Butyrate Protects Pancreatic Beta Cells from Cytokine-Induced Dysfunction

Round 1

Reviewer 1 Report

The manuscript titled “Butyrate Protects Pancreatic Beta Cells from Cytokine-induced Dysfunction” is comprehensive and well-presented. This is an interesting study with great scientific quality. The authors found that butyrate preserves beta-cell function under inflammatory conditions. I have just a few comments that may help to improve the manuscript:

1) Line 30-31, the authors stated that impaired insulin secretion from pancreatic beta cells is characteristic for ALL FORMS of diabetes. To my understanding, it is type 2 diabetes that is associated with insulin secretory defects. The authors may cite a reference to support their claim.

2) Line 394, the authors should state the number of mice from which pancreatic islets were isolated.

3)   Are there any limitations to the apoptosis assay used in the study?

Author Response

Dear Reviewer 1

Thank you for your time to review our manuscript and your insightful suggestions to improve our manuscript. 

Please se our comments below:

1) We have now changed the phrasing to "Impaired insulin secretion from pancreatic beta cells is characteristic for type 2 diabetes and is the main cause of glucose intolerance.

2) We have now included the number of animals and islets yield in material and methods. Please see line 416-418 in track changes version. In addition we have described if experiments were performed from islets from individual animals or from pools of islets.

3)   We have decided to use the Cell Death Detection Assay which measures release of cytoplasmic nucleosomes. This assay has been validated for analysis of beta cell apoptosis by cytokines previously. There are limitation using this assay in terms of timing after initiating of apoptosis. By including the positive control with cytotoxic concentrations of cytokines the assay is able to detect apoptosis at the conditions used in our protocol. Further, we carefully study our islets using microscopy and find no morphologic changes of the islet structure.

Reviewer 2 Report

The title of the article reflects the content of the article.

The keywords are selected correctly.

In the "Summary" section, the authors correctly presented the results of the study. In particular, the authors pointed out an important effect of butyrate: the compound had a positive effect on the secretion of insulin by beta cells. The second positive effect of butyrate is associated with a decrease in the expression of inflammatory genes.

It is not correct to talk about "inflammatory conditions", since this phrase also implies inflammation in vivo. Meanwhile, the authors imitated inflammation with cytokines in vitro, which reflects only part of the inflammation process. In this regard, the authors are invited to take this circumstance into account in the "Summary". The conclusion about the possibility of therapeutic use of butyrate in the treatment and prevention of T2D, it seems to me, is premature, since for this it is necessary to obtain a similar positive effect of butyrate on laboratory animals.

The purpose of this study is to determine the means of preserving or enhancing the function of beta cells and, thus, improving glucose homeostasis.The object of the study is butyrate.

The methods presented are clear and reproducible, the references to the methods are correct.

The results of the study of new ones. I would like to get some answers to some questions. Why is the scheme of cytokine exposure not general? If only IL-1β was applied to mouse islets, then a mixture of IL-1β, INF-γ and TNF-α acted on INS-1E cells and EndoC-βH1 cells?

Why is there no general standard for butyrate doses used in the experiment: doses of 0.1 mM, 0.2 mM and 0.4 mM were studied on islands, doses of 0.1 mm and 0.4 mM were studied on EndoC-βH1 cells, and 0.4 mM were studied on INS-1E cells?

The originality of the results is beyond doubt. No bias was found in the provision of results and conclusions.

In the chapter "Results", the authors showed that butyrate reduces the proliferation of cells in mouse islets and beta cells of human EndoC-βH1. In the chapter "Discussion", the authors suggested that butyrate would be appropriate to study its potential for therapeutic use in the prevention or treatment of type 2 diabetes mellitus. However, there is evidence that with the development of type 2 diabetes, the number of beta cells decreases, although not as significantly as with type 1 diabetes. Thus, the use of butyrate in conditions of a decrease in beta cells in type 2 diabetes is quite dangerous? Give an answer to this question.

The presented article is very important for the development of new strategies for restoring impaired secretion of pancreatic beta cells in diseases associated with impaired glucose metabolism.

