PONE-D-20-02951

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is under circadian and dietary control

PLOS ONE

Dear Dr. Verrey,

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 address the methodological and interpretational concerns raised by the reviewers. Specifically, add ad libitum fed animals as control group (ad libitum means that these animals have access to food over 24 hours). The data have been generated under diurnal conditions and not circadian conditions. Therefore, the conclusions should also be made under that aspect (the presence of a light/dark cycle). The experiment suggested by reviewer 1 under point 3 should be performed and added to the manuscript. Quantification of the data (point 4 reviewer 1 should also be included). ​

==============================

We would appreciate receiving your revised manuscript by Apr 17 2020 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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

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,

Urs Albrecht, 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.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.

In your revised cover letter, please address the following prompts:

a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.

b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories.

We will update your Data Availability statement on your behalf to reflect the information you provide.

3. 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: No

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: Oparija-Rogenmozere and colleagues investigated the expression and phosphorylation of essential amino acid transporter Lat4 (Slc43a2) in three regions of the intestine of mice fed from early to late night. The results indicate differential expression and phosphorylated states of Lat4 according to intestine regions (i.e. duodenum, jejunum and ileum) and amount of proteins in the diet (8, 18 or 40%).

As detailed below, the manuscript raises several methodological and interpretative issues that need to be addressed.

1. Food-entrained versus ad lib-fed mice

It is not fully clear why all mice had food access limited to 8 h, starting at dark onset. Here food access is limited to the usual feeding period which might have enhanced daily rhythmicity. Why were mice fed ad libitum not studied as control group? Otherwise, what is the advantage or the purpose to study food-restricted mice fed only at night? Please specify.

2. Circadian control

Reading the present manuscript raises comments on the concept of circadian clocks. Circadian rhythms refer to endogenous biological rhyhms that are controlled by self-sustained clocks. Such endogenopus rhythms can only be assessed in constant environmental conditions (including constant light or dark, and constant ambient temperature). When a light-dark cycle is present, as it is the case in the present study, the parameters studied over 24 h should be called « daily » or « diurnal » rhythms. As a consequence, the word « circadian » such as used in the title is inappropriate for the present experiment and should be omitted throughout the manuscript.

3. Clock-controlled versus feeding-induced regulation

This study reports daily variations of several parameters such as protein levels of LAT4 in the duodenum and LAT4-pS297 in jejunum. When higher values are found at night, the present protocol does not allow discriminating between putative clock-controlled or feeding-induced regulation.

To test for feeding-induced control, another group of mice should be fed during daytime only (with the assumption that there will be a 12-h shift in the daily variations). The same remark holds for food-anticipatory mechanisms.

To test for clock-controlled regulation, another group of mice should be fasted (with the assumption that the daily variations will persist if they are regulated by endogenous clocks).

If ever the authors cannot add these new groups of mice, they should be much more cautious in their discussion on the interpretation of their findings, so as not to mislead the reader.

4. Image analysis and quantification

In figures 9 and 11, the authors consider that « low protein diet leads to increased intracellular localization of LAT4 in jejunum » and this diet « has no strong impact on LAT4 subcellular localization in ileum ». It seems that these respective statements are based on visual inspection only. Image analysis and quantification are required for both parameters.

5. Circadian clock in the intestine

It is surprising for a study supposed to investigate clock-related variations in the intestine that the authors did not cite previous investigations of circadian oscillations in that organ. Actually, the 2 proposed citations (one original work and one review) do not deal at all with the intestinal clock (i.e. Damiola et al. 2000 and Welsh et al. 2010, see refs 25 and 26, respectively).

Alternative suggestions:

Hepatic, duodenal, and colonic circadian clocks differ in their persistence under conditions of constant light and in their entrainment by restricted feeding. Polidarová L, Sládek M, Soták M, Pácha J, Sumová A. Chronobiol Int. 2011 Apr;28(3):204-15. doi: 10.3109/07420528.2010.548615.

PMID:21452916

Clock gene expression in the murine gastrointestinal tract: endogenous rhythmicity and effects of a feeding regimen. Hoogerwerf WA, Hellmich HL, Cornélissen G, Halberg F, Shahinian VB, Bostwick J, Savidge TC, Cassone VM. Gastroenterology. 2007 Oct;133(4):1250-60. Epub 2007 Jul 12.

PMID: 17919497

6. Miscellaneous

The main text indicates that mice were fed from ZT12 to ZT20, while Figure 1 suggests that mice had food access from ZT12 to ZT18. Please correct where needed.

« Rest » or « resting » period, rather than « passive » period.

Reviewer #2: Most research on circadian entrainment to feeding has focused on neural mechanisms and the liver, so it is quite interesting to learn about changes in the intestine in the study of Oparija-Rogenmozere and colleagues. They describe the expression of uni-, sim- and anti-porters in response to scheduled feeding and increased/decreased dietary protein content, eventually focusing their study on Lat4, an amino acid transporter that they have studied previously that is regulated by phosphorylation. In the present study they examine the expression and regulation of this transporter in great deal and the study appears technically sound in all ways.

