Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced MASLD
and Vanusa Manfredini 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThank you for the opportunity to review the manuscript entitled “Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced NAFLD.”
This study investigates the dose-dependent effects of vitamin D supplementation on biochemical, inflammatory, and antioxidant markers, as well as liver histology, using an animal model comprising 60 Wistar rats equally divided by sex. The topic is of interest and potential translational value.
The Introduction is well structured, provides appropriate context, and effectively guides the reader into the subsequent sections of the manuscript. I have no major concerns regarding this part.
The Methods are described clearly and allow replication by other research groups. However, I have some reservations concerning the anesthetic protocol. The use of ketamine and xylazine, although applied uniformly to all animals, may potentially influence certain laboratory parameters. It would therefore be important for the authors to clarify whether this could have affected the results, ideally supported by references to prior studies demonstrating the absence of such an effect.
The Results are presented in a clear and detailed manner, which deserves recognition. The Conclusions are consistent with the data and appropriately formulated.
The Discussion, while somewhat lengthy, is comprehensive and not excessively repetitive. It addresses the main aspects of the findings. My only suggestion would be that, given the use of the clinically outdated concept of NAFLD, the authors might also consider referring to the more recent definitions MAFLD and MASLD, or alternatively, employ a more neutral term such as “hepatic steatosis” throughout the manuscript.
In conclusion, this is an interesting and well-conducted study. I would be pleased to review a revised version following the authors’ response.
Author Response
# Reviewer 1
Thank you for the opportunity to review the manuscript entitled “Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced NAFLD.”
This study investigates the dose-dependent effects of vitamin D supplementation on biochemical, inflammatory, and antioxidant markers, as well as liver histology, using an animal model comprising 60 Wistar rats equally divided by sex. The topic is of interest and potential translational value.
The Introduction is well structured, provides appropriate context, and effectively guides the reader into the subsequent sections of the manuscript. I have no major concerns regarding this part.
Thank you for your kind words and for taking the time to review our manuscript. We are very pleased that you found the topic relevant and the Introduction well structured. Your positive feedback motivates us to further improve the quality of our work.
The Methods are described clearly and allow replication by other research groups. However, I have some reservations concerning the anesthetic protocol. The use of ketamine and xylazine, although applied uniformly to all animals, may potentially influence certain laboratory parameters. It would therefore be important for the authors to clarify whether this could have affected the results, ideally supported by references to prior studies demonstrating the absence of such an effect.
Xylazine and ketamine are widely used for analgesia in research because they are considered safe and produce only minimal, transient cardiopulmonary effects in animal models. In Wistar rats, the intraperitoneal administration of these anesthetics is calculated based on actual body weight, which allows access to the cardiac chamber without causing harm to the animal and enables the collection of peripheral blood samples. Following the reviewer’s comments, we searched the literature for possible clinical and physiological changes associated with the use of xylazine and ketamine. We found no evidence indicating alterations in biochemical, inflammatory, or oxidative stress parameters related to their use.
The Results are presented in a clear and detailed manner, which deserves recognition. The Conclusions are consistent with the data and appropriately formulated.
We also appreciate your recognition of the clarity and detail in the Results, as well as your assessment that the Conclusions are consistent with the data. Such observations are very encouraging for our team.
The Discussion, while somewhat lengthy, is comprehensive and not excessively repetitive. It addresses the main aspects of the findings. My only suggestion would be that, given the use of the clinically outdated concept of NAFLD, the authors might also consider referring to the more recent definitions MAFLD and MASLD, or alternatively, employ a more neutral term such as “hepatic steatosis” throughout the manuscript.
Thank you for your comment. We have replaced the term NAFLD with MASLD throughout the manuscript.
In conclusion, this is an interesting and well-conducted study. I would be pleased to review a revised version following the authors’ response.
Thank you very much for your comments and positive feedback. We have carefully addressed all suggestions and highlighted them in the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript “ Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced NAFLD” by Silvia Muller de Moura Sarmento et al. analyzed the potential hepatoprotective effects of vitamin D in rats with MASLD.
Please replace NAFLD by MASLD and do not use abbreviations in the title and the abstract (ALT etc.).
“total antioxidant status (TAS)” this explanation is unnecessary in the abstract.
Line 54 – 56. Please add references.
The Introduction has to describe recent studies related to the current research project.
40,576,000 IU/g, NAFDL, with VD supplementation - please correct.
Please define all abbreviations before first use.
Figure 1, please use upper case letters for the labels. Leptin needs not to be abbreviated. Clarify ***p < 0.001; ***p < 0.0001. ? Explain abbreviations in the figure legend. Leptin and adiponectin levels should be given in ng or µg per mL.
TNF-alpha should be TNF.
What was the vitamin D level of rats with and without NAFLD. Was there a difference between males and females? What was the vitamin D levels at the end of this study for both sexes
C-reactive protein (C-RP), usually CRP is used.
The vitamin D doses used seem to be much higher than doses used in human, please explain and discuss.
It seems that significance levels are not included in e.g. figure 3a2.
Figure 4, the x-axis label has no unit. Analysis of vitamin D is not explained in more detail in the text. Here, CRP is correct.
Carbonyl and CAR are used, please decide for one abbreviation.
“Figure 5d-e, an increased degree of hepatic fibrosis was evident “ how was this detected?
“In this experimental model, oral vitamin D was administered at doses of 2,500 IU, 289 7,000 IU, 14,000 IU, and 21,000 IU, resulting in serum 25(OH)D concentrations of 152.8 290 ng/mL, 175.6 ng/mL, 193.6 ng/mL, and 275.5 ng/mL, respectively. The „ this should be included in results and has to be given for female and male rats. Please also include vitamin D levels of control rats.
“supplementation with 2,000 IU of vitamin D “ per day?
“The association between the lipid profile and vitamin D may be related to the hormone’s role in lipid metabolism and synthesis “ please describe this in more detail.
Please provide the body weights of the animals.
“C-RP in peripheral blood and adipose tissue, “ is mostly derived from the liver, please correct.
“vitamin D (2,000 IU/day) and vitamin E (0.9 g/day)” per kg?
As sex-specific studies are rare the authors should compare the levels of all of their meatabolite between male and female rats. Data should be provided as a supplementary table.
Author Response
# Reviewer 2
The manuscript “Effect of Oral Vitamin D Overdose in Male and Female Wistar Rats with Induced NAFLD” by Silvia Muller de Moura Sarmento et al. analyzed the potential hepatoprotective effects of vitamin D in rats with MASLD.
Please replace NAFLD by MASLD and do not use abbreviations in the title and the abstract (ALT etc.).
“total antioxidant status (TAS)” this explanation is unnecessary in the abstract.
Thank you for your comment. We have replaced the term NAFLD with MASLD throughout the manuscript and have avoided using these abbreviations in the abstract.
Line 54 – 56. Please add references.
Thank you for your comment. We have now cited two new references to better support the relationship between MASLD and vitamin D.
The Introduction has to describe recent studies related to the current research project.
Thank you for your comment. Recent references were added to the introduction.
40,576,000 IU/g, NAFDL, with VD supplementation - please correct.
Thank you for your comment. The correction has been made
Please define all abbreviations before first use.
Thank you for your comment. We revised the use of abbreviations.
Figure 1, please use upper case letters for the labels. Leptin needs not to be abbreviated. Clarify ***p < 0.001; ***p < 0.0001.? Explain abbreviations in the figure legend. Leptin and adiponectin levels should be given in ng or µg per mL.
Thank you for your comment. The correction has been made in Figure 1, and the abbreviations have been added to the legend. Please see that in the legend of all the images was reported: Asterisks indicate statistically significant differences compared to the control group (*p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001), so that all the comparisons were performed against the group that no received vitamin D.
TNF-alpha should be TNF.
Thank you for your comment. "TNF-alpha" has been changed to "TNF" throughout the entire manuscript, including the text and figures.
What was the vitamin D level of rats with and without NAFLD. Was there a difference between males and females? What was the vitamin D levels at the end of this study for both sexes
Thank you for your comment. In this experimental model, rats began the MASLD protocol at 60 days of age. No manipulations were performed prior to this process, as such interventions could stress the animals and compromise the induction of hepatic steatosis. Regarding the differences between males and females shown in Figures, these findings are unprecedented, which is why we analyzed the data separately by sex. In other words, the physiological differences between male and female rats may represent a source of variability, which we accounted for in our analysis.
Please note that in many cases, the male and female rats presented very similar profiles. As a result, when the data were plotted together, the profiles overlapped significantly. Initially, we attempted to plot the male and female data together, but for the reason stated above, we chose to present them separately.
Furthermore, adding comparisons with the start of the experiment and between genders would increase the complexity of interpretation and worsen the clarity of the graphs. This would provide minimal benefit for very limited results (i.e., glucose levels), as these comparisons fall outside the main focus of our investigation.
We would also like to highlight that implementing this change would require extensive modifications to our manuscript, necessitating revisions to the objectives, discussion, and results sections.
Please note that all data used in the manuscript have been shared as electronic supporting material. If readers are interested in further examining these differences for the small number of markers where they may be present, they can do so using our original data.
C-reactive protein (C-RP), usually CRP is used.
Thank you for your comment. "C-RP" has been changed to "CRP" throughout the entire manuscript, including the text and figures.
The vitamin D doses used seem to be much higher than doses used in human, please explain and discuss.
