Systematic Expression and Localization Profiling of Piezo2 in Rodent Pancreatic Islets

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work presents interesting and relatively novel results regarding the expression and potential role of the Piezo2 channel in pancreatic islets. The authors employed a broad range of methods, including gene and protein expression analysis, immunohistochemistry, immunofluorescence, and functional experiments using cell culture. The manuscript is written in sound scientific language, logically organized, and the results are presented clearly. However, the Materials and Methods section and the statistical analysis require several important clarifications.

The most significant limitation of the work is the insufficient description of the experimental group sizes. The animal section does not provide the number of mice and rats used in each experiment, making it difficult to assess the statistical power of the obtained results. The authors only state that the numbers are presented in the figure legends, but basic information should also be included in the methods section.

The description of the PCR analysis raises some concerns. The authors used classical RT-PCR and qualitative product evaluation on agarose gels. However, quantitative analysis of gene expression (qPCR) is lacking, limiting the value of conclusions regarding changes in Piezo2 levels under different experimental conditions. Furthermore, PCR reaction conditions (cycle number, annealing temperatures, and amplification parameters) were not provided, making it difficult to reproduce the experiment.

For the immunohistochemical and immunofluorescence analyses, information on antibody specificity control procedures was lacking. Negative controls and data confirming the specificity of the anti-Piezo2 antibody used were not provided, which is particularly important in protein localization studies.

The section on Western blots requires expansion. The molecular weight of the detected proteins and details regarding the amount of protein material analyzed are not provided. Furthermore, the method for quantitative signal analysis and normalization of results to α-tubulin are not described in detail.

The functional section concerning insulin secretion remains the most controversial. The experiments were conducted only on the INS-1 cell line, without confirmation of the results in primary β-cells or isolated pancreatic islets. Additionally, the biological sample size was only n = 3, which is the minimum for statistical analyses and limits the strength of the conclusions drawn.

The statistical analysis was generally described correctly, but requires further clarification. The authors used Student's t-test, one-way analysis of variance, and two-way analysis of variance with Tukey's test, but they did not specify whether the assumptions of normality of distribution and homogeneity of variance were verified. This is particularly important with small sample sizes. Furthermore, for fluorescence intensity analyses, the authors treated individual pancreatic islets as independent observations, while it is unclear how many animals the analyzed material came from. This could lead to the problem of pseudoreplication and overestimation of statistical significance.

In some of the results, the authors state that Piezo2 does not directly participate in the regulation of GSIS. However, this conclusion seems far-fetched in light of the presented data. A pharmacological approach was used exclusively, without methods for silencing or overexpressing the Piezo2 gene. Therefore, a more cautious interpretation of the results would be advisable.

In summary, the manuscript addresses an interesting and poorly understood aspect of pancreatic islet biology, and the obtained results could make a valuable contribution to research on mechanosensitive channels. However, before publication, I recommend further clarification of the methodology, a more thorough presentation of the statistical analysis, and a more cautious interpretation of the functional results. Taking these considerations into account, the work will have significantly greater scientific value.

The bibliography includes 32 entries and generally covers key aspects related to the topic of the work. The cited sources are mostly current and well-selected. However, it would be worthwhile to consider including more recent publications on the role of Piezo channels in the physiology and pathology of pancreatic islets to better contextualize the results.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

Your work is of interest and appears suitable for publication. My main concern is the absence of a control group, which would allow comparison of Piezo2 expression in the basal state versus under dietary (pathological) conditions. It would also be valuable to determine whether Piezo2 is expressed in the rodent pancreas, particularly in mice. Finally, since PIEZO2 is primarily implicated in diabetic complications, I encourage you to discuss this aspect.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript examines the expression and localization of Piezo2 in rodent pancreatic islets and explores its possible role in pancreatic beta-cell biology under metabolic stress conditions. Although the topic is potentially interesting and relevant to metabolic disease, the study has several major weaknesses that substantially limit its scientific impact and mechanistic strength.

1- The main limitation of the manuscript is that it is largely descriptive and lacks strong functional evidence. Most conclusions are based on expression patterns and colocalization experiments rather than direct mechanistic studies. The authors repeatedly suggest that Piezo2 may contribute to beta-cell adaptation and endocrine cell specialization, but no genetic manipulation experiments such as Piezo2 knockdown, knockout, or overexpression studies were performed to support these claims. Without causal experiments, the conclusions remain speculative.

2- The functional experiments are also weak and somewhat contradictory. The insulin secretion assay shows that stretch stimulation increases GSIS, but inhibition of Piezo2 with D-GsMTx4 produces a similar increase in insulin secretion. This result is difficult to interpret mechanistically and raises concerns regarding the specificity of the inhibitor or the validity of the proposed model. The manuscript ultimately concludes that Piezo2 does not directly regulate GSIS, which weakens the biological significance of the study because the work then becomes primarily descriptive rather than mechanistic. 

