Reviewer ’s report  
 
Title: Alcoholic vs Non -Alcoholic Fatty Liver in Rats: Distinct Differences in Endocytosis and 
Vesicle Trafficking despite Similar Pathology  
 
 
Version: 0 Date: 23 Nov 2015  
 
 
Reviewer: Jason Burkhead  
 
 
Reviewer's report:  
 
 
This is an interesting study that that provides new insight into differences in non -alcoholic fatty-
liver disease (NAFLD) and alcoholic fatty -liver disease (AFLD). The observation that ASGPR 
internalization as well as Rab3D and Rab7 were decreased is a novel finding. The studies appear 
to be well designed and are clearly explained for the most part , but some specific experiments 
would benefit from additional description or clarification.  
 
Most of my comments and questions are based on the explanation of the experimental 
procedures.  
 
1. It is my understanding that the generally accepted acronyms fo r non -alcoholic fatty -liver 
disease and alcoholic fatty -liver disease are NAFLD and AFLD, respectively. These are 
the designations used in the journals published by the AASLD as well as in NIH 
publications. The NAFLD and AFLD acronyms are used in at least one instance in the 
manuscript, but otherwise NAFL and AFL are used. I suggest that the authors clarify the 
most accepted usage and apply it consistently throughout the manuscript.  
 
2. The clinical distinction between non -alcoholic steatohepatitis and alcoh olic 
steatohepatitis in humans is characterized by differences between AST and ALT, with 
AST>ALT for alcoholic liver disease and ALT>AST for non -alcoholic. If this data is 
available from the Vetscan, it would be interesting to help establish the models for  
comparison to patients.  
 
3. The diets used to induce disease are Lieber DeCarli with/without ethanol as well as 
lean/high fat (60% lipid) diets. The induction of pathology is apparent from the results. It 
would be helpful to clarify if these diets are intended to be representative  of human diets 
associated with alcoholism and metabolic syndrome/NAFLD and how they are 
comparable.  
 
4. The methods note that rats were sacrificed in the morning hours, but it is not indicated 
that the rats fasted overnight or if morning hours followed th e active (dark) or inactive 
(light period). Insulin levels in HFD are significantly increased, but without more 
experimental detail it is not clear if the animals are insulin resistant. HOMA -IR, which 
has been validated for rats in other studies, or glucos e tolerance (which would be 
impractical and not really necessary) would allow for more discussion of insulin 
resistance.  
 
5. Under Methods, clinical chemistry describes a plasma profile analyzed by Vetscan. 
However, the results in the paper focus on serum p rofiles and analytes. It would be 
appropriate to clarify the biological sample that was used for the experiments.  
 
6. Were triglycerides prepared from isolated hepatocytes or homogenized liver tissue? This 
isn't quite clear from the methods.  
 
7. Under Weste rn Blot, post -nuclear supernatant (PNS) was prepared, but the methods, 
buffers, centrifugation, etc were not described.  
 
8. mRNA analysis/ gene expression: Primer sequences and Taqman probe sequences for 
target genes and controls should be provided.  
 
9. Plasma hormone quantification: again, there is a mix of reference to plasma and serum in 
 the methods and results sections.  
 
10. Statistical significance for analysis was assigned in the figures and legends with P<0.05, 
however, I was unable to find the actua l statistical significance tests in the methods or 
figure legends. There was mention of pair feeding in the animal care methods, which 
might warrant different parameters for statistical testing. The results suggest that the data 
was not analyzed as paired samples, but this is not clear. It is not exactly clear how paired 
data would be used in this study, or exactly how pairs would be assigned. It would be 
helpful to clarify the pair feeding and explain the statistical testing.  
 
11. Figure 1 does not have sc ale bars for histology, though they are present for figure 2.  
 
Overall, this work is of importance to understanding differences between modes of hepatic injury 
and provides new information that has potential to aid in diagnosis and treatment of disease.  
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 supporte d 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 a dditional 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:  
 
Acceptable  
 
 
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 gai n 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 fu ture?  
 
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.  
 
