The Emerging Role of N-Lactoyl-Phenylalanine (Lac-Phe) in Metabolic Regulation and Disease: From Exercise-Induced Metabolite to Therapeutic Candidate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript provides a concise and up-to-date overview of the emerging biological functions of N-lactoyl-phenylalanine (Lac-Phe), an exercise-induced metabolite that has recently attracted considerable interest in the fields of metabolism, exercise physiology, and immunometabolism. The review summarizes current knowledge regarding the biosynthesis of Lac-Phe, its physiological functions, and its potential relevance to metabolic and inflammatory diseases.

Overall, the manuscript is well written and addresses an interesting and rapidly evolving topic. The authors successfully integrate findings from metabolomics, neuroendocrine regulation, and immunometabolism, highlighting the potential role of lactoferrin as a mediator between exercise, appetite regulation, and metabolic homeostasis. The inclusion of recent studies and the analysis of their potential therapeutic implications further reinforce the relevance of this review.

However, several aspects of the manuscript could be improved to enhance clarity:

- The abstract contains a wealth of mechanistic and clinical information, making it somewhat dense. The authors might consider simplifying this section to highlight the most important concepts and reduce unnecessary details. In particular, the statements regarding the applications of biomarkers and their clinical implications could be shortened or refined.

- The manuscript states that lactoferrin-phenylalanine could surpass lactate as a biomarker of mitochondrial dysfunction. While this is an interesting possibility, the current evidence appears limited and largely exploratory. The authors should consider qualifying this statement or providing additional references to support its potential utility in conditions such as MELAS syndrome or sepsis.

- While the formation of Lac-Phe from lactate and phenylalanine is mentioned, the biochemical mechanism could be described more clearly. In particular, the role of CNDP2 and the physiological regulation of this pathway during exercise, feeding, and pharmacological interventions should be explained in greater detail.

Some sections of the manuscript combine findings 

Minor grammatical and stylistic revisions throughout the manuscript would further improve its clarity.

 

Author Response

We would like to thank Reviewer 1 for the extensive assessment of our manuscript, the positive feedback, and the important and helpful comments/suggestions. We have taken all the remarks into account, as detailed below, and have provided our responses to specific comments. We believe that, following the reviewers’ suggestions, our revised manuscript has improved in clarity, and we hope the changes will be considered satisfactory.

 

 

Reviewer 1

This manuscript provides a concise and up-to-date overview of the emerging biological functions of N-lactoyl-phenylalanine (Lac-Phe), an exercise-induced metabolite that has recently attracted considerable interest in the fields of metabolism, exercise physiology, and immunometabolism. The review summarizes current knowledge regarding the biosynthesis of Lac-Phe, its physiological functions, and its potential relevance to metabolic and inflammatory diseases.

Overall, the manuscript is well written and addresses an interesting and rapidly evolving topic. The authors successfully integrate findings from metabolomics, neuroendocrine regulation, and immunometabolism, highlighting the potential role of lactoferrin as a mediator between exercise, appetite regulation, and metabolic homeostasis. The inclusion of recent studies and the analysis of their potential therapeutic implications further reinforce the relevance of this review.

However, several aspects of the manuscript could be improved to enhance clarity:

 

1. The abstract contains a wealth of mechanistic and clinical information, making it somewhat dense. The authors might consider simplifying this section to highlight the most important concepts and reduce unnecessary details. In particular, the statements regarding the applications of biomarkers and their clinical implications could be shortened or refined.

 Response: We thank the reviewer for this constructive suggestion. We have revised the Abstract to improve clarity and conciseness. Specifically, mechanistic details in the biosynthesis sentence (e.g., "closely reflecting increases in glycolytic flux and lactate production") were condensed. The neuronal mechanism sentence was simplified — "inhibiting hypothalamic orexigenic Agouti-related protein (AgRP) neurons while sparing anorexigenic pro-opiomelanocortin neurons" was shortened to "inhibiting hypothalamic orexigenic AgRP neurons," as the full neuronal nomenclature is elaborated in the main text. Additionally, the peripheral effects sentence was streamlined to remove redundant pathway details (NF-κB, ROS) that are detailed in the body. The biomarker sentence was refined from listing specific disease states with mechanistic explanations to a single concise statement: "outperforms lactate in reflecting mitochondrial dysfunction across conditions such as MELAS, sepsis, and NADH-reductive stress." Besides, the final sentences were condensed — recent findings and the clinical trial description were merged into two concise sentences, and the closing summary was tightened to remove redundant phrasing.

 

2. The manuscript states that lactoferrin-phenylalanine could surpass lactate as a biomarker of mitochondrial dysfunction. While this is an interesting possibility, the current evidence appears limited and largely exploratory. The authors should consider qualifying this statement or providing additional references to support its potential utility in conditions such as MELAS syndrome or sepsis.

Response: We thank the reviewer for this important critique. We agree that the original manuscript overstated the superiority of Lac-Phe as a biomarker relative to lactate. The supporting evidence comes primarily from exploratory metabolomics studies and a small number of cohort investigations, which is insufficient to draw definitive clinical conclusions. We have revised the relevant statements throughout the manuscript to appropriately qualify these claims. Specifically, the phrase "may offer diagnostic advantages over traditional markers such as lactate" has been revised to "have shown preliminary potential as biomarkers that may complement or outperform traditional markers such as lactate, though larger prospective studies are needed to confirm their clinical utility," with updated references [1,11,12] in Introduction (Section 1).

The biomarker paragraph to the MELAS statement in Section 3 that Lac-Phe "surpasses lactate in sensitivity" was revised to "may offer a diagnostic advantage over lactate... however, this advantage has been demonstrated primarily in small-scale metabolomics studies and warrants validation in larger clinical cohorts." Reference [10] (Pek et al., 2019) was removed from this citation cluster, as it describes pro-inflammatory properties of MELAS iPS-derived endothelial cells and does not directly assess Lac-Phe as a biomarker. Additionally, the MELAS accumulation statement in Section 3 "surpass lactate as biomarkers" was revised to "may outperform lactate as biomarkers... although these findings are based on exploratory metabolomics data and require prospective clinical validation." Moreover, the claim about sepsis in Section 3 that Lac-Phe "predicts mortality" was qualified to note that "these observations derive from a single cohort study (Rogers et al. [11]) and await independent replication. These findings position Lac-Phe not only as a diagnostic biomarker" was revised to acknowledge that most evidence is preclinical or small-scale, and that further mechanistic and clinical investigations are warranted.

 

3. While the formation of Lac-Phe from lactate and phenylalanine is mentioned, the biochemical mechanism could be described more clearly. In particular, the role of CNDP2 and the physiological regulation of this pathway during exercise, feeding, and pharmacological interventions should be explained in greater detail.

Response: We thank the reviewer for this suggestion. We have substantially expanded Section 2 (Biosynthesis and Regulation of Lac-Phe) to provide a more mechanistically detailed account of CNDP2 function and the regulatory inputs governing Lac-Phe synthesis under exercise, feeding, and pharmacological conditions.

 

4. Some sections of the manuscript combine findings. 

 Response: We thank the reviewer for this observation. We agree that several paragraphs in Section 3 combined findings from distinct biological contexts, which reduced clarity. We have restructured the relevant passages in the revise manuscript.

 

5. Minor grammatical and stylistic revisions throughout the manuscript would further improve its clarity.

 Response: We thank the reviewer for this suggestion. We have carefully reviewed the entire manuscript and made the grammatical and stylistic revisions to improve clarity and precision throughout.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a clear and up-to-date mini-review on Lac-Phe, integrating recent literature on biosynthesis, physiological function, and therapeutic potential. The topic is relevant because Lac-Phe has recently emerged as a candidate exerkine mediator of the effects of exercise on food intake and metabolism. However, the manuscript presents numerous problems that need to be overcome. With substantial revisions, the article could become a useful review on Lac-Phe, but it does not yet reach the analytical level expected in journals as Antioxidants.

Major comments

- The introduction includes redundant information. I suggest that the authors restructure it into three main points: discovery of Lac-Phe, role in dietary control, and therapeutic potential.

- Much of the text presents narrative descriptions of current findings. However, the authors do not take into account methodological limitations, size of evidence, reproducibility, and differences between experimental models. Therefore, the authors should include a critical section addressing the limitations of current evidence, differences between species, and translational gaps.

- In several passages, the manuscript treats Lac-Phe as the main mediator of the effects of exercise. However, current evidence indicates that the effect is partial, occurs mainly in obesity, and depends on metabolic context. I recommend that the authors reformulate passages that suggest direct causality and include language such as "may contribute," "partially mediates," and "remains to be determined."

- The manuscript does not adequately discuss important benchmarks for the physiology of exercise, including intensity and volume, type of exercise, and nutritional status. Without discussing these parameters, the physiology of Lac-Phe is impossible to understand.

- Another problem is that the manuscript mentions clear central effects, but the molecular receptor has not yet been identified; therefore, the cellular mechanisms remain hypothetical. The discussion also treats several mechanisms as established, which needs to be presented with more caution.

- The manuscript ignores classic mediators of appetite regulation such as leptin and ghrelin. Without this context, the reader cannot assess the relative importance of Lac-Phe.

- The text states that Lac-Phe “surpasses lactate in detecting mitochondrial dysfunction.” This statement is too strong for the available evidence because the cited studies are not sufficiently conclusive and have limitations that prevent such a conclusion.

- The table presents three conceptual problems that need to be corrected: lactate is not traditionally classified as an exerkine, IL-6 has pro-inflammatory and anti-inflammatory functions, and irisin remains controversial in humans. The authors should revise the table, highlighting the level of evidence, the primary source, and the actual physiological role.

- Reference 8 (Pedersen & Febbraio 2008) is cited for a Lac-Phe clinical trial, which is incorrect. This indicates a serious problem with the literature review.

- In some sections, the manuscript mixes mechanisms related to lactate and Lac-Phe. For example, the effects via GPR81 are known for lactate, not for Lac-Phe.

- The link between Lac-Phe and redox homeostasis seems exaggerated. Most evidence shows metabolic correlation and not direct redox regulation. Correct this.

- Section 3 mixes several topics. For example, neurobiology, immunology, metabolism, exercise, and cancer. This impairs clarity. I suggest that the authors reorganize this section.

- The figure is limited as it only describes appetite suppression, while the text discusses several other aspects such as immunology, redox state, metabolism, biomarkers, and others. Why don't the authors create an integrative figure with the exercise-lactate-Lac-Phe axis, brain, immune system/inflammation, and energy metabolism?

- The authors should include a critical section on "Current controversies and limitations".

- It would be scientifically important for the authors to differentiate between evidence in rodents and evidence in humans. Perhaps creating two separate sections would increase the clarity and context of the evidence discussed so far.

- The authors should expand the translational discussion because important issues such as pharmacokinetics, stability, side effects, and safety, often tested in rodents, help explain the results in humans from studies that are unfeasible to conduct in humans.

Minor comments:

- Review grammar in several paragraphs

- Standardize Lac-Phe / N-lactoyl-phenylalanine nomenclature

- Review citations

- Reduce repetition of phrases about exercises

- Improve figure captions

- Conduct a more thorough literature review

Author Response

We would like to thank Reviewer 2 for the extensive assessment of our manuscript, the positive feedback, and the important and helpful comments/suggestions. We have taken all the remarks into account, as detailed below, and have provided our responses to specific comments. We believe that, following the reviewers’ suggestions, our revised manuscript has improved in clarity, and we hope the changes will be considered satisfactory.

 

 

Response to Reviewer 2

The manuscript presents a clear and up-to-date mini-review on Lac-Phe, integrating recent literature on biosynthesis, physiological function, and therapeutic potential. The topic is relevant because Lac-Phe has recently emerged as a candidate exerkine mediator of the effects of exercise on food intake and metabolism. However, the manuscript presents numerous problems that need to be overcome. With substantial revisions, the article could become a useful review on Lac-Phe, but it does not yet reach the analytical level expected in journals as Antioxidants.

Major comments

 

1. The introduction includes redundant information. I suggest that the authors restructure it into three main points: discovery of Lac-Phe, role in dietary control, and therapeutic potential.

