Association between Altered Thyroid Function and Prediabetes in Diet-Induced Prediabetic Male Sprague Dawley Rats
, Akinjide Akinnuga 1, Ntethelelo Sibiya 2, Phikelelani Ngubane 1 and Andile Khathi 1,*Round 1
Reviewer 1 Report
The association of prediabetes, type-2 diabetes and thyroid hormone have been studied extensively in patient population. However, very few report focused on animal study. This is a very straight forward study. However, there is a few concerns and need to be addressed.
1. What is the percentage of carbohydrate and fat in the diet to induce the pre-diabetes? Because reference no. 17 is not visible or accessible to verify the diet composition.
2. What is the rationale for using 15% fructose in water to induce the pre-diabetes?
3. Have you experienced; the rat progressed prediabetes status to diabetic at certain age or post diet weeks. This information will be helpful for readers to establish the model.
4. Method line 101- 102. What is about two different blood collections: Both sets of blood are plasma? Please delete duplication otherwise mention why you collected second set of blood.
5. Line 103: Spinning speed is very low (503g) to get a clear separation of plasma. Please make sure it is a correct number.
6. Baseline values of T4, T3 and TPO is missing in table 1. Because age & sex is already a factor to influence the thyroid hormone. Please provide the data otherwise this can be the limitation of this study.
7. The discussion is unnecessary elaborative, explaining the synthesis and function of different thyroid hormone. This needs to be focused based on the relationship between prediabetes and thyroid hormone otherwise readers will be lost. Please be concise.
8. Conclusion: No background is needed here. Be specific what this study is concluded and a line of future direction.
Author Response
The association of prediabetes, type-2 diabetes and thyroid hormone have been studied extensively in patient population. However, very few report focused on animal study. This is a very straight forward study. However, there is a few concerns and need to be addressed.
- What is the percentage of carbohydrate and fat in the diet to induce the pre-diabetes? Because reference no. 17 is not visible or accessible to verify the diet composition.
Reply: The percentage composition of the diet has been included in the Materials and Methods section under Induction of prediabetes. The reference no. 17 has been replaced in the manuscript
- What is the rationale for using 15% fructose in water to induce the pre-diabetes?
Reply: 15% of fructose was added to the high fat high carbohydrate (HFHC) diet to mimic the Western diet in the developed and developing countries. Also, most beverages in the Western societies contain at least 10 - 15% fructose. Combination of HFHC and beverages is a typical Western diet which is rampant in the developed countries and that is the type of diet we are imitating in this study
- Have you experienced; the rat progressed prediabetes status to diabetic at certain age or post diet weeks. This information will be helpful for readers to establish the model.
Reply: We are yet to extend our research on prediabetes in rats beyond 32 – 40 weeks and at this age, the animals are still prediabetic. However, we also have the intention of carrying out future research to test your suggested hypothesis
- Method line 101- 102. What is about two different blood collections: Both sets of blood are plasma? Please delete duplication otherwise mention why you collected second set of blood.
Reply: One of the blood samples was used to determine glycated haemoglobin and the other blood samples were centrifuged (Eppendorf centrifuge 5403, Germany) at 4°C, 503g for 15 minutes to obtain plasma. This has been corrected in the manuscript
- Line 103: Spinning speed is very low (503g) to get a clear separation of plasma. Please make sure it is a correct number.
Reply: We made use of 503 rcf or gravity (g) and not 503 rpm. However, 503g may be low but we used longer duration of 15 minutes for spinning to avoid lysis of the blood cells and obtain clear plasma. Also, 503g for 15 minutes has been published in most of the studies from our lab.
- Baseline values of T4, T3 and TPO is missing in table 1. Because age & sex is already a factor to influence the thyroid hormone. Please provide the data otherwise this can be the limitation of this study.
Reply: The values of T3 and T4 as well as TPO that were presented in the Figure 2 and 3 respectively in the NPD (non-prediabetic) group against PD (prediabetic) group are the baselines. Because we have presented the same data in the Figure 2 and 3 that is the reason the data did not appear in the table 1. Our study addressed prediabetes in male rats only.
- The discussion is unnecessary elaborative, explaining the synthesis and function of different thyroid hormone. This needs to be focused based on the relationship between prediabetes and thyroid hormone otherwise readers will be lost. Please be concise.
Reply: The discussion has been re-worked to be more concise and to focus more on thyroid function and prediabetes.
- Conclusion: No background is needed here. Be specific what this study is concluded and a line of future direction.
Reply: This has been corrected in the manuscript in red text
Reviewer 2 Report
This is a well designed study that suffers from some fixable flaws in the abstract and introduction. The way the data is presented could also be improved.
Specific comments:
Line 11: a comma before "however"
Line 13: the sentence should not start with a numeral thus "12" should be "Twelve" or alternatively: "Sprague Dawley rats (n=12) were randomly assigned……"
Line 23: more substantively the final conclusion needs to state that this study was done in rodents and not suggest that the results are generalizable to humans thus it needs to say "…. association between thyroid dysfunction and diet-induced prediabetes in rats."
Line 29: The authors misstate what occurs in T2DM. They say that it " occurs due to impaired insulin action on target tissues." This is incorrect. In fact the reference they cite namely Punthakee et al. notes that T2DM is characterized by predominant insulin resistance with or without impaired insulin secretion but the main factor is not the action of insulin but the existence of insulin resistance usually by downregulation of receptors. Again in lines 36-38 the authors suggest that there is something wrong with the hormones when it is the receptors that are affected.
Line 84: "The induction of prediabetes in the male rats followed a previously described experimental protocol" is all that is said about the method for induction of prediabetes. While I appreciate that the method is published given that this procedure is something used solely by the author's laboratory and at the time of publication was described as "novel" and that this method not a generally accepted method more of a description should be given here along with substantiation of validation.
In the presentation of the results given that the sample size is so small (n=6 per group) it would be better reflected by box and whisker plots so that variance of individual responses could be assessed. The responses for both the treatment and control group appear to have virtually no variance and thus the present bar charts do not adequately present the data.
There are some minor usage issues that should be corrected.
Author Response
This is a well designed study that suffers from some fixable flaws in the abstract and introduction. The way the data is presented could also be improved.
Specific comments:
Line 11: a comma before "however"
Reply: Corrected in the manuscript in red text
Line 13: the sentence should not start with a numeral thus "12" should be "Twelve" or alternatively: "Sprague Dawley rats (n=12) were randomly assigned……"
Reply: Corrected in the manuscript in red text
Line 23: more substantively the final conclusion needs to state that this study was done in rodents and not suggest that the results are generalizable to humans thus it needs to say "…. association between thyroid dysfunction and diet-induced prediabetes in rats."
Reply: Corrected in the manuscript in red text
Line 29: The authors misstate what occurs in T2DM. They say that it " occurs due to impaired insulin action on target tissues." This is incorrect. In fact the reference they cite namely Punthakee et al. notes that T2DM is characterized by predominant insulin resistance with or without impaired insulin secretion but the main factor is not the action of insulin but the existence of insulin resistance usually by downregulation of receptors. Again in lines 36-38 the authors suggest that there is something wrong with the hormones when it is the receptors that are affected.
Reply: Corrected in the manuscript in red text in the Introduction and the reference Punthakee et al has been replaced in the reference section
Line 84: "The induction of prediabetes in the male rats followed a previously described experimental protocol" is all that is said about the method for induction of prediabetes. While I appreciate that the method is published given that this procedure is something used solely by the author's laboratory and at the time of publication was described as "novel" and that this method not a generally accepted method more of a description should be given here along with substantiation of validation.
Reply: The induction of prediabetes has been explained briefly and corrected in the manuscript.
In the presentation of the results given that the sample size is so small (n=6 per group) it would be better reflected by box and whisker plots so that variance of individual responses could be assessed. The responses for both the treatment and control group appear to have virtually no variance and thus the present bar charts do not adequately present the data.
Reply: The results have been re-worked and we have incorporated whisker plots to show variance.
Round 2
Reviewer 2 Report
The authors were very responsive and have resolved all the issues that were detected in this manuscript.
There are one or two minor wording issues that can easily be resolved by the editor.
