Reviewer’s report
Title: 
HFD and HFD-provoked Hepatic Hypoxia act as Reciprocal Causation for
NAFLD via HIF-independent Signaling
Version: 
0 
Date: 
13 Jul 2020
Reviewer's report:
Zhang et al. in this manuscript provide evidence that hypoxia and lipid
accumulation form vicious cycle to promote NAFLD disease conditions in HIFs-
independent manner. 
The manuscript addresses some interesting points but appears incomplete and
inadequate at the specific important observations. 
Major criticisms;
1.The authors treated newborn mice with 75 % oxygen for 5 days followed by 21
% oxygen for another 5 days, and designated as "hypoxia treatment". Although
pimonidazole-adducts were detected in the mouse liver (Figure 4A), the reviewer
thinks that this treatment mimics hyperoxia followed by normoxia, and these
alterations do not occur in NAFLD patients. The authors should explain the
reason why you did not treat mice with 7~10 % oxygen to evoke hypoxia in
vivo, and check if the low oxygenated conditions could stimulate lipid
infiltration with altered gene expressions of lipid metabolism in mouse liver as
well. On the other hand, the authors also mentioned that intermittent, but not
chronic, hypoxia is a hallmark of OSAS, that is tightly linked with NAFLD. The
reviewer requests the authors to explain the reason why you did not reproduce
the same conditions in obese adult mice. 
2.The authors clearly showed lipid infiltration in mouse liver exposed to either
HFD (Figure 1D) or hypoxia (Figure 4B). However, there is no description about
the region (periportal, pericentral or homogenously) of lipid accumulation and
hypoxia in liver acinus. In addition, the authors should examine whether lipid
accumulation and hypoxia occur in the same region among liver acinus in each
experimental condition.
3.In mice, hypoxia increases and decreases gene expression involved in
lipogenesis and lipolysis, respectively (Figure 4D and 4E). Can hypoxia elicit the
similar changes in vitro? Based on your hypothesis, hypoxia and lipid
accumulation enhances its effects reciprocally. If so, can simultaneous exposure
to hypoxia and palmitate further deteriorate lipid accumulation? In addition, the
reviewer wonders if exposure of mice to HFD evokes the similar alterations in
the gene expressions to those in hypoxia-treated mice.
Minor criticisms;
1.There is no information about sequences of siRNAs against HIF-1alpha and -
2alpha, with transfection methods.
2.The authors should describe experimental conditions in detail. For example,
information about cell density, age and sex of mice, and timing of liver sampling
(fed or fasted conditions) is lacking.
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Yes
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 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.
I declare that I have no competing interests
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the editorial
office before completing the review.
If you wish, you can include your name in your published report. Please note
you must decide whether to include your name at the start of the process and
confirm this decision whenever prompted. If you change your decision later, we
will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Reviewer’s report
Title: 
HFD and HFD-provoked Hepatic Hypoxia act as Reciprocal Causation for
NAFLD via HIF-independent Signaling
Version: 
0 
Date: 
13 Jul 2020
Reviewer's report:
Zhang et al. in this manuscript provide evidence that hypoxia and lipid
accumulation form vicious cycle to promote NAFLD disease conditions in HIFs-
independent manner. 
The manuscript addresses some interesting points but appears incomplete and
inadequate at the specific important observations. 
Major criticisms;
1.The authors treated newborn mice with 75 % oxygen for 5 days followed by 21
% oxygen for another 5 days, and designated as "hypoxia treatment". Although
pimonidazole-adducts were detected in the mouse liver (Figure 4A), the reviewer
thinks that this treatment mimics hyperoxia followed by normoxia, and these
alterations do not occur in NAFLD patients. The authors should explain the
reason why you did not treat mice with 7~10 % oxygen to evoke hypoxia in
vivo, and check if the low oxygenated conditions could stimulate lipid
infiltration with altered gene expressions of lipid metabolism in mouse liver as
well. On the other hand, the authors also mentioned that intermittent, but not
chronic, hypoxia is a hallmark of OSAS, that is tightly linked with NAFLD. The
reviewer requests the authors to explain the reason why you did not reproduce
the same conditions in obese adult mice. 
2.The authors clearly showed lipid infiltration in mouse liver exposed to either
HFD (Figure 1D) or hypoxia (Figure 4B). However, there is no description about
the region (periportal, pericentral or homogenously) of lipid accumulation and
hypoxia in liver acinus. In addition, the authors should examine whether lipid
accumulation and hypoxia occur in the same region among liver acinus in each
experimental condition.
3.In mice, hypoxia increases and decreases gene expression involved in
lipogenesis and lipolysis, respectively (Figure 4D and 4E). Can hypoxia elicit the
similar changes in vitro? Based on your hypothesis, hypoxia and lipid
accumulation enhances its effects reciprocally. If so, can simultaneous exposure
to hypoxia and palmitate further deteriorate lipid accumulation? In addition, the
reviewer wonders if exposure of mice to HFD evokes the similar alterations in
the gene expressions to those in hypoxia-treated mice.
Minor criticisms;
1.There is no information about sequences of siRNAs against HIF-1alpha and -
2alpha, with transfection methods.
