PONE-D-21-26341Role of miR-181c in Diet-Induced Obesity Through Regulation of Lipid Synthesis in LiverPLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, Akiyoshi et al explored the role of miR-181c in diet induced obesity in vivo through both loss and gain of function assays. In miR-181c/d knockout mouse model, the authors have shown that inactivation of miR-181c/d led to glucose intolerance post 26 weeks high fat diet challenge associated with more lipid accumulation in liver and up-regulation of lipid synthesis genes potentially through up-regulation of IDH1 at protein level. Similarly, the authors have also tested the response of HFD in miR-181c overexpressing mouse model through AAV mediated gene delivery. Although no change in body weight, the authors claimed expression of miR-181c can attenuate lipid accumulation in liver post high fat diet challenge.

Previously the authors have shown inactivation of miR-181c/d protected mice against ischemia-reperfusion injury, therefore, the results presented in this manuscript are un-expected. Here are several concerns:

1. The regulatory role of miR-181c/d on IDH1

No sufficient data supporting that miR-181c directly regulates IDH1 in liver. For example, the authors have not shown mRNA expression level of IDH1 and IDH2 in miR-181c/d knockout animals or AAV8-miR-181c injected animals. There is no luciferase assay suggesting miR-181c indeed regulate IDH1 3’UTR activity. Without this evidence, the changes of IDH1 protein expression could be secondary effect.

2. Relevance of miR-181c in liver disease

The authors hypothesized that c/d KO mice would be protected from metabolic stress, so the results shown here are unexpected. Is there any change of miR-181c or miR-181d in NASH or other liver disease?

3. Characterization of mouse phenotype post high fat diet challenge

The authors should provide better phenotype characterization of the mouse model. For example, there is only one-time GTT and ITT. The authors should show baseline GTT and ITT in the mouse models (before HFD). There is no liver weight; H&E and liver fibrosis results.

Minor issue

1. The oil-red staining images have poor quality. No scale bar, Figure 4A is not white balanced.

Reviewer #2: The authors have explored the role of miR-181c in a DIO mouse model using a miR-181c -/- mouse and rescue with a AAV-8 delivering miR-181c.

Endpoints are:

Changes in target gene expression using qPCR

Indirect calorimetric measurements

Glucose tolerance tests/insulin levels

Body fat

TGs

Concluding that IDH1 is important in coordinating the changes in lipid biology related to 181c and propose that overexpression of 181c is protective

I do think the observations are of merit and warrant publication but I do have I number of reservations/comments:

- Be consistent in 181c nomenclature ie use 181c throughout

- The main text describing Figure 8, I personally found confusing in regards to glucose tolerance test and would just state no blood glucose or insulin levels were measured

- The authors measure leptin but no rationale as to why measured

- Why did the authors chose a retro-orbital route for AAV delivery, especially if aiming for liver expression, admittedly this was achieved, but tail vein injection is often used to optimize delivery to the liver.

- Could the authors explain why the numbers of mice in each group or observations, varied so much.

- Figure 4+10 (when any histology) need to include magnification

- Figure 5B +5C can you explain why in whole heart tissue you have low levels of 181c but then can find it in the mito fraction. Whole tissue still has the mito fraction within it, so would it not express 181c too?

- For first use of gene name in the manuscript define, as definition of gene names occurs later in the manuscript or in figure legends

- The evidence for IDH1 is mainly through association based on experimental observations and the literature. It would have been good if the authors had demonstrated that 181c is directly acting on the IDH1 3’UTR in a liver cell to provide more supporting evidence for the authors conclusion.

- Discussion, I would present some ideas about why 181c has a cytosolic v. mitochondrial location in the liver in contrast to the heart. Are any gender differences anticipated/known, as authors have used a male mouse model. I would be more reserved on the use of 181c as a protective agent for obesity as in the context of the liver it can act as an Oncomir too.

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Role of miR-181c in Diet-Induced Obesity Through Regulation of Lipid Synthesis in Liver

PONE-D-21-26341R1

Dear Dr. Samarjit Das,

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.

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Aijun Qiao, Ph.D.

Academic Editor

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Additional Editor Comments (optional):

Reviewers' comments:

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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

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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: Yes

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

Reviewer #1: Yes

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 #1: Yes

Reviewer #2: Yes

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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: (No Response)

Reviewer #2: The authors have addressed all my major concerns. Though the author's discussion on the organ specific differences of 181c ie heart and liver, would of benefit by being expanded on eg the metabolic function of the liver v the heart primary function of producing sufficient ATP for cardiomyocyte contractility.

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Reviewer #1: No

Reviewer #2: No