Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice

Linh V. Nguyen; Quang V. Ta; Thao B. Dang; Phu H. Nguyen; Thach Nguyen; Thi Van Huyen Pham; Trang HT. Nguyen; Stephen Baker; Trung Le Tran; Dong Joo Yang; Ki Woo Kim; Khanh V. Doan

doi:10.1371/journal.pone.0224674

Peer Review History

Original SubmissionJuly 29, 2019
5 Sep 2019 Decision Letter - Dong-Gyu Jo, Editor PONE-D-19-21383 Carvedilol Improves Glucose Tolerance and Insulin Sensitivity in Treatment of Adrenergic Overdrive in High Fat Diet-induced Obesity PLOS ONE Dear Dr. Doan, 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. We would appreciate receiving your revised manuscript by Oct 20 2019 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Dong-Gyu Jo, Ph.D 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. During your revisions, please note that a simple title correction is required: "Carvedilol Improves Glucose Tolerance and Insulin Sensitivity in Treatment of Adrenergic Overdrive in High Fat Diet-induced Obesity in mice". Please ensure this is updated both in the manuscript file and the online submission information. 3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (i) methods of sacrifice, (ii) methods of anesthesia and/or analgesia, and (iii) efforts to alleviate suffering. 4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: In the manuscript entitled “Carvedilol Improves Glucose Tolerance and Insulin Sensitivity in Treatment of Adrenergic Overdrive in High Fat Diet-induced Obesity”, Nguyen et al. have investigated the metabolic effects of carvedilol, a third-generation beta-blocker agent, in the HFD-induced obese mice in which they found that the basal plasma norepinephrine of those mice is chronically elevated and correlated to the plasma leptin concentrations. They showed that treatment of adrenergic overactivation from the excessive norepinephrine in HFD-fed mice with carvedilol enhances glucose tolerance and insulin sensitivity by blocking the glucose overproduction in the liver and increasing muscular insulin sensitivity. Overall, the manuscript appears very solid and well done. The manuscript would benefit from addressing following points: (1) The p-Creb level should be assessed together with total Creb level in addition to the loading protein in the samples. (2) In Figure 2B, G and Figure S2B, the unit of Y-axis should be provided. (3) In Figure S1D, the scale bar does not correspond to the absolute readings in the bar graphs. The authors should revise it. Reviewer #2: This study submitted by Nguyen et al. claims that adrenergic overdrive such as high level of catecholamine link to plasma leptin level, consequently it contributes to the genesis of metabolic disorder. Nguyen et al. found that basal norepinephrine level is increased with a strong correlation of plasma leptin concentration in high-fat diet (HFD)-induced obese mouse model. By treating carvedilol, a third-generation beta type blocker, in HFD-induced obese mouse, the glucose tolerance and insulin sensitivity were improved. This study is well designed and formed. Particularly Nguyen et al. carefully propose important point regarding potential causation of metabolic disorder which is the correlation between adrenergic overdrive and plasma leptin level, and they verified the effect of carvedilol against adrenergic overdrive. I have an only minor comment regarding this study. Figure legend should describe how many (N=??) repeat each experiment regardless of its type including western blot and its analysis. Although figure itself has displayed a number which I assume is an experimental number. ****** 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: No [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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0224674.r001
Revision 1
26 Sep 2019 Author Response Rebuttal Letter (PONE-D-19-21383) We sincerely appreciate the Academic Editor and Reviewers for taking your valuable time to consider our work. Here, we would like to address the journal requirements from the academic editor and the reviewer’s comments as follows: Journal 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: We have formatted the manuscript following PLOS ONE's style requirements. Additionally, for this reason, we have rearranged the Figure 3 and Supplemental Figure 3 to make 04 main figures in the revised manuscript. We have therefore updated the Figs 3 and 4 and S3 Fig and the text in the “Results” and “Figure legends” sections in the revised manuscript correspondingly (lines 273-291, 537-553 and 584-587). 2. During your revisions, please note that a simple title correction is required: "Carvedilol Improves Glucose Tolerance and Insulin Sensitivity in Treatment of Adrenergic Overdrive in High Fat Diet-induced Obesity in mice". Please ensure this is updated both in the manuscript file and the online submission information. Response: We have corrected and updated the title “Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice” both in the revised manuscript (line 2) and the online submission information as the Academic Editor suggested. We sincerely appreciate the Academic Editor for this insightful correction to better clarify our findings. 3. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information on the animal research and ensure you have included details on (i) methods of sacrifice, (ii) methods of anesthesia and/or analgesia, and (iii) efforts to alleviate suffering. Response: We apologize the Academic Editor for the missing information of animal research on (i) methods of sacrifice, (ii) methods of anesthesia and/or analgesia, and (iii) efforts to alleviate suffering in the “Methods” section. We sacrificed mice by decapitation after anesthetizing with ketamine. We have provided this detail information in the “Methods” section in the revised manuscript (lines 131-132). 4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Response: We have provided the original uncropped and unadjusted images of all blots/gels in the S4 Fig of the Supporting Information file. 