A new model of insulin-deficient diabetes: male NOD mice with a single copy of Ins1 and no Ins2
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- Babaya, N., Nakayama, M., Moriyama, H. et al. Diabetologia (2006) 49: 1222. doi:10.1007/s00125-006-0241-4
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We describe a novel model of insulin-deficient diabetes with a single copy of the gene encoding insulin 1 (Ins1) and no gene encoding insulin 2 (Ins2).
Materials and methods
We constructed five lines of mice: mice with two copies of Ins1 (NODIns1+/+,Ins2−/−), mice with a single copy of Ins1 (NODIns1+/−,Ins2−/−), mice with two copies of Ins2 (NODIns1−/−,Ins2+/+), mice with a single copy of Ins2 (NODIns1−/−,Ins2+/−) and NODIns1+/−,Ins2−/− mice with a transgene encoding B16:Ala proinsulin.
By 10 weeks of age, all male NODIns1+/−,Ins2−/− mice were diabetic, whereas all female NODIns1+/−,Ins2−/− were not diabetic (p<0.0001). In contrast, neither male nor female NODIns1−/−,Ins2+/− with a single copy of Ins2 (rather than single copy of Ins1) developed early diabetes and no mice with two copies of either gene developed early diabetes. Islets of the diabetic male NODIns1+/−,Ins2−/− at this early age had no lymphocyte infiltration. Instead there was heterogeneous (between islet cells) weak staining for insulin. Although only male NODIns1+/−,Ins2−/− mice developed diabetes, both male and female NODIns1+/−,Ins2−/− mice had markedly decreased insulin content. In NODIns1+/+,Ins2−/−, there was also a significant decrease in insulin content, whereas NODIns1−/−,Ins2+/+ mice, and even NODIns1−/−,Ins2+/− mice, were normal. Male NODIns1+/−,Ins2−/− mice were completely rescued from diabetes by introduction of a transgene encoding proinsulin. On i.p. insulin tolerance testing, male mice had insulin resistance compared with female mice.
These results suggest that Ins1 is a ‘defective gene’ relative to Ins2, and that the mouse lines created provide a novel model of sex-dimorphic insulin-deficient diabetes.
KeywordsGender differenceInsulin biosynthesis/secretionInsulin geneInsulin resistanceKnock-out mouseMetabolic diabetesNonobese diabetic mouseSex differenceTransgenic mouse
gene encoding insulin 1
gene encoding insulin 2
intraperitoneal glucose tolerance test
insulin tolerance test
Insulin is synthesised by pancreatic beta cells and plays a predominant role in glucose homeostasis. Mice have two genes encoding insulin, insulin 1 (Ins1) on chromosome 19, and insulin 2 (Ins2) on chromosome 7. Ins1 differs from Ins2 by two amino acids at positions B9 and B29 and there are additional differences in the leader sequence and the connecting peptide. In addition, Ins1 lacks an intron present in Ins2. These structural features suggest that Ins1 was generated by an RNA-mediated duplication–transposition event involving a transcript of Ins2, which was reinserted into the genome (retroposon) .
In a previous study, we created Ins1 knock-out (Ins1-KO) and Ins2-KO mice on the nonobese diabetes (NOD) background. The Ins1-KO prevented the majority of progression to autoimmune type 1 diabetes, whereas the Ins2-KO accelerated the development of type 1 diabetes . Similarly, Thebault-Baumont et al. have shown that Ins2-KO mice bred onto the NOD background develop accelerated insulitis and diabetes . These studies have suggested that the role of each gene encoding insulin in autoimmune diabetes is different.
More recently, we have described double insulin KO (Ins1-KO and Ins2-KO) NOD mice with a mutated gene encoding preproinsulin (B16:Ala) that rescues the mice from metabolic diabetes . These mice lacking a native B:9–23 sequence do not develop autoimmune diabetes. During evaluation and creation of these mice, we unexpectedly found that male NOD mice with the genotype Ins1+/−, Ins2−/− (NODIns1+/−,Ins2−/−) developed early onset of diabetes (<10 weeks of age). In this study, we describe the NODIns1+/−,Ins2−/− mice, a novel strain with non-autoimmune insulin-deficient diabetes.
Materials and methods
We constructed four lines of mice: (1) mice with two copies of Ins1 (NODIns1+/+,Ins2−/−; Ins2-KO); (2) mice with a single copy of Ins1 (NODIns1+/−,Ins2−/−); (3) mice with two copies of Ins2 (NODIns1−/−,Ins2+/+; Ins1-KO); and (4) mice with a single copy of Ins2 (NODIns1−/−,Ins2+/−). The mice were established by breeding the original insulin knock-outs kindly provided by J. Jami (Cochin and Saint Vincent de Paul Hospital, Paris, France) on to NOD/Bdc mice using marker-assisted congenic methods . NODIns1+/−,Ins2−/− and NODIns1−/−,Ins2+/− were produced by mating (Ins1-KO×Ins2-KO)F1 with Ins2-KO and mating (Ins1-KO×Ins2-KO)F1 with Ins1-KO, respectively. NODIns1+/−,Ins2−/− with a mutated transgene encoding proinsulin (NODIns1+/−,Ins2−/−,Tg(+)) were constructed by mating with NODIns1+/+,Ins2−/− and NODIns1−/−,Ins2−/− with the transgene encoding B16:Ala proinsulin [4, 5]. Mice were housed in a pathogen-free animal colony at Barbara Davis Center for Childhood Diabetes with an approved protocol from the University of Colorado Health Sciences Center Animal Care and Use Committee. All mice had free access to tap water in an air-conditioned room (22–25°C) with a 12-h light–darkness cycle (06.00–18.00 h). In addition, we produced control strains with the genetic region of 129S1/SvImj mice surrounding Ins1 and Ins2 bred onto the NOD mice and these mice did not develop ‘early’ diabetes but typical insulitis associated with later onset of diabetes as reported .
Genomic DNA was extracted from mouse tails. The genotyping for the Ins1-knock-out gene, the Ins1-wild-type gene, the Ins2-knock-out gene and the Ins2-wild-type gene was performed by using PCR . The PCR products were electrophoresed on 2% agarose gels and visualised by ethidium bromide staining.
Diagnoses of diabetes
Glucose was measured weekly with the FreeStyle blood glucose monitoring system (TheraSense, Alameda, CA, USA), and the mice were considered diabetic after two consecutive blood glucose values >13.9 mmol/l. After development of diabetes, the mice were killed immediately and the pancreas was fixed in 10% formalin to perform histological analysis.
Insulin autoantibody (IAA) assay
IAA was measured with a 96-well filtration plate micro-IAA assay as previously described  and expressed as an index. A value of 0.01 or greater is considered positive and exceeds the 99th percentile of normal controls.
Insulin content in pancreas
Mice were analysed for insulin content at the age of 4–5 weeks, and blood glucose was measured before and after overnight fasting to confirm lack of diabetes. Insulin was extracted with 4 ml acid–ethanol with an overnight incubation at 4°C. Insulin concentration of the supernatant after centrifuging and diluting (×1,000) was measured with an ELISA-based insulin kit (Mercodia, Uppsala, Sweden). Calculated insulin content was corrected for pancreatic weight or body weight.
Response to exogenous insulin
To detect insulin resistance, an insulin tolerance test (ITT) was performed by injecting human insulin (0.75 IU/kg) i.p. into overnight-fasted mice at the age of 10 weeks and blood glucose levels were measured at 0, 15, 30, 45 and 60 min.
Intraperitoneal glucose tolerance test (ipGTT)
To assess glucose tolerance in NODIns1−/−,Ins2+/+ and NODIns1−/−,Ins2+/−, an ipGTT (2 g glucose/kg body weight) was performed in overnight-fasted mice, and blood glucose levels were measured at 0, 30, 60, 90 and 120 min. We calculated the glucose AUCs according to the trapezoidal rule from the glucose measurements at baseline (0 min), 30, 60, 90 and 120 min.
The pancreata obtained from the mice were fixed in 10% formalin and paraffin-embedded. Paraffin-embedded tissue sections were stained with a monoclonal mouse anti-insulin antibody (Sigma, St Louis, MO, USA) followed by incubation with a peroxidase-labelled anti-mouse IgG antibody (DakoCytomation, Carpinteria, CA, USA), and also with a peroxidase-labelled broad-spectrum secondary antibody (Zymed/Invitrogen Corporation, Carlsbad, CA, USA) for peroxidase staining on adjacent sections. For immunofluorescence staining, the secondary antibody incubation took place with anti-guinea-pig AMCA (blue)-, anti-mouse Texas Red (red)-, and anti-rabbit Cy2 (green)-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA).
Data are shown as means±SEM. Statistical analyses of insulin content, body weight, ITTs and ipGTTs were performed by Mann–Whitney’s U test. Survival curves were analysed with the log-rank test. Statistical tests used PRISM software (Graphpad, San Diego, CA, USA). p<0.05 was regarded as significant.
After 10 weeks of age, female Ins2-KO (NODIns1+/+,Ins2−/− and NODIns1+/−,Ins2−/−) mice had a high prevalence of diabetes (Fig. 1b) and severe insulitis. However, no Ins1-KO (NODIns1−/−,Ins2+/+ and NODIns1−/−,Ins2+/−) mice in either the males or females developed diabetes until 36 weeks of age (Fig. 1).
In this study, we report that male NOD mice with only a single copy of Ins1 (NODIns1+/−,Ins2−/−) develop ‘metabolic diabetes’ at less than 10 weeks of age. The pathogenesis is probably a defect of insulin production with low and heterogeneous expression of insulin that can be corrected by additional transgenic expression of proinsulin. A single copy of Ins2 gene is sufficient to prevent diabetes and is associated with normal insulin content. This suggests that Ins1 gene is a functionally defective gene relative to Ins2 gene and interestingly, there is no compensation sufficient to prevent diabetes with only a single Ins1 gene.
It has been reported that Ins1-KO and Ins2-KO, when on a C57BL/6 strain background and with two copies of each gene encoding insulin, have insulin content in the pancreas similar to wild-type mice, in addition to normal glucose tolerance, because of the compensatory response of insulin transcription [7, 8]. In contrast to these studies, our NODIns1+/+,Ins2−/− mice have lower insulin content compared with wild-type NOD mice. A major difference between these studies may be related to the background genome, namely NOD background in our study vs C57BL/6 background. We are surprised that there is not compensation for insulin deficiency with the single copy of Ins1 and with two copies of Ins1. However, a strain derived from the same colony as the NOD mouse, the Nagoya–Shibata–Yasuda mouse [9, 10], a model of type 2 diabetes, which may share background genome with NOD [11, 12], has no compensational hypertrophy of pancreatic islets despite increasing insulin resistance with ageing . In addition, Kulkarni et al. reported the importance of background genome in the induction of type 2 diabetes .
Male and female NODIns1+/−,Ins2−/− mice had similarly decreased levels of pancreatic insulin content in young non-diabetic mice. However, diabetes incidence between male and female NODIns1+/−,Ins2−/− mice was significantly different. We hypothesise that small but statistically significant differences in response to insulin as evidenced by our i.p. ITT may relate to sex differences in development of overt diabetes of these insulin-deficient NODIns1+/−,Ins2−/− mice. We believe that greater insulin resistance for male mice with marginal insulin production leads to hyperglycaemia. In fact, most animal models of type 2 diabetes (including Nagoya–Shibata–Yasuda mice) show a high prevalence of type 2 diabetes in male mice [9, 10].
In humans, a polymorphism upstream of the INS promoter correlates with insulin expression in the thymus, but has little effect on pancreatic insulin expression [14, 15]. Therefore, loci other than the insulin locus may contribute to pancreatic insulin expression. Further analysis of crosses of NOD and B6 mice with insulin gene knock-outs may help clarify such genetic loci that possibly relate to regulation of insulin production. Of note, humans have only the insulin 2 homologous gene.
Analysing structural features of the gene encoding insulin, Soares et al. reported that Ins1 was generated by an RNA-mediated duplication–transposition event involving a transcript of Ins2 . In addition, only Ins2 is expressed in the NOD thymus with both genes expressed in islet beta cells [3, 16, 17]. This study clearly indicates that Ins1 (on the NOD genetic background) is a functionally defective gene compared with Ins2, and provides a novel model of sex-dimorphic insulin-deficient diabetes.
This work was supported by grants from the National Institutes of Health (DK32083, DK55969, DK62718), Diabetes Endocrine Research Center (P30 DK57516), the American Diabetes Association, the Juvenile Diabetes Foundation, and the Children’s Diabetes Foundation. N. Babaya was supported by the Osaka Medical Research Foundation for Incurable Diseases. M.Nakayama was supported by a fellowship from the Juvenile Diabetes Foundation.