Author Response

Dear Reviewer 2

Thank you very much, for your critical reviewing of our manuscript “Butyrate protects pancreatic beta cells from cytokine-induced dysfunction” and for the excellent questions raised.

We are very pleased with you positive thoughts regarding our research and in the following section we will respond to your questions.

• It is not correct to talk about "inflammatory conditions", since this phrase also implies inflammation in vivo. Meanwhile, the authors imitated inflammation with cytokines in vitro, which reflects only part of the inflammation process. In this regard, the authors are invited to take this circumstance into account in the "Summary".

Thank you for noticing this incorrect phrasing, we agree with your opinion. We will change this to                    “In summary, Butyrate prevents pro-inflammatory cytokine induced beta cell dysfunction”.

• The conclusion about the possibility of therapeutic use of butyrate in the treatment and prevention of T2D, it seems to me, is premature, since for this it is necessary to obtain a similar positive effect of butyrate on laboratory animals.

We do believe that butyrate could be potential for therapeutic use of T2D. However, we agree that this is a premature statement that of course need further experiments in vivo. We therefore, remove this statement from our abstract.

• The results of the study of new ones. I would like to get some answers to some questions. Why is the scheme of cytokine exposure not general? If only IL-1β was applied to mouse islets, then a mixture of IL-1β, INF-γ and TNF-α acted on INS-1E cells and EndoC-βH1 cells?

In our experiments we have used IL-1β only to induce dysfunction in mouse islets and rat INS-1E cells, whereas we have used a combination of the three cytokines IL-1β, TNF-α and INF-γ in human EndoC-βH1 cells. It is well known that humane beta cells requires exposure to several different pro-inflammatory cytokines to become dysfunctional, whereas rodent beta cells respond to IL-1β alone. A new reference addressing this has been included in the manuscript at line 433 in the method section (L A Berchtold et al 2016).

• Why is there no general standard for butyrate doses used in the experiment: doses of 0.1 mM, 0.2 mM and 0.4 mM were studied on islands, doses of 0.1 mm and 0.4 mM were studied on EndoC-βH1 cells, and 0.4 mM were studied on INS-1E cells?

We did dose response analysis of GSIS for all the three beta cell models used in this manuscript. For GSIS experiments with mouse islets we tested the doses 0.1-0.6 mM butyrate on IL-1b induced dysfunction and found as showed in Figure 1 that 0.2 mM butyrate significantly increased insulin secretion with out affecting apoptosis in the islets. Further increase of butyrate in the mouse islets experiments did not significantly increase insulin secretion. Therefore, the concentration of 0.2 mM butyrate was selected. This is stated in line 94 in the manuscript.

Next, we tested 0.1-0.4 mM butyrate on IL-1β induced dysfunction in INS-1E cells and found that 0.4 mM was needed to revert the detrimental effects of IL-1β. An explanation for the higher concentration of butyrate needed in this cell line protocol might be explained by the high proliferation rate of INS-1E cell lines vs primary cells.

With this observation in hand, we tried out 0.1-0.4 mM butyrate in the EndoC-βH1 as these cells are also cell lines. We found, as shown in figure 2A, that 0.4 mM butyrate was sufficient in increasing insulin secretion. We believe that these minor differences in butyrate concentrations needed to protect beta cells reflect the fact that we are using different cell types (primary vs cell lines) as well as different species (rat, mouse and human).

• In the chapter "Results", the authors showed that butyrate reduces the proliferation of cells in mouse islets and beta cells of human EndoC-β In the chapter "Discussion", the authors suggested that butyrate would be appropriate to study its potential for therapeutic use in the prevention or treatment of type 2 diabetes mellitus. However, there is evidence that with the development of type 2 diabetes, the number of beta cells decreases, although not as significantly as with type 1 diabetes. Thus, the use of butyrate in conditions of a decrease in beta cells in type 2 diabetes is quite dangerous? Give an answer to this question.

It is generally believed that beta cell proliferation in adults play a minor role in maintain beta cell insulin secretory capacity. It has recently been shown that the main mechanism for reduced beta cell function in T2D is not loss of beta cell as such but rather dedifferentiation into a less mature and functional beta cell. Thus proliferation plays at most a minor role in reestablishing beta cell function.