Novel Bacillus ginsengihumi CMRO6 Inhibits Adipogenesis via p38MAPK/Erk44/42 and Stimulates Glucose Uptake in 3T3-L1 Pre-Adipocytes through Akt/AS160 Signaling

Round 1

Reviewer 1 Report

The study entitled “ A noveType of the Paperl Bacillus ginsengihumi CMRO6 inhibites adipogenesis via p38MAPK/Erk 44/42 and stimulates glucose uptake in 3T3-L1 pre-adipocytes through Akt/AS160 signaling” by Lee et al., defines the role of CMRO6 metabolites on adipogenesis. Overall, the manuscript is good and will be helpful for further studies to evaluate its impact in preclinical models. However, there are few concerns and some of them are mentioned below. If authors clarify and reframe the manuscript that would be great.

 

1. Manuscript has many typological errors (for eg. 16SrRNA instead of 16S rRNA, 150rpm to 150 rpm) line 80, 86, 87, 106-108. A proofreading will help to remove all the typological errors.
2. Source of Oil Red O Stain (if purchased and process if made in lab) 112
3. How the percentage of lipid content was determined. Is it relative to control/vehicle or absolute value? Please clarify. 116
4. How much protein was used for immunoblotting and what was dilution of antibody?
5. In fig 1, what is ab and cd and what is P value of ab and cd? Please be specific.
6. What is BCMRO6? Is it CMRO6? line 164
7. Did authors use any positive control for lipid accumulation treatment?
8. Is it possible for authors to provide high resolution images for Fig. 2b in supplementary data, because the staining is not clear?
9. Again, in Fig 2 what is ab? Are all the asterisks are with P<0.05 or some have lower p values?
10. What is RO6 in fig 3f? is it same CMRO6? If yes, please be consistent.
11. Treatment with CMRO6 downregulated the expression of adipogenic and lipogenic proteins and authors also quantified the western blots. Are the differences statistically significant in fig 4a and 4b?
12. Section 3.6 should be reframed properly.
13. In Fig 5a, basal bar graph showing asterisks c, it is compared to what? It would be great if authors clearly mention about asterisks what is compared to what.
14. Discussion should also be reframed.

Author Response

We thank the reviewer for providing useful comments about our research paper, which will greatly improve the quality of the manuscript. We ask apology for typographical mistakes and grammatical errors.   In response to the reviewers' suggestions, we have read through the entire manuscript and modified it accordingly. Please note that changes have been made in red across the manuscript.

1. Manuscript has many typological errors (for eg. 16SrRNA instead of 16S rRNA, 150rpm to 150 rpm) line 80, 86, 87, 106-108. A proofreading will help to remove all the typological errors.

Yes, we have agreed with the reviewer's comment.  We have carefully and thoroughly read the whole manuscript and have taken the time to edit and correct all typographical and grammatical errors.   

2. Source of Oil Red O Stain (if purchased and process if made in lab) 112

We have included details of Oil Red O stain. Catalogue #O9755-25g (0.35% in isopropyl alcohol, Sigma Aldrich, MO, USA)

3. How the percentage of lipid content was determined. Is it relative to control/vehicle or absolute value? Please clarify. 116

By comparing the treatment to the control sample, the percentage of lipid content was calculated. Percentage of lipid=Test/control X100

4. How much protein was used for immunoblotting and what was dilution of antibody?

Equal amounts (15μg/well) of experimental proteins were separated by SDS-PAGE (Mini Protean Pre-casting gels, 12% Biorad, Hercules, USA) and the separated proteins were then transferred onto PVDF (polyvinylidene difluoride (PVDF) membranes) membrane by Turbo Transfer Gel method (BioRad, Hercules, USA). The membranes were immunoblotted with respective primary antibodies 1:1000 dilutions

5. In fig 1, what is ab and cd and what is P value of ab and cd? Please be specific.

Thank you for your kind words. We have provided an explanation of the alphabets included in the bar graph. They were highlighted by yellow shadows. The data were presented as the mean ± STD for six replicates (n=6). abcd different alphabets within the figure indicate significant differences between groups (p<0.05).

7. What is BCMRO6? Is it CMRO6? line 164

We apologize for this careless error. They are the same. It has been revised in its entirety.

8. Did authors use any positive control for lipid accumulation treatment?

The rosiglitazone is a positive control that we used for the differentiation as well as lipid accumulation. It is well known that rosiglitazone (RGZ) is a PPAR-γ agonist which induces lipid accumulation during the differentiation of adipocytes via increases in the key transcriptional factor PPAR-γ (Figure 3).

9. Is it possible for authors to provide high resolution images for Fig. 2b in supplementary data, because the staining is not clear?

Thanks for your suggestion. We have provided high resolution images for figure 2a-b based on a suggestion from asper reviewers (please see the original file submission).

10. Again, in Fig 2 what is ab? Are all the asterisks are with P<0.05 or some have lower p values?
11.  

Thank you for your kind words. We have provided an explanation of the alphabets included in the bar graph. They were highlighted by yellow shadows.

The data were presented as the mean ± STD for six replicates (n=6). abc different alphabets within the figure indicate significant differences between groups (p<0.05).

12. What is RO6 in fig 3f? is it same CMRO6? If yes, please be consistent.

We apologize for this careless error. They are the same. It has been revised in its entirety.

13. Treatment with CMRO6 downregulated the expression of adipogenic and lipogenic proteins and authors also quantified the western blots. Are the differences statistically significant in fig 4a and 4b?

The statistical analysis was performed for all experimental data and the figures are now arranged according to the analysis results. Also, the protein intensity was quantified by ImageJ software. CMRO6 treatment during differentiation reduced the translation level of C/CEB-β, C/CEB-α, PPAR-γ2, SREBP-1c, FAS, ACC, and FABP4. Results were expressed as the mean ± STD, n= 3 *p<0.05; **p<0.01 control vs treatment by an independent t-test;

14. Section 3.6 should be reframed properly.

Thank you for your valuable suggestion. In response to a reviewer's suggestion, we have reframed 3.6 section writing part.

15. In Fig 5a, basal bar graph showing asterisks c, it is compared to what? It would be great if authors clearly mention about asterisks what is compared to what.

Thank you for your kind words. We have provided an explanation of the alphabets included in the bar graph. They were highlighted by yellow shadows.

The data were presented as the mean ± STD for six replicates (n=6). abc different alphabets within the figure indicate significant differences between groups at p<0.05 level

16. Discussion should also be reframed.

Thank you for your valuable suggestion. In response to a reviewer's suggestion, we have reframed discussion section.

 

Reviewer 2 Report

Certain metabolites and proteins from bacteria may impact the differentiation and functions of adipose cells. In the manuscript, Lee et al. treated 3T3-L1 preadipocytes with the cell-free medium from CMRO6 and showed the treatment inhibits cells’ differentiation and lipid accumulation and increases glucose uptake. Although the effect of the metabolites from CMRO6 against adipogenesis and lipogenesis is associated with p38MAPK and Erk44/42 pathways, the authors need more evidence to support their conclusions.

Comments:

1. There are several mistakes in the text. The authors need to correct the critical information.
2. As the authors mentioned the cell-free metabolites from CMRO6 have anti-obesity functions, then what are the metabolites in the cell-free supernatant that play a role in regulating lipid synthesis and glucose uptake? How do they regulate the cellular pathways?
3. In figure 4a, some of the proteins seem not to change a lot. Can the authors use qPCR to test the genes expression?
4. If CMRO6 functions through p38MAPK and Erk1/2, can p38MAPK specific activator and Erk44/42 activator reverse the effect of CMRO6 on differentiation and lipid accumulation?
5. If CMRO6 induces glucose uptake, does the Akt inhibitor reverse the glucose uptake in CMRO6 treatment?
6. Quantification of the western blot bands could help to show the difference.

Author Response

Reviewer 2

 

We thank the reviewer for providing useful comments about our research paper, which will greatly improve the quality of the manuscript. We ask apology for typographical mistakes and grammatical errors.   In response to the reviewers' suggestions, we have read through the entire manuscript and modified it accordingly. Please note that changes have been made in red across the manuscript.

 

The antiobesity effects of CMRO6 have been confirmed by fat deposition in adipocytes in response to treatment and compared to control cells. As part of this study, fat content was determined in an experimental sample to confirm the CMRO6's ability to inhibit lipid accumulation, as demonstrated by the staining method of oil red O. The next key players for adipocyte differentiation such as PPAR C/EBPs, SREBP1c, as well as lipogenic markers such as FAS, ACC, and FABP4 were also analyzed. This also confirmed the important role that CMRO6 plays in fat deposition. Furthermore, the molecular mechanism behind the inhibition effect of CMRO6 has also been identified. The data has clearly indicated that the CMRO6 has the ability to inhibit obesity related makers in adipocytes and, therefore, proves that the probiotic used for its antiobesity properties. As for glucose uptake, we determined the glucose uptake level by the cells in response to CMRO6 or insulin  and it  has provided us with a clear understanding as to how the CMRO6 could facilitate glucose uptake via insulin related signaling pathways. These data are sufficient for asserting the antiobesity property of CMRO6 and its glucose uptake properties.  The paper has been modified accordingly with relevant changes appended separately. We do hope that the revised manuscript is now suitable for publication in molecules

 

 

1. There are several mistakes in the text. The authors need to correct the critical information.

 

We apologize for careless mistakes. Throughout the whole manuscript, all typos and grammatical errors have been carefully checked and revised the same.

 

2. As the authors mentioned the cell-free metabolites from CMRO6 have anti-obesity functions, then what are the metabolites in the cell-free supernatant that play a role in regulating lipid synthesis and glucose uptake? How do they regulate the cellular pathways?

 

Antiobesity properties of probiotics have been associated with   their ability to modulate the intestinal microbiota, energy metabolism, genes related to thermogenesis glucose metabolism, lipid metabolism and parasympathetic nerve activity.  The probiotics can produce several branched-chain fatty acids which have anti- obesity properties (den Besten et al., 2013). Butyrate and propionate reduce the appetite, inhibiting inflammatory phenotype, increasing adiponectin(Sivamaruthi et al., 2019), SCFAs produced by probiotics exert antiobesity effects through modulation of lipid and glucose metabolism (Schwiertz et al., 2010; Wang et al., 2015), resulting in a decrease in adipocyte size (Lin et al., 2012; Takemura et al., 2010).In particular, SCFAs reduce fat deposition in adipose tissue by accelerating the oxidation of fatty acids (den Besten et al., 2015a; Takemura et al., 2010) and switching the switch the metabolic state from lipogenesis to fat oxidation by regulation of PPAR γ(den Besten et al., 2015b; Vieira et al., 2013). We found that treatment with the CMRO6 inhibited adipocyte differentiation and lipid accumulation by down regulating adipogenic and lipogenic markers. This effect may be a result of SCFA production by Bacillus ginsengihumi.  In order to address this, we must further investigate the mechanisms by which SCFAs produced by Bacillus ginsengihumi modulate fat accumulation and differentiation of adipocytes.  Definitely we consider the reviewer suggestion and will try to find out the SCFAs production and its effects on molecular mechanisms of adipocyte differentiation and lipid accumulation. The reviewer's suggestion will definitely be taken into account and we will try to figure out the types of SCFAs that are produced by Bacillus ginsengihumi and what their effects are on adipocyte differentiation and lipid synthesis.   

 

3. In figure 4a, some of the proteins seem not to change a lot. Can the authors use qPCR to test the genes expression?

 

Thank s for your valuable suggestion.  Now we have analyzed the intensity of immunoblot with help of ImageJ software as well as performed statistical analysis between experimental data, it confirmed that the control and treatment has statistically significant at the values between 0.05 and 0.01 level.

 

4. If CMRO6 functions through p38MAPK and Erk1/2, can p38MAPK specific activator and Erk44/42 activator reverse the effect of CMRO6 on differentiation and lipid accumulation?

 

Yes, we strongly agreed with reviewer comment. Inhibitors of MAPKs and ERK1/2 could result in a significant reduction in differentiation and lipid accumulation in adipocytes (Bost et al., 2005; Gwon et al., 2013; Tang et al., 2003). These finding confirm that both MAPKs and ERK1/2 actication are essential for adipogenesis and lipogenesis.  However, we did not use these agonists and antagonists in this study.  We will take the suggestions made by the reviewers into consideration, and we will subsequently use different kinds of inhibitors and activators in future experiments.

 

5. If CMRO6 induces glucose uptake, does the Akt inhibitor reverse the glucose uptake in CMRO6 treatment?

Yes, we strongly agree with the reviewer's suggestion.  MK2206 is an Akt inhibitor, which has the ability to inhibit glucose metabolism and protein synthesis induced by insulin. In this study, however, we used insulin instead of MK2206 as a positive regulator for glucose uptake and insulin-related signaling modulation in differentiated adipocytes.

 

6. Quantification of the western blot bands could help to show the difference.

Yes, we have quantified protein bands in experimental samples using imagj software and calculated the intensities based on a housekeeping gene

Bost, F., Aouadi, M., Caron, L., Binétruy, B. 2005. The role of MAPKs in adipocyte differentiation and obesity. Biochimie, 87(1), 51-6.

den Besten, G., Bleeker, A., Gerding, A., van Eunen, K., Havinga, R., van Dijk, T.H., Oosterveer, M.H., Jonker, J.W., Groen, A.K., Reijngoud, D.-J., Bakker, B.M. 2015a. Short-Chain Fatty Acids Protect Against High-Fat Diet–Induced Obesity via a PPARγ-Dependent Switch From Lipogenesis to Fat Oxidation. Diabetes, 64(7), 2398-2408.

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den Besten, G., van Eunen, K., Groen, A.K., Venema, K., Reijngoud, D.J., Bakker, B.M. 2013. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J Lipid Res, 54(9), 2325-40.

Gwon, S.Y., Ahn, J.Y., Jung, C.H., Moon, B.K., Ha, T.Y. 2013. Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells. BMC Complement Altern Med, 13, 207.

Lin, H.V., Frassetto, A., Kowalik Jr, E.J., Nawrocki, A.R., Lu, M.M., Kosinski, J.R., Hubert, J.A., Szeto, D., Yao, X., Forrest, G., Marsh, D.J. 2012. Butyrate and Propionate Protect against Diet-Induced Obesity and Regulate Gut Hormones via Free Fatty Acid Receptor 3-Independent Mechanisms. PLOS ONE, 7(4), e35240.

Schwiertz, A., Taras, D., Schäfer, K., Beijer, S., Bos, N.A., Donus, C., Hardt, P.D. 2010. Microbiota and SCFA in Lean and Overweight Healthy Subjects. Obesity, 18(1), 190-195.

Sivamaruthi, B.S., Kesika, P., Suganthy, N., Chaiyasut, C. 2019. A Review on Role of Microbiome in Obesity and Antiobesity Properties of Probiotic Supplements. BioMed Research International, 2019, 3291367.

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Round 2

Reviewer 2 Report

The authors made significant improvements in the revised manuscript. However, there are still some mistakes, for example, the title of Figure 5. 

Author Response

We thank the reviewer for providing useful comments about our research paper, which will greatly improve the quality of the manuscript. We ask apology for typographical mistakes and grammatical errors.   In response to the reviewers' suggestions, we have read through the entire manuscript and modified it accordingly. Please note that changes have been made in red across the manuscript.

 

 

1. The authors made significant improvements in the revised manuscript. However, there are still some mistakes, for example, the title of Figure 5. 

Thanks for your valuable information. We have carefully and thoroughly read the whole manuscript again and have taken the time to edit and correct all typographical and grammatical errors.   We revised the conclusion to include all the essential information as suggested by the reviewer. Our conclusion has now been presented clearly, and we hope that it will assist the readers in understanding. Most of the experimental design and protocols used in the present study were published previously by our research team, therefore citing references for all experimental designs and protocols has been part of the citations. 

Author Response File: Author Response.docx