KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not on whole-body insulin sensitivity in rats

Gagandeep Mann; Stephen Mora; Olasunkanmi A. John Adegoke

doi:10.1371/journal.pone.0309324

Peer Review History

Original SubmissionFebruary 23, 2024
3 May 2024 Decision Letter - Md. Wasim Khan, Editor PONE-D-24-07286KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not whole-body insulin sensitivity in ratsPLOS ONE Dear Dr. Adegoke, 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 Jun 17 2024 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. 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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. Additional Editor Comments: Dear Dr Adegoke The reviewers have submitted their critiques. As you will see, Rev 2 has proposed that the manuscript be rejected in its current form. However, although I agree with all the comments of both Revs 1 and 2, I feel that if given the opportunity, this manuscript could be improved. Particularly, the comment in Line 373 has caught my attention - S6K immunoblots were not possible due to technical difficulties. There are hundreds of published articles showing S6K immunoblots, and I do not think this can be a challenge, especially given the premise of this manuscript. I invite you to revise the manuscript according to the comments. [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: No ******** 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: Very nice piece of work, well written and communicated. However, why didn't the authors look at PI3K and GLUT4 to confirm the insulin signaling pathway?. Also, to check if there is any interaction with PTEN since the author was looking at the activation of IRS-1,? There was no mention of the number, ethics clearance, month, or year of approval. Why did the author use HPLC to detect BCAAs and not conventional UV detectors or capillary electrophoresis (CE) coupled with conventional UV detectors? Reviewer #2: This is a well written paper with some well thought out work, but there are few flaws that prevent it from receiving an acceptance. 1) The authors clearly demonstrate that leucine and KIC levels are elevated at 30 minutes and significantly decrease in most tissues by 60 minutes following gavage, yet the insulin tolerance tests performed occurred at 60 minutes. The authors argue based on these tolerance tests that there is no insulin resistance caused by leucine and KIC, yet their time point is clearly after levels have returned to normal physiological levels and no longer match levels cited in their discussion as being pathological. 2) The signaling pathway for IRS phosphorylation and regulation in the liver is incomplete due to the omission of IRS-2 phosphorylation status. While IRS-2 does not play a pronounced role in muscle insulin sensitivity and corresponding glucose tolerance, it plays a crucial role in the liver. 3) The statement in line 373-374 regarding the missing S6K1 and proxy evidence of S6 phosphorylation at S235/236 is not adequate, as this site has been shown to be phosphorylated by multiple other kinases. This figure needs the addition for S6K1 for any claim that the observed S6 phosphorylation is driven via AKT-mTOR1-S6K1 activity. ****** 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: Patrick Nwabueze Okechukwu 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.0309324.r001
Revision 1
11 Jun 2024 Author Response We thank the associate editor and the reviewers for carefully reading our manuscript and the detailed comments submitted. We have thoroughly and substantially reviewed the manuscripts to reflect the concerns raised. In the responses below, references to page and line numbers are for the clean copy of the revised manuscript. Associate Editor: 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 Authors’ Response: We thank the associate editor for this comment and have ensured the manuscript is correctly formatted. 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Authors’ Response: We thank the associate editor for this comment. We have indicated on page 7 line 168 that we euthanized the rats via decapitation. On page 7 line 159-60 we had already indicated that rats were handled 2-3 times weekly to reduce stress on the day of the gavage. We also included on page 8 lines 178-180, that we waited 1 hour between the first gavage and the insulin injection to reduce the stress on the animals. 3. Please expand the acronym “NSERC” (as indicated in your financial disclosure) so that it states the name of your funders in full. This information should be included in your cover letter; we will change the online submission form on your behalf. Authors’ Response: Thank you: we have made these changes in the cover letter. 4. Thank you for stating the following financial disclosure: "NSERC" 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. Authors’ Response: Thank you: we have made these changes in the cover letter. 5. 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. Authors’ Response: Thank you for this comment: we have included the uncropped and unadjusted images underlying all blot results in the supporting information files. 