PONE-D-25-12994Glucagon dysfunction in the liver induces hyperplasia of PP cells and the production of glucagon and pancreatic polypeptide double-positive cellsPLOS ONE

Dear Dr. Fujitani,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has great opportunity to publish in our Journal. However, we recommend some minor corrections to 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.

Please submit your revised manuscript by May 16 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Mohammad Sarif Mohiuddin, MD, PhD 

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“This work was supported by Grants-in-Aid for Scientific Research (18K08501 and 21K08547 to YN) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This work was supported by Grants-in-Aid for Scientific Research (18K08501 and 21K08547 to YN) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We extend our heartfelt gratitude to W. Mizutani, Y. Tamura, A. Suda, N. Kagami, K. Iizuka, A. Fukaishi, Y. Miyazaki, H. Netsu, and T. Ishizaka of the Fujitani Laboratory (Gunma University, Gunma, Japan) for their invaluable technical assistance. We are also deeply grateful to Dr. H.A. Popiel (Tokyo Medical University, Tokyo, Japan) for critically reviewing the manuscript. Our sincere thanks go to the members of Dr. Y. Kitamura’s laboratory and Dr. J. Shirakawa’s laboratory (both from IMCR, Gunma University) for their insightful discussions. We also thank Dr. A. Oue, Dr. T. Kakinuma, and other members of the Bioresource Center at Gunma University for their generous support of general research on mice.”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“This work was supported by Grants-in-Aid for Scientific Research (18K08501 and 21K08547 to YN) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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: Yes

**********

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: This manuscript provides an excellent investigation into the physiological link between glucagon signaling and pancreatic polypeptide (PP) cell hyperplasia using two or more different mouse models with glucagon signaling deficiency. However, there are a few minor issues in the structure and presentation of the manuscript that need attention. I would recommend revising these to improve the clarity and flow of the paper. Additionally, it might be beneficial to add a few more data or analyses to further support the conclusions, particularly in regard to the molecular mechanisms underlying the observed phenotypes.

In the Methods section, under "Immunohistochemistry," the fixation method through perfusion is described. However, there is an inconsistency between this description and the handling of tissues in the "Quantification of α- and PP-cell mass" section. Please revise the manuscript to ensure consistency between these two sections.

In the "Islet culture" section, there is insufficient information about how islets were collected. A brief additional explanation would be helpful. Also, at the end of this section, the embedding with OCT compound is mentioned, but there is no further description about immunostaining. Please either add this explanation to this section or include it in the "Immunohistochemistry" section.

In the "Metabolomic analysis" methods, there is no explanation about which tissues' "frozen samples" were used, and the Results section does not mention the results obtained using this method. This should be clarified and included.

In Figure 2B, the ratio of Gcg/PP double-positive cells is quantified. It would be helpful to perform the same quantification in Figure 1B for consistency. Additionally, in Figure 1B, SST staining is shown; please include SST staining in Figure 2B as well and provide the ratios of single, double, and triple-positive cells for each marker.

In the lines after 305, the term "self-proliferation" is used multiple times, but since it has not been evaluated whether the proliferation is truly "self" or not, it would be more appropriate to simply use the term "proliferation."

In Figures 3D and 3F, the ratios are used for quantification. However, as with Figure 3B, showing mass or the ratio relative to the total islet cells would help enhance the reader's understanding.

In Figure 5F, including the Ctrl+Rap group would make the comparison with the control group clearer.

Reviewer #2: 1)The authors identify GCG+ PP+ and PP+ INS+ cells but do not assess whether these cells are functional. Do they secrete both hormones?

Authors can try hormone secretion assays like ELISA for Glucagon or PP after sorting the cells.

2)The origin of double-positive cells remains unclear. Are α cells adopting PP identity, or are they a distinct lineage?

3)While mTOR activation is implicated, the downstream effectors remain undefined. Authors should check at least mRNA expression of mTOR pathway targeted genes.

4)There is no information about the sex of the mice. Did the study design with both mice? If both types of mice are used for the study, then author should observe whether the phenotypes are sex dependent or not?

5)Provide a graphical model summarizing the proposed mechanism.

6)Although the current title is technically accurate, it is somewhat complex and may be difficult for readers to grasp easily. I recommend simplifying the title to enhance clarity and accessibility, particularly for a broad, interdisciplinary readership. Consider rephrasing to more directly convey the central findings, such as: “Liver-specific glucagon dysfunction promotes PP-cell hyperplasia and formation of glucagon+ PP+ endocrine cells.”

**********

6. 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 #1: No

Reviewer #2: Yes:  Rashu Barua

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Liver-specific glucagon dysfunction promotes PP-cell hyperplasia and formation of glucagon and PP double-positive cells

PONE-D-25-12994R1

Dear Dr. Fujitani,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Mohammad Sarif Mohiuddin, MD, PhD 

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: