High Glucose Concentration on the Metabolic Activity of C6 Glia Cells: Implication in Alzheimer’s Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript "High glucose concentration on the metabolic activity of C6 glia 2 cells: Implication in Alzheimer's disease" seems interesting in its title however, the whole manuscript is very poorly written. A lot of abnormally long, complex sentences make it hard to understand author's point of view, particularly in the Introduction section. In addition to inappropriate sentences, a lot of spelling mistakes, lack of coherence in the flow of information, and repetition of the same terms, especially in the results section, are compromising the quality of manuscript presentation.

Scientific flaws: It is well reported that high glucose concentration in cell culture affects all intracellular molecule activities and phosphorylates many proteins, leading to abnormal pathways. what is new in this study? I suggest that the authors should add some in-vivo experiments to support their hypothesis.

 

Comments on the Quality of English Language

There are lot of spelling mistakes and very complex sentences that need to be rewritten in a meaningful form, especially the abstract and introduction parts.

Author Response

The authors thank reviewer 1 for his/her invaluable comments that were very important in improving the manuscript.

The manuscript "High glucose concentration on the metabolic activity of C6 glia 2 cells: Implication in Alzheimer's disease" seems interesting in its title however, the whole manuscript is very poorly written. A lot of abnormally long, complex sentences make it hard to understand author's point of view, particularly in the Introduction section. In addition to inappropriate sentences, a lot of spelling mistakes, lack of coherence in the flow of information, and repetition of the same terms, especially in the results section, are compromising the quality of manuscript presentation.

Scientific flaws: It is well reported that high glucose concentration in cell culture affects all intracellular molecule activities and phosphorylates many proteins, leading to abnormal pathways. what is new in this study? I suggest that the authors should add some in-vivo experiments to support their hypothesis.

Comments on the Quality of English Language

There are lot of spelling mistakes and very complex sentences that need to be rewritten in a meaningful form, especially the abstract and introduction parts.

Thanks for the suggestion, changes have been done.

We rewrote the abstract, introduction, discussion and conclusion so that it was more comprehensible.

To address the question:

1. Novelty of the Study:
   This study introduces new insights into the molecular mechanisms underlying the DM-AD link, specifically focusing on the impact of high glucose concentration (HGC) on astrocyte-like C6 cells. Unlike previous studies that broadly report cellular responses to HGC, this research investigates specific functional and metabolic alterations, such as mitochondrial activity, oxidative stress, and the expression of AD-related proteins like β-amyloid and hyperphosphorylated tau (pTau). It highlights the temporal relationship between pTau and Aβ production under HGC conditions, which has not been thoroughly explored in earlier research.
2. Value of the Model:
   By using astrocyte-like C6 cells as a model, the study offers a controlled environment to dissect the role of astrocytes in the DM-AD connection. This approach is particularly valuable because astrocytes are key regulators of glucose metabolism in the brain and are implicated in AD pathology.

3. Regarding In Vivo Studies:
While in vivo experiments would provide additional validation, the in vitro model serves as a crucial first step for isolating and understanding specific cellular and molecular mechanisms. Future research could build on these findings by incorporating in vivo studies to confirm the relevance of these mechanisms in the context of the entire organism.

Reviewer 2 Report

Comments and Suggestions for Authors

The study, entitled 'High glucose concentration on the metabolic activity of C6 glia 2 cells: Implication in Alzheimer's disease', describes a possible link between the development of AD and the role of high glucose concentration. Some items listed below might aid in increasing the quality of the paper:

- Abstract does not give sufficient information on what has been aimed and obtained.

- Introduction lacks many information such as pathophysiology of AD, current treatment options and previous work on the topic.

- Overall the link of mitochondrial activity and AD has not been proven under the assay conditions. Therefore I strongly suggest a change in the title.  

Author Response

The authors thank reviewer 2 for his/her invaluable comments that were very important in improving the manuscript.

The study, entitled 'High glucose concentration on the metabolic activity of C6 glia 2 cells: Implication in Alzheimer's disease', describes a possible link between the development of AD and the role of high glucose concentration. Some items listed below might aid in increasing the quality of the paper:

- Abstract does not give sufficient information on what has been aimed and obtained.

Thanks for the suggestion, changes have been done.

- Introduction lacks many information such as pathophysiology of AD, current treatment options and previous work on the topic.

Thank you very much for the suggestion.

We rewrote the abstract, introduction, discussion and conclusion so that it was more comprehensible.

Overall the link of mitochondrial activity and AD has not been proven under the assay conditions. Therefore I strongly suggest a change in the title.  

We appreciate the reviewer’s observation regarding the link between mitochondrial activity and AD under the assay conditions. Our study primarily investigates the impact of high glucose concentrations (HGC) on mitochondrial function and its association with early markers of AD, such as pTau production. While the direct link to AD progression might not have been explicitly demonstrated in the current work, our findings provide foundational insights into the metabolic alterations that precede AD pathology, particularly under diabetic conditions.

Reviewer 3 Report

Comments and Suggestions for Authors

Interesting work on the AD-DM link.

1. row 29 - please revise the expression: ”AD represents 70%....the etiology of this disease..” 

2. at Results, 3.1. Cell viability and proliferation: please clarify if the cells exposed to HGC showed a higher tendency to late apoptosis or necrosis? 

3. at Discussions, rows 361 to 370: please clarify  if HGC decrease the GFAP expression in C6 cells (361-364) or increase the GFAP expression (372-374)

4. row 418: ACG is, I think, HCG

5. at Conclusions: row482: 72 h exposure of astrocite-like cells......please add ”exposire to HGC”...

6. All precise explanations of the results are provided in the Figures legend. It will be usefull to add them also in the text, it provides more clarity

7. Please use shorter phrases, it will, in my opinion, make the data and the mechanisms more easy to understand and more easy to follow...

8. Please make the Conclusions more precise ant systematic; maybe more direct related to the results of your study

 

Comments on the Quality of English Language

Shorter phrases will improve the accessibility of the content

Author Response

The authors thank reviewer 3 for his/her invaluable comments that were very important in improving the manuscript.

interesting work on the AD-DM link.

1. row 29 - please revise the expression: ”AD represents 70%....the etiology of this disease..” 

Thanks for the suggestion, changes have been done.

2. at Results, 3.1. Cell viability and proliferation: please clarify if the cells exposed to HGC showed a higher tendency to late apoptosis or necrosis? 

The annexin V and propidium iodide staining used in our study primarily distinguishes early apoptotic, late apoptotic, and necrotic cells. In the HGC group, the data indicated a higher tendency toward late apoptosis compared to the control group, as evidenced by the significant increase in annexin V+/PI+ cells. However, there was no corresponding increase in necrotic cells (annexin V-/PI+), suggesting that the HGC-induced cellular stress predominantly leads to apoptotic pathways rather than necrosis.

 

3. at Discussions, rows 361 to 370: please clarify  if HGC decrease the GFAP expression in C6 cells (361-364) or increase the GFAP expression (372-374)

We appreciate the reviewer highlighting the apparent contradiction in the discussion regarding GFAP expression under HGC conditions. To clarify:

1. Short-Term Exposure (72 Hours):
   Our study found no statistically significant increase in GFAP expression after 72 hours of HGC exposure compared to the control group. However, trends observed in the data suggest that prolonged HGC exposure may lead to an increase in GFAP expression, consistent with other studies reporting delayed astrocytic reactivity.
2. Longer-Term Considerations:
   It has been documented in the literature that GFAP expression initially decreases under short-term HGC conditions, potentially due to delayed cell maturation and stress-related alterations. Over longer periods, however, astrocyte-like cells exhibit increased GFAP expression, coinciding with morphological changes indicative of reactive astrogliosis.
3. row 418: ACG is, I think, HCG

Thanks for the suggestion, changes have been done.

5. at Conclusions: row482: 72 h exposure of astrocite-like cells......please add ”exposire to HGC”...
6. All precise explanations of the results are provided in the Figures legend. It will be usefull to add them also in the text, it provides more clarity.

Thanks for the suggestion, we consider leaving the figure captions as they are so as not to be repetitive in the results section.

7. Please use shorter phrases, it will, in my opinion, make the data and the mechanisms more easy to understand and more easy to follow...
8. Please make the Conclusions more precise ant systematic; maybe more direct related to the results of your study

Thanks for the suggestion, changes have been done.

Comments on the Quality of English Language

Shorter phrases will improve the accessibility of the content

Thanks for the suggestion, changes have been done.

We rewrote the abstract, introduction, discussion and conclusion so that it was more comprehensible.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the changes. The presentation of the scientific work has improved a lot.

Author Response

Dear Reviewer 3, the authors thank you for your valuable time and suggestions for improving the manuscript.