Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1

Patrick Mordel; Fanny Fontaine; Quentin Dupas; Michael Joubert; Stéphane Allouche

doi:10.1371/journal.pone.0289475

Peer Review History

Original SubmissionAugust 31, 2022
14 Oct 2022 Decision Letter - Rodrigo Franco, Editor PONE-D-22-24271Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1PLOS ONE Dear Dr. Allouche, 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 Nov 28 2022 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, Rodrigo Franco 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. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by Université Caen Normandie (Unicaen – France)." We note that you have provided funding information that is not 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 Université Caen Normandie (Unicaen – France)." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Thank you for stating the following in the Competing Interests section: "PM, QD and SA declare that they have no conflict of interest. MJ has received research grants and/or advisory fees from Astrazeneca, Bayer, Boehringer Ingelheim, Lilly and Novonordisk. " Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 6. 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. 7. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Partly Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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: No ****** 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: The manuscript of Hernandez et al. aims to evaluate the impact of glucose swings on mitochondrial functions in the cardiomyocyte cell line HL-1. Exposure of HL-1 cells to glucose swings promoted time-dependent mitochondrial dysfunctions suggesting a deleterious effect of such condition in patients with diabetes that could contribute to diabetic cardiomyopathy. The manuscript is well written. However, a few comments and questions are provided for clarification and some additional evidence is encouraged. 1. The authors used four different groups such as (LG, 2.8 mmol/l), normal (NG, 5.5 mmol/l), high (HG, 25 mmol/l) or intermittent high glucose (IHG, swing between low and high). It would be good to use another group as no glucose to measure the glucose consumption. 2. Glucose fluctuation promotes mitochondrial dysfunctions, to confirm that authors need to measure the product ATP levels. 3. There is no molecular mechanism established in this manuscript. The authors need to discuss the future studies warranted to identify the mechanism involving mitochondrial dysfunction promoted by glucose fluctuation. Reviewer #2: This manuscript discusses how glycemic variability has been suggested as a risk factor for diabetes complications; however, these mechanisms remain poorly understood. The authors demonstrated that glucose swings in HL-1 cells promote time-dependent mitochondrial dysfunctions suggesting a harmful effect of such condition in patients with diabetes that could contribute to diabetic cardiomyopathy. In figure 2A, the authors do not explain why the graph has negative values and the standard error is overlapping in the insulin treatment and no insulin treatment; it looks like there is no significant difference. The following graphs are similar, but the authors do not explain why the graphs of glucose concentration have negative values; neither is it clear whether all experiments with glucose were performed in the presence of insulin because most of the patients are treated with insulin. So the cellular model does not fit to compare partially at the human level. The authors did not measure any GLUT transporter, whether oxidized or reduced; GLUT transporter status is not shown, and neither is cell death, which means there was no cell death in the hypoglycemic phase. How was mitochondria function affected to produce superoxide anion instead of water? The authors should perform WB for Glut-1 and 4 to see which one is involved and fluorescent microscopy to see how cells are affected morphologically. At this point, the manuscript is very confusing and hard to follow. ****** 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: Yes: Anandhan Annadurai 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.] 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. https://doi.org/10.1371/journal.pone.0289475.r001
Revision 1
1 Mar 2023 Author Response Response to Reviewers Caen, France 23/02/2023 Dear Editor, Thank you for giving us the opportunity to submit a revised version of our manuscript entitled “Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1” by Mordel and collaborators. We also would like to thank reviewers for their useful and detailed comments that have improved the quality of our manuscript. Responses to the Editor : Point 1 : We have made changes as requested in the revised version. Points 2 and 3: Concerning the Financial Disclosure, we had no grant numbers for the awards we received from the Université Caen Normandie (Unicaen – France). We don’t know where to mention this information. Point 4 : We have updated the statement about conflict of interest in our revised manuscript as follows : Conflict of interest: PM, FF, QD and SA declare that they have no conflict of interest. MJ has received research grants and/or advisory fees from Astrazeneca, Bayer, Boehringer Ingelheim, Lilly and Novonordisk. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Point 5 : Upon re-submitting our revised manuscript, we upload all data obtained and presented in this study. Point 6 : Western blot and gel image data were included in the supporting information file. Point 7 : We removed the phrase “data not shown” in the revised manuscript and replaced by data. All the data obtained in the present work were included in the supporting information file. Responses to the Reviewer #1: 1. The authors used four different groups such as (LG, 2.8 mmol/l), normal (NG, 5.5 mmol/l), high (HG, 25 mmol/l) or intermittent high glucose (IHG, swing between low and high). It would be good to use another group as no glucose to measure the glucose consumption. Response : A total glucose withdrawal from the culture medium would have been too deleterious for HL-1 cells to conduct experiments mainly during 72h. Most of the cell lines used in labs, including HL-1 cells, are highly dependent on glucose oxidation and culture in glucose-free medium leads to a major reduction in cell proliferation and in cell death. This is the main reason why we did not include this group “no glucose” in our experiments. In addition, we wanted to mimic physiological or pathological settings with our experimental conditions, what a free-glucose group would have not allowed. 2. Glucose fluctuation promotes mitochondrial dysfunctions, to confirm that authors need to measure the product ATP levels. As recommended by the reviewer, we conducted experiments to measure ATP production upon 12 and 72h treatment under the different conditions : LG, NG, HG and IHG. Those data are included in the revised version. 