The combination of zalfermin and semaglutide has additive therapeutic effects in a diet-induced obese and biopsy-confirmed mouse model of MASH

Jenny Norlin; Maria Dermit; Nikos Sidiropoulos; Elisabeth D. Galsgaard; Henrik H. Hansen; Michael Feigh; Sanne S. Veidal; Markus Latta; Emma Henriksson; Birgitte Andersen

doi:10.1371/journal.pone.0331665

Peer Review History

Original SubmissionFebruary 3, 2025
7 Apr 2025 Decision Letter - Habib Yaribeygi, Editor Dear Dr. Andersen, 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. Please submit your revised manuscript by May 22 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. 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'. 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Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: This preclinical study investigated the effects of zalfermin, a novel long-acting FGF21 analogue, both alone and in combination with semaglutide, in the AMLN DIO-MASH mouse model—a well-established, biopsy-confirmed model of MASH and fibrosis. The findings highlight the additive benefits of a combined low-dose zalfermin and semaglutide treatment, which led to metabolic, biochemical, and histological improvements comparable to or exceeding those observed with high-dose monotherapies. Hepatic transcriptome analyses suggest that zalfermin’s effects, both as a monotherapy and in combination, were partially mediated by weight loss–independent molecular mechanisms. The data indicate that treatment improved liver metabolic markers, inflammation, and ER stress pathways, with these changes largely attributable to weight loss. However, a subset of ECM-related genes remained suppressed after adjusting for body weight in DIO-MASH mice, while PSR staining results suggested no significant improvement in liver fibrosis. Given the growing interest in FGF21 and incretin-based therapies for MASH, this study’s focus on the combination of an FGF21 analogue with semaglutide is particularly relevant to a broad audience. The manuscript is well-written and the data well-organized, with only a few minor comments: 1. In Fig. 1C, the Y-axis unit should be "%" instead of "g." 2. Some scale bars are difficult to see; please correct them, such as in the right panels of Fig. 3 and Fig. S3. 3. In Fig. S1B, label the group information. Reviewer #2: Really interesting article and a pleasure to read. The study is well-executed, and you successfully integrate different aspects—pharmacotherapy, genetics, and histology. It makes me curious about how humans would respond to this protocol. I have just four remarks: 1) The abstract would be more easy to read if you add the sections "background, methods, results, conclusion" 2) There is a typo in the title: "has additive therapeutic" instead of "hasadditivetherapeutic" 3) In the conclusion, you should emphasize more strongly that combination therapy is effective for both weight loss and histological changes. Otherwise, one might be inclined to think that a high dose of either zalfermin or semaglutide could be an alternative. 4)How do you explain the increased food intake observed with zalfermin while still achieving weight loss? In your discussion, you mention the preference for protein sources, but mice still consume more overall. ****** what does this mean? ). 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Revision 1
20 May 2025 Author Response REBUTTAL LETTER Dear editor, Thank you for the swift and positive handling of our manuscript entitled “The combination of zalfermin and semaglutide has additive therapeutic effects in a diet-induced obese and biopsy-confirmed mouse model of MASH.” We sincerely appreciate the reviewers’ insightful and constructive comments, which have helped us improve the manuscript. We have carefully addressed each point raised and provided our detailed responses below (indicated in blue italics). Revisions in the manuscript are highlighted in yellow for clarity. We believe the editorial process has significantly strengthened the manuscript, and we hope it is now suitable for publication. As requested, please see the amended statements below. Competing Interests Statement: The study was funded by Novo Nordisk A/S. The funder was involved in the study design, data collection and analysis, decision to publish, and preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. The RNA sequencing data are available in the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/; accession number GSE256063). The code used to generate the results is available in the GitHub repository (https://github.com/novonordisk-research/Zalfermin_liver_RNAseq). On behalf of all co-authors, Yours sincerely, Birgitte Andersen, Scientific Vice President, Diabetes, Obesity & MASH, Novo Nordisk REVIEWER 1 This preclinical study investigated the effects of zalfermin, a novel long-acting FGF21 analogue, both alone and in combination with semaglutide, in the AMLN DIO-MASH mouse model, a well-established, biopsy-confirmed model of MASH and fibrosis. The findings highlight the additive benefits of a combined low-dose zalfermin and semaglutide treatment, which led to metabolic, biochemical, and histological improvements comparable to or exceeding those observed with high-dose monotherapies. Hepatic transcriptome analyses suggest that zalfermin’s effects, both as a monotherapy and in combination, were partially mediated by weight loss independent molecular mechanisms. The data indicate that treatment improved liver metabolic markers, inflammation, and ER stress pathways, with these changes largely attributable to weight loss. However, a subset of ECM-related genes remained suppressed after adjusting for body weight in DIO-MASH mice, while PSR staining results suggested no significant improvement in liver fibrosis. Given the growing interest in FGF21 and incretin-based therapies for MASH, this study’s focus on the combination of an FGF21 analogue with semaglutide is particularly relevant to a broad audience. The manuscript is well-written and the data well-organized, with only a few minor comments: 1. In Fig. 1C, the Y-axis unit should be "%" instead of "g." 2. Some scale bars are difficult to see; please correct them, such as in the right panels of Fig. 3 and Fig. S3. 3. In Fig. S1B, label the group information. Response: Thank you for bringing these issues to our attention. The typographical error has been corrected (Fig. 1C), scale bars have been enhanced for better visibility (Fig. 3, Fig. S3), and legends have been added where needed (Fig. S1B). REVIEWER 2 Really interesting article and a pleasure to read. The study is well-executed, and you successfully integrate different aspects, pharmacotherapy, genetics, and histology. It makes me curious about how humans would respond to this protocol. I have just four remarks: 1) The abstract would be more easy to read if you add the sections "background, methods, results, conclusion" Response: Although the abstract is structured for clarity, we note that PLOS ONE does not permit subheadings in abstracts. 2) There is a typo in the title: "has additive therapeutic" instead of "has additive therapeutic" Response: The manuscript title reads ‘The combination of zalfermin and semaglutide has additive therapeutic effects in a diet-induced obese and biopsy-confirmed mouse model of MASH’. We have reviewed it carefully and did not identify any typographical errors. 3) In the conclusion, you should emphasize more strongly that combination therapy is effective for both weight loss and histological changes. Otherwise, one might be inclined to think that a high dose of either zalfermin or semaglutide could be an alternative. Response: We thank the reviewer for this insightful comment. In our preclinical study, the low-dose combination of zalfermin and semaglutide significantly improved both metabolic parameters and liver histology in MASH. However, since high-dose zalfermin alone demonstrated comparable efficacy across most endpoints, these findings also support the potential of zalfermin as a monotherapy. We have revised the Conclusion section to reflect both therapeutic options (p. 28). 4) How do you explain the increased food intake observed with zalfermin while still achieving weight loss? In your discussion, you mention the preference for protein sources, but mice still consume more overall. Response: We speculate that zalfermin-induced changes in nutrient preference could support beneficial metabolic adaptations despite increased caloric intake. Increased protein intake has been associated with fat mass loss which contributes to reduced body weight and improved metabolic health. This aspect is now considered in the Discussion (p.25). https://doi.org/10.1371/journal.pone.0331665.r002
20 Aug 2025 Decision Letter - Nobuyuki Takahashi, Editor The combination of zalfermin and semaglutide has additive therapeutic effects in a diet-induced obese and biopsy-confirmed mouse model of MASH PONE-D-25-05768R1 Dear Dr. Andersen, 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. For questions related to billing, please contact billing support . 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, Nobuyuki Takahashi, Ph.D. Academic Editor PLOS ONE Additional Editor Comments: I apologize for the delay in the review process. Although one reviewer provided no response regarding a minor revision, I have decided to accept your revision as an academic editor. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: (No Response) ****** 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 ******** https://doi.org/10.1371/journal.pone.0331665.r003
Formally Accepted
Acceptance Letter - Nobuyuki Takahashi, Editor PONE-D-25-05768R1 PLOS ONE Dear Dr. Andersen, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Nobuyuki Takahashi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0331665.r004

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