Verapamil Restores β-Cell Mass and Function in Diabetogenic Stress Models via Proliferation and Mitochondrial Respiration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Arefanian et al. described the effects of verapamil on pancreatic β-cells using the Min6 cell line and a zebrafish model system. The authors demonstrated that verapamil promoted Min6 cell proliferation by enhancing CCK and PCNA expression, and that verapamil protected Min6 cells while enhancing mitochondrial function against STZ and T1D cytokine mixture stressors. Additionally, the authors showed that verapamil also protects zebrafish β-cells against MTZ treatment. I found these results quite interesting. However, this manuscript has not yet reached publication standards for Cells due to poor data organization and inadequate experimental descriptions. The authors should address the following concerns:

(1) Figures 1A and B: The y-axis is labeled only as "% of Untreated," making it impossible to understand what parameter is being measured. Consequently, these data cannot be properly interpreted.

(2) Figure legend formatting is inconsistent throughout the manuscript. Figure 1 legends describe methods but not results, Figure 2 legends describe results only, and Figure 3 legends describe objectives, methods, and results. The authors should adopt a unified format. I recommend following the Figure 3 format, which includes objectives, methods, and results, as this provides the most comprehensive information for readers.

(3) Figures 3D-L: To better demonstrate verapamil's effects, the authors should perform untreated control and verapamil treatment experiments simultaneously and present both conditions within the same graphs for direct comparison.

(4) Figure 4C: The authors presented mCherry fluorescence intensity data in zebrafish. While fluorescent protein intensity reflects gene expression levels, the experimental objectives require quantification of β-cell numbers rather than expression intensity alone. The authors should perform β-cell counting analysis. Moreover, the authors should investigate both cell death and proliferation in zebrafish β-cells to determine verapamil's in vivo effects. Furthermore, glucose assays would provide functional readouts of β-cell performance following verapamil treatment. Therefore, I recommend that the authors should perform these experiments as well.

(5) Figure 4B: Yolk autofluorescence varies considerably between samples, potentially compromising accurate fluorescence measurements. Samples with strong yolk autofluorescence should be excluded from analysis, or the yolk region should be physically removed post-fixation before quantitative measurements.

(6) The mechanism underlying verapamil's protective effects remains unclear. Is the observed phenotype solely attributable to L-type calcium channel blockade, or are additional mechanisms involved? The authors should discuss these possibilities, including potential off-target molecules and signaling pathways that could contribute to the observed β-cell protection and proliferation.

 

Author Response

Comments and Suggestions for Authors

Arefanian et al. described the effects of verapamil on pancreatic β-cells using the Min6 cell line and a zebrafish model system. The authors demonstrated that verapamil promoted Min6 cell proliferation by enhancing CCK and PCNA expression, and that verapamil protected Min6 cells while enhancing mitochondrial function against STZ and T1D cytokine mixture stressors. Additionally, the authors showed that verapamil also protects zebrafish β-cells against MTZ treatment. I found these results quite interesting.

 

We would like to thank the reviewer for the time and effort. In the revised version, we addressed all the reviewers’ concerns.

 

However, this manuscript has not yet reached publication standards for Cells due to poor data organization and inadequate experimental descriptions. The authors should address the following concerns:

 

• Figures 1A and B: The y-axis is labeled only as "% of Untreated," making it impossible to understand what parameter is being measured. Consequently, these data cannot be properly interpreted.

We would like to thank the reviewer for the notification. Figure 1A and 1B were corrected.

 

• Figure legend formatting is inconsistent throughout the manuscript. Figure 1 legends describe methods but not results, Figure 2 legends describe results only, and Figure 3 legends describe objectives, methods, and results. The authors should adopt a unified format. I recommend following the Figure 3 format, which includes objectives, methods, and results, as this provides the most comprehensive information for readers.

We would like to thank the reviewer for the notification. We agree with the reviewer, accordingly, the figure legends were correct as recommended. Please see Figure legends, Lanes 274-306, 339-352, and 385-398.  

 

 

• Figures 3D-L: To better demonstrate verapamil's effects, the authors should perform untreated control and verapamil treatment experiments simultaneously and present both conditions within the same graphs for direct comparison.

We would like to thank the reviewer for the notification. We agree with the reviewer, accordingly, the figure legends were correct as recommended. Please see Figures and their legends Lanes 274-306, 339-352, and 385-398.  

 

• Figure 4C: The authors presented mCherry fluorescence intensity data in zebrafish. While fluorescent protein intensity reflects gene expression levels, the experimental objectives require quantification of β-cell numbers rather than expression intensity alone. The authors should perform β-cell counting analysis. Moreover, the authors should investigate both cell death and proliferation in zebrafish β-cells to determine verapamil's in vivo effects. Furthermore, glucose assays would provide functional readouts of β-cell performance following verapamil treatment. Therefore, I recommend that the authors should perform these experiments as well.

