Insulin Secretion by β-Cell-Like Cells Derived from Pulp Stem Cells Depends on Augmented Cytosolic Zinc Levels than GABA Levels
Round 1
Reviewer 1 Report
The present study is very interesting but to improve the overall quality of the manuscript are needed the following results:
A western blot analysis of GABAA receptor and ZIP3 is needed in Figure 1.
An immunofluorescence analysis revealing the ZIP3 localization.
the manuscript is very similar to the prevoius published one https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187892/. Please highlight the novelty.
Author Response
Comments and Suggestions for Authors
The present study is very interesting but to improve the overall quality of the manuscript are needed the following results:
A western blot analysis of GABAA receptor and ZIP3 is needed in Figure 1.
An immunofluorescence analysis revealing the ZIP3 localization.
Response:
To complied with the critiques, Fig. 1rev and associated text have been added and Fig.3B has been revised.
the manuscript is very similar to the prevoius published one https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187892/
. Please highlight the novelty.
Response:
The current report used the same presentation format as the previous report. However, as denoted, the current report focused on elucidating the relationship between function of GABA and zinc levels as insulin secretagogues. This study is an independent new study with different perspectives compared to our previous publication at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187892/. Novelty is highlighted with new sentences. See newly added highlighted-bold sentences below.
See page 4, Lines 82-84:
“We sought to investigate if ZIP3 could be another principal zinc uptake transporter in addition to ZIP8 in SHED β-cells. Further, we examined the effects of increased zinc levels in the cytosol on insulin secretion and on GABA-induced activation in SHED β-cells.”
Submission Date
03 September 2020
Date of this review
21 Sep 2020 11:09:16
Reviewer 2 Report
In this manuscript, Gyuyoup Kim et al, found that in SHED derived β-cells, knockdown of ZIP3 reduced intracellular zinc level, and concomitantly reduced insulin secretion. Moreover, Zinc pretreated SHED β-cells exhibited a GABA-induced increase in Ca2+ influx. The idea is novel and interesting, however, some conclusions in this manuscript are not fully supported by the experiment. The following is a few concerns raised by this work.
1, The author stated that zinc enhances GABA-induced membrane depolarization of SHED β-cells in Abstract and Conclusions, however, the depolarization phenotype or marker of the SHED β-cells is not detected all over the Results.
2, SLC39A family of zinc transporters have more than 10 members, why the author chose ZIP3? Does the author detect the expression of SLC39A family in SHED β-cells? Is ZIP3 expressed relatively higher than other zinc transporters?
3, The quality of western blot of ZIP3 in Figure 2 is very bad. The author did not show their western blot method in the manuscript. For the transmembrane transporter, regular RIPA buffer does not work good. The best way is to use a transmembrane protein extraction reagent and follow the protocol exactly, then usually you can get a specific band and a complete KO by siRNA. The reagent for transmembrane protein extraction is https://fivephoton.com/pdfs/Transmembrane%20Protein%20Ext.pdf
4, The method for western blot and the catalog number for all the antibodies should be added in Methods.
5, Some writing should follow the formal guidelines. For example, the symbols for genes are italicized in qRT-PCR analysis.
Author Response
In this manuscript, Gyuyoup Kim et al, found that in SHED derived β-cells, knockdown of ZIP3 reduced intracellular zinc level, and concomitantly reduced insulin secretion. Moreover, Zinc pretreated SHED β-cells exhibited a GABA-induced increase in Ca2+ influx. The idea is novel and interesting, however, some conclusions in this manuscript are not fully supported by the experiment. The following is a few concerns raised by this work.
1, The author stated that zinc enhances GABA-induced membrane depolarization of SHED β-cells in Abstract and Conclusions, however, the depolarization phenotype or marker of the SHED β-cells is not detected all over the Results.
Response:
We have revised the word “depolarization” to “increased calcium influx”. We exhibited “increased Ca2+ levels in the cytosol of SHED β-cells in response to addition of zinc or GABA plus zinc into the medium.
2, SLC39A family of zinc transporters have more than 10 members, why the author chose ZIP3? Does the author detect the expression of SLC39A family in SHED β-cells? Is ZIP3 expressed relatively higher than other zinc transporters?
Response:
Studies on SLC39A family of zinc transporters in rodent beta-cells had been published by many other researchers. We previously detected ZIP8 transporter in SHED β-cells for the first time. Based upon the previous findings, the current report newly detected ZIP3. Due to technical difficulties to obtain fully differentiated SHED β-cells, not many other research groups have been performing similar studies.
3, The quality of western blot of ZIP3 in Figure 2 is very bad. The author did not show their western blot method in the manuscript. For the transmembrane transporter, regular RIPA buffer does not work good. The best way is to use a transmembrane protein extraction reagent and follow the protocol exactly, then usually you can get a specific band and a complete KO by siRNA. The reagent for transmembrane protein extraction is https://fivephoton.com/pdfs/Transmembrane%20Protein%20Ext.pdf
Response:
We understand reviewer critiques on quality of Fig. 2B. We think that the low quality is not from buffer, but mainly from quantity of proteins. Obtaining stem-cells from the pulp of exfoliated deciduous teeth is the first step of this long process. SHED does not yield β-cell-like cells easily, and differentiation steps are long and difficult. Amount of proteins extracted from the cells has reduced quickly during process and senescence occurs quickly. For these reasons, we had to use primary rodent β-cells to build up the rationale toward SHED β-cells as shown in New Fig. 1rev.
We also briefly added subcellular fractionation method as well.
4, The method for western blot and the catalog number for all the antibodies should be added in Methods.
Complied.
5, Some writing should follow the formal guidelines. For example, the symbols for genes are italicized in qRT-PCR analysis.
Complied.
Submission Date
03 September 2020
Date of this review
21 Sep 2020 08:03:44
Reviewer 3 Report
- Line 66: please first give a short description of SHED β-Cells methodology and then Differentiation of SHED β-Cells as this section must be improved.
- Line 92: chang ΔCt to –ΔCt
- Line 127: All t-tests must undergo Bonferroni correction to avoid any false statistical significance. Please justify your data analysis and describe this section in detail.
- It is not clear what the error bars in the figures account for, means? Medians? SEM or SD.
- Line 193: please give a full name of any abbreviation mentioned in the figure in the legend and apply this comment to all figures.
Author Response
Comments and Suggestions for Authors
Line 66: please first give a short description of SHED β-Cells methodology and then Differentiation of SHED β-Cells as this section must be improved.
Response:
We added suggested information as follow:
2.1 Differentiation of SHED β-cells
First, dental pulp tissue extracted from exfoliated deciduous teeth was dissolved in cold saline. Then, SHED cell culture was initiated in α-minimum essential media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), 5 μg/mL gentamicin sulfate (Gemini Bio-Products, West Sacramento, CA, USA), and 20 mmol/L l-glutamine (Invitrogen). SHED cells were amplified to passage 5 in the media, and then were differentiated to SHED β-cells using previously established protocol and specific media.10 SHED cells from deciduous dental pulp proliferate as an adherent monolayer, which cluster into spherical aggregates of cells when transferred to non-adherent tissue culture conditions as previously shown. We followed the previously published differentiation protocol faithfully.21 On Day 10 of growth in non-adherent conditions to induce islet-like cellular aggregates (ICA), the majority of SHED ICA stained weakly positive with dithizone. Day-10 SHED β-cells were supplemented as needed with 100 µM GABA and 50 µM zinc.
2. Line 92: chang ΔCt to –ΔCt
Please note the revised sentence.
3. Line 127: All t-tests must undergo Bonferroni correction to avoid any false statistical significance. Please justify your data analysis and describe this section in detail.
Complied as required. See highlighted and bold sentences.
4.It is not clear what the error bars in the figures account for, means? Medians? SEM or SD.
Complied as required. See the revision.
5.Line 193: please give a full name of any abbreviation mentioned in the figure in the legend and apply this comment to all figures.
Complied as required.
Submission Date 03 September 2020
Date of this review 16 Sep 2020 09:44:25
Round 2
Reviewer 1 Report
Authors improved the manuscript according to my suggstions.