3- Another major weakness is the limited quantitative analysis. The immunofluorescence data are mostly qualitative, and the manuscript lacks rigorous quantification of colocalization, cell population percentages, and fluorescence intensity normalization. The evidence supporting localization in PPy-lineage beta cells is therefore not fully convincing. Furthermore, the sample sizes in several experiments are relatively small, reducing statistical power and confidence in the findings. Statistical methods are also insufficiently detailed in some figure legends.

4- Methodological limitations are also present. Only male animals were used, and the absence of female animals is not adequately justified or discussed despite known sex differences in pancreatic islet biology and diabetes progression. The study relies heavily on pharmacological inhibitors, yet concerns regarding inhibitor specificity are not sufficiently addressed. In addition, no validation experiments are shown for antibody specificity in the immunostaining studies, which is particularly important for ion channel proteins.

5- The novelty of the manuscript is moderate rather than high. While Piezo2 localization in pancreatic islets has not been well characterized previously, the study does not substantially advance understanding of beta-cell physiology or diabetes mechanisms because it fails to demonstrate a clear functional role for Piezo2. 

Overall, although the manuscript provides some potentially useful descriptive observations regarding Piezo2 expression in pancreatic endocrine cells, the study lacks sufficient mechanistic depth, functional validation, and experimental rigor.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors, thank you for your responses. However, the manuscript must clearly explain why a control group was not included. The additional paragraph you provided does not adequately address this point, and further clarification is required.

Author Response

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Reviewer 3 Report

Comments and Suggestions for Authors

I would like to thank the authors for their careful revision of the manuscript and for providing detailed responses to my previous comments. The manuscript has improved compared with the original version. The authors have moderated several of their conclusions, added methodological details, and better acknowledged the limitations of the study. These changes make the manuscript more balanced and easier to interpret.

However, several important concerns remain.

1. The study is still largely descriptive. Although the conclusions have been moderated, the manuscript still does not provide direct functional evidence supporting the biological role of Piezo2 in pancreatic β-cells. The proposed role of Piezo2 remains speculative because no genetic loss-of-function or gain-of-function experiments were performed. I understand that generating new animal models is not feasible during the revision period, but this limitation should remain clearly emphasized throughout the manuscript.
2. Quantitative analysis of the immunofluorescence data remains limited. The additional methodological details improve the presentation of the data. However, the localization results are still mainly based on representative images. More rigorous quantitative analyses, such as the percentage of Piezo2-positive endocrine cells, quantitative colocalization analysis, or other objective measurements, would substantially strengthen the conclusions.
3. Antibody validation remains insufficient. The authors state that antibody specificity was confirmed by the manufacturer and by a previous publication. While this information is helpful, it does not replace experimental validation in the current study. Because Piezo2 is a membrane ion channel and antibody specificity can be challenging, this limitation should be clearly acknowledged in the Discussion.
4. The functional significance of Piezo2 remains uncertain. The revised manuscript now concludes that Piezo2 may not play a major role in glucose-stimulated insulin secretion under the experimental conditions used. This interpretation is more consistent with the presented data. However, the biological significance of the observed expression pattern remains unclear, and this point should be discussed more carefully without implying functions that have not been experimentally demonstrated.
5. The original Western blot images were not included in the raw data provided for review. Since the Western blot is an important part of the evidence supporting Piezo2 protein expression, the authors should provide the complete, uncropped original blot images for both Piezo2 and the loading control (α-tubulin). The original images should include all lanes, molecular weight markers, and the full membrane before cropping. This information is important for evaluating band specificity, protein loading, and overall data quality.
6. Western blot quality and antibody specificity. The manuscript reports a protein band corresponding to Piezo2; however, without the original uncropped blot images, it is not possible to determine whether additional nonspecific bands are present or whether the selected bands accurately represent the complete experiment. Providing the original blots would improve the transparency and reliability of the data. In addition, the statement that antibody specificity was confirmed by the manufacturer and a previous publication is helpful but does not fully validate antibody specificity in the current study. Because Piezo2 antibodies have been reported to show nonspecific binding in some applications, the limitation of antibody specificity should be more clearly acknowledged in the Discussion. If additional validation data are available, they should be included.
7. Data transparency. To improve the transparency and reproducibility of the study, I recommend that all original source data, including uncropped Western blots and any other raw images used for quantitative analysis, be provided as supplementary material. This has become common practice in many journals and allows readers to better evaluate the quality of the experimental data.

Author Response

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Author Response File: Author Response.pdf