Pachyrhizus erosus Inhibits Adipogenesis via the Leptin-PPARγ-FAS Pathway in a High-Fat Diet-Induced Mouse Model

Round 1

Reviewer 1 Report (New Reviewer)

Blood chemistry analysis methods should be explained in more detailed way and conclusions presented at the end of the paper. 

Author Response

Blood chemistry analysis methods should be explained in more detailed way and conclusions presented at the end of the paper. 

Ans) Thank you so much for generous comments and according to the comments we amended the manuscript.

Reviewer 2 Report (New Reviewer)

Comments to the Author

 

The authors verified the potency of P. erosus as an AOM and aimed to discover the potential mechanism. It is an interesting study but there are problems need to be revised. The manuscript submitted by the authors is an obvious draft and needs substantial revision to improve quality and readability. Specific comments and questions are listed below.

 

Major concerns

1. The authors should seek for help of a native speaker to revise language and grammar. 

2. The authors indicated that P. erosus inhibited the adipogenesis-related markers such as leptin level, expressions of C/EBPα and PPARγ. Leptin is known as an adipocytokine, so how does P. erosus regulate the leptin level?  

3. The mice were supplied with 250 mg/kg/day P. erosus for treatment. Why did authors use this dosage? 

 

Minor concerns

1. The formation of manuscript looked a mess, such as the different line spacing, the remaining correcting trace. And the tables were embedded in pictures without headings.

2. P7 L241 The gene name should be 'PPARγ', not 'PPRγ'.

Author Response

The authors verified the potency of P. erosus as an AOM and aimed to discover the potential mechanism. It is an interesting study but there are problems need to be revised. The manuscript submitted by the authors is an obvious draft and needs substantial revision to improve quality and readability. Specific comments and questions are listed below.

 

Major concerns

1. The authors should seek for help of a native speaker to revise language and grammar. 

Ans) Thank you so much and I amended the manuscript via MDPI.

 

2. The authors indicated that P. erosusinhibited the adipogenesis-related markers such as leptin level, expressions of C/EBPα and PPARγ. Leptin is known as an adipocytokine, so how does P. erosusregulate the leptin level?  

Ans) Thank you so much for informative comments. Leptin is one of the important energy balance regulators and obese adipocytes increase the releasing level of leptin (Farooqi, I.S. Defining the neural basis of appetite and obesity: from genes to behaviour. Clin Med 2014, 14 (3), 286-289.). I guessed that P. erosus directly controlled the releasing level of leptin. Although P. erosus inhibited fat tissue increment and in this study we didn’t find the regulation mechanism of leptin level but showed only the down-regulated level of leptin. And then we suggested the down-regulated C/EBPa and PPARγ through the decreased leptin level. That meant that although we couldn’t find the controlling cause/pathway of leptin level we could suggest only the down-regulation pathway of C/EBPa - PPARγ-mediated adipogenesis via leptin and the modulation pathway of C/EBPα - PPARγ via leptin has been repoted (Palhinha, L.; Liechocki, S.; Hottz, E.D.; Pereira J.A.da.S.; de Almeida, C.J.; Moraes-Vieira, P.M.M.; Bozza, P.T.; Maya-Monteiro, C.M. Leptin induces proadipogenic and proinflammatory signaling in adipocytes. Front Endocrinol. 2019, 10, 841.).

 

3. The mice were supplied with 250 mg/kg/day P. erosusfor treatment. Why did authors use this dosage? 

 Ans) Thank you so much for informative comments. As the reviewer knows although it is different between human and mouse we should conduct experiment using with animals without human because of the latent toxicity of candidates. And then based on the interspecific diversity the result should be translated (extrapolated) using with the Adjustment Factor and that of mouse is 13.3 (Baird, S.J.S.; Cohen, J.T., Graham, J.D. Noncancer risk assessment: a probabilistic alternative to current practice. Hum Ecol Risk Assess. 1996, 2 (1), 79-102.). We think that when the natural product-extracted functional foods could be applied on the human with 2.5 mg (ordinary dose) but in this study we applied the half of ordinary dose, 250 mg/kg/day for mouse.

