Establishing a Female Animal Model of Prediabetes Using a High-Carbohydrate, High-Fat Diet

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study is interesting but it lacks a sound justification/rationale for the study. Methodology needs much more details about the process. Results can be improved including the discussion.

 

Line 11-12: “This condition is well established in male animal  models for diabetes; however, few female models exist.” – This is not a strong justification and can you revise it accordingly.

 

Line 15-16: “Female Sprague Dawley rats were randomly allocated to one of two groups: the standard diet (SD) group fed the standard diet with water, and the high-carbohydrate, high-fat group (HCHF) fed a high-carbohydrate, high-fat diet supplemented with fructose.” Please revise the sentence to make sure what those two diets are. Its clear that you used SD and HCHF but what is high-fat diet supplemented with fructose. Is it the same as HCHF diet?

 

Introduction is one whole paragraph. Try to break it down to small paragraphs based on the subtopics for easy reading. 

 

Line 98: Using a previously reported protocol, the animals were induced with experimental prediabetes [20]. You may have to briefly mention what this procedure was in here.

 

Have you included any acclimatisation period? These detailed are missing in the methods.

 

Table 1 summarises the ingredients while Table 2 has given the macro-nutrient composition. What are the compositions of HCHF diet?

 

Inclusion of a figure for study design would be helpful to summarise all the details such as number of mice, interventions, time duration etc.

 

How was the food intake measured?

 

Line 109: “After prediabetes was confirmed in the animal…” mention how was this confirmed.

 

Line 120: how did you measure carbohydrate loading?

 

Information about Fasting Blood Glucose measurements are missing.

 

Line 130: need to mention the details of animal scarification.

 

Fasting Glucose vs FBG: be consistent with wording

 

Figure 1: what was the baseline values? What were the baseline values for other parameters (body weight etc.) as well?

 

Table 3 should ideally come before the Figure 1. This applies to the other figures and corresponding tables as well.

 

Line 271: What were the baseline values?

Are there any baseline values for insulin and other hormones? 

 

The first paragraph of the discussion is sort of repetitive as most of the information has been already mentioned in the introduction. Include a summary paragraph of your results in the first para.

 

Line 371: “A limited physiological range of 3.5–5.5 mmol/l should be maintained for blood glucose levels, including a post-prandial blood glucose level of <7.8 mmol/l and a FBG below 5.6 mmol/l “. This opening paragraph does not make any sense without any context. 

 

Discussion is lacking the justifications for the results obtained in detail.

 

What are the strengthens, limitations of this study including the future directions?

 

Conclusion must be precise.

 

 

 

 

 

 

 

Author Response

RESPONSE TO REVIEWER 1 COMMENTS

We would like to express our gratitude by thanking you for taking the time to review this manuscript. Please find the detailed responses below and a highlighted copy of the manuscript with the corresponding revisions in the re-submitted files. We have amended the overall structure, format and presentation of the manuscript according to your suggestions. Please note that the results (graphs and tables) have been altered according to each reviewer's suggestions.

 

A point-by-point response to comments and suggestions for authors

 

• Line 11-12: "This condition is well established in male animal models for diabetes; however, few female models exist." – This is not a strong justification and can you revise it accordingly.

 

Response: This has been noted and revised accordingly under the abstract section as follows; 'prediabetes is a condition that often precedes the onset of type 2 diabetes and is characterized by moderate levels of insulin resistance. This condition is well established in male animal models for diabetes; however, few female models exist. There is accumulating evidence that sex variations affect the pathogenesis, treatment, and consequences of numerous diseases, such as type 2 diabetes. Therefore, we sought to develop a female diet-induced prediabetic animal model to better understand prediabetes development and its effects in females' (lines 11-16).

 

 

• Line 15-16: "Female Sprague Dawley rats were randomly allocated to one of two groups: the standard diet (SD) group fed the standard diet with water, and the high-carbohydrate, high-fat group (HCHF) fed a high-carbohydrate, high-fat diet supplemented with fructose." Please revise the sentence to make sure what those two diets are. Its clear that you used SD and HCHF but what is high-fat diet supplemented with fructose. Is it the same as HCHF diet?

 

Response: The HCHF diet is a combination diet which consists of a high-carbohydrate and high-fat diet combined with drinking water supplemented with fructose.

 

To improve clarity, the statement has been amended under the abstract section to 'female Sprague Dawley rats were randomly allocated to one of two groups: the standard diet (SD) group fed the standard diet with normal drinking water, and the high-carbohydrate, high-fat group (HCHF) fed a high-carbohydrate and high-fat diet with drinking water supplemented with fructose' (lines 16-19).

 

• Introduction is one whole paragraph. Try to break it down to small paragraphs based on the subtopics for easy reading. 

 

Response: The introduction has been altered and sectioned into smaller paragraphs to improve overall presentation and understanding (Introduction section: lines 38-84).

 

• Line 98: Using a previously reported protocol, the animals were induced with experimental prediabetes [20]. You may have to briefly mention what this procedure was in here.

 

Response: The induction of prediabetes section has been corrected to 'Using a previously reported protocol, the animals were induced with experimental prediabetes' [20]. Briefly, prediabetes was induced in female Sprague Dawley rats by allowing the animals to consume a high-carbohydrate, high-fat (HCHF) diet with drinking water supplemented with 15% fructose for 36 weeks. Prediabetes was confirmed in the animals, using the American Diabetes Association criteria for prediabetes, which is defined by the presence of impaired glucose tolerance (IGT) between 7.8-11.0mmol/L and/or impaired fasting glucose (IFG) between 6.1-6.9mmol/L [21]' (lines 113-119).

 

Reference: Luvuno M, Mabandla M, Khathi A. Voluntary ingestion of a high-fat high-carbohydrate diet: a model for prediabetes. Ponte Int Sci Res J. 2018;74.

 

• Have you included any acclimatization period? These detailed are missing in the methods.

 

Response: The methods and materials section has been amended and includes details on the acclimatization period (lines 98-101). The section on animals and housing has been amended as follows: 'The animals were acclimatized to their new environment for 1 week before the commencement of the study. The animals were administered a normal diet (EPOL, Pretoria West, South Africa) and normal drinking water for acclimatization' (lines 98-101).

 

• Table 1 summarises the ingredients while Table 2 has given the macro-nutrient composition. What are the compositions of HCHF diet?

 

Response: Table 2 has been amended and includes the percentage compositions of the HCHF diet (lines 135-137).

 

• Inclusion of a figure for study design would be helpful to summarise all the details such as number of mice, interventions, time duration etc.

Response: A diagram displaying the experimental/study design has been added to the material and methods section (experimental design- Figure 1).

 

• How was the food intake measured?

 

Response: The methods and materials section has been amended and includes an additional sub-section titled 'Calculation of food and water intake' (lines 161-166). Briefly, food intake was recorded every four weeks during the experimental period. The animals were placed in specialized metabolic cages to measure food and water intake for 24 hours. Each animal was provided with 100g of their respective diet and 100ml of their respective water type. After 24 hours, the remaining grams of food and the volume of water that the animals had not consumed were measured.

 

• Line 109: "After prediabetes was confirmed in the animal…" mention how was this confirmed.

 

Response: This statement has been amended under the sub-section, induction of prediabetes and includes details regarding prediabetes diagnosis and confirmation.

The statement has been corrected as follows, 'Prediabetes was confirmed in the animals, using the American Diabetes Association criteria for prediabetes, which is defined by the presence of impaired glucose tolerance (IGT) between 7.8-11.0mmol/L and/or impaired fasting glucose (IFG) between 6.1-6.9mmol/L [21]' (lines 114-119).

 

• Line 120: how did you measure carbohydrate loading?

 

Response: Details regarding carbohydrate loading have been included in the section' oral glucose tolerance response' (lines 170-173).

 

• Information about Fasting Blood Glucose measurements are missing.

Response: This has been noted, and the methods and material section has been altered and includes information regarding the measurements (lines 131-133) (lines 170- 173).

 

Briefly, animals were fasted overnight for 18 hours. After the 18-hour fasting period, blood was drawn from each animal using the tail-prick technique, and an OneTouch select glucometer was used to measure the fasting blood glucose concentrations before carbohydrate loading and OGTT.

 

• Line 130: need to mention the details of animal scarification.

 

Response: This has been noted, and the methods and materials section has been amended as follows, 'All the animals were sacrificed by decapitation at the end of week 36 using a sharpened guillotine. Blood was collected into pre-cooled EDTA tubes. After 15 minutes of centrifuging the blood at 4 °C, 503 g (Eppendorf centrifuge 5403, Germany), the plasma was collected and kept at -80 °C in a Bio Ultra freezer until it was required for biochemical analysis. Plasma samples were used to measure insulin, ghrelin, and leptin concentrations. Furthermore, the skeletal muscle and liver tissues were excised, weighed, and stored in pre-cooled Eppendorf containers and snap-frozen in liquid nitrogen before storage in a Bio Ultra freezer (Snijers Scientific, Holland) at -80 °C' (lines 181-189).

 

 

• Fasting Glucose vs FBG: be consistent with wording

 

Response: This was corrected to fasting blood glucose throughout the manuscript (highlighted in yellow).

 

• Figure 1: what was the baseline values? What were the baseline values for other parameters (body weight etc.) as well?

 

Response: Due to the long duration of the study, the authors chose to only present results from the main/essential stages of the induction period.

The animals used in this study were all bred in a controlled environment (UKZN, BRU animal facility) and reflected similar weights and blood glucose levels prior to induction of prediabetes.

 

• Table 3 should ideally come before the Figure 1. This applies to the other figures and corresponding tables as well.

 

Response: Thank you for your advice. This has been implemented for all applicable tables and figures in the results section.

 

• Line 271: What were the baseline values? Are there any baseline values for insulin and other hormones? 

 

Response: The glycated hemoglobin, insulin, glycogen, HOMA-IR, ghrelin and leptin tests were conducted terminally; therefore, no baseline values are available.

 

• The first paragraph of the discussion is sort of repetitive as most of the information has been already mentioned in the introduction. Include a summary paragraph of your results in the first para.

 

Response: This has been noted and the first paragraph of the discussion has been partially amended (lines 474-490).

 

• Line 371: "A limited physiological range of 3.5–5.5 mmol/l should be maintained for blood glucose levels, including a post-prandial blood glucose level of <7.8 mmol/l and a FBG below 5.6 mmol/l ". This opening paragraph does not make any sense without any context. 

 

Response: This paragraph has been amended as follows; 'glucose, a necessary fuel, must constantly be present in the blood at a sufficient quantity for every living organism [62]. A physiological range of 3.5–5.5 mmol/l should be maintained for blood glucose levels, including a post-prandial blood glucose level of <7.8 mmol/l and a fasting blood glucose (FBG) below 5.6 mmol/l [32, 33].' (lines 492-495).

 

Reference: Abbas, Z., Sammad, A., Hu, L., Fang, H., Xu, Q. and Wang, Y., 2020. Glucose metabolism and dynamics of facilitative glucose transporters (GLUTs) under the influence of heat stress in dairy cattle. Metabolites, 10(8), p.312.

 

• Discussion is lacking the justifications for the results obtained in detail.

 

Response: The discussion has been partial reworded to improve clarity (discussion section).

 

• What are the strengthens, limitations of this study including the future directions?

 

Response: The discussion and conclusion have been partially reworded to improve clarity and includes information regarding the strengths, limitations and future studies (lines 586-602) (lines 604-618) (highlighted in yellow).

 

• Conclusion must be precise.

 

Response: The conclusion has been improved and partially reworded to improve clarity (lines 604-624).

 

Reviewer 2 Report

Comments and Suggestions for Authors

This study is an attempt to develop a female diet-induced prediabetic animal model through high-fat and high-carbohydrate intake and is expected to be helpful for diabetes treatment research. However, correction and supplementation of the following matters are required.

 

The unit presentation of the results presented in the table should be added accurately.

There is a need to rewrite the Figures so that it is clear and easy for readers to understand. In particular, it would be a good idea to rewrite from Fig. 6 to Fig. 10 in a different format.

Author Response

RESPONSE TO REVIEWER 2 COMMENTS

We want to express our gratitude by thanking you for taking the time to review this manuscript. Please find the detailed responses below and a highlighted manuscript copy with the corresponding revisions in the re-submitted files. We have amended the overall structure and presentation of the manuscript according to your suggestions. Please note that the results (graphs and tables) have been altered according to each reviewer’s suggestions.

 

Point-by-point response to comments and suggestions for authors

 

• The unit presentation of the results presented in the table should be added accurately.

 

Response: The unit presentation of the results was amended in each applicable table (Results section).

 

• There is a need to rewrite the Figures so that it is clear and easy for readers to understand. In particular, it would be a good idea to rewrite from Fig. 6 to Fig. 10 in a different format.

 

Response: The figures and descriptions have been partially amended to improve clarity (Results section- Figure 1 to Figure 10).

 

Reviewer 3 Report

Comments and Suggestions for Authors

Overall Nkosi et al try to describe a new female animal model for prediabetes. Which is overall an interesting topic. However, they fail to meet standard scientific practices on a experimental and also formel level.

The following list is not including every single point but gives hints what the authors really need to improve:

1. Statistics performed were not related to gold standard tests like Welch-test and Annova --> There was no justification why non-parametric tests were used. I assume this was due to not normal distributed values - However, If a animal model is reliable in terms of reproduceability a normaldistribution of values, should be given otherwise, the model would behaive sometimes artifically.

2.Another non standardized way is the order of the samples normally controls are ordered on the left side and treatment groups are on the rigth side.

3. In the discussion no comparisons to other models of T2D or prediabetes were done and it is not discussed how this model compares to male models.

4. Furthermore, I'am missing any phenotypes related to insulin sensitivity how can the authors be sure, that this is really pre-diabetes.

5. There is no explanation given why the food intake also of control mice is going up or is this total food intake?

6. Using BMI in animal models is not very appropriate.

7. Overall there a set of standard phenotypes measured but no conclusion from all the data together is drawn. If the authors want to state that this is a good model for human prediabetes they need to draw this conclusion and compare this to human prediabetes.

8. For example they need to answer which tissue is mostly effected in their animal model and how well is this translateable to the human situation.

 

Author Response

RESPONSE TO REVIEWER 3 COMMENTS

We want to express our gratitude by thanking you for taking the time to review this manuscript. Please find the detailed responses below and a highlighted copy of the manuscript with the corresponding revisions in the re-submitted files. We have amended the overall structure, format and presentation of the manuscript according to your suggestions. Please note that the results (graphs and tables) have been altered according to each reviewer's suggestions.

 

Point-by-point response to comments and suggestions for authors

 

• Statistics performed were not related to gold standard tests like Welch-test and Annova --> There was no justification why non-parametric tests were used. I assume this was due to not normal distributed values - However, If a animal model is reliable in terms of reproduceability a normal distribution of values, should be given otherwise, the model would behaive sometimes artifically.

 

Response: Thank you for your comment. To assist with the analysis of the results presented in this manuscript, a statistician was consulted. The statistical tests performed were on the advice of the statistician. Since the outcomes measured were numeric/continuous, we first examined the distribution of the data using a box plot and then employed the Shapiro test to confirm or assess the normality of the data. Secondly, to evaluate whether there are statistically significant differences between the groups for each outcome, we utilized either a parametric or non-parametric test, depending on the distribution of the data. Since the data was collected from two independent samples, the SD and HCHF groups, we employed an independent two-sample t-test (or its non-parametric counterpart, the Wilcoxon Rank Sum test) to assess statistical differences. We employed a non-parametric Friedman test to assess potential variations within the same groups across the various time points (week 12, 24 and 36). The outcomes measured such as liver glycogen, muscle glycogen, Hb1Ac, insulin and ghrelin hormone, did not violate the normality assumption. Descriptive statistics for these outcomes were reported as mean and standard deviation with 95% confidence interval for the mean. The outcomes such as HOMA-IR, leptin hormone, body weight, BMI, fasting glucose, food intake and ogtt violated the normality assumption and therefore descriptive statistics for these outcomes were reported using median and interquartile range.

 

• Another non standardized way is the order of the samples normally controls are ordered on the left side and treatment groups are on the right side.

 

Response: All graphs and tables have been amended accordingly in the results section.

 

• In the discussion no comparisons to other models of T2D or prediabetes were done and it is not discussed how this model compares to male models.

 

Response: Thank you for your comment. The authors would like to bring to the reviewer's attention that comparisons to previously used models of prediabetes and our previously established novel HCHF diet-induced prediabetic male animal model were either mentioned within the text or referenced in the discussion. Comparisons to the prediabetic male animal model previously established in our laboratory were made in the discussion. The references and their corresponding reference number for the published studies using the HCHF diet conducted by our laboratory are indicated below. The authors have also compared the previously established human model of Type 2 Diabetes Mellitus in the discussion and conclusion (lines 610-612).

However, if the reviewer, after taking into consideration all the changes and additions the authors implemented in the revised manuscript and still wishes for the authors to further elaborate with regards to comparing human models, our previously established male prediabetic animal model and the current female prediabetic animal model, then the authors are willing to implement these changes in the second revised manuscript.

 

References:

- (Ref:20):  Luvuno M, Mabandla M, Khathi A. Voluntary ingestion of a high-fat high-carbohydrate diet: a model for prediabetes. Ponte International Science Research Journal. 2018 May;74.

- (Ref: 25): Gamede M, Mabuza L, Ngubane P, Khathi A. The effects of plant-derived oleanolic acid on selected parameters of glucose homeostasis in a diet-induced prediabetic rat model. Molecules. 2018;23(4):794.

-(Ref:39): Naidoo K, Ngubane PS, Khathi A. Investigating the effects of diet-induced prediabetes on the functioning of calcium-regulating organs in male Sprague Dawley rats: effects on selected markers. Frontiers in Endocrinology. 2022 11 July;13:914189.

 

 

• Furthermore, I'am missing any phenotypes related to insulin sensitivity how can the authors be sure, that this is really prediabetes.

 

Response: In order to ensure that the animals were prediabetic, the authors used the American Diabetes Association criteria for prediabetes which is defined by the presence of impaired glucose tolerance (IGT) between 7.8-11.0mmol/L and/or impaired fasting glucose (IFG) between 6.1-6.9mmol/L (lines 116-119) (lines 510-512). In addition, the following parameters were also assessed either during the induction period or terminally: fasting blood glucose, oral glucose tolerance test, food intake, body weights, BMI, glycated haemoglobin, glycogen, HOMA-IR, plasma insulin, plasma ghrelin and plasma leptin. These tests were carefully chosen by utilizing the male diet induced prediabetic animal model that was previously established in our laboratory as a reference (Luvuno et al., 2018). Subsequently multiple studies have been published in a variety of journals using this exact male diet induced prediabetes animal model. This particular diet (HCHF) composition accurately resembles the current modern dietary patterns and has been proven numerous times to be successful in inducing prediabetes in male rat models however no female models exist. Therefore to fill in this knowledge gap, this study was carried out to determine if this particular diet can also successfully induce prediabetes in female Sprague Dawley rats. Thereby creating a successfully animal model of female diet induced prediabetes.

 

Reference:

- Luvuno, M., Mabandla, M. and Khathi, A., 2018. Voluntary ingestion of a high-fat high-carbohydrate diet: a model for prediabetes. Ponte Int. Sci. Res. J, 74.

-American diabetes association professional practice committee, american diabetes association professional practice committee. Classification and diagnosis of diabetes: Standards of Medical Care in Diabetes-2022. Diabetes Care. 2022 Jan 1;45(Supplement 1):S17-38.

-Mabuza LP, Gamede MW, Maikoo S, Booysen IN, Ngubane PS, Khathi A. Effects of a Ruthenium Schiff Base Complex on Glucose Homeostasis in Diet-Induced Prediabetic Rats. Molecules. 2018;23(7).

-Mabuza LP, Gamede MW, Maikoo S, Booysen IN, Ngubane PS, Khathi A. Cardioprotective effects of a ruthenium (II) Schiff base complex in diet-induced prediabetic rats. Diabetes Metab Syndr Obes. 2019;12:217-23.

- Siboto A, Akinnuga AM, Khumalo B, et al. The effects of a [3+1] oxo-free rhenium (V) compound with uracil-derived ligands on selected parameters of glucose homeostasis in diet-induced prediabetic rats. Obesity Medicine. 2020;19:100258.

 

• There is no explanation given why the food intake also of control mice is going up or is this total food intake?