I have several suggests for edits and clarification below:

Introduction, line 1, the word “laying” should be deleted

Most of the uses of the word “already” are unnecessary and it can be deleted without changing the meaning of the sentences

Introduction sentence: “Small intestine also possesses a self-sustaining circadian rhythm for which the most powerful entrainment is food intake (25, 26).” Reference 26 is not correct.

Introduction, last paragraph, please consider citing some of the studies that cast some doubt on the necessity of ghrelin peptide as the mechanism of behavioral anticipation of scheduled meals:

Gunapala et al https://doi.org/10.1371/journal.pone.0018377, which demonstrates that ghrelin deletion does not alter food anticipatory activity in mice

And also Daily et al 2012 doi: 10.1210/en.2011-1464, which dissociates gut peptide entrainment from behavioral entrainment

Also state whether that the ghrelin receptor is expressed in the intestine and that you were seeking to test for a direct action of ghrelin on the intestinal cells

Proofread by a native speaker--many sentences are missing the article “The”

Table I, replace “gender” with “sex”

Methods, was one week of inverted LD really enough to shift light entrained rhythms? Was this checked behaviorally?

Methods, It is not necessary to denote “JAX™” when describing C57BL/6J mice as the Jax is implied

Methods, “45oC”, remove “C”

Results, “Thus, the expression of LAT4 protein could be regulated at the level of translation or post-translationally (e.g. protein stability and/or translocation to membrane).”. This later bit about translocation should be removed as it doesn’t appear that the authors specifically isolated membrane fraction (versus other intracellular compartments) with the low speed centrifugation step that was applied. If more effort was taken to isolate membranes than what is described in the Methods section then it should be described there

What is the meaning of “Swiss roll”? Please define

There are at least two references to unpublished manuscripts. I believe that it is against the policy of PLoS ONE to cite unpublished manuscripts. At least that was the case the last time I published in PLoS ONE in 2018.

Link to phospho web domains needed only in methods section

For the “box and whisker” plots, it is only necessary to describe their meaning once instead of repeatedly in each figure legend.

Please attempt to fit a sine wave to the data that you believe to be showing a circadian rhythm and describe the goodness of fit to a sine wave. I appreciate that the data points are shown but it would be nice to see if these are rhythmic or not as many of them just appear noisy.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Andrew Steele

[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.

Attachments

Attachment

Submitted filename: plos one review.pdf

PONE-D-20-02951R1

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is controlled by food-entrained diurnal rhythm and dietary proteins

PLOS ONE

Dear Dr. Verrey,

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 Jun 11 2020 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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

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,

Urs Albrecht, Ph.D.

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 #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: Yes

Reviewer #2: (No Response)

**********

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

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

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: (No Response)

Reviewer #2: (No Response)

**********

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: (No Response)

**********

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: (No Response)

Reviewer #2: Excellent job on revising the manuscript. I'm very sorry that I did not mention this upon my first review, but the style that is used in parts of the results section seems inappropriate. For example, parts of the results section read like Figure legends in stead of as a narrative that references Figures parenthetically to support the description.

This is a copy-past of an early paragraph within the results section:

Fig 1. mRNAs of nutrient uniporters in mice duodenum show little or no food 504

entrained diurnal regulation, in contrast to symporters and antiporters. A: Food505

entrainment regimen used for all mice experiments. ZT0: start of the resting period; ZT12:

506 start of the active period. Mice fed 18% protein diet only from ZT12 to ZT20 for at least

507 14 consecutive days. Euthanasia at ZT12 was done before the feeding starts. B: mRNA

508 expression of different nutrient transporters in duodenum measured by real time PCR.

One cannot write a results section in this manner--this should be in the figure legends and the results should be a narrative written with proper sentences in paragraph form. For example, "The mRNA of nutrient uniporters in the duodenum of mice showed little or no food entrained diurnal regulation, in contrast to x (Figure 1A)... (Figure 1B) and so on.

**********

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: No

Reviewer #2: Yes: Andrew Steele

[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.

PONE-D-20-02951R2

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is controlled by food-entrained diurnal rhythm and dietary proteins

PLOS ONE

Dear Dr. Verrey,

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 Jun 21 2020 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.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

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,

Urs Albrecht, Ph.D.

Academic Editor

PLOS ONE

Additional academic editor comments;

Reviewer two had a minor comment on the R1, and this second new comment has to be added to the revision. Both reviewers have been invited to look at R1, the first one accepted it and the second one had a minor comment. These are the standings. So the authors should correct the second comment of reviewer 2.

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

[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.

Phosphorylation of mouse intestinal basolateral amino acid uniporter LAT4 is controlled by food-entrained diurnal rhythm and dietary proteins

PONE-D-20-02951R3

Dear Dr. Verrey,

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,

Urs Albrecht, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for your revisions and resolve the misunderstanding.

Reviewers' comments: