LCHP Diet Enriched with Cholesterol Promotes Non-Alcoholic Fatty Liver Disease in Wistar Rats
Round 1
Reviewer 1 Report
In the manuscript entitled "LCHP diet enriched with cholesterol promotes non-alcoholic fatty liver disease in Wistar rats", the authors investigated how low carbohydrate high protein diet enriched with cholesterol (LCHPch) affects the health of rats. Through the study, they found LCHPch diets impaired liver function, kidney weight and significantly changed fatty acid profile. These data are original, however, I have some minor concerns regarding current manuscript:
1) For the three different diets, how did you decide the cdifferent concentrations of corn starch, caseine, sucrose and fat?
2) Both the text Table 3 mentioned that LCHPch diet increased kidney weight. Is it functional hypertrophy? Can you add HE histological results?
3) What do the marks "a" and "b" extactly mean in the tables?
4) Add SD bars to the data in Figure 1.
5) Correct the writing of numbers in Figure 2, eg, should be "50.0" other than "50,0".
6) Please add what kind of staining it is to the figure legend in Figure 5.
7) The Materials and Methods part mentioned "The ratio ORO area to tissue area [%] counted in 20x fields was calculated and compared to steatosis grades where < 5% means 0 (none), whereas 5-33% means 1 (mild), 34-66% means 2 (moderate) and > 66% means 3 (severe)." Where are the results? Can you add?
8) What concentration did you use for ORO? Please clarify.
Author Response
Response to Reviewer 1 Comments
Point 1: “For the three different diets, how did you decide the different concentrations of corn starch, caseine, sucrose and fat?”
The experimental diets were based on AIN-93G diets (Reeves et al. 1993). The semisynthetic AIN-93G diet served as a ”positive control” diet consists of 7% fat and 20% of protein. Western diet as a “negative control” contains 21% fat and 20% protein. In turn, LCHP diet contained 21% fat and 52.4% protein. We used cornstarch to balance the diets. Cholesterol (1%) was added to all diets. The compositions of the experimental WD and LCHP diets were based on Foo et al. 2009.
Foo, S.Y.; Heller, E.R.; Wykrzykowska, J.; Sullivan, C.J.; Manning-Tobin, J.J.; Moore, K.J.; Gerszten, R.E.; Rosenzweig, A. Vascular effects of a low-carbohydrate high-protein diet. Proc Natl Acad Sci USA 2009, 8, 106, (36): 15418-1523.
Point 2: “Both the text Table 3 mentioned that LCHPch diet increased kidney weight. Is it functional hypertrophy? Can you add HE histological results?”
Thank you for this suggestion. However, we have not the HE histological staining of kidneys. Despite the increased kidney weight in LCHPch group there was no significant difference between groups in the amount of fat in kidneys. The average values were as follows: AINch 7.2 ± 2.7 [%], WDch 8.5 ± 2.9 [%], LCHPch 6.9 ± 3.3 [%]. Therefore, the increased kidney weight is not the result of the fat concentration.
Point 3: “What do the marks "a" and "b" extactly mean in the tables?”
The means marked with letters “a” and “b” (in each line) are significantly different (p<0.05).
Point 4: “Add SD bars to the data in Figure 1”
According to Reviewer’s suggestion we added SD of each value in Figure 1. However, in our opinion the figure has become unreadable (see below, please).
Point 5: “Correct the writing of numbers in Figure 2, eg, should be "50.0" other than "50,0".
Thank you very much for the comment. As required we corrected the Figure 2.
Point 6: “Please add what kind of staining it is to the figure legend in Figure 5”.
Thank you very much for the comment. As required we completed the information about ORO staining in Figure 5 legend.
Point 7: “The Materials and Methods part mentioned "The ratio ORO area to tissue area [%] counted in 20x fields was calculated and compared to steatosis grades where < 5% means 0 (none), whereas 5-33% means 1 (mild), 34-66% means 2 (moderate) and > 66% means 3 (severe)." Where are the results? Can you add?”
The ratio ORO area to tissue area [%] counted in 20x fields results was shown in the text of Results section. According to Reviewer suggestion we added this results as a Figure 6.
Point 8: “What concentration did you use for ORO? Please clarify”.
To stain liver’s sections we mixed 24 ml of saturated solution of ORO with 16 ml of distilled water. As required we added this information to the manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
LCHP diet is known to have diverse effect from metabolism perspective. The rationale for the study makes sense, however, there is not enough scientific rigor in the experimental design and results illustrated. The sample size is too less and probably we can trying to read too much into the cohort of 18 mice. I would extending the experimental cohort and extend the experimental study design which would come across as a more promising study.
Author Response
Response to Reviewer 2 Comments
“LCHP diet is known to have diverse effect from metabolism perspective. The rationale for the study makes sense, however, there is not enough scientific rigor in the experimental design and results illustrated. The sample size is too less and probably we can trying to read too much into the cohort of 18 mice. I would extending the experimental cohort and extend the experimental study design which would come across as a more promising study”.
Thank you very much for the Reviewer 2 Comments and statement that our study makes sense. We checked and improved results illustration (Figure 1). Additionally, Figure 6 presented the ratio of ORO staining/tissue area [%] of liver has been added. Generally, in nutritional studies 6-12 rats are used. A wide analysis of research reported in the literature, and the knowledge of ethical rules allowed to design our experiment with respect to “3R” rules, where “reduce” means responsibility for considering reducing the number of animals. It is also to be mentioned that our experimental design, including the number of animals in each group (n=6) was approved by the Local Ethics Committee. The decision of the Local Ethics Committee is based on the detailed analysis of research leaders and professionals who pay particular attention to the “3R” rules. Taking the above into consideration, we decided to used 18 rats in our cohort as a sufficient amount of animals to test our hypothesis. In that cohort we received the statistical significant results.
Moreover, we would like to add that our aim was not to “read too much into the cohort of 18 mice”. It was an attempt to obtain a wider view of the question concerned on possible benefits and risks of LCHP diet. We also realize that further studies in this important area are strongly needed.
Author Response File: Author Response.pdf
