Reviewer's report
Title:Deficiency of iNOS-derived NO accelerates lipid accumulation-independent
liver fibrosis in non-alcoholic steatohepatitis mouse model
Version:2Date:17 September 2014
Reviewer: Mariana Machado
Reviewer's report:
The authors present a study that evaluates the role of inducible NO synthase
(iNOS) in the development and severity of NAFLD, taking into consideration that
its deficiency was shown to be protective from diet-induced metabolic syndrome,
but it has been associated to higher fibrosis in other animal models of liver
disease. They fed WT and iNOS KO mice with regular high fat diet (not western
diet) for 10 and 48 weeks and found a decrease in liver steatosis but an increase
in fibrosis and inflammation. They also showed activation of NFkB pathway and,
when treating the KO mice with a proteasome inhibitor (epoxomicin), which also
inhibits NFkB (decreases the degradation of its inhibitor), they found a decrease
in gene expression of several pro-inflammatory and pro-fibrogenic cytokines.
The concept is interesting and it is well documented. I have however some
considerations:
Major points:
1. There should be a discussion on the fact that KO mice have more
injury/fibrosis despite the fact that they have less hepatic steatosis. I do not agree
with the observation that it could be burnout NASH with loss of steatosis in the
sequence of advanced liver disease, since at 10 weeks of diet already less
steatosis was found. Fat accumulation in the liver may in fact be a protective
mechanism, with detoxification of biologically active lipids in the inert
triglycerides. In fact, KO mice had less triglycerides in the liver, but had more free
fatty acids in the liver and serum! Also, I would like the authors to comment the
downregulation of SREBP1c, since it has been shown by several other authors to
be upregulated in the liver in animals fed with high fat diet (mimicking what
happens with human obesity-associated NAFLD). It would also be interesting to
comment the discrepancy between insulin resistance and NASH in animal
models.
2. Another major point relates to the experiment with epoxomicin. First it was not
specified how many mice were used. Were those mice accounted when they
contabilized the animals used in the high fat diet experiment. Also, clearly this
agent is not specific for NFkB and, in fact, deregulation of proteasome has been
described in NASH. In that way the authors cannot deduce causality, they can
only say that treatment associated with decrease in NFkB nuclear staining, which
associated with decrease in those cytokines. Could not be done a more careful
analysis, at least in terms of fibrosis and steatosis in those treated animals? Also,
what happens in WT mice fed HFD and treated with epoxomicin? It would be an
important control, since the effect of drug may be independent of iNOS
deficiency.
3. Still regarding NFkB, the authors stated that activation of NFkB occurred in
hepatocytes, hepatic stellate cells and Kupffer cells, however they did not show
evidence of that. I think that to say that, the authors should do double staining, at
least for desmin and a macrophage marker.
Minor point:
4. A minor question – the magnitude of oil red images does not seem the same
between groups, mainly between KO and WT fed 48 weeks.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no competing interests.
Reviewer's report
Title:Deficiency of iNOS-derived NO accelerates lipid accumulation-independent
liver fibrosis in non-alcoholic steatohepatitis mouse model
Version:2Date:17 September 2014
Reviewer: Mariana Machado
Reviewer's report:
The authors present a study that evaluates the role of inducible NO synthase
(iNOS) in the development and severity of NAFLD, taking into consideration that
its deficiency was shown to be protective from diet-induced metabolic syndrome,
but it has been associated to higher fibrosis in other animal models of liver
disease. They fed WT and iNOS KO mice with regular high fat diet (not western
diet) for 10 and 48 weeks and found a decrease in liver steatosis but an increase
in fibrosis and inflammation. They also showed activation of NFkB pathway and,
when treating the KO mice with a proteasome inhibitor (epoxomicin), which also
inhibits NFkB (decreases the degradation of its inhibitor), they found a decrease
in gene expression of several pro-inflammatory and pro-fibrogenic cytokines.
The concept is interesting and it is well documented. I have however some
considerations:
Major points:
1. There should be a discussion on the fact that KO mice have more
injury/fibrosis despite the fact that they have less hepatic steatosis. I do not agree
with the observation that it could be burnout NASH with loss of steatosis in the
sequence of advanced liver disease, since at 10 weeks of diet already less
steatosis was found. Fat accumulation in the liver may in fact be a protective
mechanism, with detoxification of biologically active lipids in the inert
triglycerides. In fact, KO mice had less triglycerides in the liver, but had more free
fatty acids in the liver and serum! Also, I would like the authors to comment the
downregulation of SREBP1c, since it has been shown by several other authors to
be upregulated in the liver in animals fed with high fat diet (mimicking what
happens with human obesity-associated NAFLD). It would also be interesting to
comment the discrepancy between insulin resistance and NASH in animal
models.
