Accepted Article
DR. NOBUYA SASAKI (Orcid ID : 0000-0003-2617-1468)
Article type
: Original Article
Title: A novel murine model of congenital diabetes: the insulin hyposecretion mouse
Running Head: Novel mouse model of congenital diabetes
Authors: Kenta Nakano1, 2), Rieko Yanobu-Takanashi2), Yuki Takahashi1), Hayato Sasaki1), Yukiko Shimizu2), Tadashi Okamura2, 3)* and Nobuya Sasaki1)*
1) Laboratory of Laboratory Animal Science and Medicine, School of Veterinary Medicine, Kitasato University, Towada, Japan 2) Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine (NCGM), Tokyo 162-8655, Japan 3) Section of Animal Models, Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan
*Co-Corresponding author:
Nobuya Sasaki Laboratory of Laboratory Animal Science and Medicine School of Veterinary Medicine, Kitasato University, 35-1, Higashi-23, Towada, Aomori 034-8628 Japan Phone/Fax: +81-176-24-9496
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/jdi.12895 This article is protected by copyright. All rights reserved.
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E-mail:
[email protected]
Tadashi Okamura Department of Laboratory Animal Medicine, Research Institute, National Center for Global Health and Medicine (NCGM), 1-21-1, Toyama, Shinjuku-ku, Tokyo 162-8655 Japan Phone/Fax: +81-3202-7181(ext. 2748)/+81-3202-7364 E-mail:
[email protected]
ABSTRACT Aims/Introduction Diabetic animal models have made an enormous contribution to our understanding of the etiology of diabetes and the development of new medications. The aim of this study was to develop and characterize a novel, non-obese murine strain with spontaneous diabetes, the insulin hyposecretion (ihs) mouse. Materials and Methods During the development of ICGN.B6-Tns2WT strain as the control for ICGN-Tns2nph congenital nephrotic strain, diabetic mice were discovered and named ihs mice. Intraperitoneal insulin tolerance and oral glucose tolerance tests and insulin secretion experiment by pancreas perfusion system were performed on ihs mice. The pancreatic islets were examined histologically, and the mRNA expression of pancreatic β-cell-specific genes or genes associated with monogenic diabetes was examined by RT-qPCR. Results The ihs mice exhibited several distinctive diabetes-related characteristics: (1) the onset of diabetes was observed only in the male mice; (2) there were no differences in insulin content between the ihs and control mice; (3) impaired insulin secretion was elicited by glucose, potassium chloride and sulfonylureas; (4) there is a significant reduction of relative β-cell volume with no signs of inflammation or fibrosis; (5) they exhibited a normal glycemic response to exogenous insulin; and (6) the mice were not obese. Conclusion The ihs mouse provides a novel murine model of congenital diabetes that exhibits insulin secretion failure. This model allows not only an analysis of the progression of diabetes, but also the identification of unknown genes involved in insulin secretion. KEY WORDS non-obese diabetic mouse, hypoinsulinemia, hyperglycemia
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Accepted Article
INTRODUCTION Type 2 diabetes (T2D) is a metabolic disorder that is characterized by abnormal glucose
homeostasis due to some defect in the secretion and/or action of insulin. There has been a dramatic increase in T2D patients in East Asian countries, which now represent a fourth of the global diabetes population1. Although the precise mechanisms that underlie the
development and progression of T2D have not been fully elucidated, it is thought that a
combination of multiple genetic and environmental factors contribute to the pathogenesis of the disease2. There has therefore been increased interest in reappraising animal models of T2D in which genetic and environmental factors that may influence the
development of the disease and related complications can be precisely controlled in vivo to help elucidate the etiology of diabetes and develop new medications. Animal models have made an enormous contribution to the study of diabetes mellitus, providing new
information on its management and treatment in humans. Animal models for T2D have been widely used for elucidating the genes responsible for the development of T2D, the physiological course of the disease, and related complications3, 4.
T2D in East Asian countries is characterized by a lower level of obesity compared to T2D in Caucasians, as well as a younger age of onset5, 6 and β-cell dysfunction6-8. Relatively few people with diabetes in Japan are obese, and the impairment of insulin secretion often develops before the onset of diabetes9. Thus, non-obese animal models are needed to elucidate the complete pathogenesis of diabetes. To this end, we have established a novel, non-obese murine strain with spontaneous diabetes: the insulin hyposecretion (ihs) mouse. The ihs mouse allows not only the analysis of the progression of diabetes, but also the identification of unknown genes involved in insulin secretion. This report describes the development and characterization of the ihs mouse.
MATERIALS AND METHODS Ethical statement All animal experiments were approved by the President of Kitasato University and NCGM, following consideration by the Institutional Animal Care and Use Committee of Kitasato University (Approval ID: No. 17-099) and NCGM (Approval ID: No. 17056), and were conducted in accordance with institutional procedures, national guidelines and the relevant national laws on the protection of animals.
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Breeding of congenic mice
Accepted Article
The ICGN mouse provides a model of glomerular dysfunction that shows gross morphologic changes in the podocyte foot processes that accompany proteinuria (Appendix S1). Previously, we demonstrated that proteinuria in ICGN mice was caused by a deletion mutation in the tensin2 (Tns2) gene (designated Tns2nph)10, 11. As the original ICGN mouse is a spontaneous mutant derived from closed colony ICR mice, there is no control strain. We therefore created a control strain by using classical breeding methods to introgress the wild-type Tns2 gene (Tns2WT) from the C57BL/6J (B6) mouse into the inbred ICGN strain (ICGN.B6-Tns2WT)12. Then, the F1 (B6 × ICGN) mice heterozygous for Tns2 mutation were backcrossed to ICGN mice for 10 generations, and subsequently homozygous ICGN.B6-Tns2WT/Tns2WT mice were produced by sib-mating. During this strain breeding process, we discovered mutant mice with polydipsia, polyuria, hyperglycemia, and hypoinsulinemia, which we named ihs mice (Figure 1). As ihs mice are spontaneous mutants derived from ICGN mice, which are an animal model for congenital nephrosis, B6 mice were used in this study as a reference.
Phenotype measurements The mice were weighed weekly from 5 weeks of age. The nasal-anal length was measured and the body mass index (BMI) was calculated at 12, 25 and 52 weeks by taking the weight (in grams) and dividing it by the square of the nasal-anal length (in cm). Body composition was measured by NMR (LF50 BCA-Analyzer, BRUKER, Billerica, MA, USA).
Blood and urine analysis Urine glucose levels were measured semiquantitatively using Uro-paper III (Eiken, Tokyo, Japan). To obtain paired measurements with urine glucose levels, blood was collected by tail snip immediately after urine collection. The blood glucose levels were measured using Glutest Ace and Glutest Sensor (Sanwa Chemical Co., Tokyo, Japan). Diabetes was diagnosed when the glucose level was higher than 250 mg/dL under ad libitum feeding conditions.
Oral glucose and insulin tolerance tests The mice were fasted for 16 h and tail blood glucose was measured at 0, 30, 60, 90, and 120 min after the oral administration of glucose (2 g/kg body weight; Otsuka Pharmaceutical, Tokyo, Japan) by gavage. Plasma samples were collected from the retro-orbital venous plexus at 0, 15, and 30 min for insulin measurement. Plasma insulin levels were determined by ultra-sensitive mouse insulin kit (Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). The area under the curve (AUC) of insulin secretion was calculated after subtraction of the baseline insulin level at the time the glucose was administered. An insulin tolerance test was performed after the mice had fasted for 3 h. Insulin (0.75 unit/kg body weight; Humulin, Lilly, Indianapolis, IN, USA) was injected into the intraperitoneal space and blood glucose levels were measured with a glucose monitor at 0, 30, 60, 90, and 120 min after the injection.
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Other analyses Non-fasting plasma insulin and active GLP-1 levels were measured by ELISA (Appendix S2 and
Accepted Article
S3). Insulin secretion from in situ perfused pancreata (Appendix S4) and insulin secreted by islets isolated from ihs mice (Appendix S5) were measured. Pancreata were subjected to histological analysis, including an analysis of islet morphology, as described previously13, 14. The details are given in Appendix S6. RT-qPCR for genes associated with monogenic diabetes were performed. Details are presented in Appendix S7 and Table S1.
Statistical analysis The results are expressed as means ± SEM. Significant differences in AUC or reverse AUC were analyzed using unpaired Student’s t-tests. The results for insulin secretion from the in situ perfused pancreas analysis were analyzed using repeated-measures ANOVA and Student’s t-test. Student’s t-test was used for comparisons of two independent groups, respectively. A P value
