Review Reports - Phenotype-Specific Mitochondrial Responses to Mediterranean Diet and Exercise in Elderly Obesity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well structured and addresses a timely and clinically relevant topic by investigating phenotype-specific mitochondrial responses to lifestyle modification in elderly individuals with obesity. The integration of clinical, nutritional, physical activity, and mitochondrial parameters is a clear strength of the study. Overall, the data are solid and the conclusions are generally supported by the results. However, several methodological clarifications and minor improvements in data presentation and interpretation would further strengthen the manuscript.

Clarification of MHO and MUO Classification

The criteria used to define metabolically healthy obesity (MHO) and metabolically unhealthy obesity (MUO) should be stated more explicitly in the Methods section. Although reference is made to previous studies, briefly reporting the number and type of metabolic alterations required for classification would help readers better contextualize the phenotype-specific mitochondrial differences observed.

PBMC Heterogeneity

In Section 2.5, it would be useful to clarify whether PBMCs were analyzed as an unfractionated heterogeneous population or whether any normalization for cell subtype composition (e.g., lymphocytes vs. monocytes) was considered. Additionally, a short note in the Discussion addressing the limitations of using PBMCs as a surrogate tissue for systemic mitochondrial function would improve transparency.

mRNA–Protein Discordance

The frequent discordance between mRNA and protein levels (e.g., OPA1, MFN2, TFAM, COXIV) represents an important biological observation. While this aspect is discussed later in the manuscript, I suggest highlighting it more explicitly in the Results section to better guide the reader through the interpretation of the molecular data. In addition, the authors may consider briefly discussing whether post-transcriptional or translational regulatory mechanisms could account for the observed discrepancies between mRNA and protein expression levels.

Statistical Power, Sample Size, and Data Presentation
Several correlation analyses were performed in relatively small subgroups (in some cases N = 8–14). The authors should explicitly acknowledge, either in the Results section or in the figure legends, that these analyses are exploratory and limited by sample size, in order to avoid potential overinterpretation. In addition, some results—such as OPA1 protein levels assessed by western blot—appear to be repeated across figures (e.g., Figures 1 and 2). The authors are encouraged to clarify the rationale for this presentation and to better explain the analytical strategy used to distinguish baseline differences from longitudinal changes. Specifically, a clearer explanation is needed to help the reader understand why the changes observed after 12 months of intervention are relatively modest in certain analyses (e.g., Figure 1), and how these results should be interpreted in the context of the study design.

Comments on the Quality of English Language

Minor Language and Typographical Issues

There are several minor grammatical and typographical issues throughout the manuscript (e.g., inconsistent use of LSM/LMS, “PBMCS,” “centrifugated,” “regarding to”). A final careful language revision is recommended to improve clarity and overall presentation. In addition, the nomenclature of PINK1 isoforms is not always consistent between the main text and the corresponding figures. Harmonizing the terminology across text, figures, and figure legends would greatly improve readability and avoid potential confusion for the reader.

Author Response

In regard to the manuscript entitled “Phenotype-specific mitochondrial responses to Mediterranean diet and exercise in elderly obesity” submitted to Nutrients for consideration for the special issue “Nutritional and Metabolic Biomarkers in Obesity,” we would like to thank you for giving us the opportunity to revise our manuscript.

We have carefully considered the comments made by the reviewers and agree with most of them. Each comment has been addressed and we have modified the manuscript accordingly.

We sincerely hope that the current version of the manuscript will be acceptable for publication in your journal.

All changes are shown in red so that they may be easily seen.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thank you to give the oportunity to review your manuscript. I have some sugestions to improve it.

Abstract

Clarify the design at the beginning, e.g. “secondary analysis of a 12-month lifestyle intervention trial” and explicity state that the sample size was 43 elferly individuals with obesity, stratified into MHO and MUO according to WHO criteria.

Introduction

Expand the introduction to briefly summarize the heterogeneity of MHO definitions and justify the chosen WHO-based operational definition, providing key components.

Metods

Provide more detail on the intervention: number and type of visits, who delivered counseling, use of written materials, and whether any specific caloric targets were set.

Present baseline and 12‑month MedDiet and RAPA scores (means ± SD/SEM) in a table (e.g., Table 1 or a new table) for MHO and MUO separately, including p values and effect sizes, given that adherence is central to interpreting mitochondrial changes.

Results

Reformat Table 1 for clarity: separate columns for baseline and 12‑month values per group, with Δ and Cohen’s d in additional columns or a separate table.

Ensure all abbreviations in the table are defined in the footnote and consistent with the main text (MHO, MUO).

For correlations, consider reporting 95% confidence intervals along with rho and p, at least for the main associations emphasized in the Discussion.

Discussion

Structure the Discussion to first address the predefined primary outcomes (phenotype differences in fusion/mitophagy markers and their changes with LSM), then discuss secondary correlation analyses as exploratory.

Clarify that the concept of phenotype‑tailored lifestyle interventions is promising but remains to be tested in interventional studies specifically designed for this purpose.

References

Run the reference list through the Nutrients reference manager or style check to ensure full consistency with MDPI guidelines (abbreviations, italics, DOIs, etc.).

Best regards,

Author Response

We have carefully considered the comments made by the reviewers and agree with most of them. Each comment has been addressed and we have modified the manuscript accordingly.

We sincerely hope that the current version of the manuscript will be acceptable for publication in your journal.

All changes are shown in red so that they may be easily seen.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Revision of abbreviation is needed (when are they first introduced etc.).

Please emphasize the clinical revelance of Your study and what is new regarding the groups of metabolically healthy and unhealthy obese.

The results section could use some clarification. There is too many content (including pictures) and it is difficult to follow.

Comments on the Quality of English Language

Could use some revision.

Author Response

We have carefully considered the comments made by the reviewers and agree with most of them. Each comment has been addressed and we have modified the manuscript accordingly.

We sincerely hope that the current version of the manuscript will be acceptable for publication in your journal.

All changes are shown in red so that they may be easily seen.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Overall, the study is well designed, methodologically sound, and clearly written, and the results are internally consistent and biologically plausible. The longitudinal 12-month intervention, combined dietary and physical activity assessment, and integration of molecular, nutritional, and clinical outcomes represent clear strengths. The findings that MUO participants exhibit more pronounced mitochondrial remodeling and clinical improvements than MHO participants are novel and add meaningful insight to the field.

That said, several conceptual, methodological, and interpretive issues should be addressed to strengthen the manuscript and improve clarity and rigor. These points can be resolved with moderate revisions, rather than additional experimentation. Here are the comments and suggestions.

Although the manuscript occasionally uses language implying intervention effects, the study is described as an observational cohort and a secondary analysis of a prior trial. This limits causal inference. Please clarify more explicitly in the Methods and Discussion that causal conclusions should be interpreted cautiously and replace wording such as “LSM improves” with “LSM is associated with” where appropriate. It would be better to discuss briefly how the lack of a non-intervention control group may influence interpretation.

The criteria used to classify MHO and MUO are referenced but not fully detailed in this manuscript, and it is unclear whether participants changed phenotype during follow-up. Please list the metabolic criteria used to define MHO and MUO in the Methods (even if previously published). Also, please state whether any participants transitioned between phenotypes during the 12-month period, and if not assessed, acknowledge this as a limitation. Also, please discuss how phenotype instability could influence longitudinal mitochondrial outcomes.

The molecular analyses are conducted in relatively small subgroups, with further reductions due to missing data, increasing the risk of type I and type II errors, particularly for correlation analyses. Please add a brief power or sample size consideration (even post hoc) to contextualize null findings, especially in MHO. Also, please consider clearly labeling correlation findings as hypothesis-generating.

The identification of the ~60 and ~45 kDa MFN2 bands as degradation products is plausible but inferential. Please explicitly state that these bands are putative degradation products. If possible, cite literature supporting MFN2 proteolysis patterns or briefly discuss alternative explanations (e.g., splice variants or antibody cross-reactivity). I also recommend avoiding definitive mechanistic claims without confirmatory assays.

While PBMCs are practical, they may not fully reflect mitochondrial dynamics in metabolically active tissues such as muscle or adipose tissue. Please expand the Discussion to more clearly delineate what PBMC mitochondrial changes can and cannot represent. It would be better to clarify that findings reflect systemic or immune-cell mitochondrial adaptations rather than direct tissue-specific metabolism.

It may be editorial issue, but Table 1 is difficult to read. Please align.

Author Response

We have carefully considered the comments made by the reviewers and agree with most of them. Each comment has been addressed and we have modified the manuscript accordingly.

We sincerely hope that the current version of the manuscript will be acceptable for publication in your journal.

All changes are shown in red so that they may be easily seen.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The changes have been applied correctly.

Best regards,

Author Response

Thank you so much for your comments.

These cooments have helped improve the quality of our manuscript.

Best regards

ROSA