2. Another major point relates to the experiment with epoxomicin. First it was not
specified how many mice were used. Were those mice accounted when they
contabilized the animals used in the high fat diet experiment. Also, clearly this
agent is not specific for NFkB and, in fact, deregulation of proteasome has been
described in NASH. In that way the authors cannot deduce causality, they can
only say that treatment associated with decrease in NFkB nuclear staining, which
associated with decrease in those cytokines. Could not be done a more careful
analysis, at least in terms of fibrosis and steatosis in those treated animals? Also,
what happens in WT mice fed HFD and treated with epoxomicin? It would be an
important control, since the effect of drug may be independent of iNOS
deficiency.
3. Still regarding NFkB, the authors stated that activation of NFkB occurred in
hepatocytes, hepatic stellate cells and Kupffer cells, however they did not show
evidence of that. I think that to say that, the authors should do double staining, at
least for desmin and a macrophage marker.
Minor point:
4. A minor question – the magnitude of oil red images does not seem the same
between groups, mainly between KO and WT fed 48 weeks.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Deficiency of iNOS-derived NO accelerates lipid accumulation-independent
liver fibrosis in non-alcoholic steatohepatitis mouse model
Version:2Date:17 September 2014
Reviewer: Mariana Machado
Reviewer's report:
The authors present a study that evaluates the role of inducible NO synthase
(iNOS) in the development and severity of NAFLD, taking into consideration that
its deficiency was shown to be protective from diet-induced metabolic syndrome,
but it has been associated to higher fibrosis in other animal models of liver
disease. They fed WT and iNOS KO mice with regular high fat diet (not western
diet) for 10 and 48 weeks and found a decrease in liver steatosis but an increase
in fibrosis and inflammation. They also showed activation of NFkB pathway and,
when treating the KO mice with a proteasome inhibitor (epoxomicin), which also
inhibits NFkB (decreases the degradation of its inhibitor), they found a decrease
in gene expression of several pro-inflammatory and pro-fibrogenic cytokines.
The concept is interesting and it is well documented. I have however some
considerations:
Major points:
1. There should be a discussion on the fact that KO mice have more
injury/fibrosis despite the fact that they have less hepatic steatosis. I do not agree
with the observation that it could be burnout NASH with loss of steatosis in the
sequence of advanced liver disease, since at 10 weeks of diet already less
steatosis was found. Fat accumulation in the liver may in fact be a protective
mechanism, with detoxification of biologically active lipids in the inert
triglycerides. In fact, KO mice had less triglycerides in the liver, but had more free
fatty acids in the liver and serum! Also, I would like the authors to comment the
downregulation of SREBP1c, since it has been shown by several other authors to
be upregulated in the liver in animals fed with high fat diet (mimicking what
happens with human obesity-associated NAFLD). It would also be interesting to
comment the discrepancy between insulin resistance and NASH in animal
models.
2. Another major point relates to the experiment with epoxomicin. First it was not
specified how many mice were used. Were those mice accounted when they
contabilized the animals used in the high fat diet experiment. Also, clearly this
agent is not specific for NFkB and, in fact, deregulation of proteasome has been
described in NASH. In that way the authors cannot deduce causality, they can
only say that treatment associated with decrease in NFkB nuclear staining, which
associated with decrease in those cytokines. Could not be done a more careful
analysis, at least in terms of fibrosis and steatosis in those treated animals? Also,
what happens in WT mice fed HFD and treated with epoxomicin? It would be an
important control, since the effect of drug may be independent of iNOS
deficiency.
3. Still regarding NFkB, the authors stated that activation of NFkB occurred in
hepatocytes, hepatic stellate cells and Kupffer cells, however they did not show
evidence of that. I think that to say that, the authors should do double staining, at
least for desmin and a macrophage marker.
Minor point:
4. A minor question – the magnitude of oil red images does not seem the same
between groups, mainly between KO and WT fed 48 weeks.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Acceptable
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:
I have no competing interests.
assistant