PONE-D-20-11137

β-adrenergic receptor signaling evokes the PKA-ASK axis in mature brown adipocytes

PLOS ONE

Dear Dr. Hattori,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your manuscript was reviewed by two knowledgeable referees in this area and their comments are appended. As you will see while they find your work of interest, they both have raised several points that will need to be properly addressed by the authors before I can proceed further. In particular, Reviewer#1 raised issues regarding potential adverse effects of viral transfection on brown adipocyte differentiation as well as the traits of ASKKO cells. The authors need to address all their comments to fully satisfy both reviewers.

We would appreciate receiving your revised manuscript by Jun 22 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Makoto Kanzaki, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the present manuscript, the authors identified the ASK family as a crucial signaling molecule bridging PKA and MAPK in mature brown adipocytes. Mechanistically, the phosphorylation of ASK1 at threonine 99 and serine 993 is critical in PKA-dependent ASK1 activation. Additionally, PKA also activates ASK2, which contributes to MAPK regulation. However, a couple of issues need to be addressed.

Major points:

1. In this study, immature brown adipocytes were treated with adenovirus-mediated PKIα, WT mouse ASK1, mouse ASK1 S973A, and mouse ASK2, on day 2. The authors should check the effect of the viral infection (PKIα, ASK1, S973A, and ASK2) on the differentiation of brown fat cells.

2. As the author mentioned, the expression of ASK2 alone is unstable and because ASK1 can prevent degradation. However, in Figure 1C, p38 and JNK activation could be observed in ASK1KO cells. The authors should check the expression of the other ASKs in ASK1KO cells.

3. In Figure 4B,C,D, the authors used the antibodies for phospho-ASK (T845) and phospho-ASK (T807) to indicate the activation of ASK, respectively. What is the difference between these two phosphorylation sites? The authors should add information about these two phosphorylation sites.

4. The authors should check the differentiation makers (aP2, PPARγ, c/EBPα, PGC1α) of ASKKO adipocytes by western blot and qPCR.

Reviewer #2: In this manuscript, authors further studied the function of ASK1 on beta3 adrenergic signaling pathway in mature brown adipocyte. Same with immature brown adipocyte, ASK1 was also required in this pathway. Besides, they found that the phosphorylation of Thr99 and Ser33 was critical for PKA-dependent AKS1 activation. The knockout of ASK1 or ASK2 both reduced the expression of UCP1 in mature brown adipocyte. These experiments were well designed and performed.

But it is not clear why authors did not continue to investigate the effect of ASK on thermogenesis in mature adipocyte but to examine its function in glucose clearance. It is confusing that why the specific deletion of ASK1 in adipose tissue had no effect on glucose clearance, since adipose tissue is also responsible to glucose absorbance.

1. ASK1 can upregulate the expression of Ucp1 in immature brown adipocyte, and it also promoted Ucp1’s expression in mature brown adipocyte. Is there any mechanic difference?

2. Blood glucose concentrations should be tested in ASK1 AKO mice after CL injection, since CL is helpful to clear glucose.

3. How about glucose uptake in ASK KO adipocyte in vitro?

4. Gene names need to be italic. All characters in a protein name need to be capitalized.

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Reviewer #1: No

Reviewer #2: No

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β-adrenergic receptor signaling evokes the PKA-ASK axis in mature brown adipocytes

PONE-D-20-11137R1

Dear Dr. Hattori,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Makoto Kanzaki, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Your revised manuscript was sent to the original referees, but one reviewer was unfortunately unable to review the revised manuscript. Upon careful reading by myself, I and another reviewer found that the revised manuscript has been much improved by addressing most of concerns raised by both reviewers. So I made this decision without sending it to new reviewer to avoid further delay. 

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Responses are satisfactory. The manuscript has been improved properly and can be considered to be accepted.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No