6) The reviewers have submitted their critiques. As you will see, Rev 2 has proposed that the manuscript be rejected in its current form. However, although I agree with all the comments of both Revs 1 and 2, I feel that if given the opportunity, this manuscript could be improved. Particularly, the comment in Line 373 has caught my attention - S6K immunoblots were not possible due to technical difficulties. There are hundreds of published articles showing S6K immunoblots, and I do not think this can be a challenge, especially given the premise of this manuscript. I invite you to revise the manuscript according to the comments. Authors’ Response: We thank the associate editor for this comment: As can be seen in the original manuscript, we obtained phosphorylated S6K1 and showed that it responded to leucine gavage in ALL the three muscles that we studied (gastrocnemius, soleus and EDL) (Fig 2C-D, Fig 3C-D, and Fig 4C-D). So, the issue was not that we were unable to obtain phosphorylated S6K1 signal per se. Rather, our challenge was with obtaining phosphorylated S6K1 signal in liver and heart samples. Hence our resolve to use the phosphorylated S6 as a surrogate for phosphorylated S6K1 (again, only for liver and heart samples). We acknowledge the fact that it would be good to have phosphorylated S6K1 data for liver and heart, and we attempted to do so but were unsuccessful. But even if we had succeeded, given the fact that in liver and heart samples, IRS-1 serine phosphorylation (which would result from the action of phosphorylated (activated) S6K1) is NOT altered (Fig 5C, E, and Fig 6C, E), nor was there treatment effect on AKT (Fig 5C, F and 6C, F), it is our opinion that any treatment effect on S6K1 phosphorylation would be inconsequential to the conclusion of the manuscript. Nevertheless, and to further address this, we prepared fresh heart and liver samples, loaded higher amounts of proteins per well than we did before, and obtained new antibodies to repeat the western blot. Unfortunately, there was again no signal for phosphorylated S6K1 in either heart or liver samples. Because we loaded control samples provided by the antibody manufacturer and successfully detected signals for phosphorylated S6K1 in those control samples, we believe the inability to detect phosphorylated S6K1 signal in those samples was because of the transient nature of S6K1 phosphorylation in response to leucine/KIC gavage in heart and liver as compared to muscle samples (please see the blots from these new analyses in the supporting information; the blots are also pasted below). Please note that unlike the signals for phosphorylated S6K1, we robustly detected total S6K1 in these samples (please see supporting information; the blots are also pasted below). Reviewer #1: Very nice piece of work, well written and communicated. However, why didn't the authors look at PI3K and GLUT4 to confirm the insulin signaling pathway?. Authors’ Response: We thank the reviewer for these comments. Akt is downstream of PI3K, and Akt phosphorylation is a strong indicator of insulin signaling (Diabetes Metab Syndr Obes. 2014; 7: 55–64) as it is involved in GLUT4 translocation (Molecular Endocrinology, Volume 19, Issue 4, 1 April 2005, Pages 1067–1077). Akt was unaltered in response to KIC or leucine gavage, which is consistent with other studies that showed an increase in the activation of the mTORC1-S6K1-IRS-1 axis but without an effect on Akt in response to leucine/protein ingestion (Diabetes. 2015 May;64(5):1555-63). Given the absence of treatment effect on Akt, we do not think measuring PI3K would add much to the manuscript. The half-life of GLUT4 protein is 8-10 hours (Biochem Biophys Rep. 2015 Jul; 2: 45–49), longer than our treatment times. GLUT4 translocation too can be affected by treatments. However, since this translocation is regulated by AKT (World J Biol Chem. 2020 Nov 27; 11(3): 76–98), but Akt phosphorylation is unchanged in every tissue reported in this study, we think it unlikely that GLUT4 translocation would be affected under the conditions studied. One study shows that supraphysiological levels of leucine (10 mM) can reduce GLUT4 translocation in cardiomyocytes. However, this study used 6-7x greater amount of leucine, and they saw a consistent reduction in Akt phosphorylation with reduced GLUT4 (Am J Physiol Heart Circ Physiol 313: H432–H445, 2017). Thus, the lack of effect on phosphorylated Akt in our study suggests that GLUT4 translocation was not changed. GLUT4 translocation can also occur in response to exercise, but this was not studied this manuscript. Also, to check if there is any interaction with PTEN since the author was looking at the activation of IRS-1? Authors’ Response: Thank you for this comment. The half-life of PTEN is 7-8 hours (Cold Spring Harb Perspect Med. 2020 May; 10(5): a036079), so we think it unlikely that 0.5-3 h of leucine/KIC gavage would affect PTEN abundance. Also, PTEN is downstream of IRS-1 and is involved in negatively regulating PI3K. If PTEN protein level/interaction were changed, this might result in changes in the phosphorylation of Akt, which we did not see. Consistent with the reviewer’s comment, there is evidence that PTEN can dephosphorylate tyrosine residues of IRS-1 (Nat Struct Mol Biol. 2014 Jun; 21(6): 522–527). However, although a few studies examined the effects of nutrients on PTEN (Discov Oncol. 