Review for 10.1186/s12876-015-0298-y (2015)

Reviewer's report
Title:Differential hepatotoxicity of dietary and DNL-derived palmitate in the
methionine-choline-deficient model of steatohepatitis
Version:2Date:19 March 2015
Reviewer: Joern M. Schattenberg
Reviewer's report:
The authors address an important issue – the role of saturated fatty acids derived
from the diet or de-novo lipogenesis in the pathogenesis of NASH. The study of
Pierce et al. builds directly on two earlier studies, in which they have shown that
enriching the diet with simple sugar enhances steatohepatitis, whereas
substituting dietary sugar with complex carbohydrate reduces liver injury
(Pickens et al., 2009 and 2010). The question of the present study is well
defined, and as a whole contemplated, this manuscript including the single parts
of the text (title, abstract, background, methods, results/discussion and
conclusion) and figures is proficient, perfectly understandable and clearly
structured. The presented data are sound, and well discussed. Nevertheless
there are small points of criticism and suggestions for revision. Above all, I would
suggest few additional experiments in order to explain the different effects of
dietary and DNL-derived saturated fatty acids on liver cells/hepatocytes and to
provide mechanistic insights into their metabolic impacts in-vivo. Major
comments are listed below.
Major comments
Pierce et al. convincingly demonstrate by appropriate methods (histological
stainings, triglyceride and fatty acid analysis) that feeding of MCD diets
containing sucrose induced the most pronounced hepatic steatosis after 21 days
regardless of the accompanying type of dietary fat. The combination of sucrose
and palmitate caused worst steatosis, although all mice fed MCD diets lost
comparable amounts of body weight regardless of the macronutrient composition
of the diet (Figure 1 and 2). As theses weight changes occur universally and are
not related to the mechanism observed, this information does not add meaningful
and can be omitted.
Moreover, liver injury and accompanying infiltration of inflammatory CD11b+
leukocytes correlated positively with the degree of hepatic lipid accumulation as
shown by well done TUNEL and CD11b stainings, serum ALT measurements
and qRT-PCR analyses (Figure 3).
At this point it is essential to note, that CD11b is mainly expressed on F4/80+
Kupffer cells/macrophages and infiltrating monocytes, which are known to trigger
NASH via secretion of cytokines and chemokines including TNF-#, CCL2,
CXCL2 and CXCL10. Therefore, the authors should also investigate the hepatic
mRNA expression of the key cytokine TNF-#. Are there differences concerning
the activation of caspases and MAP kinases, esp. JNK? Did the different MCD
formulas affect other metabolic parameters of the mice (e.g. glucose levels)?
Furthermore Pierce et al. show, that feeding of MCD diets containing sucrose
and/or palmitate caused hepatic palmitate accumulation - preferentially in hepatic
triglycerides -, which seemed to correlate positively with the degree of liver injury
(Figure 4). However, mice fed MCD sucrose-oleate accumulated no more
palmitate than those fed MCD starch-palmitate, although their ALT levels were
significantly higher, suggesting that palmitate arising from sucrose in the MCD
diet (DNL palmitate) is more hepatotoxic than palmitate itself. Interestingly, the
combined feeding of sucrose and palmitate induced more hepatic palmitate
accumulation and higher ALT levels than would have been predicted by a mere
additive effect of both nutrients. Therefore the authors conclude that dietary
sucrose - through DNL conversion to palmitate in the liver - (DNL palmitate) is an
important inducer of liver injury in the MCD model and more hepatotoxic than
dietary palmitate, which becomes toxic only in combination with dietary sugar.
This part is most interesting and I would suggest some further mechanistic
analysis as detailed above. Are injurious pathways differentially activated? Does
lipoautophagy contribute to the observed differences? These findings also
suggests different metabolic fates for DNL vs. exogenous palmitate. Are key
enzymes of the hepatic de novo lipogenesis and their regulators regulated
differentially in the experimental groups? Are important transcription factors (e.g.
PPARs) differently regulated?
Minor essential revisions:
- Please add scale bars to all microscopic images (Figure 2A and 3A).
- How did the authors assess TUNEL+ and CD11b+ cells in figures 3B and D).
Please include this information in the Methods/figure legend
Level of interest: An article of outstanding merit and interest 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