Sex-Specific and Reproductive Status-Dependent Effects of Liraglutide on Metabolic Disorders Associated with Prediabetes

Round 1

Reviewer 1 Report

In this study by Lebertová and colleagues titled “Sex-specific and reproductive status dependent effect of liraglutide on metabolic disorders associated with prediabetes,” the effect of liraglutide was analyzed in male, female, and ovariectomized female rats with hereditary hypertriglyceridemia (HHTg) as a model of prediabetes. Here are my comments related to this manuscript.

-The abstract section needs improvement, including liraglutide dose and the period of time with treatment. 

-Materials and methods section should be improved in several sections where there is missing information. Include information about the bodyweight of group rats at the beginning of the study, including the type of diet that rats were fed. 

-There is not enough information about group rats in the materials and methods section.

-Please clarify how tissues were analyzed for each parameter evaluated (e.g., liver, myocardium, and skeletal muscle), including more detail regarding the amount of tissue and its dissection. 

-How many hours the rats were fasting before the sacrifice.

-Please clarify if the integrity of the total mRNA was verified.

-The main concern related to this study is that female group rats were not synchronized with the estrous cycle before sacrifice. Studies suggest that rat estrous cycles (proestrus, estrus, metestrus, and diestrus) significantly influence metabolic parameters at the time of sacrifice.

-In the results section the authors should describe some important results with p-values. 

-Why were liver histologies with hematoxylin and eosin staining not included in the study? These liver histologies are important to analyze the lipid accumulation and focus of inflammation.

-In this study key genes were analyzed at the level of mRNA expression related to lipid metabolism. However, the authors should include the expression of some key proteins for lipid metabolism using Western blot assays.

Based on the expression of Pparα and Pparγ mRNA in the liver of rat groups, discuss how the expression patterns of these genes influence lipid metabolism.

-This study reported that liraglutide had more beneficial effects on metabolic parameters in female rats compared to male rats. However, the study of Milani et al. (reference 28) reported that “men achieved greater weight loss (WL), BMI reduction, %WL, WL > 5%, and >10% than women, and they also showed more significant improvements in metabolic parameters (total and LDL cholesterol, Fibrosis-4 Index FIB-4).”

-This study must include a limitations section. 

-Update MASLD Prevalence in the following sentence related to the introduction section: “Metabolic dysfunction-associated steatosis liver disease (MASLD) affects more than 30% of the global population [1].”  

Author Response

Dear reviewer,

Thank you for your extensive and professional comments. In line with your recommendations, we have improved the abstract. However, it was necessary to adhere to the maximum word count of 200 according to the author instructions. In the Methods section, we have provided a more detailed description of the animals and their diet, and clarified that the animals were decapitated in a non-fasted state, which better reflects the metabolic changes associated with prediabetes. RNA quality and concentration were determined spectrophotometrically (Nanodrop One, Thermo Fisher Scientific, MA, USA). Samples with RNA A₂₆₀/A₂₈₀ >1.9 and A₂₆₀/A₂₃₀ >1.8 were used in qPCR.

In the Discussion section, we added a paragraph on the limitations of the study, including the fact that the females were not synchronised in their estrous cycles. We also made significant changes to the Discussion section and slightly altered the conclusion to provide a better commentary on and interpretation of the results. According to your recommendation, comments about PPARs have been added. The authors thank you for bringing citation 28 to their attention; this has now been replaced with a more appropriate one.

The p-values were added to the Result section of the text. Furthermore, the authors would like to point out that they were unable to determine protein expression using Western blot assays. A short statement acknowledging the absence of protein-level validation forms part of the limitations of the study at the end of the discussion. Histological examination of the liver was not performed in this study. The rat strain used only exhibits simple hepatic steatosis, which is not reflected in the histological assessment, as evidenced by our previous results. Therefore, it is unlikely that liraglutide treatment would show any changes. For this reason, the assessment was not performed.

Once again, we thank you for your comments and we hope that now the revised manuscript will comply with your remarks.

Reviewer 2 Report

I. Overall Assessment

 

This study used non-obese prediabetic HHTg rats as models to investigate gender- and reproductive status-related differences in the effects of liraglutide on improving glucose and lipid metabolism and MASLD-associated phenotypes. The topic has clinical translational value, with well-designed experimental groups and comprehensive evaluation parameters. However, the manuscript has major shortcomings in methodological rigor, consistent statistical approaches, rigorous result interpretation, in-depth mechanistic analysis, as well as figure, table and text presentation. It fails to meet the journal’s publication requirements and can only be reconsidered for publication after substantial revisions.

 

Major Issues (Required Revisions)

Inconsistencies in Statistical Methods

1. The Methods section states Tukey’s post-hoc test was applied, whereas all figure legends refer to Fisher’s LSD test, leading to contradictions.
2. Two-way ANOVA was used to analyze the interaction of sex/reproductive status and treatment. However, only treatment effects were described without clarification on the significance of interactions, resulting in insufficient statistical evidence for sex-related differences.
3. Sample size was rationalized in accordance with the 3Rs principle, yet no sample size calculation or statistical power analysis was provided, indicating inadequate methodological rigor.

Inaccurate Presentation of Key Results and Overstated Conclusions

1. Hepatic TAG levels were significantly reduced only in female and ovariectomized female rats, with no remarkable changes observed in male rats. This core discrepancy was not highlighted in the abstract or main text, which may mislead readers.
2. Conclusions of improvement were drawn based on trends of certain indicators instead of strict significance criteria of P < 0.05.
3. Intergroup differences in oxidative stress and anti-inflammatory markers were mild, while their effects were overemphasized. Evidence supporting mechanistic correlations was insufficient.

Insufficient Methodological Details Hindering Experimental Reproducibility

1. Critical details regarding animal husbandry, drug administration schedule and sample processing were vaguely described.
2. The qPCR protocol lacked detailed validation data on reference gene stability, amplification efficiency and quality control.
3. Only the dosage was specified for the oral glucose tolerance test (oGTT), with no clear specifications on fasting duration, blood collection time points and blind trial design.
4. Descriptions of core biochemical assays including lipid extraction and enzyme activity detection were overly brief.

Insufficient Depth in Discussion

1. No clear explanation was provided for the superior efficacy in female rats relative to males, and discussions on the crosstalk mechanism between estrogen and GLP-1 pathway remained superficial.
2. The upstream and downstream causal relationships among hepatic lipid metabolism-related genes, inflammation and oxidative stress were undefined, with complete mechanistic pathways missing.
3. Study limitations were not fully discussed, including only acute/subacute intervention design, absence of dose gradient setting, lack of long-term liver fibrosis observation and independent experimental verification.

Poor Figure Quality and Inconsistent Labeling

1. Abbreviations including OVX, TAG, DAG and HOMA-IR were not fully defined upon first appearance in figure titles.
2. Statistical symbols and letters were too small, and asterisk markers were inconsistent, failing to meet journal publication standards.
3. Inconsistent significant figures and disordered layout existed in Table 1 and Table 2, and missing data were not annotated accordingly.
4. Inconsistent labeling was found in error bars, group divisions and statistical significance markers.
5. Partial axes in bar graphs were indistinct.

Linguistic and Formatting Defects

1. Grammatical errors existed in titles, including missing hyphens and incorrect singular-plural usage.
2. The abstract was redundant and lacked key quantitative results.
3. Abbreviations were used inconsistently throughout the manuscript with no dedicated abbreviation list attached.
4. Several sentences suffered from poor logical coherence and non-standard academic English expression

Minor Issues (Suggested Revisions)

1. The introduction failed to fully elaborate the clinical background of sex differences and menopausal impacts, and existing research gaps were not sufficiently emphasized.
2. The Results section was verbose and unfocused, which should be streamlined following the logical order: phenotype → metabolism → molecular mechanism → oxidative stress and inflammation.
3. The Discussion section showed excessive overlap with published literature, lacking innovative interpretations and suggestions for clinical translational application.
4.  Ethics approval information is complete.

Revision Requirements and Publication Recommendations

1. Statistical methods must be standardised; Tukey’s or LSD tests must be used consistently throughout the manuscript, and interaction effects must be reported in full.

2. All results must be rewritten strictly according to p-values, without exaggerating or omitting key gender differences.