We would like to thank the reviewer for the notification. We fully agree that β-cell number, cell death/proliferation assays, and functional glucose measurements would provide a more comprehensive assessment of verapamil’s in vivo effects. Our present study, however, was designed as a first-line in vivo screening approach in zebrafish, using mCherry fluorescence intensity as a rapid, non-invasive readout of β-cell mass and gene expression changes in living embryos. While fluorescence intensity correlates with β-cell content, it does not distinguish between cell number and expression level per cell, as correctly noted. Due to technical limitations using living zebrafish embryos, we are unable to incorporate β-cell counting, apoptosis/proliferation markers, or glucose tolerance assays in the current study. Nevertheless, we have added a statement in the Discussion to clarify these limitations and highlight them as important next steps. We agree that future studies combining direct β-cell enumeration, proliferation/death assays, and glucose assays will be critical to fully establish the mechanistic and functional outcomes of verapamil treatment in vivo. We appreciate the reviewer’s suggestions, which will guide the design of our future work, and we believe that our present findings still provide valuable initial evidence of verapamil’s effects on β-cells in the zebrafish model. Please, see lanes 515-546

 

• Figure 4B: Yolk autofluorescence varies considerably between samples, potentially compromising accurate fluorescence measurements. Samples with strong yolk autofluorescence should be excluded from analysis, or the yolk region should be physically removed post-fixation before quantitative measurements.\

We thank the reviewer for raising this important point. We agree that yolk autofluorescence can compromise fluorescence quantification in zebrafish embryos. In our study, however, the experiments were conducted in living embryos, not fixed samples, and we employed specific imaging techniques to measure fluorescence intensity selectively in the pancreatic region while excluding signals from the surrounding yolk autofluorescence. This approach allowed us to focus on β-cell–specific fluorescence and minimize background noise. We have clarified this point in the revised Materials and Methods section (please, see lines 224-231, and Supplementary Figure S2), where we now detail the imaging procedure used to define pancreatic fluorescence from adjacent auto-fluorescent regions. We believe this addition will better inform readers of our strategy to ensure accurate quantification of living embryos.

 

 

 

• The mechanism underlying verapamil's protective effects remains unclear. Is the observed phenotype solely attributable to L-type calcium channel blockade, or are additional mechanisms involved? The authors should discuss these possibilities, including potential off-target molecules and signaling pathways that could contribute to the observed β-cell protection and proliferation.

We thank the reviewer for this thoughtful and important comment. We agree that the precise mechanisms underlying verapamil’s protective effects on β-cells remain incompletely understood. While L-type calcium channel blockade is the primary and best-characterized action of verapamil, we acknowledge that additional mechanisms may contribute to the observed phenotype. Indeed, potential involvement of other signaling pathways and off-target effects, including modulation of ER stress responses, antioxidant defenses, and regulation of pro-survival transcriptional programs. In the revised Discussion section, we have expanded our text to address these possibilities. We highlight that while our data are consistent with a beneficial effect of verapamil on β-cell preservation, the exact molecular mediators cannot be conclusively identified from our current study. We emphasize the need for further mechanistic investigations to distinguish between calcium channel–dependent and calcium channel–independent pathways, and to identify additional molecular targets that may underlie β-cell protection and proliferation. We appreciate the reviewer’s suggestion, which has strengthened the scope of our discussion and contextualized our findings within the broader literature. Please see Lanes 513-544.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Title: Verapamil Restores β-Cell Mass and Function in Diabetogenic Stress Models via Proliferation and Mitochondrial Respiration

The study presents significant findings on the effects of verapamil on β-cell mass and function, showcasing a promising avenue for diabetes treatment. Overall, the manuscript presents important findings that warrant publication after addressing the following concerns.
I look forward to reviewing the revisions.

Below are my comments and suggestions to enhance the manuscript:

1. The introduction is compelling, but the Author should add recent literature on complications related to β-cell preservation to broaden the context and highlight the relevance of the author's findings.

2. Simplify Technical Jargon: The manuscript effectively conveys complex concepts, but simplifying some language would help. Providing brief explanations for terms like "apoptosis" or "cholecystokinin" would make the content more accessible to all readers.

3. Methodology: The methodology is thorough, but providing specifics on the dosages of verapamil used in MIN6 cells and zebrafish models would enhance reproducibility. Additionally, explaining the rationale for the chosen dosages and including more details about the concentrations used in the experiments would be beneficial.

4. Discussion of Mechanisms: The section effectively situates the author's findings in the literature. They should expand on how verapamil influences β-cell function, as this would enhance the relevance of the findings to clinical practice.

5. Future Directions and Clinical Relevance: The conclusion summarizes your findings well, but it could better highlight their clinical relevance. Discuss potential future research directions and their implications for diabetes treatment to clarify how your work may impact patient care.

 

Author Response

Comments and Suggestions for Authors

Title: Verapamil Restores β-Cell Mass and Function in Diabetogenic Stress Models via Proliferation and Mitochondrial Respiration

The study presents significant findings on the effects of verapamil on β-cell mass and function, showcasing a promising avenue for diabetes treatment. Overall, the manuscript presents important findings that warrant publication after addressing the following concerns.
I look forward to reviewing the revisions.

We would like to thank the reviewer for the time and effort. The reviewers’ suggestions and comments were useful to improve our manuscript. In the revised version, we addressed all the reviewers’ concerns.