3. There is no molecular mechanism established in this manuscript. The authors need to discuss the future studies warranted to identify the mechanism involving mitochondrial dysfunction promoted by glucose fluctuation. Response : We agree with the reviewer’s comment. Our data are mainly descriptive and the molecular mechanisms involved in mitochondrial dysfunction upon IHG are speculative. We think that complementary experiments would be necessary to go further in those mechanisms. For instance, we could measure the mitochondrial permeability transition pore and mitochondrial Ca2+ during exposure to different concentrations of glucose, which are major regulators of the electrons flux through the respiratory chain. We could also conduct transcriptomic experiments to examine the regulation of genes encoding for enzymes implicated in the metabolism (beta-oxidation, Krebs, respiratory chain complexes…) and in ROS detoxification. As suggested by the reviewer, we added this short paragraph in the perspective of this work. Responses to the Reviewer #2: 1. In figure 2A, the authors do not explain why the graph has negative values and the standard error is overlapping in the insulin treatment and no insulin treatment; it looks like there is no significant difference. Response : Values in the fig 2A are negative since we observed glucose consumption by HL-1 cells. As indicated in the materials and methods, we measured glucose concentrations in culture medium at t=0 and t=12 or 72h then we determined glucose consumption as follows : glucose t=12 or 72h - glucose t=0. We agree with the reviewer that values obtained with and without insulin are overlapping but statistical analysis using the Wilcoxon test indicated a significant difference between the 2 groups (-4.84 +/- 0.75 vs -6.01+/-0.82; p = 0.031). In previous papers, insulin was demonstrated to promote glucose uptake but with more sensitive methods (3H-deoxyglucose) (Chaudary et al., 2002; Shuralyova et al., 2004). 2. The following graphs are similar, but the authors do not explain why the graphs of glucose concentration have negative values; neither is it clear whether all experiments with glucose were performed in the presence of insulin because most of the patients are treated with insulin. Response : Except for experiments showed in the Fig.2A and B, we did not add additional insulin in the culture medium. As described by White et al. (2004), the Claycomb medium has already human recombinant insulin (15 𝜇g/l) and this could explain why we did not obtain a huge glucose consumption when we added further insulin. This point was added to the discussion. 3. So the cellular model does not fit to compare partially at the human level. Response : When using an experimental model, we should keep in mind that it has limitations to represent a pathophysiological state in humans. However, to elucidate molecular mechanisms they are very useful since we can precisely place cells in specific conditions (ex : a precise glucose swing between high and low glucose concentration). This point was clearly indicated in the discussion (lines 603-607). 4. The authors did not measure any GLUT transporter, whether oxidized or reduced; GLUT transporter status is not shown. Response : Since the goal of our study was to evaluate mitochondrial functions upon different glucose exposure, it did not seem relevant to us to study this aspect. However, the first experiences were conducted to check that the different glucose treatments had metabolic impact; by measuring lactate production under glucose treatments (S1 Fig), we can state indirectly that GLUTs were not limiting factors. 5. And neither is cell death, which means there was no cell death in the hypoglycemic phase. Response : We did not specifically measure cell death when using the different glucose treatments. As cell death can be easy detectable by microscopy since cells are floating in the culture medium, we can affirm that low glucose exposure either for 12 or 72h did not promote HL-1 cell death. However, when cultured for long time (>3 weeks) in normal glucose medium (1 g/l), we observed a decrease in cell proliferation compared to the high glucose medium. 6. How was mitochondria function affected to produce superoxide anion instead of water? Response : This is a difficult question to answer; from our data and in our experimental conditions, we observed a higher superoxide anion production and protein oxidation upon high glucose (HG) and intermittent high glucose (IHG) compared to low and normal glucose conditions. We can hypothesize that when increasing electron flow through the respiratory chain, we increase the probability of electron leak to generate reactive oxygen species. This could be deleterious for mitochondrial respiratory chain as demonstrated by Zuo et al. (2009) after ischemia/reperfusion when ROS production is highly increased; furthermore, those mitochondrial alterations can be reversed by the addition of an antioxidant. However, we showed that only IHG treatment but not HG was able to impact the mitochondrial respiration indicating that superoxide anion production is probably not the only mechanism to promote mitochondrial dysfunction (Fig 4A, succinate). 7. The authors should perform WB for Glut-1 and 4 to see which one is involved and fluorescent microscopy to see how cells are affected morphologically. At this point, the manuscript is very confusing and hard to follow. Response : Since Glut-1 and 4 expression in HL-1 cells was previously studied (Chaudary et al., 2002; Shuralyova et al., 2004) and we used the same cells and cell culture conditions, we did not perform such experiments. We rather used indirect parameters (glucose consumption and lactate production) even if there are less sensitive than a direct determination of glucose uptake. Once again, the goal of our study was to examine mitochondria under different glucose exposure but not the metabolic flux. Those references were added in the discussion. Once again, we thank both reviewers for their useful comments and we appreciate your time reading our manuscript. Looking forward to reading your response. Yours sincerely, Pr Stéphane Allouche Attachments Attachment Submitted filename: response to reviewers Mordel.docx https://doi.org/10.1371/journal.pone.0289475.r002
20 Jul 2023 Decision Letter - Kanhaiya Singh, Editor Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1 PONE-D-22-24271R1 Dear Dr. Allouche, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Kanhaiya Singh, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): 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 #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 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 #1: Partly Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 #1: Yes Reviewer #2: Yes ****** 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 #1: Yes Reviewer #2: Yes ****** 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 #1: The authors respond most of the critics in a logical manner. I would recommend to consider it for publication. Reviewer #2: (No Response) ****** 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 #1: Yes: Anandhan Annadurai Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0289475.r003
Formally Accepted
14 Sep 2023 Acceptance Letter - Kanhaiya Singh, Editor PONE-D-22-24271R1 Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1 Dear Dr. Allouche: I'm 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 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. If we can help with anything else, please email us at plosone@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. Kanhaiya Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289475.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .