Reviewer’s report
Title: Fructo-Oligosaccharides Ameliorate Steatohepatitis, Visceral Adiposity, and Associated
Chronic Inflammation via Increased Production of Short-Chain Fatty Acids in a Mouse Model of
Non-alcoholic Steatohepatitis
Version: 0 Date: 27 Nov 2019
Reviewer: Emanuele Albano
Reviewer's report:
Please include all comments for the authors in this box rather than uploading your report as an
attachment. Please only upload as attachments annotated versions of manuscripts, graphs, supporting
materials or other aspects of your report which cannot be included in a text format.
Please overwrite this text when adding your comments to the authors.
The manuscript by Takai and co-workers investigates the effects of fructo-oligosaccharides (FOS) on
steatohepatitis and visceral adiposity in a model of NASH based on the induction of over-feeding by
the  administration  of  monosodium  glutamate  (MSG)  to  newborn  C57BL/6J  mice.   The  authors
observed that FOS treatment improved steatosis, inflammation and hepatocyte ballooning in the liver of
mice with NASH. These effects were associated with a lowering in the hepatic mRNA expression of
fatty acid synthase and glycerol-3-phosphate acyltransferase. Furthermore, FOS inhibited adipocyte
enlargement and macrophage recruitment in epididymal fat in parallel with changes in gut microbiota
composition and increased fecal concentrations of short chain fatty acids (SCFA) suggesting that FOS
action  on  dysbiosis  might  influence  the  development  of  metabolic  derangements  leading  to
steatohepatitis.
The work is interesting and has a potential translational impact. However, additional data are required
to support the author's conclusions. The following points need attention:
a) Figure 1 shows that FOS supplementation improves body weight as well as epididymal fat expansion
and inflammation. Nonetheless, the effects on adipose tissue require a better characterization from the
metabolic  and  inflammatory point of view  by measuring  glucose  tolerance  and insulin  response,
assessing adipose tissue expression of pro-inflammatory cytokines and the production of adipokines.   
b) The characterization of FOS action on NASH is also insufficient. Liver triglyceride content need to
be shown along with the liver or circulating levels of pro-inflammatory cytokines.
c) The authors have previously shown that FOS supplementation improves intestinal barrier function in
MCD fed mice.   It would be important to evaluate how much such an effect accounts for ameliorating
NASH in this model.  The measurement of circulating LPS levels can address this issue.
d) It is interesting that FOS reduced inflammatory cells infiltration in the adipose tissue, but the data
presented are insufficient to substantiate histology. Flow cytometry should be used to show the changes
in the prevalence of F4/80+/CD11b+ macrophages and of B and T lymphocytes.  Furthermore, M1
macrophages are identified as F4/80+/CD11b+/Ly6Chigh cells.  The use of CD11c is not a suitable
marker.
e) The overall impact of the work would be increased by investigating whether FOS supplementation is
also effective in ameliorating dybiosis and liver alterations with more widely used model of NASH
such as the high fat western diet.   
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you recommend an
additional statistical review?
If an additional statistical review is recommended, please specify what aspects require further
assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?
6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below.
If your reply is yes to any, please give details below.
No complelling interests
I agree to the open peer review policy of the journal. I understand that my name will be included
on my report to the authors and, if the manuscript is accepted for publication, my named report
including any attachments I upload will be posted on the website along with the authors'
responses. I agree for my report to be made available under an Open Access Creative Commons
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments
which I do not wish to be included in my named report can be included as confidential comments
to the editors, which will not be published.
I agree to the open peer review policy of the journal
Reviewer’s report
Title: Fructo-Oligosaccharides Ameliorate Steatohepatitis, Visceral Adiposity, and Associated
Chronic Inflammation via Increased Production of Short-Chain Fatty Acids in a Mouse Model of
Non-alcoholic Steatohepatitis
Version: 0 Date: 27 Nov 2019
Reviewer: Emanuele Albano
Reviewer's report:
Please include all comments for the authors in this box rather than uploading your report as an
attachment. Please only upload as attachments annotated versions of manuscripts, graphs, supporting
materials or other aspects of your report which cannot be included in a text format.
Please overwrite this text when adding your comments to the authors.
The manuscript by Takai and co-workers investigates the effects of fructo-oligosaccharides (FOS) on
steatohepatitis and visceral adiposity in a model of NASH based on the induction of over-feeding by
the  administration  of  monosodium  glutamate  (MSG)  to  newborn  C57BL/6J  mice.   The  authors
observed that FOS treatment improved steatosis, inflammation and hepatocyte ballooning in the liver of
mice with NASH. These effects were associated with a lowering in the hepatic mRNA expression of
fatty acid synthase and glycerol-3-phosphate acyltransferase. Furthermore, FOS inhibited adipocyte
enlargement and macrophage recruitment in epididymal fat in parallel with changes in gut microbiota
composition and increased fecal concentrations of short chain fatty acids (SCFA) suggesting that FOS
action  on  dysbiosis  might  influence  the  development  of  metabolic  derangements  leading  to
steatohepatitis.