2023 Feb 23;14(1):25.doi: 10.1007/s12672-023-00634-1; Nutr. 2021 Sep 4;151(9):2636-2645.doi: 10.1093/jn/nxab190), much of what is known about the phosphatase relates to its regulation by growth factors. In future, it would be interesting to study whether amino acids regulate its interactions and whether such a regulation is functionally consequential. There was no mention of the number, ethics clearance, month, or year of approval. Authors’ Response: Thank you. On page 6 lines 136-138 we included an ethics statement, the animal use protocol number, month and year of approval. Why did the author use HPLC to detect BCAAs and not conventional UV detectors or capillary electrophoresis (CE) coupled with conventional UV detectors? Authors’ Response: We thank the reviewer for this comment. Although amino acids may be measured using different systems, liquid chromatography (HPLC) is one of the common systems and has been our preferred approach (Am J Physiol Cell Physiol. 2024 Mar 1;326(3):C866-C879, Physiol Rep. 2024 Apr; 12(8): e16003, Physiol Rep. 2021 Jan; 9(1): e14673). We have now included this in the methods section on page 9 lines 202. Reviewer #2: 1) The authors clearly demonstrate that leucine and KIC levels are elevated at 30 minutes and significantly decrease in most tissues by 60 minutes following gavage, yet the insulin tolerance tests performed occurred at 60 minutes. The authors argue based on these tolerance tests that there is no insulin resistance caused by leucine and KIC, yet their time point is clearly after levels have returned to normal physiological levels and no longer match levels cited in their discussion as being pathological. Authors’ Response: We thank the reviewer for taking the time to read our manuscript and for the reviewer’s comments. Regarding when the ITT was done, firstly, the institutional animal care committee had us wait an hour after the first gavage before doing the ITT in order to reduce the amount of stress on the animals. Secondly, although leucine levels dropped after 60 min in most tissues, the level was still quite high in plasma (~100�M), a level not far from those seen in insulin resistance as discussed on pages 21-22, lines 485-490. Even if there was an insulin resistance at an earlier time point that we missed, this would still suggest that, at best, such an effect was very transient and might be inconsequential. 2) The signaling pathway for IRS phosphorylation and regulation in the liver is incomplete due to the omission of IRS-2 phosphorylation status. While IRS-2 does not play a pronounced role in muscle insulin sensitivity and corresponding glucose tolerance, it plays a crucial role in the liver. Authors’ Response: We thank the reviewer for this comment. We agree that IRS-2 is crucial in the liver, to our knowledge IRS-2 is not phosphorylated by S6K1 in the liver in response to leucine or KIC (or any BCAA/BCKAs), the nutrients that we studied. However, S6K1 liver-specific knockdown resulted in increased total IRS-2 protein, suggesting that S6K1 could impact IRS-2 (J Biol Chem. 2012 May 25; 287(22): 18769–18780), thus we have included the omission of IRS-2 as a limitation in our discussion on page 26 line 588. Others have looked at how S6K1 phosphorylates IRS-1 serine residues as reviewed (Nutrients. 2016 Jul; 8(7): 405), and specifically in the liver (Proc Natl Acad Sci USA. 2007 Aug 28;104(35):14056-61). Although one study shows that mTORC1 is required for insulin stimulation of serine phosphorylation of IRS-2 in Fao cells (hepatic carcinoma cells), S6K1 is not required for this effect. Furthermore, this phosphorylation had no effect on the downstream PI3K/Akt pathway (Am J Physiol Endocrinol Metab. 2011 May;300(5):E824-36). We have included this in the discussion section on page 26, lines 588-592. 3) The statement in line 373-374 regarding the missing S6K1 and proxy evidence of S6 phosphorylation at S235/236 is not adequate, as this site has been shown to be phosphorylated by multiple other kinases. This figure needs the addition for S6K1 for any claim that the observed S6 phosphorylation is driven via AKT-mTOR1-S6K1 activity. Authors’ Response: We thank the reviewer for this comment. As can be seen in the original manuscript, we obtained phosphorylated S6K1 and showed that it responded to leucine gavage in ALL the three muscles that we studied (gastrocnemius, soleus and EDL) (Fig 2C-D, Fig 3C-D, and Fig 4C-D). So, the issue was not that we were unable to obtain phosphorylated S6K1 signal per se. Rather, our challenge was with obtaining phosphorylated S6K1 signal in liver and heart samples. Hence our resolve to use the phosphorylated S6 as a surrogate for phosphorylated S6K1 (again, only for liver and heart samples). We acknowledge