 Response: We have condensed the first paragraph of the Introduction to eliminate information that duplicates Section 2. The restructured Introduction now addresses three focused points: (1) the discovery of Lac-Phe as an exercise-inducible metabolite; (2) its roles in appetite suppression and metabolic control; and (3) its therapeutic and biomarker potential. A cross-reference to Section 2 directs readers to detailed mechanistic content.

 

2. Much of the text presents narrative descriptions of current findings. However, the authors do not take into account methodological limitations, size of evidence, reproducibility, and differences between experimental models. Therefore, the authors should include a critical section addressing the limitations of current evidence, differences between species, and translational gaps.

Response: We have added a dedicated paragraph at the end of Section 5 ("Current Controversies and Limitations") that explicitly addresses: the predominance of rodent-based evidence; the uncertain translational magnitude of appetite suppression in humans; the absence of an identified Lac-Phe receptor; the correlative —rather than causal — nature of redox associations; and the limitations of the exerkine classification given non-exercise stimuli that also elevate Lac-Phe.

 

3. In several passages, the manuscript treats Lac-Phe as the main mediator of the effects of exercise. However, current evidence indicates that the effect is partial, occurs mainly in obesity, and depends on metabolic context. I recommend that the authors reformulate passages that suggest direct causality and include language such as "may contribute," "partially mediates," and "remains to be determined."

Response: We have revised all relevant passages to use appropriately hedged language. "Potent appetite suppression" has been replaced with "notable appetite suppression (observed primarily in obese or diet-induced obese models)"; "partial mediation" has been qualified as "partial and context-dependent mediation"; and "central mediator" has been softened to "potential integrator."

 

4. The manuscript does not adequately discuss important benchmarks for the physiology of exercise, including intensity and volume, type of exercise, and nutritional status. Without discussing these parameters, the physiology of Lac-Phe is impossible to understand.

Response: We have added to Section 2 a discussion of exercise type (sprint vs. aerobic), volume, and nutritional status as modulators of Lac-Phe biosynthesis, alongside a note that these parameters remain incompletely studied.

 

5. Another problem is that the manuscript mentions clear central effects, but the molecular receptor has not yet been identified; therefore, the cellular mechanisms remain hypothetical. The discussion also treats several mechanisms as established, which needs to be presented with more caution.

Response: We explicitly state that "the cognate receptor for Lac-Phe on hypothalamic neurons has not yet been identified, and the precise downstream signaling cascade remains to be determined." Similar caveats have been added throughout, and the receptor gap is highlighted in the new Controversies paragraph in Section 5.

 

6. The manuscript ignores classic mediators of appetite regulation such as leptin and ghrelin. Without this context, the reader cannot assess the relative importance of Lac-Phe.

Response: We have added a paragraph in Section 3 contextualizing Lac-Phe within the classical leptin/ghrelin appetite-regulatory framework, noting that Lac-Phe appears to act acutely and in an obesity-dependent manner even under leptin-resistant conditions, and that interactions with ghrelin signaling remain unexamined.

 

7. The text states that Lac-Phe “surpasses lactate in detecting mitochondrial dysfunction.” This statement is too strong for the available evidence because the cited studies are not sufficiently conclusive and have limitations that prevent such a conclusion.

Response: Already addressed in Reviewer 1 revisions; further strengthened here by noting that supporting evidence derives "predominantly from small patient cohorts" and that prospective validation is required "before diagnostic claims can be made."

 

8. The table presents three conceptual problems that need to be corrected: lactate is not traditionally classified as an exerkine, IL-6 has pro-inflammatory and anti-inflammatory functions, and irisin remains controversial in humans. The authors should revise the table, highlighting the level of evidence, the primary source, and the actual physiological role.

 Response: We have added an explicit caveat sentence after Table 1 noting: (1) lactate is included as a precursor/metabolic signal, not as a canonical exerkine; (2) IL-6 exerts both pro- and anti-inflammatory effects depending on context; and (3) the human relevance of irisin remains contested owing to historical antibody cross-reactivity issues.

 

9. Reference 8 (Pedersen & Febbraio 2008) is cited for a Lac-Phe clinical trial, which is incorrect. This indicates a serious problem with the literature review.

