PONE-D-24-57032Cnot4 heterozygosity attenuates high fat diet-induced obesity in mice and impairs PPARγ-mediated adipocyte differentiation.PLOS ONE

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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 publication criteria as it currently stands. The shortcomings of this paper needs to be worked out before it can be considered for publication. Therefore, we invite you to resubmit a revised version of the manuscript that addresses the points raised during the review process.

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Specific concerns expressed during peer review were:

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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: 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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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

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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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: This manuscript investigates the role of CNOT4, an E3 ubiquitin ligase, in adipocyte differentiation and obesity, using Cnot4 heterozygous knockout mice. The study finds that Cnot4 heterozygosity attenuates high-fat diet-induced obesity by impairing adipocyte differentiation, and that this effect is mediated by the reduced transcriptional activity of PPARγ. While the study presents some interesting findings, several concerns arise regarding its originality, methodological rigor, and interpretation of results.

While the manuscript claims that the role of CNOT4 in obesity is unexplored, the introduction cites studies on other components of the CCR4-NOT complex (CNOT3, CNOT6L, and CNOT7) that are involved in obesity and energy metabolism, which raises questions about the novelty of exploring another member of this complex. Although this is by itself not a huge issue.

The study suggests that up-regulating CNOT4 might benefit lipodystrophy patients, which seems counterintuitive given their finding that CNOT4 promotes adipocyte differentiation. If CNOT4 is necessary for adipocyte differentiation, then, it is not clear how its upregulation would treat lipodystrophy, which is characterized by loss of adipose tissue. The authors need to clarify this point.

The study proposes that understanding CNOT4's mechanism in activating PPARγ could lead to new drugs for obesity, diabetes, and cardiovascular disease. However, the study does not show a direct interaction between CNOT4 and PPARγ. The assertion that CNOT4’s role in PPARγ regulation could lead to new therapies requires stronger evidence than what the manuscript currently provides.

The study concludes that Cnot4 heterozygosity leads to reduced adipocyte differentiation using in vitro MEF experiments, but the study shows that Cnot4 Het mice did not show significant change in the expression of metabolic genes in WATs and livers. This needs to be explained.

The authors claim that Cnot4 heterozygous deletion suppressed adipocyte differentiation partly through reduced transcriptional activity of PPARγ. However, Cnot4 Het mice did not show improved dyslipidemia or glucose intolerance. If PPARγ is crucial for adipogenesis, it is not clear why dyslipidemia or glucose intolerance were not affected despite changes in PPARγ activity.

The study does not investigate the specific mechanisms behind how CNOT4 regulates PPARγ transcriptional activity. The study suggests a potential role of epigenetic modification or RNAPII regulation, however, these suggestions remain purely speculative.

The methods section lacks detail regarding the specific procedures for measuring mRNA expression, protein expression and luciferase activity. The manuscript mentions that 'qRT-PCR analysis was conducted as previously described' citing another paper, but this doesn’t allow the reader to properly assess the rigor of those methods.

The figure legends include some statistical tests. However, the data provided in the figures are sometimes not sufficient to justify the statistical tests they claim to have used.

There is an inconsistency in the data presented. For example, Figure 2A shows mRNA expression of Cnot4 in mouse livers for WT and Cnot4 Het1920, but that data is not used anywhere else in the results.

Figure 1: The figure legend describes the knockout strategy and shows genotyping results, but it doesn't include the genotypes of the parents of the embryos. This omission makes it difficult to assess whether the correct genotypes were obtained for the different crosses.

Figure 4: The representative images of adipocytes stained with Oil Red O are not clear. Although the areas of Oil Red O staining are quantified in a bar graph, the image clarity makes it difficult to ascertain the level of lipid accumulation in the cells.

Overall Recommendation: The study presents some interesting observations regarding CNOT4's role in adipocyte differentiation and obesity. However, there are significant concerns about the originality, the methodological rigor, the interpretation of results, and the statistical analysis. The manuscript requires major revisions as outlined above.

Reviewer #2: This study by Yamaguchi et al. investigates the role of CNOT4 in adipocyte differentiation and its impact on diet-induced obesity using a KO mouse model. The authors provide evidence that the KO mice exhibit resistance to diet-induced obesity, attributed to suppressed adipocyte hyperplasia by regulating PPAR� transcriptional activity. These findings suggest altered adipose tissue functionality. However, I have several concerns that require further clarification and discussion.

1. The authors do not address the fate of dietary lipids in the KO mice, which is crucial given their resistance to diet-induced obesity and smaller adipocyte size. If adipocyte differentiation and lipid storage are impaired, it is important to discuss the potential lipid redistribution and its implications for systemic metabolic health. The data indicate that ectopic lipid accumulation in the liver is absent and circulating lipid levels remain unaltered in the KO mice. These observations suggest that lipid oxidation or energy expenditure might be enhanced. Further investigation or at least a discussion of these possibilities is needed.

2. The quantification of adipocyte size should be presented more comprehensively (e.g., frequency histograms or size range percentages) to better illustrate the observed differences. Additionally, the authors should specify how many fields of view per slide and how many mice were analyzed. The same level of detail is necessary for Figure 4C.

3. In the second paragraph of the Discussion, the authors mention that there were no differences in the mRNA levels of Ucp1, Gdf15, and Fgf21 in Cnot4 Het mice (lines 171–172). However, I could not find any corresponding data in the manuscript. If these results have been published elsewhere, the authors should provide an appropriate citation. If the data are unpublished, they should explicitly state that it is an unpublished observation or “data not shown”.

4. The last paragraph of Discussion part, lines 207–209, does not make sense. It implies a causal relationship between the lack of improvement in glucose and lipid levels and the ineffectiveness of CNOT4 suppression for obesity treatment.

5. Please specify the name and version of the statistical software used for data analysis.

6. There are some typos in the manuscript. Line 188, the authors probably meant “down-regulate”, and line 422 (E) probably means weight of “hearts”.

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Reviewer #1: No

Reviewer #2: No

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Cnot4 heterozygosity attenuates high fat diet-induced obesity in mice and impairs PPARγ-mediated adipocyte differentiation.

PONE-D-24-57032R1

Dear Dr. Kuba,

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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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

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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

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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

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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: Some minor remaining concerns:

The manuscript continues to suggest that CNOT4 regulates PPARγ activity through recruitment to chromatin, but no direct or indirect co-factor interaction is shown. The absence of direct interaction is acknowledged, but the discussion still speculates heavily about epigenetic or polymerase-associated mechanisms based on yeast orthologs. This should be clearly labeled as a hypothesis.

The authors wisely toned down their claims, but occasional phrases still hint at clinical relevance (e.g., implications for obesity treatment) that go beyond the evidence.

While addressed, the manuscript would benefit from a clearer, less technical explanation of why Cnot4 Het affects differentiation in MEFs but not mature adipocyte gene expression in vivo. This distinction is central to interpreting the model.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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