the fact that it would be good to have phosphorylated S6K1 data for liver and heart, and we attempted to do so but were unsuccessful. But even if we had succeeded, given the fact that in liver and heart samples, IRS-1 serine phosphorylation (which would result from the action of phosphorylated (activated) S6K1) is NOT altered (Fig 5C, E, and Fig 6C, E), nor was there treatment effect on AKT (Fig 5C, F and 6C, F), it is our opinion that any treatment effect on S6K1 phosphorylation would be inconsequential to the conclusion of the manuscript. Nevertheless, and to further address this, we prepared fresh heart and liver samples, loaded higher amounts of proteins per well than we did before, and obtained new antibodies to repeat the western blot. Unfortunately, there was again no signal for phosphorylated S6K1 in either heart or liver samples. Because we loaded control samples provided by the antibody manufacturer and successfully detected signals for phosphorylated S6K1 in those control samples, we believe the inability to detect phosphorylated S6K1 signal in those samples was because of the transient nature of S6K1 phosphorylation in response to leucine/KIC gavage in heart and liver as compared to muscle samples (please see the blots from these new analyses in the supporting information; the blots are also pasted below). Please note that unlike the signals for phosphorylated S6K1, we robustly detected total S6K1 in these samples (please see supporting information; the blots are also pasted below). Attachments Attachment Submitted filename: Mann et al -Response to reviewers-oa.docx https://doi.org/10.1371/journal.pone.0309324.r002
16 Jul 2024 Decision Letter - Owen Ngalamika, Editor PONE-D-24-07286R1KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not on whole-body insulin sensitivity in ratsPLOS ONE Dear Dr. Adegoke, 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. The reviewer has raised an important issue that needs to be addressed. In particular, the timing of insulin tolerance tests relative to detection of KIC and Leucine levels has not been adequately addressed in the revised manuscript. We are therefore requesting the authors to address this and the other comments from the reviewer in the discussion and study limitations sections. Please submit your revised manuscript by Aug 30 2024 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, Owen Ngalamika Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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: Partly ****** 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 ****** 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 ****** 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: I thank the authors for their additional experiments and revisions from the previous review. For the most part all comments were addressed except for the following: The authors clearly demonstrate that leucine and KIC levels are elevated at 30 minutes and significantly decrease in most tissues by 60 minutes following gavage, yet the insulin tolerance tests performed occurred at 60 minutes. The authors argue based on these tolerance tests that there is no insulin resistance caused by leucine and KIC, yet their time point is clearly after levels have returned to normal physiological levels and no longer match levels cited in their discussion as being pathological. The response provided to this comment does not adequately address the concern provided. As demonstrated by the data provided, levels of circulating leucine concentrations decrease at 60min from the peak at 30min, and in tissues the levels return to the same as control (water gavage). The argument that leucine levels remaining at 100 umole/liter is still quite high and thus similar to those seen with obesity and insulin resistance contradicts the findings in the works cited. Citation 45 shows that circulating leucine levels in nonobese subjects were 112uM and citation 46 shows that in normoglycemic that it was 113uM. In both citations levels were above 120uM if not above 130uM in groups with obesity and/or insulin resistance, suggesting that there is likely a minimum threshold to impact physiology, a threshold not being met at 60 minutes. This implication is supported by your findings as levels dramatically reduce at the tissue level when circulating levels decrease below this threshold. It is completely reasonable based on the veterinary recommendation that this limitation can not be addressed via experimentation, but this needs to be addressed in the discussion as the current study only suggests that the effects of leucine are transient and likely concentration dependent, and not the conclusion reached in lines 491-495. ****** 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 ******** [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.0309324.r003
Revision 2
19 Jul 2024 Author Response We thank the associate editor and the reviewers for carefully reviewing the edits we made to our manuscript and the opportunity to fix this revision. We have thoroughly reviewed the manuscript to reflect the concern raised. In the response below, references to page and line numbers are for the clean copy of the revised manuscript. Reviewer 2: I thank the authors for their additional experiments and revisions from the previous review. For the most part all comments were addressed except for the following: The authors clearly demonstrate that leucine and KIC levels are elevated at 30 minutes and significantly decrease in most tissues by 60 minutes following gavage, yet the insulin tolerance tests performed occurred at 60 minutes. The authors argue based on these tolerance tests that there is no insulin resistance caused by leucine and KIC, yet their time point is clearly after levels have returned to normal physiological levels and no longer match levels cited in their discussion as being pathological. The response provided to this comment does not adequately address the concern provided. As demonstrated by the data provided, levels of circulating leucine concentrations decrease at 60min from the peak at 30min, and in tissues the levels return to the same as control (water gavage). The argument that leucine levels remaining at 100 umole/liter is still quite high and thus similar to those seen with obesity and insulin resistance contradicts the findings in the works cited. Citation 45 shows that circulating leucine levels in nonobese subjects were 112uM and citation 46 shows that in normoglycemic that it was 113uM. In both citations levels were above 120uM if not above 130uM in groups with obesity and/or insulin resistance, suggesting that there is likely a minimum threshold to impact physiology, a threshold not being met at 60 minutes. This implication is supported by your findings as levels dramatically reduce at the tissue level when circulating levels decrease below this threshold. It is completely reasonable based on the veterinary recommendation that this limitation can not be addressed via experimentation, but this needs to be addressed in the discussion as the current study only suggests that the effects of leucine are transient and likely concentration dependent, and not the conclusion reached in lines 491-495 Authors’ Response We thank the reviewer for taking the time to read the revisions we made to the manuscript and for the reviewer’s comment. Although plasma leucine level was still significantly much higher at the time of the ITT (at least 3X the values in the water gavage group), we agree with the reviewer that there might be a threshold that is higher than what was observed at 60 min after gavage, especially in the tissues. Therefore, we have edited lines 491-498 (Discussion section) and lines 600-605 (Limitation section) to reflect this possibility. These sections now read: Lines 491-498: “The fact that we observed no significant effect of leucine/KIC gavage on whole body insulin sensitivity in this study even with circulating levels of leucine and KIC that were much higher than basal (especially for leucine) suggests that altered leucine and KIC levels are a product of insulin resistance instead of causative. However, because the insulin tolerance test was carried out 60 min post gavage, at a time point when circulating and tissue levels of leucine and KIC were off their peak values (attained at 30 minutes), it is possible that the effects of leucine and KIC on insulin sensitivity are concentration dependent and transient.” Lines 600-605: “Additionally, we performed the insulin tolerance test 60 min after gavage instead of at 30 min (when circulating and tissue leucine and KIC levels peaked). This decision was made based on the recommendation of the institutional animal care committee in order to reduce the stress of consecutive gavages/injections in a close time frame. By so doing however, we might have missed a critical time point because it is possible that the effect of the gavage on insulin sensitivity is concentration dependent and transient.” Attachments Attachment Submitted filename: Mann et al - response to reviewers-July19-24.docx https://doi.org/10.1371/journal.pone.0309324.r004
7 Aug 2024 Decision Letter - Owen Ngalamika, Editor KIC (ketoisocaproic acid) and leucine have divergent effects on tissue insulin signaling but not on whole-body insulin sensitivity in rats PONE-D-24-07286R2 Dear Dr. Adegoke, 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, Owen Ngalamika 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 #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 #2: Yes ****** 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 ****** 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 ****** 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: (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 #2: No ******** https://doi.org/10.1371/journal.pone.0309324.r005
Formally Accepted
12 Aug 2024 Acceptance Letter - Owen Ngalamika, Editor PONE-D-24-07286R2 PLOS ONE Dear Dr. Adegoke, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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. Owen Ngalamika Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0309324.r006

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