 Response: Reference 8 has been corrected to cite the NCT06743009 trial registration directly.

 

10. In some sections, the manuscript mixes mechanisms related to lactate and Lac-Phe. For example, the effects via GPR81 are known for lactate, not for Lac-Phe.

Response: We have revised the relevant sentence to explicitly state that GPR81-dependent NF-κB suppression is an effect of lactate, not Lac-Phe, and that any potentiating role of Lac-Phe through this pathway "has not been directly demonstrated and remains speculative."

 

11. The link between Lac-Phe and redox homeostasis seems exaggerated. Most evidence points to metabolic correlation rather than direct redox regulation. Correct this.

Response: We have added a clarifying statement: "the causal relationship between Lac-Phe and redox regulation remains largely correlative; most evidence reflects metabolic associations rather than direct mechanistic regulation of redox state. Rigorous functional studies are needed to establish whether Lac-Phe is a driver or a consequence of the observed redox changes."

 

12. Section 3 mixes several topics. For example, neurobiology, immunology, metabolism, exercise, and cancer. This impairs clarity. I suggest that the authors reorganize this section.

Response: Already substantially addressed in Reviewer 1 revisions; Section 3 was reorganized into distinct paragraphs for central neuroendocrine effects, peripheral immune effects, systemic metabolic effects, biomarker function, anti-inflammatory mechanisms, and exercise-redox connections.

 

13. The figure is limited as it only describes appetite suppression, while the text discusses several other aspects such as immunology, redox state, metabolism, biomarkers, and others. Why don't the authors create an integrative figure with the exercise-lactate-Lac-Phe axis, brain, immune system/inflammation, and energy metabolism?

Response: We acknowledge this and plan to create an integrative figure for the revised submission.

 

 

14. The authors should include a critical section on "Current controversies and limitations".

Response: We have added a "Current Controversies and Limitations" paragraph in Section 5.

 

 

15. It would be scientifically important for the authors to differentiate between evidence in rodents and evidence in humans. Perhaps creating two separate sections would increase the clarity and context of the evidence discussed so far.

Response: Throughout the revised manuscript, we have added explicit qualifiers distinguishing preclinical rodent findings from human evidence (e.g., "in rodent models," "in diet-induced obese mice," "human data are limited to correlational findings"). The Controversies paragraph in Section 5 also explicitly addresses this species gap.

 

16. The authors should expand the translational discussion because important issues such as pharmacokinetics, stability, side effects, and safety, often tested in rodents, help explain the results in humans from studies that are unfeasible to conduct in humans.

Response: We have substantially expanded Section 4 to include discussion of: gastrointestinal peptidase susceptibility limiting oral bioavailability; the clinical inapplicability of intraperitoneal administration; the absence of human plasma half-life data; the conceptual status of prodrug strategies; and the need for systematic safety evaluation of potential off-target effects on phenylalanine homeostasis and insulin signaling.

 

 

Minor comments:

 

1. Review grammar in several paragraphs

Response: We thank the reviewer for this suggestion. We have carefully reviewed the entire manuscript and made the grammatical and stylistic revisions to improve clarity and precision throughout.

 

2. Standardize Lac-Phe / N-lactoyl-phenylalanine nomenclature

Response: We thank the reviewer for this suggestion. We have standardized the Lac-Phe/N-lactoyl-phenylalanine nomenclature in our revised manuscript.

 

3. Review citations

Response: We thank the reviewer for this suggestion. We have checked citations in our revised manuscript.

 

4. Reduce repetition of phrases about exercises

Response: We thank the reviewer for this suggestion. We have reduced the repetition of phrases about exercises in our revised manuscript.

 

5. Improve figure captions

Response: We thank the reviewer for this suggestion. We have improved figure captions, allowing readers to understand the data without referring to the main text in our revised manuscript.

 

6. Conduct a more thorough literature review

Response: We thank the reviewer for this suggestion. Relevant literatures have been supplemented to address topics in our revised manuscript.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors attempted to address the suggestions. The authors made changes to the text and the manuscript was improved. Therefore, I believe the manuscript has reached the level required for publication and recommend its acceptance for publication.