 

 

Minor concerns

1. The formation of manuscript looked a mess, such as the different line spacing, the remaining correcting trace. And the tables were embedded in pictures without headings.

 Ans) Thank you so much for your generous comments and we resubmitted the clean version of the revised manuscript.

 

2. P7 L241 The gene name should be 'PPARγ', not 'PPRγ'.

 Ans) We amended the word which was just typo.

Round 2

Reviewer 2 Report (New Reviewer)

The authors improved the manuscript largely, but there remains questions needed to be solved.

 

1. The authors thought that when the natural product-extracted functional foods could be applied on the human with 2.5 mg, but in this study they applied 250 mg/kg/day for mouse. The dosage they used looks like not well-grounded.

 

2. All the tables were embedded in pictures without headings. They should be displayed as independent files, and the headings are needed.

Author Response

The authors improved the manuscript largely, but there remains questions needed to be solved.

 

1. The authors thought that when the natural product-extracted functional foods could be applied on the human with 2.5 mg, but in this study they applied 250 mg/kg/day for mouse. The dosage they used looks like not well-grounded.

Ans) Thank you so much for the question but I mistook the answer about that. According to the U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research (CDER) (U.S. Department of Health and Human Services Food and Drug Administration, Center for Drug Evaluation and Research, 2005. Guidance for industry, estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers.) the human equivalent dose (HED) of mouse is 12.3 the used dose was 250 mg/kg/day and then the human dose can be converted below.

 

 250 (mouse dose)/ 12.3 (HED) ´ 65 (adult bodyweight) = 1,219 mg = 1.2 g.

 

We are so much sorry that we made you be confused.

 

 

2. All the tables were embedded in pictures without headings. They should be displayed as independent files, and the headings are needed.

Ans) Thank you so much for the generous comments. When we described the manuscript as the tables in pictures could make the readers to understand easily the Figures all the tables were put in pictures. However, it is not necessary to exist and depending on the comments I omitted all the tables in pictures.

 

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Overall, a good manuscript is submitted. The title is a subject for revision because it does not show that the experiment is with mice. The figures and tables are informative and of good quality. English is acceptable, but native speakers should revise the whole text, including the abstract where "healthy problems" should be replaced by "health problems". The methods section needs to explain which results are presented and discussed: from the initial experiment or the repeated one. The Latin name of an enzyme is not acceptable. The paper could be accepted after minor revisions.

Author Response

Reviewer #1’s comments

Overall, a good manuscript is submitted.

The title is a subject for revision because it does not show that the experiment is with mice.

Ans) Thank you so much for your generous comment and I amended the manuscript title.

 

The figures and tables are informative and of good quality. English is acceptable, but native speakers should revise the whole text, including the abstract where "healthy problems" should be replaced by "health problems".

Ans) Thank you so much for the comment and this manuscript was edited by professional company.

 

The methods section needs to explain which results are presented and discussed: from the initial experiment or the repeated one.

Ans) Thank you so much for the comment and added the explain in the method section.

 

The Latin name of an enzyme is not acceptable.

Ans) Thank you so much for your generous comment about the type and I corrected that.

 

The paper could be accepted after minor revisions.

Reviewer 2 Report

In the current study, the authors found that P. erosus could reduce body weight and regulate liver hepatic lipogenesis in diet-induced obese mice. Mechanistically, treatment of P. erosus could inhibit both C/EBP level and PPAR level to reduce FAS expression. The studies were well designed and performed, I do have some concerns:

1. Did  P. erosus affect the food intake in mice?

2. P. erosus regulated the expression of FAS, similarly, acetyl-CoA carboxylase (ACCα) is the rate-limiting enzyme catalyzing the synthesis of fatty acid, whether P. erosus influence ACC expression.

3. Which ingredient might be the main active ingredient in P. erosus according to your study?  Whether the vitro experiments were used to further verify the validity of the results?

4. Bar figures have to be changed to dot plots to show the data distribution.

Author Response

Reviewer #2’s comments

 

In the current study, the authors found that P. erosus could reduce body weight and regulate liver hepatic lipogenesis in diet-induced obese mice. Mechanistically, treatment of P. erosus could inhibit both C/EBP level and PPAR level to reduce FAS expression. The studies were well designed and performed, I do have some concerns:

1. Did  P. erosus affect the food intake in mice?

Ans) Thank you so much for your question. I described that in 2.2. Animal experiments and bodyweight measurement, “After 7 days acclimation for 56 days all mice were treated depending on each group’s treatment and P. erosus treatments (28 mL/g of body weight) were conducted during 14:00 to 16:00 using with feeding needle.”

 

2. P. erosus regulated the expression of FAS, similarly, acetyl-CoA carboxylase (ACCα) is the rate-limiting enzyme catalyzing the synthesis of fatty acid, whether P. erosus influence ACC expression.

Ans) ACC can inhibit from Acetyl-CoA to Malonyl-CoA and then can block the FAS activation (Liu T, et al., 2020, Exp Ther Med. DOI: 10.3892/etm.2020.8700). Although we didn’t define the ACC stimulation effect by P. erosus I think that the reviewer’s hypothesis can be right. I think we need to prove the relation of ACC and P. erosus’s effect.

 

3. Which ingredient might be the main active ingredient in P. erosus according to your study?  Whether the vitro experiments were used to further verify the validity of the results?

Ans) Thank you so much for your generous comment and I agree with your opinion. As I think the main active ingredient in P. erosus might an inulin and I described that in discussion part, “Although there are several anti-obesity-related reports about inulin as an active compound in P. erosus almost of them are relevant with prebiotic inulin [32, 33]. In this study we evaluated the anti-obesity effect of P. erosus extract and then we checked up the inulin content in P. erosus extract (Table 1). The inulin content per 1 mg/mL of P. erosus extract was 0.38 ± 0.029 mg/mL and in the further study the relation of this result and inulin need to be defined.”   

 

4. Bar figures have to be changed to dot plots to show the data distribution.

Ans) I appreciated the comment, but I think that in order to clearly show the results to the readers bar figures might be more useful than dot plots. Please understand my opinion.

Reviewer 3 Report

Authors attempted to stud anti-obesity potential of Pachyrhizus erosus.

Comments:

 

The manuscript requires extensive language revision for improving clarity. 

 

Section 1. Introduction pg 3: 'In this study I verified the potency of P. erosus as an AOM including assuring the anti-83 obesity mechanism of Pachyrhizus erosus. '  

It is a multi-author manuscript, then why is it stated as 'Í'?

 

Adequate reasoning for the choice of the plant, for anti-obesity testing is not provoided.

 

 

Section 2.2: 

What is the rationale for using as many as 36 rats, for only three exeprimental groups? Considering the disease model is not a cytotoxic or cancer model, where there may be chances of mortality, it is not clear why authors chose n=36.

Authors state "In order to guarantee the reproducibility of the result the 103 animal study was conducted twice." Does it mean, another 36 rats were again divided into 3 groups? 

 

2.7. "Real time-poly chain reaction (RT-PCR) analysis" --- rewrite - it should be 'Polymerase' (not poly)

 

The table in Figure 1 is hazy, cannot read the numbers / symbols clearly. Same is the case with Fig 2 table clarity.

 

In all the mean values, what is n=?

 

Figure 5D: The top 6 photomicrographs are not revealing anything clearly --- its all dark - c/EBP and PPAR - the differnces between the exeprimental groups are not clearly visible.

 

Why was the Inulin analysis done? What are its results and implications?

 

 

4. Discussion

"Obesity is not just a physiological uncomfortable symptom but as the prevalence and 262 the health risk of that faces on the disease [3]..." statement does not convey the right message.

 

"Especially, by 2030 20% of female and 14% of male could be 265 prospected to become obesity patients [5]..."  --- globally? percentage of what sample population?

 

The section '5. Conclusions'  is mentioned without any content written under that section.

 

Author Response

Reviewer #3’s comments

 

Authors attempted to stud anti-obesity potential of Pachyrhizus erosus.

Comments:

 

The manuscript requires extensive language revision for improving clarity. 

 Ans) Thank you so much for your comment but this manuscript had been already edited by professional company.

 

Section 1. Introduction pg 3: 'In this study I verified the potency of P. erosus as an AOM including assuring the anti-83 obesity mechanism of Pachyrhizus erosus. '  

It is a multi-author manuscript, then why is it stated as 'Í'?

