Review for 10.1186/s12876-015-0269-3 (2015)

Reviewer's report
Title:Deficiency of iNOS-derived NO accelerates lipid accumulation-independent
liver fibrosis in non-alcoholic steatohepatitis mouse model
Version:2Date:16 September 2014
Reviewer: Manlio Vinciguerra
Reviewer's report:
This interesting manuscript reports an accelerated fibrosis and inflammation
(NASH) in the liver of mice devoid of iNOS, without an increased lipid
accumulation (NAFL), compared to normal mice. The model used is a long term
high fat diet treatment (up to 48 weeks = nearly one year). The group is
experienced in the field.
Except the final set of experiments, the injection of NF-kB inhibitor to monitor the
gene expression of inflammatory molecules, which is not informative for the
phenotype, the study is well designed and solid. It contributes to a highly debated
area, i.e. the role on NO in NAFLD/NASH progression. The data are convincing.
Major compulsory revision
The study fails to integrate/discuss recent advancements on NO research in
NAFLD/NASH and the role of aging that, given the long term HFD model
proposed, is compelling. I suggest a revision where the authors suppress not
informative experiments, and integrate the data in a bigger frame, considering
also new findings and the role of aging. In other words, the manuscript would
need to be largely re-written starting with the Introduction without necessarily
doing new experiments. In details:
1) Gu et al. recently demonstrated by knock-out and knock-down approaches the
existence of a nitric oxide (NO)-signaling pathway that is crucial for the control of
hepatic fatty acid oxidation in zebrafish, in mice and in cultured human
hepatocytes, which is in turn under the control of SLC7A3, an essential
membrane transporter for cationic amino acids such as arginine (Qilin Gu, Xiaojie
Yang, Li Lin, Shaoyang Li, Qing Li, Shan Zhong, Jinrong Peng and Zongbin Cui.
Genetic ablation of solute carrier family 7a3a leads to hepatic steatosis in
zebrafish during fasting. Hepatology 2014). However, the role of NO as a
protector from NAFLD is debatable. It has been reported that an increase, rather
than a decrease, of NO production accompanies NAFLD onset (Ibrahim M,
Farghaly E, Gomaa W, Kelleni M, Abdelrahman AM. Nitro-aspirin is a potential
therapy for non alcoholic fatty liver disease. Eur J Pharmacol 2011 Jun
1;659(2-3):289-295. ). Accordingly, in the pathogenesis of NAFLD/NASH, NO
potentiates cytotoxicity by reacting with superoxide anions to form peroxynitrite, a
strong oxidant that promotes nitration of tyrosine to form nitrotyrosine (Silibinin
modulates lipid homeostasis and inhibits nuclear factor kappa B activation in
experimental nonalcoholic steatohepatitis). This is consistent with the fact that
nitrosative stress developed upon an excess in NO content correlates with
NAFLD severity: NO-mediated nitration of hepatocellular proteins is markedly
induced in the inflamed liver tissue from patients with chronic liver disease
(Intrahepatic accumulation of nitrotyrosine in chronic viral hepatitis is associated
with histological severity of liver disease). How to reconcile the results of the
present study of decreased NAFL/increased NASH in iNOS KO mice under HFD
with the dicothomy in the literature that inhibition/depletion of intracellular NO
production and, on the opposite, enzymatic NO overproduction, could both lead
to aggravating lipid accumulation as in NAFLD? This literature must be discussed
originally in light of the data.
2) Aging. It is known that aging is associated to an increased production of NO
by iNOS (http://www.sciencedirect.com/science/article/pii/S0531556510001932).
This trend is also evident in Figure 3.3 of this work. The authors deliberately used
a long term model (48 weeks) to study NAFLD to NASH progression using a 60%
energy from fat HFD model. By the end point, animals are 1 year old, which is
equivalent to 50 human years, a middle aged men. In absence of iNOS, also the
lack of NO-associated age-dependent changes are playing a factor. To avoid
making a new study using an informative short term model of NAFLD-NASH
(such as the Stam-Stelic, MCD diet or carbon tetrachloride) the authors should
really make a point of young versus old in genereal in liver diseases and the role
of NO citing relevant literature (http://www.ncbi.nlm.nih.gov/pubmed/23815295;
http://www.sciencedirect.com/science/article/pii/S0531556510001932). NO has
been reported also to increase life span in model organisms. Do iNOS -/- live
less?
3) iNOS +/+ and iNOS -/- mice are shown to have similar body weight upon HFD
and similar adiposity. Considering that the liver is a major depot of fat during
obesity, where the fat is gone during this "burned-out NASH"? Is it all
transformed in NEFA? Did the authors perform metabolic cages experiments to
study if iNOS -/- actually move more, as it has been reported for these animals in
other contexts
(http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0010962)?
4) why NAS score the same in the two groups, despite the iNOS -/- having less
NAFL?
5) I suggest to suppress the experiments with epoxomicin. A time-limited
reduction in inflammatory gene expression is not very informative. It would be
informative a regression of NASH with systemic prolonged administration of
epoxomicin or a NF-kB inhibitor, which has not been done.
Minor
Figure 6: Scheme, not Schema.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests