Sex-Specific Metabolic Footprint of Ketogenic Diet in C57BL/6J Mice

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Sex-specific metabolic footprint of ketogenic diet in C57BL/6J 2 mice” by Sablić et al. shows the effect of the keto diet in different mouse tissues after 3 months of dietary intervention. Although authors bring a different perspective to the clinical practice for clinicians, warning about the deleterious effects of long-term use, there are some caveats about its suitability for publication at this stage.

General comments:

-          Professional English-language editing is recommended for better readability.

-          Abstract needs to be checked to show the hypothesis (based on the literature background), research question, how the authors answer those questions, and main outcomes and conclusions.

-          Statistical methods could be simplified. Use the appropriate section to describe tissue or data processing and selection, and keep stat paragraph simple, only mentioning the tests used in any case (if different analyses were performed due to the different nature of the dataset).

-          Results would benefit from being more concise. Particularly, I would avoid adding extra info about stats, which breaks the flow of the reading.

Specific comments:

-          Figure 2 shows the body weight change in percentage values. I would suggest that authors describe in the appropriate method section the equation used for calculating that percentage. Is there any specific reason why the author chose a percentage instead of an absolute value?

-          Histological analysis:

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• I would suggest that authors include information about the number of slices analysed per animal in the methods section.

• Were there tissues from all animals analysed? If not, please add the n used in every figure legend of the manuscript.

• The representative images in figure 4 are not clear, reflecting the results. If the staining that matters is the one surrounding the central vein, I would suggest using magnification images for that area.

-          Figure 7 might show the tentative metabolite annotation instead of the number for readability.

-          If repeated measure ANOVA were employed for tissue comparisons, why does Figure 9 show the data in different graphs per tissue?

-          From the introduction, it is clear the importance of liver and skeletal muscle, and it is quite easy to understand the selection of serum as the third tissue to explore since it is the main connection between organs; however, is there any specific reason why the authors also selected spleen among the rest of the peripheral organs? If so, the introduction needs to be updated to introduce the importance of the spleen.

-          Due to the adiposity sex differences pointed out in the discussions as the main explanation for the delay in body weight increase of females, did the author check adipose tissue weight?

-          I am not sure whether authors could talk about sex specific changes in skeletal muscle metabolism and sterol suppression, as the two groups (males vs females) were not statistically different (Figures 7 and 9). I would suggest that authors be cautious about the emphasis they use in the discussion to avoid misleading readers' conclusions.

Minor comments:

-          The first sentence of the abstract seems incomplete. Please check it. As it is read, it is confusing: “Ketogenic diets (KD) induce profound metabolic shifts but their long-term tissue-specific 26 effects, particularly sex-dependent adaptations in skeletal muscle metabolome, hepatic lipid handling, and sterol homeostasis”.

-          Results paragraph starts with “Therefore”, I would suggest that authors revise the English connectors used throughout the manuscript and ask for advice from an English speaker to improve readability.

-          Legend figure 9 needs to be updated with the whole name for the different metabolite acronyms.

-          Legend figures need to display the tissue used for the specific analysis.

Author Response

Dear reviewer, the authors appreciate your valuable comments and recommendations. They were helpful and substantialy improved our manuscript. We have prepared our response in the attachment. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your submission,

 

My main concerns with the paper are that

• The background does not provide a holistic background of the field and mostly discusses the negatives of KD
• The rationale for the study could be improved
• No clear hypothesis provided
• The result section does not include all relevant inferential statistics such as F values, main effects, interactions
• how does this translate to humans KD has been used safe and effectively for over 100 years without showing these side effects, might these effects be strain/species specific and if so what is the value of this study.

 

Line by line comments:

Line 63- It is a very big claim to state that Ketogenic diet leads to metabolic dysfunction, please provide more evidence or state this more tentatively

Line 79 – the rationale provided for sub aims of the study is poorly expressed – other than that current evidence is limited but what would be the gain

Line 88- Reference here is unclear, as this is not a KD related reference

General introduction- The introduction seems rather brief for many concepts to be introduced. The rationale for the individual markers has somewhat been provided, however a clear picture as what the sum of the findings will contribute is not clear. The sex -differences are at the centre of investigation but no clear rationale has been provided

Line 100- It appears strange as to why one group KDM have a reduced sample size

Line 101- What was the rationale for 12 weeks?

Line 111- should this read “at 3000rpm for 10 min”

Line 184- statistical analysis was not clear. It seems as if only t-test were conducted here you are interested in a Diet* sex effect. These were not reported in the results section line 266 and following. Main effects were not reported

Line 248- please provide actual grams in text as body weight changes can look more dramatic than they actually are.

Line 248- Why was food intake not provided or measured to see calorie intake

Line 353- none of the main effects presented

Line 376- it states reduced metabolites but not in relation to what- what was the comparison here i.e KDMis reduced for dodecanedioylcarnitine in relation to SDM? What was KDF compared to and what was SDF compared to?

Line 413- t-test without an ANOVA are not appropriate. Please specify

Line 441 – calorie spelling

Line 454 – HFD and ketogenic diet are two very different diet profiles and should not be mixed reference 32 is a HFD

Line 457- Reference which would be relevant to support claims such as Long-term ketogenic diet contributes to glycemic control but promotes lipid accumulation and hepatic steatosis in type 2 diabetic mice - ScienceDirect, The ketogenic diet prevents steatosis and insulin resistance by reducing lipogenesis, diacylglycerol accumulation and protein kinase C activity in male rat liver - Jani - 2022 - The Journal of Physiology - Wiley Online Library

Line 447 – this paragraph is very biased towards a negative view and does not present a evenly distributed view of the current kd literature within that area

Line 473- big statements are being made without presenting all literature as stated in previous comment

Line 544- sample size limitation seems contradictory to the a priori sample size calculation. The minimum was 8, which was achieved. Therefore how is this a limitation. The a priori should have been a 2 way ANOVA

Line 547- 12 week old C57 are equivalent to adults it is unclear why it would be more important to test this in aged mice – due to its use in neurodegenerative disorders but what about neurodevelopmental disorders (younger people) or epilepsy

Line 548- if the strain is a issue why was this strain chosen in the first place?

 

 

 

Author Response

Dear reviewer, we are grateful for your valuable time and effort to review the manuscript. We have addressed all comments and revised the manuscript which is now stronger. The response to the comments is attached. 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the revisions. The Authors have addressed all comments appropriately