 

Response: The induction period took place over a duration of 36 weeks (9 months). The induction period was carried throughout multiple rat life stages; adolescence then teenager, young adult, adult and finally matured adult at termination of the study. At the beginning of the induction period, all animals weighed between 150-180g and were approximately 4 weeks old (adolescent). If we take into account the body weight and the life stage of the animals at 12, 24 and 36 weeks, then the increase in food intake reported in the control diet group can be regarded as normal for the growth of the animal and the corresponding nutritional requirements for those life stages. The increase in the control group food intake at week 12, 24 and 36 are significantly lower than the HCHF group. The control group food intake at week 36, is still marginal lower than the HCHF group at week 12. The trends seen in the food intake between the control and HCHF groups are similar to the trends reported from our laboratory in studies carried out using male rat models.

 

• Using BMI in animal models is not very appropriate.

 

Response: Thank you for your advice. We chose to use BMI as one of the additional parameters to assess for prediabetes as BMI has been most commonly used as a non-invasive tool to assist in diagnosing those whom are at a higher risk for developing prediabetes. BMI, as a risk factor for prediabetes has been shown to contribute to 30.8% of deaths worldwide (Mkhize et al., 2023). In our laboratory, we have previously investigated the effects of prediabetes on BMI and have established that this attribute is indeed a risk factor for prediabetes. Therefore, it was imperative that we observed and reported the BMI in our animal model.

 

Reference: Cassandra Mkhize, B., Mosili, P., Sethu Ngubane, P. and Khathi, A., 2023. The relationship between adipose tissue RAAS activity and the risk factors of prediabetes: a systematic review and meta-analysis. Adipocyte, 12(1), p.2249763.

 

• Overall there a set of standard phenotypes measured but no conclusion from all the data together is drawn. If the authors want to state that this is a good model for human prediabetes they need to draw this conclusion and compare this to human prediabetes.

 

Response: The discussion and conclusion have been partially reworded to improve clarity (highlighted in yellow).

 

• For example they need to answer which tissue is mostly effected in their animal model and how well is this translateable to the human situation.

 

Response: This study focused on establishing a female model of prediabetes only. An in depth evaluation of particular tissues were not presented in this manuscript as it is a part of another manuscript that is currently under peer review. The authors currently have numerous papers under peer review using the female prediabetic animal model presented in this manuscript.  This manuscript is the main paper for all the other studies.

 

The diet induced prediabetic male animal model that was previously established in our laboratory was used as a reference (Luvuno et al., 2018) for establishing a female prediabetic model. Multiple studies have been published in a variety of journals using this novel diet induced prediabetic male animal model (Luvuno et al., 2018, Mabuza et al., 2018, Mabuza et al., 2019, Siboto et al., 2020). This particular diet (HCHF) composition accurately resembles the modern human dietary patterns and has been proven numerous times to be successful in inducing prediabetes in male rats as well as resemble human prediabetes characteristics (Discussion and conclusion section).

However, if the reviewer, after taking into consideration all the changes and additions the authors implemented in the revised manuscript and still wish for the authors to further elaborate, then the authors are willing to implement these changes in the second revised manuscript to the satisfaction of the reviewer.

 

Reference:

- Luvuno, M., Mabandla, M. and Khathi, A., 2018. Voluntary ingestion of a high-fat high-carbohydrate diet: a model for prediabetes. Ponte Int. Sci. Res. J, 74.

 -Mabuza LP, Gamede MW, Maikoo S, Booysen IN, Ngubane PS, Khathi A. Effects of a Ruthenium Schiff Base Complex on Glucose Homeostasis in Diet-Induced Prediabetic Rats. Molecules. 2018;23(7).

-Mabuza LP, Gamede MW, Maikoo S, Booysen IN, Ngubane PS, Khathi A. Cardioprotective effects of a ruthenium (II) Schiff base complex in diet-induced prediabetic rats. Diabetes Metab Syndr Obes. 2019;12:217-23.

- Siboto A, Akinnuga AM, Khumalo B, et al. The effects of a [3+1] oxo-free rhenium (V) compound with uracil-derived ligands on selected parameters of glucose homeostasis in diet-induced prediabetic rats. Obesity Medicine. 2020;19:100258.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Mostly, all comments were adressed by the authors.