3. All methodological details must be supplemented to meet reproducibility standards.

4. The discussion section must be rewritten to strengthen explanations of mechanisms, clinical significance and study limitations.

5. Figures, tables, abbreviations, formatting and language must be fully standardised.

This study features robust data and clear points of innovation; it has publication potential following revision, but the current version requires substantial revision.

 

Author Response

Dear reviewer,

Thank you for your extensive and professional comments.

Inconsistencies in Statistical Methods: The authors apologise for incorrectly stating the post hoc test in the figure legends. This has been corrected in all figures; however, in Figure 6, a one-way ANOVA and Fisher's post hoc test were used. This is stated in the description of the statistical method. In accordance with your recommendation to declare the appropriate number of animals in the experimental groups, a power analysis has been provided and added to the statistical method.

The authors would like to provide a clearer explanation of the statistical method used, as well as of how it is expressed in the individual graphs. In all figures, based on two-way ANOVA method used, the blue lines represent significance of liraglutide in general treatment across all groups as a result of the two-way ANOVA (Ptreatment). The green lines represent significances of liraglutide treatment depending on sex as P interaction of treatment between males and females (showed as Psex). In the same way, the red lines represent significances of liraglutide treatment depending on menopause as P interaction of treatment between females and OVX female (showed as Pmenopause). Color coding was used for better clarity. The black bars represent multiple comparrison between untreated and treated groups, for better clarity only the results of these multiple comparisons are presented in figures. It was not possible to present all these P-values ​​in the tables, therefore only the Ptreatment and Pinteraction were shown.

Inaccurate Presentation of Key Results and Overstated Conclusions: In line with your recommendations, we have made slight changes to the conclusion to improve the commentary and interpretation of the results. We have also corrected some parts of the results description, including the titles.

Insufficient Methodological Details Hindering Experimental Reproducibility: The method section has been updated to include all the necessary details about the animals, their diet, extraction and precise measurement of enzyme activities and oGTT. Data on gene stability, amplification efficiency and quality control has also been added.

Data on reference gene stability, amplification efficiency and quality control:

The housekeeping gene Hprt1 was selected for normalising RT-qPCR data based on its high stability across a wide variety of tissue types and under different experimental conditions (Svingen T. et al. (2015), Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions. PeerJ 3:e855; DOI 10.7717/peerj.855).

The quantitative real-time PCR was performed on LightCycler 1536 designated for massive screening (1536 wells per plate), reaction volumes were 1ml. Amplification and quality control were validated using integrated LightCycler 1536 software. The technical correctness of the run was verified by an internal pipetting control. The biological positivity of the samples and the specific signal increase were confirmed by analysis of the kinetics of the amplification curves (evaluating the Slope and EPF parameters) within a technical quadruplicate.

Insufficient Depth in Discussion: According to your recommendations, we made significant changes to the Discussion section, and the conclusion was also slightly amended to provide a clearer interpretation of the results. We added a paragraph about the limitations of the study and provided further comments on the interaction between estrogen and GLP-1 and the interpretation of the results including the PPARs. Some parts of the discussion were shortened, especially the section on oxidative stress and inflammation, with speculative and repetitive parts removed.

Poor Figure Quality and Inconsistent Labeling: We have corrected the figure legends and improved the formal quality of all figures.

Linguistic and Formatting Defects: In line with your recommendation, we have corrected the titles and abstract, as well as any inaccurate expressions. We have also checked the abbreviations used throughout the manuscript. The manuscript has been proofread for English language and style.  

Once again, we thank you for your comments and we hope that now the revised manuscript will comply with your remarks.

Reviewer 3 Report

The manuscript investigates the sex-specific and reproductive status-dependent effects of liraglutide in hereditary hypertriglyceridemic rats, used as a non-obese prediabetic model. The topic is relevant, since the metabolic effects of GLP-1 receptor agonists may differ according to sex and menopausal status, and this aspect remains insufficiently explored. The inclusion of male, fertile female, and ovariectomized female rats represents an interesting experimental design, particularly in the context of prediabetes, hepatic steatosis, insulin resistance, and metabolic dysfunction-associated steatotic liver disease.

Overall, the study is well conceived and includes a broad metabolic characterization, covering glucose tolerance, insulin sensitivity in peripheral tissues, visceral adiposity, hepatic lipid accumulation, fatty acid composition, hepatic gene expression, inflammatory mediators, and oxidative stress markers. The finding that liraglutide improves glucose tolerance in all groups, while exerting stronger effects on hepatic triglyceride accumulation, visceral adiposity, and lipid-related gene expression in females and ovariectomized females, is potentially valuable and may support the relevance of sex and reproductive status in modulating the metabolic response to GLP-1 receptor agonists.

However, several aspects require further refinement before publication.

Mechanistic interpretation of sex-specific effects

The authors should better clarify whether the stronger response observed in females is mainly related to sex hormones, differences in food intake reduction, changes in adiposity, or potential pharmacokinetic/pharmacodynamic differences. The current interpretation is biologically plausible, but in several sections it remains speculative. Since estradiol levels did not significantly change after liraglutide treatment, the authors should be cautious in attributing the observed effects directly to estrogen-related mechanisms. The discussion should more clearly distinguish between demonstrated findings and mechanistic hypotheses.

Food intake as a potential confounder

Liraglutide markedly reduced food intake in all groups. Therefore, it is difficult to separate direct pharmacological effects from secondary effects related to caloric restriction and weight loss. This point should be discussed more explicitly. Ideally, the authors should clarify whether pair-fed controls were considered or explain this limitation in the Discussion. Without pair-fed controls, conclusions regarding direct effects of liraglutide on hepatic lipid metabolism, oxidative stress, and gene expression should be presented with caution.

Gene expression data require more cautious interpretation

The hepatic mRNA expression results are interesting, particularly regarding Scd1, Srebf1, Srebf2, Pparα, Pparγ, Hmgcr, and Tgfβ. However, mRNA levels do not necessarily reflect protein expression or enzymatic activity. The authors should avoid overinterpreting these findings as definitive evidence of pathway activation or inhibition. A short statement acknowledging the absence of protein-level validation would improve the balance of the interpretation.

Language and terminology

The manuscript requires moderate language revision. Some expressions should be corrected or refined, for example “beneficial effects of T2D” should be “beneficial effects in T2D,” and “metabolic dysfunction-associated steatosis liver disease” should be corrected to “metabolic dysfunction-associated steatotic liver disease.” The text should also consistently use “triacylglycerols” or “triglycerides” and define abbreviations only once.

Conclusion

This is an interesting and potentially valuable experimental study showing that liraglutide improves several metabolic alterations in a non-obese prediabetic rat model, with relevant differences according to sex and reproductive status.. After these revisions, the study could provide a meaningful contribution to the understanding of sex-specific metabolic responses to GLP-1 receptor agonists.

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Author Response

Dear reviewer,

Thank you for your extensive professional comments. In line with your recommendations, we have added a paragraph to the Discussion section regarding study limitations, including a short statement acknowledging the absence of protein-level validation. Unfortunately, we haven't had the opportunity to measure protein expression by Western blotting. Furthermore, due to the study's scope, it was not possible to use pair-fed controls, which is mentioned in the study limitations.

In addition, significant changes were made to the Discussion section, and the conclusion was slightly altered to provide a more accurate interpretation of the results.

Throughout the manuscript, we corrected any incorrect or inaccurate expressions, improving the English language and style in the process.

Once again, we thank you for your comments and we hope that now the revised manuscript will comply with your remarks.

 

Round 2

Reviewer 1 Report

I have no comments; my suggestions were taken into account. 

I have no comments; my suggestions were taken into account. 

Author Response

Dear reviewer,

we did not find any additional comments from your side, so we would like to thank you once again for your positive evaluation of our manuscript. We greatly appreciate the time and effort you devoted to reviewing our work.

Reviewer 2 Report

The author’s revisions largely meet my expectations. 

Following minor amendments, such as adjusting the citation format and appropriately deepening the discussion (e.g. by drawing comparisons with similar studies), the manuscript is acceptable.

Author Response

Dear reviewer,

thank you for your evaluation and helpful suggestions. The citation format has been checked, and the Discussion section has been expanded to include comparision with similar studies, as recommended. We aprpreciate your valuable comments, which have helped improve the quality of the manuscript.