Caveolin-1 Modulates Islet Amyloid Polypeptide Expression Through Interaction with TXNIP in Murine Pancreatic β-Cells
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsLiu and colleagues explored the function of caveolin-1 (CAV-1) in the regulation of IAPP synthesis and secretion. They have generated some convincing data showing that CAV-1 knockdown leads to a reduction of IAPP expression and release from beta-cells and provide some detail of a potential mechanism which may underlie this. However, this mechanism is based primarily on a series of observations that the expression of other proteins linked to IAPP synthesis are also changed following CAV-1 knockdown. As such, whilst these experiments support the suggested mechanism, without, for example, rescue experiment, they cannot conclusively confirm the mechanism responsible. Furthermore, these data are exclusively generated in rodent models, and nothing has been replicated in any human model (e.g. EndoC cell or human islets). As such, this reduces the potential impact of the work and its relevance to human diabetes.
Additionally, there are several more specific issues noted with the manuscript which require attention. I would also suggest that the authors go through the text carefully to correct some minor issues in grammar and formatting. That said, the majority of the text reads well.
- Line 72. The CAV-1 insulinoma cells were not ‘CAV-1 knockouts’ as stated, rather they were CAV-1 knockdowns as siRNA was used, and this would only deplete CAV-1 protein levels rather being a genetic deletion. Please amend the terminology used here.
- Line 131. For complete clarity, could the dilutions of the primary antibodies used in this study be stated – for both Westerns and IF experiments.
- Line 147. Supplementary table S1 does not appear to have been submitted, please ensure this is included.
- Line 169. Can you please provide some further detail on the image analysis. Elsewhere it states that it is the ‘red fluorescence per β-cell area’ but it is presented as an ‘area %’. Is this saying it is the percentage of beta-cells which have over a certain threshold of fluorescence signal for the target protein? It is not clear to me. Some information on the analysis pipeline would be helpful.
- Please ensure all abbreviations (e.g. CD) are expanded in the figure legends.
- Result 3.1 – What was the level of Cav-1 knockdown? In the methods section you mention that expression levels of Cav-1 were quantified, however these data have not been reported.
- Result 3.1-3.3. How long after siRNA treatment did you measure mRNA and protein expression? Please include in figure legends.
- There is a lack of consistency with the loading controls used for Westerns; you use both Actin and GAPDH. Was there a rationale for this?
- Line 224. There is an error with figure legend 3. The order in which the figure panels are described does not match the order that they are presented.
- Results 3.4-onwards – Again there is no validation of the CAV-1 KO in these mouse islets. This needs to be included to be confident in the results.
- Figure 4. In the text you refer to Fig 4A as data from mice given a HFD after CAV-1 deletion, and Fig 4B was from mice in which CAV-1 had been knocked down after the HFD. However, these descriptions are reversed in the legend. Can you please clarify which is correct.
- Result 3.4. The serum IAPP value in NC+HFD in both 4A and 4B is identical (3.49±0.15) despite these being two independent sets of experiments – this surely has to be an error. Can you please check this?
- Figure 4C onwards please ensure that scale bars are easier to see on the images and state the scale bar size in all relevant legends.
- Figure 4D (and later figures). When quantifying image data, how many islets per mouse were included in the analysis?
- Line 240. You state that ‘..red fluorescence per β-cell area decreased by 65%, regardless of diet sequence, while insulin staining remained comparable between genotypes..’. However you only present one set of data here and don’t state which diet sequence was followed to generate it. Please clarify this.
- Line 314-320. I struggled to see anything from your data suggesting that a high fat diet increased IAPP as implied in your conclusion. If anything, HFD had reduced serum IAPP vs mice on a control diet, please amend your conclusions accordingly.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study investigates the role of Cav-1 in regulating IAPP expression and metabolism in pancreatic β-cells using in vitro and in vivo models. It suggests that Cav-1 silencing reduces IAPP levels and may influence pathways involved in protein processing and degradation. However, the findings are mainly based on expression data and lack sufficient functional validation. A few clarifications are needed.
What is the rationale for selecting NIT-1 cells instead of human β-cell models? NIT-1 cells are a murine insulinoma cell line that expresses murine IAPP, which is non-amyloidogenic, unlike human IAPP. As a result, this model has poor amyloid-forming capacity, limiting its relevance for studying IAPP aggregation and toxicity.
How was Cav-1 silencing efficiency validated at both mRNA and protein levels?
The mRNA expression data are presented; however, it is unclear whether the results are expressed as relative fold change. Please clarify how the data were normalized, including the reference gene used and the control group against which the fold change was calculated.
The Cav-1–TXNIP axis is proposed in this study. However, functional validation experiments are lacking.
The link between Cav-1 and β-cell apoptosis or functional outcomes is not directly demonstrated in this study.
Why were only serum IAPP levels measured and not pancreatic tissue levels?
Were histological analyses performed to assess amyloid deposition in pancreatic islets?
How was β-cell function evaluated in vivo?
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn the manuscript " Caveolin 1 modulates the synthesis and degradation of Islet Amyloid Polypeptide via TXNIP in murine pancreatic beta cells" by Liu et.al., the authors have identified a new mechanism for IAPP regulation that is driven by the Caveolin-1 protein. This study has major implications in the field of diabetes and islet cell transplantation, where IAPP deposits cause significant damage to islets. The results show a promising trend that Caveolin-1 may be involved in the IAPP regulation, however, some data need further validation that can further bolster the conclusion of the manuscript.
1) The Authors show in figure 4C via immunofluorescence staining that islets have high expression of IAPP, however, this staining does not show deposits rather, it shows the expression of amylin protein. Using a more specific staining such as Congo Red or Thioflavin S to show amyloid deposits would have strengthened the overall conclusion of the paper.
Overall, it was a well drafted and organized manuscript.
Author Response
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Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsThe paper entitled “Caveolin-1 modulates the synthesis and degradation of islet amyloid polypeptide via TXNIP in murine pancreatic β-cells” investigates the effect of Cav-1 depletion in NIT-1 cells and in an already established inducible β-cell-specific Cav-1 knockout mouse model, deepening the results of a previous genome-wide profiling of primary mouse islets bearing Cav-1 knockdown in which a significant reduction in IAPP mRNA expression was evidenced.
The paper is well written and documents the effect of Cav-1 depletion on enzymes involved in IAPP synthesis and degradation, but the results shown don’t clarify the modality of interaction with TXNIP.
Indeed in Fig. 3 authors show that in NIT-1 cells Cav-1 depletion reduces TXNIP mRNA expression and protein synthesis whereas in Figure 7 show also a possible physical interaction of Cav-1 with TXNIP.
The authors should clarify therefore how Cav-1 regulates TXNIP activity, through a transcriptional mechanism or through a functional inhibitory interaction with the protein? How do they explain these results?
The interaction between Cav-1 and TXNIP is inferred by the spatial correlation observed in immunofluorescence analysis, one possibility to confirm this hypothesis would be verifying the co-immunoprecipitation of the proteins.
The physical interaction of TXNIP with Cav1 is not related to a physical interaction with insulin, how do the authors explain this result?
In figure 5 panel E and in figure 7 panel B the dimensions of the islets in KO+HFD mice appear significantly smaller, how do the authors explain this occurrence?
Minor points
Fig. 3: according to the caption of the figure panel B and C should be exchanged. Moreover, according to the supplementary data, the standardization of the expression of TXNIP in panel B should have considered GAPDH expression.
Fig. 7: in panel B, KO+HFD and WT+HFD indication is missing
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have done an excellent job in responding to my comments. I was pleased to see additional mechanistic data included, limitations of the work acknowledged and the conclusions tempered. I still maintain that the impact of the work is limited due to the lack of human data, but this is now acknowledged in the discussion. I feel the manuscript is much stronger with these changes and I am happy to recommend this for acceptance in Biomedicines. I wish the authors the best of luck with their future studies.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors made necessary changes and addressed the reviewers’ comments. This manuscript may be accepted for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have addressed the issue of non-formation of IAPP deposits in their current mouse model and thus alluded to the future study using a humanized IAPP mouse model to study the pathological variant further. Since this paper's focus is just related to the regulation of IAPP expression, it is acceptable for publication in its current form.
Reviewer 4 Report
Comments and Suggestions for AuthorsThe paper is now suitable for pubblication