 

 

Below are my comments and suggestions to enhance the manuscript:

1. The introduction is compelling, but the Author should add recent literature on complications related to β-cell preservation to broaden the context and highlight the relevance of the author's findings.

We would like to thank the reviewer for the notification. At the introduction, we added a paragraph that describes the complications associated with β-cell preservation. Please, see Lanes 45-59

 

2. Simplify Technical Jargon: The manuscript effectively conveys complex concepts, but simplifying some language would help. Providing brief explanations for terms like "apoptosis" or "cholecystokinin" would make the content more accessible to all readers.

We would like to thank the reviewer for the notification. We clarified the used terminologies. Please see lanes 73-77.

 

3. Methodology: The methodology is thorough, but providing specifics on the dosages of verapamil used in MIN6 cells and zebrafish models would enhance reproducibility. Additionally, explaining the rationale for the chosen dosages and including more details about the concentrations used in the experiments would be beneficial.

We would like to thank the reviewer for the notification, which helped to clarify the rational of using verapamil doses in this study. We justify this issue at the Material and Methods section. Please see Lanes 220-231.

 

 

4. Discussion of Mechanisms: The section effectively situates the author's findings in the literature. They should expand on how verapamil influences β-cell function, as this would enhance the relevance of the findings to clinical practice.

We would like to thank the reviewer for the notification, which helped to improve our manuscript. We added the different other signaling pathways that are regulated by verapamil based on our previous transcriptomic and proteomic data as well as the literature. Please see lanes 449-470.

 

5. Future Directions and Clinical Relevance: The conclusion summarizes your findings well, but it could better highlight their clinical relevance. Discuss potential future research directions and their implications for diabetes treatment to clarify how your work may impact patient care.

We would the to thank the reviewer for the available recommendations to improve our manuscript. We expanded the Future directions section and added recommendations for prospective future study conductions.  Please see lanes 513-544

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This article reveals the mechanism by which the high efficacy, affordability, and broad mechanisms of action make verapamil a desirable therapeutic candidate for diabetes. I see that the issues raised by other reviewers have been adequately addressed. I suggest the authors add some interesting content to the ”Introduction“ and "Discussion” section (PMID: 40111679, https://www.oaepublish.com/articles/mtod.2024.15, https://www.oaepublish.com/articles/mtod.2022.26). Additionally, further embellish the language of this manuscript to improve clarity, flow and readability. I have no further comments.

Author Response

Comments and Suggestions for Authors

This article reveals the mechanism by which the high efficacy, affordability, and broad mechanisms of action make verapamil a desirable therapeutic candidate for diabetes. I see that the issues raised by other reviewers have been adequately addressed. I suggest the authors add some interesting content to the ”Introduction“ and "Discussion” section (PMID: 40111679, https://www.oaepublish.com/articles/mtod.2024.15, https://www.oaepublish.com/articles/mtod.2022.26). Additionally, further embellish the language of this manuscript to improve clarity, flow and readability. I have no further comments.

 

We would the to thank the reviewer for the available recommendations to improve our manuscript. We answered the comments by the other reviewers and expanded the Future directions section and added recommendations for prospective future study conductions.  Please see lanes 449-470 and 513-544

The English language will be improved through a specialized Editing company after the final accepted version.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Arefanian et al. investigated the effects of Verapamil on β cells using both in vitro (cell line) and in vivo (zebrafish) models. The authors have provided satisfactory responses to the comments regarding the in vitro experiments raised in the previous review. However, concerning the in vivo part, they did not provide convincing answers to the experimental suggestions, such as glucose assays to support the recovery of β-cell function or analyses evaluating the effects on cell death, which seem feasible within their research environment.

Overall, while the in vitro data are acceptable, the in vivo studies are insufficient in both quality and quantity to meet the publication standards of Cells. Therefore, I cannot recommend publication of this manuscript in its current form.

Author Response

Comments and Suggestions for Authors

Arefanian et al. investigated the effects of Verapamil on β cells using both in vitro (cell line) and in vivo (zebrafish) models. The authors have provided satisfactory responses to the comments regarding the in vitro experiments raised in the previous review. However, concerning the in vivo part, they did not provide convincing answers to the experimental suggestions, such as glucose assays to support the recovery of β-cell function or analyses evaluating the effects on cell death, which seem feasible within their research environment.

Overall, while the in vitro data are acceptable, the in vivo studies are insufficient in both quality and quantity to meet the publication standards of Cells. Therefore, I cannot recommend publication of this manuscript in its current form.

 

We would like to thank the Reviewer for complementing our in vitro data. Regarding the in vivo data, we would like to notify the reviewer that we used zebrafish embryos in the study. These animal models still have limitations including the requested glucose and insulin levels in each single embryo. The use of adult fish could help but still has limitations observing pancreas development after MTZ treatment. Therefore, we added the limitations section and please see lines: 549-556 

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The responses to my comments have been addressed.

Author Response

Comments and Suggestions for Authors

The responses to my comments have been addressed.

 

We would like to thank the Reviewer for his/her kind support.

 

Author Response File: Author Response.docx