I declare that I have no competing 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 C reative Commons  
CC-BY licens e (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: Alcoholic vs Non -Alcoholic Fatty Liver in Rats: Distinct Differences in Endocytosis and 
Vesicle Trafficking despite Similar Pathology  
 
 
Version: 0 Date: 23 Nov 2015  
 
 
Reviewer: Jason Burkhead  
 
 
Reviewer's report:  
 
 
This is an interesting study that that provides new insight into differences in non -alcoholic fatty-
liver disease (NAFLD) and alcoholic fatty -liver disease (AFLD). The observation that ASGPR 
internalization as well as Rab3D and Rab7 were decreased is a novel finding. The studies appear 
to be well designed and are clearly explained for the most part , but some specific experiments 
would benefit from additional description or clarification.  
 
Most of my comments and questions are based on the explanation of the experimental 
procedures.  
 
1. It is my understanding that the generally accepted acronyms fo r non -alcoholic fatty -liver 
disease and alcoholic fatty -liver disease are NAFLD and AFLD, respectively. These are 
the designations used in the journals published by the AASLD as well as in NIH 
publications. The NAFLD and AFLD acronyms are used in at least one instance in the 
manuscript, but otherwise NAFL and AFL are used. I suggest that the authors clarify the 
most accepted usage and apply it consistently throughout the manuscript.  
 
2. The clinical distinction between non -alcoholic steatohepatitis and alcoh olic 
steatohepatitis in humans is characterized by differences between AST and ALT, with 
AST>ALT for alcoholic liver disease and ALT>AST for non -alcoholic. If this data is 
available from the Vetscan, it would be interesting to help establish the models for  
comparison to patients.  
 
3. The diets used to induce disease are Lieber DeCarli with/without ethanol as well as 
lean/high fat (60% lipid) diets. The induction of pathology is apparent from the results. It 
would be helpful to clarify if these diets are intended to be representative  of human diets 
associated with alcoholism and metabolic syndrome/NAFLD and how they are 
comparable.  
 
4. The methods note that rats were sacrificed in the morning hours, but it is not indicated 
that the rats fasted overnight or if morning hours followed th e active (dark) or inactive 
(light period). Insulin levels in HFD are significantly increased, but without more 
experimental detail it is not clear if the animals are insulin resistant. HOMA -IR, which 
has been validated for rats in other studies, or glucos e tolerance (which would be 
impractical and not really necessary) would allow for more discussion of insulin 
resistance.  
 
5. Under Methods, clinical chemistry describes a plasma profile analyzed by Vetscan. 
However, the results in the paper focus on serum p rofiles and analytes. It would be 
appropriate to clarify the biological sample that was used for the experiments.  
 
6. Were triglycerides prepared from isolated hepatocytes or homogenized liver tissue? This 
isn't quite clear from the methods.  
 
7. Under Weste rn Blot, post -nuclear supernatant (PNS) was prepared, but the methods, 
buffers, centrifugation, etc were not described.  
 
8. mRNA analysis/ gene expression: Primer sequences and Taqman probe sequences for 
target genes and controls should be provided.  
 
9. Plasma hormone quantification: again, there is a mix of reference to plasma and serum in 
 the methods and results sections.  
 
10. Statistical significance for analysis was assigned in the figures and legends with P<0.05, 
however, I was unable to find the actua l statistical significance tests in the methods or 
figure legends. There was mention of pair feeding in the animal care methods, which 
might warrant different parameters for statistical testing. The results suggest that the data 
was not analyzed as paired samples, but this is not clear. It is not exactly clear how paired 
data would be used in this study, or exactly how pairs would be assigned. It would be 
helpful to clarify the pair feeding and explain the statistical testing.  
 
11. Figure 1 does not have sc ale bars for histology, though they are present for figure 2.  
 
Overall, this work is of importance to understanding differences between modes of hepatic injury 
and provides new information that has potential to aid in diagnosis and treatment of disease.  
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 supporte d 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 a dditional 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:  
 
Acceptable  
 
 
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 gai n 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 fu ture?  
 
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.  
 
I declare that I have no competing 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 C reative Commons  
CC-BY licens e (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: Alcoholic vs Non -Alcoholic Fatty Liver in Rats: Distinct Differences in Endocytosis and 
Vesicle Trafficking despite Similar Pathology  
 
 
Version: 0 Date: 23 Nov 2015  
 
 
Reviewer: Jason Burkhead  
 
 
Reviewer's report:  
 
 
This is an interesting study that that provides new insight into differences in non -alcoholic fatty-
liver disease (NAFLD) and alcoholic fatty -liver disease (AFLD). The observation that ASGPR 
internalization as well as Rab3D and Rab7 were decreased is a novel finding. The studies appear 
to be well designed and are clearly explained for the most part , but some specific experiments 
would benefit from additional description or clarification.  
 
Most of my comments and questions are based on the explanation of the experimental 
procedures.  
 
1. It is my understanding that the generally accepted acronyms fo r non -alcoholic fatty -liver 
disease and alcoholic fatty -liver disease are NAFLD and AFLD, respectively. These are 
the designations used in the journals published by the AASLD as well as in NIH 
publications. The NAFLD and AFLD acronyms are used in at least one instance in the 
manuscript, but otherwise NAFL and AFL are used. I suggest that the authors clarify the 
most accepted usage and apply it consistently throughout the manuscript.  
 
2. The clinical distinction between non -alcoholic steatohepatitis and alcoh olic 
steatohepatitis in humans is characterized by differences between AST and ALT, with 
AST>ALT for alcoholic liver disease and ALT>AST for non -alcoholic. If this data is 
available from the Vetscan, it would be interesting to help establish the models for  
comparison to patients.  
 
3. The diets used to induce disease are Lieber DeCarli with/without ethanol as well as 
lean/high fat (60% lipid) diets. The induction of pathology is apparent from the results. It 
would be helpful to clarify if these diets are intended to be representative  of human diets 
associated with alcoholism and metabolic syndrome/NAFLD and how they are 
comparable.  
 
4. The methods note that rats were sacrificed in the morning hours, but it is not indicated 
that the rats fasted overnight or if morning hours followed th e active (dark) or inactive 
(light period). Insulin levels in HFD are significantly increased, but without more 
experimental detail it is not clear if the animals are insulin resistant. HOMA -IR, which 
has been validated for rats in other studies, or glucos e tolerance (which would be 
impractical and not really necessary) would allow for more discussion of insulin 
resistance.  
 
5. Under Methods, clinical chemistry describes a plasma profile analyzed by Vetscan. 
However, the results in the paper focus on serum p rofiles and analytes. It would be 
appropriate to clarify the biological sample that was used for the experiments.  
 
6. Were triglycerides prepared from isolated hepatocytes or homogenized liver tissue? This 
isn't quite clear from the methods.  
 
7. Under Weste rn Blot, post -nuclear supernatant (PNS) was prepared, but the methods, 
buffers, centrifugation, etc were not described.  
 
8. mRNA analysis/ gene expression: Primer sequences and Taqman probe sequences for 
target genes and controls should be provided.  
 
9. Plasma hormone quantification: again, there is a mix of reference to plasma and serum in 
 the methods and results sections.  
 
10. Statistical significance for analysis was assigned in the figures and legends with P<0.05, 
however, I was unable to find the actua l statistical significance tests in the methods or 
figure legends. There was mention of pair feeding in the animal care methods, which 
might warrant different parameters for statistical testing. The results suggest that the data 
was not analyzed as paired samples, but this is not clear. It is not exactly clear how paired 
data would be used in this study, or exactly how pairs would be assigned. It would be 
helpful to clarify the pair feeding and explain the statistical testing.  
 
11. Figure 1 does not have sc ale bars for histology, though they are present for figure 2.  
 
Overall, this work is of importance to understanding differences between modes of hepatic injury 
and provides new information that has potential to aid in diagnosis and treatment of disease.  
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
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 supporte d 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 a dditional 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:  
 
Acceptable  
 
 
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 gai n 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 fu ture?  
 
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.  
 
I declare that I have no competing 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 C reative Commons  
CC-BY licens e (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