The work is interesting and has a potential translational impact. However, additional data are required
to support the author's conclusions. The following points need attention:
a) Figure 1 shows that FOS supplementation improves body weight as well as epididymal fat expansion
and inflammation. Nonetheless, the effects on adipose tissue require a better characterization from the
metabolic  and  inflammatory point of view  by measuring  glucose  tolerance  and insulin  response,
assessing adipose tissue expression of pro-inflammatory cytokines and the production of adipokines.   
b) The characterization of FOS action on NASH is also insufficient. Liver triglyceride content need to
be shown along with the liver or circulating levels of pro-inflammatory cytokines.
c) The authors have previously shown that FOS supplementation improves intestinal barrier function in
MCD fed mice.   It would be important to evaluate how much such an effect accounts for ameliorating
NASH in this model.  The measurement of circulating LPS levels can address this issue.
d) It is interesting that FOS reduced inflammatory cells infiltration in the adipose tissue, but the data
presented are insufficient to substantiate histology. Flow cytometry should be used to show the changes
in the prevalence of F4/80+/CD11b+ macrophages and of B and T lymphocytes.  Furthermore, M1
macrophages are identified as F4/80+/CD11b+/Ly6Chigh cells.  The use of CD11c is not a suitable
marker.
e) The overall impact of the work would be increased by investigating whether FOS supplementation is
also effective in ameliorating dybiosis and liver alterations with more widely used model of NASH
such as the high fat western diet.   
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you recommend an
additional statistical review?
If an additional statistical review is recommended, please specify what aspects require further
assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?
6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below.
If your reply is yes to any, please give details below.
No complelling interests
I agree to the open peer review policy of the journal. I understand that my name will be included
on my report to the authors and, if the manuscript is accepted for publication, my named report
including any attachments I upload will be posted on the website along with the authors'
responses. I agree for my report to be made available under an Open Access Creative Commons
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments
which I do not wish to be included in my named report can be included as confidential comments
to the editors, which will not be published.
I agree to the open peer review policy of the journal
Please review this paper.
user
Reviewer’s report
Title: Fructo-Oligosaccharides Ameliorate Steatohepatitis, Visceral Adiposity, and Associated
Chronic Inflammation via Increased Production of Short-Chain Fatty Acids in a Mouse Model of
Non-alcoholic Steatohepatitis
Version: 0 Date: 27 Nov 2019
Reviewer: Emanuele Albano
Reviewer's report:
Please include all comments for the authors in this box rather than uploading your report as an
attachment. Please only upload as attachments annotated versions of manuscripts, graphs, supporting
materials or other aspects of your report which cannot be included in a text format.
Please overwrite this text when adding your comments to the authors.
The manuscript by Takai and co-workers investigates the effects of fructo-oligosaccharides (FOS) on
steatohepatitis and visceral adiposity in a model of NASH based on the induction of over-feeding by
the  administration  of  monosodium  glutamate  (MSG)  to  newborn  C57BL/6J  mice.   The  authors
observed that FOS treatment improved steatosis, inflammation and hepatocyte ballooning in the liver of
mice with NASH. These effects were associated with a lowering in the hepatic mRNA expression of
fatty acid synthase and glycerol-3-phosphate acyltransferase. Furthermore, FOS inhibited adipocyte
enlargement and macrophage recruitment in epididymal fat in parallel with changes in gut microbiota
composition and increased fecal concentrations of short chain fatty acids (SCFA) suggesting that FOS
action  on  dysbiosis  might  influence  the  development  of  metabolic  derangements  leading  to
steatohepatitis.
The work is interesting and has a potential translational impact. However, additional data are required
to support the author's conclusions. The following points need attention:
a) Figure 1 shows that FOS supplementation improves body weight as well as epididymal fat expansion
and inflammation. Nonetheless, the effects on adipose tissue require a better characterization from the
metabolic  and  inflammatory point of view  by measuring  glucose  tolerance  and insulin  response,
assessing adipose tissue expression of pro-inflammatory cytokines and the production of adipokines.   
b) The characterization of FOS action on NASH is also insufficient. Liver triglyceride content need to
be shown along with the liver or circulating levels of pro-inflammatory cytokines.
c) The authors have previously shown that FOS supplementation improves intestinal barrier function in
MCD fed mice.   It would be important to evaluate how much such an effect accounts for ameliorating
NASH in this model.  The measurement of circulating LPS levels can address this issue.
d) It is interesting that FOS reduced inflammatory cells infiltration in the adipose tissue, but the data
presented are insufficient to substantiate histology. Flow cytometry should be used to show the changes
in the prevalence of F4/80+/CD11b+ macrophages and of B and T lymphocytes.  Furthermore, M1
macrophages are identified as F4/80+/CD11b+/Ly6Chigh cells.  The use of CD11c is not a suitable
marker.
e) The overall impact of the work would be increased by investigating whether FOS supplementation is
also effective in ameliorating dybiosis and liver alterations with more widely used model of NASH
such as the high fat western diet.   
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
Yes
Are you able to assess any statistics in the manuscript or would you recommend an
additional statistical review?
If an additional statistical review is recommended, please specify what aspects require further
assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following questions:
1. Have you in the past five years received reimbursements, fees, funding, or salary from an
organisation that may in any way gain or lose financially from the publication of this
manuscript, either now or in the future?
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose
financially from the publication of this manuscript, either now or in the future?
3. Do you hold or are you currently applying for any patents relating to the content of the
manuscript?
4. Have you received reimbursements, fees, funding, or salary from an organization that holds
or has applied for patents relating to the content of the manuscript?
5. Do you have any other financial competing interests?
6. Do you have any non-financial competing interests in relation to this paper?
If you can answer no to all of the above, write 'I declare that I have no competing interests' below.
If your reply is yes to any, please give details below.
No complelling interests
I agree to the open peer review policy of the journal. I understand that my name will be included
on my report to the authors and, if the manuscript is accepted for publication, my named report
including any attachments I upload will be posted on the website along with the authors'
responses. I agree for my report to be made available under an Open Access Creative Commons
CC-BY license (http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments
which I do not wish to be included in my named report can be included as confidential comments
to the editors, which will not be published.
I agree to the open peer review policy of the journal
assistant