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 Response: We sincerely appreciate the reviewer’s comment and suggestion on our manuscript. 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Response: We sincerely appreciate the reviewer’s comment and suggestion on our manuscript. 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 Response: We sincerely appreciate the reviewer’s comment and suggestion on our manuscript. 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 Response: We sincerely appreciate the reviewer’s comment and suggestion on our manuscript. 5. Review Comments to the Author Reviewer #1: In the manuscript entitled “Carvedilol Improves Glucose Tolerance and Insulin Sensitivity in Treatment of Adrenergic Overdrive in High Fat Diet-induced Obesity”, Nguyen et al. have investigated the metabolic effects of carvedilol, a third-generation beta-blocker agent, in the HFD-induced obese mice in which they found that the basal plasma norepinephrine of those mice is chronically elevated and correlated to the plasma leptin concentrations. They showed that treatment of adrenergic overactivation from the excessive norepinephrine in HFD-fed mice with carvedilol enhances glucose tolerance and insulin sensitivity by blocking the glucose overproduction in the liver and increasing muscular insulin sensitivity. Overall, the manuscript appears very solid and well done. The manuscript would benefit from addressing following points: (1) The p-Creb level should be assessed together with total Creb level in addition to the loading protein in the samples. Response: We agree with the reviewer that the p-Creb level should be measured together with total Creb. We have therefore run the samples to check total Creb levels and provided the results in the Figs 2A, F and S2A Fig in the revised manuscript. Similar to the loading protein GAPDH in the samples, the levels of total Creb were comparable between control and carvedilol treatment groups. Further, we have updated the Figs 2A-B, F-G and S2A and B Fig using the ratios of p-Creb/total Creb correspondingly. We thank the reviewer for this insightful suggestion. (2) In Figure 2B, G and Figure S2B, the unit of Y-axis should be provided. Response: We apologize the reviewer for the missing information on the unit of Y-axis in the Figure 2B, G and Figure S2B. We have updated the Fig 2B, G and S2B Fig providing the unit of Y-axis in the revised manuscript. We sincerely thank the reviewer for this comment. (3) In Figure S1D, the scale bar does not correspond to the absolute readings in the bar graphs. The authors should revise it. Response: We apologize the reviewer for the confusing correspondence between scale bar and absolute readings in the bar graphs in Figure S1D. We have revised the quantification of histological images and updated the results in the S1D Fig in the revised manuscript correspondingly. We sincerely appreciate the reviewer for this insightful comment to improve our manuscript. Reviewer #2: This study submitted by Nguyen et al. claims that adrenergic overdrive such as high level of catecholamine link to plasma leptin level, consequently it contributes to the genesis of metabolic disorder. Nguyen et al. found that basal norepinephrine level is increased with a strong correlation of plasma leptin concentration in high-fat diet (HFD)-induced obese mouse model. By treating carvedilol, a third-generation beta type blocker, in HFD-induced obese mouse, the glucose tolerance and insulin sensitivity were improved. This study is well designed and formed. Particularly Nguyen et al. carefully propose important point regarding potential causation of metabolic disorder which is the correlation between adrenergic overdrive and plasma leptin level, and they verified the effect of carvedilol against adrenergic overdrive. I have an only minor comment regarding this study. Figure legend should describe how many (N=??) repeat each experiment regardless of its type including western blot and its analysis. Although figure itself has displayed a number which I assume is an experimental number. Response: We apologize the reviewer for missing information on the experimental replication in the figure legends. We performed the experiments of hormone measurement, histological and western blot analyses in triplicate. We have updated the figure legends to describe information on the replication of each experiment in the revised manuscript correspondingly (lines 518, 534-535, 546, 569-570 and 581-582). We appreciate the reviewer for this insightful and constructive suggestion. 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: No Response: We sincerely appreciate the reviewer’s comment and suggestion on our manuscript. We sincerely thank the reviewers for the careful reading and insightful comments on our manuscript. Sincerely, Khanh V. Doan, Ph.D., Department of Pharmacology, School of Medicine, Tan Tao University. Address: Tan Tao University Ave., Tan Duc E.City, Duc Hoa, Long An 850000, Viet Nam. Tel: (+84-272)-376-9216 Fax: (+84-272)-376-9208 Cell: (+84-91)-185-1177 Email: doankhanh.pharm@gmail.com Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0224674.r002
21 Oct 2019 Decision Letter - Dong-Gyu Jo, Editor Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice PONE-D-19-21383R1 Dear Dr. Doan, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Dong-Gyu Jo, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0224674.r003
Formally Accepted
24 Oct 2019 Acceptance Letter - Dong-Gyu Jo, Editor PONE-D-19-21383R1 Carvedilol improves glucose tolerance and insulin sensitivity in treatment of adrenergic overdrive in high fat diet-induced obesity in mice Dear Dr. Doan: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Dong-Gyu Jo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0224674.r004

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