Development and characterisation of a rat model that exhibits both metabolic dysfunction and neurodegeneration seen in type 2 diabetes

Abstract Accurate modelling type 2 diabetes and diabetic complications in rodents has proven a challenge, largely as a result of the long‐time course of disease development in humans. In the present study, we aimed to develop and comprehensively characterise a new rodent model of type 2 diabetes. To do this, we fed Sprague–Dawley rats a high fat/high sugar diet (HFD) to induce obesity and dyslipidaemia. After 3 weeks, we s.c. implanted osmotic mini pumps to enable a 14 day, slow infusion of streptozotocin (STZ; lower dose = 100 mg kg−1; higher dose = 120 mg kg−1) to dose‐dependently reduce pancreatic beta cell mass. After removing the mini pumps, we monitored animals for 4 months using a battery of tests to assess both metabolic and neurodegenerative changes across time. Our data demonstrate the combination of the HFD and lower dose STZ leads to induction of early‐stage type 2 diabetes defined by moderate hyperglycaemia, hyperinsulinaemia and impaired glucose tolerance, at the same time as the retention of an obese phenotype. By contrast, combining the HFD and higher dose STZ leads to induction of later‐stage type 2 diabetes defined by frank hyperglycaemia, hypoinsulinaemia (but not insulin depletion) and severely impaired glucose tolerance, at the same time as retaining an obese phenotype. Regardless of dose of STZ (and level of hyperglycaemia), all diabetic rats exhibited signs of peripheral neurodegeneration in the skin and muscle. Thus, this model recapitulates many of the complex metabolic disturbances seen in type 2 diabetes and provides an excellent platform for investigating the pathophysiological mechanisms that lead to diabetic complications such as peripheral neuropathy. Key points Type 2 diabetes is a major health concern and markedly increases risk cardiovascular and neurodegenerative diseases. Accurate modelling of type 2 diabetes is a major challenge and has impeded our ability to understand the mechanisms that contribute to complications of type 2 diabetes. We have developed a method of inducing different stages of type 2 diabetes using a high fat/high sugar diet and 14 day infusion of streptozotocin to dose‐dependently destroy pancreatic beta cell mass. Over 4 months, we comprehensively characterised these animals and confirmed that they develop sustained metabolic dysfunction and progressive peripheral neurodegeneration as seen in type 2 diabetes. This new model will improve our ability to investigate the pathophysiological mechanisms that link type 2 diabetes with complications such as neurodegeneration.

The authors developed a new rat model that exhibits both metabolic dysfunction and neurodegeneration seen in humans with type 2 diabetes. This was accomplished by using a high fat/high sugar diet and delayed-onset infusion of streptozotocin to dose-dependently destroy pancreatic beta cell mass. The authors found that the combination of the diet and low dose streptozotocin leads to obesity combined with moderate fasting hyperglycaemia, hyperinsulinaemia, and impaired glucose tolerance. Combining the diet with high dose streptozotocin leads to hypoinsulinaemia (but not insulin depletion) and severe hyperglycemia (fasted and postprandially). Both models (low and high dose streptozotocin) demonstrated signs of peripheral neurodegeneration in the skin and muscle. This model offers advantages over existing models that either lead to quick destruction of beta cell mass or fail to cause hyperglycemia and impaired glucose tolerance. It is expected that this new rat model will be useful for the study of metabolic and neurological alterations in the progression from normoglycemia to hyperglycemia-hyperinsulinemia to type 2 diabetes.
The experimental design is appropriate. The characterization of metabolic function is detailed and the addition of features of neuropathy is valuable. The paper is overall well written, although the results are a little lengthy, and the data are nicely presented.
I have no specific comments. The authors did a great job, aside from the somewhat lengthy results section.
Referee #2: Southam et al. sought to induce different stages of type 2 diabetes (T2DM) using a high fat/high sugar diet and 14-day infusion of streptozotocin to dose-dependently destroy pancreatic beta cell mass. During a four month follow-up period, the authors attempted to characterise this model and demonstrated that they develop sustained metabolic dysfunction and progressive peripheral neurodegeneration.
The authors appear to have performed a robust set of experiments and characterisation work in this current manuscript, and should be commended for their experimental and technical skill.
The authors are correct that current models of T2DM do not fully recapitulate the disease progression, and symptoms observed in patients. However, I do feel that the author's claim that this model is able to "reproduce the complex metabolic disturbances associated with early-stage type 2 diabetes in humans" is overstated give the relatively limited characterisation work performed in this rodent model (compared to the totality of human T2DM characterisation and investigation). Additionally, attempting to recreate the full spectra of symptoms and co-morbidities observed in human disease, is for me, of questionable value given recent work demonstrating that T2D is a highly heterogenous disease (e.g. https://doi.org/10.2337/dbi20-0001 ). Thus my main comments are related to tempering of certain claims made by the authors.

Comments
Title: "Development and characterisation of a rat model that exhibits both metabolic 2 dysfunction and neurodegeneration seen in humans with type 2 diabetes" I don't think that the manner of metabolic dysfunction and neurodegeneration is precisely observed in humans. Therefore the authors should remove "seen in humans with type 2 diabetes" from the title.
"we wanted to understand whether these animals developed the classic neurological complications of type 2 diabetes such as peripheral neuropathy -a major cause of morbidity that effects approximately 50% of people with type 2 diabetes" The prevalence of neuropathy in T2DM appears to vary in the literature e.g. Feldman et al., 2019 suggests 8-51% prevalence. Perhaps the authors could briefly discuss the range of reported prevalence, and any relevance this might have to their model.

12-Oct-2021
The authors appear to highlight certain aspects of their model's timeline of symptom progression when it suits, and 'playdown' other aspects when it doesn't. E.g. altered hind paw withdrawal threshold was observed already at week 9, whereas, in the human condition neuropathic symptoms are progressive and occur secondary, often over a long period of time, to poor glucoregulatory control. From Feldman et al., 2019 : "Indeed, the prevalence of diabetic neuropathy increased from 8% to 42% in patients with T2DM when patients were monitored for 10 years". The authors should be clear that the timeline of development of peripheral neuropathy in their model does not recapitulate disease progression in many people with T2DM.
Given the comments above, it is clear that this concluding sentence is overstated and should be revised: "Therefore, this model recapitulates the complex and multiple metabolic disturbances seen in people with type 2 diabetes that will improve our understanding of the mechanisms that lead to neurodegeneration as well as providing a powerful platform for testing new therapeutics to limit nervous system damage in type 2 diabetes."

16-Dec-2021 1st Authors' Response to Referees
Re: JP-TFP-2021-282454 "Development and characterisation of a rat model that exhibits both metabolic dysfunction and neurodegeneration seen in humans with type 2 diabetes" by Katherine Southam, Chantal de Sousa, Abraham Daniel, Bruce Taylor, Lisa Foa, and Dino Premilovac Dear Professor Barrett, The authors would like to thank the reviewers and editors for giving us the opportunity to revise and resubmit our manuscript to the Journal of Physiology. Below is a point-by-point summary of the alterations that have been made to the manuscript based on the review comments provided.

Reviewer 1.
The authors developed a new rat model that exhibits both metabolic dysfunction and neurodegeneration seen in humans with type 2 diabetes. This was accomplished by using a high fat/high sugar diet and delayed-onset infusion of streptozotocin to dose-dependently destroy pancreatic beta cell mass. The authors found that the combination of the diet and low dose streptozotocin leads to obesity combined with moderate fasting hyperglycaemia, hyperinsulinaemia, and impaired glucose tolerance. Combining the diet with high dose streptozotocin leads to hypoinsulinaemia (but not insulin depletion) and severe hyperglycemia (fasted and postprandially). Both models (low and high dose streptozotocin) demonstrated signs of peripheral neurodegeneration in the skin and muscle. This model offers advantages over existing models that either lead to quick destruction of beta cell mass or fail to cause hyperglycemia and impaired glucose tolerance. It is expected that this new rat model will be useful for the study of metabolic and neurological alterations in the progression from normoglycemia to hyperglycemia-hyperinsulinemia to type 2 diabetes.
The experimental design is appropriate. The characterization of metabolic function is detailed and the addition of features of neuropathy is valuable. The paper is overall well written, although the results are a little lengthy, and the data are nicely presented.
I have no specific comments. The authors did a great job, aside from the somewhat lengthy results section.
Authors response: we thank reviewer 2 for the positive comments provided regarding our work.

Reviewer 2.
Southam et al. sought to induce different stages of type 2 diabetes (T2DM) using a high fat/high sugar diet and 14-day infusion of streptozotocin to dose-dependently destroy pancreatic beta cell mass. During a four-month follow-up period, the authors attempted to characterise this model and demonstrated that they develop sustained metabolic dysfunction and progressive peripheral neurodegeneration.
The authors appear to have performed a robust set of experiments and characterisation work in this current manuscript, and should be commended for their experimental and technical skill.
The authors are correct that current models of T2DM do not fully recapitulate the disease progression, and symptoms observed in patients. However, I do feel that the author's claim that this model is able to "reproduce the complex metabolic disturbances associated with early-stage type 2 diabetes in humans" is overstated give the relatively limited characterisation work performed in this rodent model (compared to the totality of human T2DM characterisation and investigation). Additionally, attempting to recreate the full spectra of symptoms and co-morbidities observed in human disease, is for me, of questionable value given recent work demonstrating that T2D is a highly heterogenous disease (e.g. https://doi.org/10.2337/dbi20-0001 ). Thus my main comments are related to tempering of certain claims made by the authors.
Authors response: we thank reviewer 2 for the positive comments provided regarding our manuscript. We agree that some of the statements made were perhaps too broad in nature and have revised the manuscript based on the individual comments provided below. Comments 1. Title: "Development and characterisation of a rat model that exhibits both metabolic 2 dysfunction and neurodegeneration seen in humans with type 2 diabetes" I don't think that the manner of metabolic dysfunction and neurodegeneration is precisely observed in humans. Therefore the authors should remove "seen in humans with type 2 diabetes" from the title.
Authors response: we agree, and we have removed the word human from the title to ensure we are more accurately representing the nature of the work in the manuscript. We hope this is enough of a change to satisfy the reviewers concerns regarding the comparisons/conclusions regarding human type 2 diabetes.
Changes to manuscript: the title of the manuscript has been changed to "Development and characterisation of a rat model that exhibits both metabolic dysfunction and neurodegeneration seen in type 2 diabetes" 2. "we wanted to understand whether these animals developed the classic neurological complications of type 2 diabetes such as peripheral neuropathy -a major cause of morbidity that effects approximately 50% of people with type 2 diabetes" The prevalence of neuropathy in T2DM appears to vary in the literature e.g. Feldman et al., 2019 suggests 8-51% prevalence. Perhaps the authors could briefly discuss the range of reported prevalence, and any relevance this might have to their model. 4. Given the comments above, it is clear that this concluding sentence is overstated and should be revised: "Therefore, this model recapitulates the complex and multiple metabolic disturbances seen in people with type 2 diabetes that will improve our understanding of the mechanisms that lead to neurodegeneration as well as providing a powerful platform for testing new therapeutics to limit nervous system damage in type 2 diabetes." Authors response: this is in line with the other comments provided and we have revised the final sentence of the manuscript and the final line of the abstract accordingly. In a number of other sentences in the manuscript, we have also replaced 'human type 2 diabetes' and with just 'type 2 diabetes' to limit some of the overstated outcomes from the new animal model as it relates to the human condition.

Changes to manuscript: we have modified language in a number of sections of the manuscript as below:
Line 42-44 (key points): "Over four months, we comprehensively characterised these animals and confirmed they develop sustained metabolic dysfunction and progressive peripheral neurodegeneration as seen in type 2 diabetes" Lines 63-65 (abstract): "Thus, this model recapitulates many of the complex metabolic disturbances seen in type 2 diabetes and provides an excellent platform to investigate the pathophysiological mechanisms that lead to diabetic complications like peripheral neuropathy." Lines 655-659 (discussion): "Therefore, this model recapitulates many of the key metabolic disturbances seen in type 2 diabetes and provides an excellent platform for testing new therapeutics to prevent or reverse complications of type 2 diabetes such as peripheral neuropathy."

20-Jan-2022 1st Revision -Editorial Decision
Dear Dr Premilovac, Re: JP-TFP-2021-282454R1-A "Development and characterisation of a rat model that exhibits both metabolic dysfunction and neurodegeneration seen in type 2 diabetes" by Katherine Southam, Chantal de Sousa, Abraham Daniel, Bruce Taylor, Lisa Foa, and Dino Premilovac Thank you for submitting your manuscript to The Journal of Physiology. It has been assessed by a Reviewing Editor and by 2 expert referees and I am pleased to tell you that it is considered to be acceptable for publication following satisfactory revision.
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Could you please address the following two comments in the updated version of the manuscript? 1) In the statistical analysis section, you mentioned that you performed a Kruskal-Wallis test to check for normality. Could you please verify that this is correct?
2) The colors used for the figures (particularly red and green) may not be visible to colorblind people. I suggest selecting alternative colors that can be visible to colorblind readers.