2.The authors should describe experimental conditions in detail. For example,
information about cell density, age and sex of mice, and timing of liver sampling
(fed or fasted conditions) is lacking.
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons upon publication of the manuscript. If you have already registered at
Publons, they will notify you of the receipt of this review and update your profile
as per your settings and their policy. If you are not registered with Publons, you
will receive an email from them asking you to register in order for them to be
able to recognize your review on your new profile page. Publons may use the
Review Data to generate derivative metadata for the benefit of Publons and you
as a reviewer, carefully considering the sensitivity of such information. For
example, Publons may verify your record as a reviewer by updating your profile
published on its webservice if you have registered for such service or help
editors to identify candidate reviewers. Please find the details of processing in
Publons’ privacy policy 
https://publons.com/about/terms
Yes
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 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.
I declare that I have no competing interests
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the editorial
office before completing the review.
If you wish, you can include your name in your published report. Please note
you must decide whether to include your name at the start of the process and
confirm this decision whenever prompted. If you change your decision later, we
will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
Please review this paper.
user
Reviewer’s report
Title: 
HFD and HFD-provoked Hepatic Hypoxia act as Reciprocal Causation for
NAFLD via HIF-independent Signaling
Version: 
0 
Date: 
13 Jul 2020
Reviewer's report:
Zhang et al. in this manuscript provide evidence that hypoxia and lipid
accumulation form vicious cycle to promote NAFLD disease conditions in HIFs-
independent manner. 
The manuscript addresses some interesting points but appears incomplete and
inadequate at the specific important observations. 
Major criticisms;
1.The authors treated newborn mice with 75 % oxygen for 5 days followed by 21
% oxygen for another 5 days, and designated as "hypoxia treatment". Although
pimonidazole-adducts were detected in the mouse liver (Figure 4A), the reviewer
thinks that this treatment mimics hyperoxia followed by normoxia, and these
alterations do not occur in NAFLD patients. The authors should explain the
reason why you did not treat mice with 7~10 % oxygen to evoke hypoxia in
vivo, and check if the low oxygenated conditions could stimulate lipid
infiltration with altered gene expressions of lipid metabolism in mouse liver as
well. On the other hand, the authors also mentioned that intermittent, but not
chronic, hypoxia is a hallmark of OSAS, that is tightly linked with NAFLD. The
reviewer requests the authors to explain the reason why you did not reproduce
the same conditions in obese adult mice. 
2.The authors clearly showed lipid infiltration in mouse liver exposed to either
HFD (Figure 1D) or hypoxia (Figure 4B). However, there is no description about
the region (periportal, pericentral or homogenously) of lipid accumulation and
hypoxia in liver acinus. In addition, the authors should examine whether lipid
accumulation and hypoxia occur in the same region among liver acinus in each
experimental condition.
3.In mice, hypoxia increases and decreases gene expression involved in
lipogenesis and lipolysis, respectively (Figure 4D and 4E). Can hypoxia elicit the
similar changes in vitro? Based on your hypothesis, hypoxia and lipid
accumulation enhances its effects reciprocally. If so, can simultaneous exposure
to hypoxia and palmitate further deteriorate lipid accumulation? In addition, the
reviewer wonders if exposure of mice to HFD evokes the similar alterations in
the gene expressions to those in hypoxia-treated mice.
Minor criticisms;
1.There is no information about sequences of siRNAs against HIF-1alpha and -
2alpha, with transfection methods.
2.The authors should describe experimental conditions in detail. For example,
information about cell density, age and sex of mice, and timing of liver sampling
(fed or fasted conditions) is lacking.
Publons Reviewer Recognition
. Springer Nature can send verification of this
review directly to Publons (a subsidiary of Clarivate Analytics). If you would
like to take advantage of this service, please click on the “Yes” option below.
Your name, email address, title of the reviewed manuscript, name of the journal,
and date of your review submission (the “Review Data”) will then be transmitted
to Publons upon publication of the manuscript. If you have already registered at
Publons, they will notify you of the receipt of this review and update your profile
as per your settings and their policy. If you are not registered with Publons, you
will receive an email from them asking you to register in order for them to be
able to recognize your review on your new profile page. Publons may use the
Review Data to generate derivative metadata for the benefit of Publons and you
as a reviewer, carefully considering the sensitivity of such information. For
example, Publons may verify your record as a reviewer by updating your profile
published on its webservice if you have registered for such service or help
editors to identify candidate reviewers. Please find the details of processing in
Publons’ privacy policy 
https://publons.com/about/terms
Yes
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 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.
I declare that I have no competing interests
Reviewer Publication Consent.
 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
) if this manuscript is accepted for
publication. Any comments that I do not wish to be included in the published
report have been included as confidential comments to the editor, which will not
be published.
If you are not happy for us to publish this report, please contact the editorial
office before completing the review.
If you wish, you can include your name in your published report. Please note
you must decide whether to include your name at the start of the process and
confirm this decision whenever prompted. If you change your decision later, we
will not be able to publish your name.
I agree to the terms of the CC-BY 4.0 license; please 
do not
 publish my name
with my report. (default)
assistant