysfunction, such as glucose/insulin intolerance and hyperlipidemia. The authors attribute most of the observed phenotypes to enhanced brown fat function, notably fatty acid catabolism and the thermogenic capacity. Mechanistically, the authors propose that CnAb1KO increases FoxO1 transcriptional activity, as a result of reduced mTOR/Akt signaling, which in turn mediates the hyper-catabolism of BAT in CnAb1KO mice.

    Major comments:

    1. The main issue of the study is it's not hypothesis driven. Based on high fat diet-induced metabolic phenotype of the whole body CnAb1KO mice, the authors put together a mechanism focusing on potential roles of CnAb1 in BAT functions that affect systemic metabolic homeostasis. However, the rationales to establish this link were based largely on correlative results and at times incorrect data interpretation (for instance, using the expression of Myf5 and Pax7 as markers for brown adipocyte differentiation). The sequential event from CnAb1 loss of function to reduced mTOR signaling and increased FoxO1 activity (or conversely, how CnAb1 increases mTOR signaling to reduce FoxO1 activity) has not been mechanistically characterized. There are also no studies to explain how FoxO1 is involved in brown fat differentiation and hyper-catabolism of BAT downstream of the CnAb1-mTOR pathway. In addition, the UCP-1 FoxO1KO experiment in Fig. 6 fails to provide strong evidence to support the claim. Thus, there are many gaps between the observed phenotype and the proposed mechanism.
    2. A second issue is that most of the phenotypes can be explained by the difference in weight gain. With the available data, it's difficult to pinpoint the tissue origin(s) mediating the weight gain/loss phenotype. The authors would first need to generate a BAT-CnAb1KO mouse line to convincingly show a main role for BAT CnAb1 in systemic metabolic homeostasis. There are also many problems with data presentations/interpretations of the metabolic phenotyping studies. For example, Fig. 1A shows that CnAb1KO mice are about 5 g lighter than controls. However, Fig. 1G indicates a 10 g difference in fat mass. The EM images in Fig. 3B are of poor quality, which seems to suggest that HFD fed CnAb1KO mice have the highest mitochondrial density. Lastly, in Fig. 4C/D, the authors interpret the reduced FFA and glycerol levels in CnAb1KO after b3-agonist injection as increased fatty acid burning by BAT, which is incorrect. If anything, the reduced glycerol release in the KO mice would suggest a reduction in lipolysis. However, the most likely explanation is that WT mice have more fat mass and as such, more fat hydrolysis.
    3. The authors should take a fresh, unbiased look at existing data, form a testable hypothesis and design a series of new experiments (including new tissue-specific KO mice) to assess the function of CnAb1 in BAT or other tissues responsible for the metabolic phenotype. If BAT is indeed involved, the authors need to mechanistically determine the role of CnAb1 in brown adipocyte differentiation vs BAT function and explain why the ratio of CnAb1/CnAb2 ratio matters in this context, as this is the basis for the entire study. A revision addressing main issues of the manuscript will not likely to be completed in a typical revision time (e.g. 3 months).

    Significance

    The thermogenic capacity of brown and beige adipocytes has shown promise as a means to reduce fat burden to treat obesity and related metabolic diseases. Identification of brown/beige adipocyte promoting mechanisms may provide druggable targets for therapeutic development. As such, the topic and findings of the current study would be of interest to researchers in the metabolism and drug development fields. The weakness of the study is that it's descriptive and the authors jump to conclusions without strong supporting evidence. Most of the metabolic phenotypes associated with CnAb1KO mice are likely secondary to the weight difference. The rationale to focus on BAT is not well justified. A well-thought-out approach would be needed to identify the tissue origins mediating the metabolic phenotypes of CnAb1KO mice and to dissect the underlying mechanisms.

    Reviewer's field of expertise: adipose tissue biology, systemic metabolic regulation, immunometabolism

  4. Version published to 10.1101/2025.05.09.653054 on bioRxiv