 Ans) Thank you so much for your generous comment and we amended that.

 

Adequate reasoning for the choice of the plant, for anti-obesity testing is not provoided.

 Ans) In discussion part we described the reason of the anti-obesity testing of P. erosus and in this study we tried to define the anti-obesity mechanism of that. “Although there are several anti-obesity-related reports about inulin as an active compound in P. erosus almost of them are relevant with prebiotic inulin [32, 33]. In this study we evaluated the anti-obesity effect of P. erosus extract and then we checked up the inulin content in P. erosus extract (Table 1). The inulin content per 1 mg/mL of P. erosus extract was 0.38 ± 0.029 mg/mL and in the further study the relation of this result.”

 

Section 2.2: 

What is the rationale for using as many as 36 rats, for only three exeprimental groups? Considering the disease model is not a cytotoxic or cancer model, where there may be chances of mortality, it is not clear why authors chose n=36.

Authors state "In order to guarantee the reproducibility of the result the 103 animal study was conducted twice." Does it mean, another 36 rats were again divided into 3 groups? 

Ans) Now we apologized that the descriptions made you to be confused. In two consecutive studies 36 mice were used for 3 groups, in the first study 18 mice were used for 3 groups, and in the second experiment 18 animals were done for 3 group. The reason of two consecutive studies was for guarantee the reproducibility of the result.

 

2.7. "Real time-poly chain reaction (RT-PCR) analysis" --- rewrite - it should be 'Polymerase' (not poly)

Ans) Thank you so much for your kindly comment and we amended the typo.

 

The table in Figure 1 is hazy, cannot read the numbers / symbols clearly. Same is the case with Fig 2 table clarity.

 Ans) We changed Figure 1 and Figure 2.

 

In all the mean values, what is n=?

 Ans) Thank you so much for your comment and we added the number in each group.

 

Figure 5D: The top 6 photomicrographs are not revealing anything clearly --- its all dark - c/EBP and PPAR - the differnces between the exeprimental groups are not clearly visible.

 Ans) We changed Figure 5D.

 

Why was the Inulin analysis done? What are its results and implications?

 Ans) In the natural products research it might be important to define the active compound for special effect and we thought although it was difficult to accurately evaluate the biological effect of specific compound, we should suggest the possible active compound in P. erosus. And we replied on your above question.

 

4. Discussion

"Obesity is not just a physiological uncomfortable symptom but as the prevalence and 262 the health risk of that faces on the disease [3]..." statement does not convey the right message.

Ans) Thank you so much for your comments and we changed the sentence. 

 

"Especially, by 2030 20% of female and 14% of male could be 265 prospected to become obesity patients [5]..."  --- globally? percentage of what sample population?

 Ans) According to the World Obesity Atlas 2022 (World Obesity Federation 2022, March 2022) by 2030 1 in 5 females and 1 in 7 males will be obese (BMI ³ 30 kg/m²) in the worldwide. And then we shifted from the number to the percentage. We added the words, “in the worldwide”.

 

The section '5. Conclusions'  is mentioned without any content written under that section.

Ans) Thank you so much for your generous comment and we omitted that.

Round 2

Reviewer 2 Report

The authors gave a response, but I want to explain my question further:

1. The authors described in 2.2, “Two times per a week the body weight and the food consumption were measured for 56 days”, my question is whether P. erosus affect the food consumption in mice, there is no relative data.

2. Data can be presented as mean ± SD in bar graphs, meanwhile, scatter dots could be added to show the data distribution.

Author Response

#2 Reviewer’s comments

The authors gave a response, but I want to explain my question further:

1. The authors described in 2.2, “Two times per a week the body weight and the food consumption were measured for 56 days”, my question is whether P. erosus affect the food consumption in mice, there is no relative data.

Ans) Thank you so much for the comments and we added the paragraph about the food intake and Figure 2.

 

2. Data can be presented as mean ± SD in bar graphs, meanwhile, scatter dots could be added to show the data distribution.

Ans) Thank you so much for comments and we changed the graph type from bar graph to scattered dot plot.

Author Response File: Author Response.docx