Thank you for your comment. Regarding the dose selection, we based our considerations on the LD50 of vitamin D in rats, which is 300 µg/kg/day (12.000 IU/kg/day). This information can be verified in the reference: SJÖDEN, Göran et al. 1α-Hydroxyvitamin D2 is less toxic than 1α-hydroxyvitamin D3 in the rat. Proceedings of the Society for Experimental Biology and Medicine, 178(3), 432–436, 1985. While our doses are higher than the clinical doses used in humans, the focus of this investigation was to explore the physiological effects of vitamin D overdose.
It seems that significance levels are not included in e.g. figure 3a2.
Thank you for your comment. All the Figures were carefully reviewed and the significance levels were added when missing.
Figure 4, the x-axis label has no unit. Analysis of vitamin D is not explained in more detail in the text. Here, CRP is correct.
Thank you for your comment. Please note that the figure legend contains the text: "The x-axis in each plot represents the vitamin D concentration." For aesthetic reasons and to make better use of the journal's page layout, we chose not to repeat the same x-axis label on all 23 individual graphs.
Carbonyl and CAR are used, please decide for one abbreviation.
Thank you for your comment. The use of abbreviation was standardized.
“Figure 5d-e, an increased degree of hepatic fibrosis was evident “how was this detected?
Thank you for your comment. We have revised the text to clarify that our histological slides are suggestive of hepatic fibrosis, rather than providing a definitive diagnosis.
In this experimental model, oral vitamin D was administered at doses of 2,500 IU, 289 7,000 IU, 14,000 IU, and 21,000 IU, resulting in serum 25(OH)D concentrations of 152.8 290 ng/mL, 175.6 ng/mL, 193.6 ng/mL, and 275.5 ng/mL, respectively. This should be included in results and has to be given for female and male rats. Please also include vitamin D levels of control rats.
Thank you for your comment. The baseline vitamin D values have been added to the text and are now highlighted
“supplementation with 2,000 IU of vitamin D “per day?
Thank you for your comment. The missing details have been added to the text.
“The association between the lipid profile and vitamin D may be related to the hormone’s role in lipid metabolism and synthesis “please describe this in more detail.
Thank you for your comment. We have revised the text to provide a more detailed explanation.
Please provide the body weights of the animals.
Thank you for your comment. The weight of the animals was monitored; however, as no significant differences were observed among the groups, we decided not to include this information in the manuscript in order to optimize the use of space within the journal’s page.
“C-RP in peripheral blood and adipose tissue, “is mostly derived from the liver, please correct.
Thank you for your comment. The sentence was removed.
“vitamin D (2,000 IU/day) and vitamin E (0.9 g/day)” per kg?
Thank you for your comment, the suggestion was considered. The unit was changed by “g/day/kg of diet”.
As sex-specific studies are rare the authors should compare the levels of all of their meatabolite between male and female rats. Data should be provided as a supplementary table.
Thank you for your comment. Please note that in many cases, the male and female rats presented very similar profiles. As a result, when the data were plotted together, the profiles overlapped significantly. Initially, we attempted to plot the male and female data together, but for the reason stated above, we chose to present them separately.
Furthermore, adding comparisons with the start of the experiment and between genders would increase the complexity of interpretation and worsen the clarity of the graphs. This would provide minimal benefit for very limited results (i.e., glucose levels), as these comparisons fall outside the main focus of our investigation.
We would also like to highlight that implementing this change would require extensive modifications to our manuscript, necessitating revisions to the objectives, discussion, and results sections.
Please note that all data used in the manuscript have been shared as electronic supporting material. If readers are interested in further examining these differences for the small number of markers where they may be present, they can do so using our original data.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn the present work, the authors evaluate the role of different doses of vitamin D supplementation in rats with non-alcoholic fatty liver disease (NAFLD). In general terms, the paper is interesting and provides further insights into the central role of vitamin D in the treatment of this condition. However, i have some issues that should be addressed: 1) In the discussion section, I missed a further explanation about the possible pathogenic mechanisms linking high vitamin D levels with NAFLD progression. What is currently known in the available literature? 2) Histopathologically, clear differences between males and females rats with NAFLD are presented, with female rats showing hepatic fibrosis at high vitamin D doses and males showing enhanced fat accumulation. However, in the discussion section, no explanations are given to these differences. Please, provide a possible hypothesis explaining these changes. 3) What are the translational applications and future paths to cover in humans derived from the results in this work? Emphasize it in the discussion section please 4) Finally, a section discussing strengths and limitations should be included at the end of the discussion.
Minor:
1) For improve the quality of the presentation, i think it should be better to compare each cytokine in males and females rats instead of separating all cytokines by sex (Place together IL-1beta in female with IL-1beta in male)
Author Response
# Reviewer 3
In the present work, the authors evaluate the role of different doses of vitamin D supplementation in rats with non-alcoholic fatty liver disease (NAFLD). In general terms, the paper is interesting and provides further insights into the central role of vitamin D in the treatment of this condition. However, i have some issues that should be addressed:
1) In the discussion section, I missed a further explanation about the possible pathogenic mechanisms linking high vitamin D levels with NAFLD progression. What is currently known in the available literature?
Thank you for your comment. These aspects were better explored in our discussion.
2) Histopathologically, clear differences between males and females rats with NAFLD are presented, with female rats showing hepatic fibrosis at high vitamin D doses and males showing enhanced fat accumulation. However, in the discussion section, no explanations are given to these differences. Please, provide a possible hypothesis explaining these changes.
Thank you for your comment. These findings were unexpected and will be further investigated by our group in order to achieve a better understanding of the results.
3) What are the translational applications and future paths to cover in humans derived from the results in this work? Emphasize it in the discussion section please
Regarding the dose selection, we based our considerations on the LD50 of vitamin D in rats, which is 300 µg/kg/day (12.000 IU/kg/day). This information can be verified in the reference: SJÖDEN, Göran et al. 1α-Hydroxyvitamin D2 is less toxic than 1α-hydroxyvitamin D3 in the rat. Proceedings of the Society for Experimental Biology and Medicine, 178(3), 432–436, 1985. While our doses are higher than the clinical doses used in humans, the focus of this investigation was to explore the physiological effects of vitamin D overdose.
4) Finally, a section discussing strengths and limitations should be included at the end of the discussion.
Thank you for your comment. As suggested, we have added a section addressing the strengths and limitations of the study at the end of the Discussion.
Minor:
1) For improve the quality of the presentation, i think it should be better to compare each cytokine in males and females rats instead of separating all cytokines by sex (Place together IL-1beta in female with IL-1beta in male)
Thank you for your comment. Please note that in many cases, the male and female rats presented very similar profiles. As a result, when the data were plotted together, the profiles overlapped significantly. Initially, we attempted to plot the male and female data together, but for the reason stated above, we chose to present them separately.
Furthermore, adding comparisons with the start of the experiment and between genders would increase the complexity of interpretation and worsen the clarity of the graphs. This would provide minimal benefit for very limited results (i.e., glucose levels), as these comparisons fall outside the main focus of our investigation.
We would also like to highlight that implementing this change would require extensive modifications to our manuscript, necessitating revisions to the objectives, discussion, and results sections.
Please note that all data used in the manuscript have been shared as electronic supporting material. If readers are interested in further examining these differences for the small number of markers where they may be present, they can do so using our original data.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have responded correctly to the previous comments - I have no additional comments.
Author Response
We thank the reviewer for their final approval of our manuscript. We are pleased that the revisions have addressed all previous comments satisfactorily.
Reviewer 2 Report
Comments and Suggestions for AuthorsRecently, a comprehensive study reported the role of vitamin D overdose in rats; 67 however, the effects of these doses in MASLD models are not yet available in the literature“ What does this mean?
Comment: The Introduction has to describe recent studies related to the current research project. Answer: Recent references were added to the introduction. But the authors added only 1 study, I think there are more.
“(*p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001“ please correct
In the manuscript “Data will be made available on request.” And “Please note that all data used in the manuscript have been shared as electronic supporting material. “ as response to my comments, please clarify. If there are supplementary data, this has to be clear from the manuscript.
Author Response
Recently, a comprehensive study reported the role of vitamin D overdose in rats; 67 however, the effects of these doses in MASLD models are not yet available in the literature “ What does this mean?
Thank you for your comment. We have revised the sentence for greater clarity and precision, explicitly stating the dose range to eliminate any ambiguity. The text now clearly indicates that the effects of these specific doses (2,500 to 21,000 IU/kg/week) in MASLD models are unknown.
Comment: The Introduction has to describe recent studies related to the current research project. Answer: Recent references were added to the introduction. But the authors added only 1 study, I think there are more.
Thank you for your comment. We have added recent references to the introduction.
“(*p < 0.05; **p < 0.01; ***p < 0.001; ***p < 0.0001“ please correct
Thank you for your comment. The correction was performed.
In the manuscript “Data will be made available on request.” And “Please note that all data used in the manuscript have been shared as electronic supporting material. “as response to my comments, please clarify. If there are supplementary data, this has to be clear from the manuscript.
Thank you for your comment. The sentence “All data generated or analyzed during this study are included in this published article and its supplementary information files” was added to the availability statement.
Reviewer 3 Report
Comments and Suggestions for AuthorsAccept in present form.
Author Response
We thank the reviewer for their final approval of our manuscript. We are pleased that the revisions have addressed all previous comments satisfactorily.
