Phenotypic Characterization of Diabetic INSC94Y Transgenic Pigs

Abstract

Background and aims: Islet allotransplantation is already successfully performed for the treatment of type 1 diabetes. However, due to a limited availability of human organs, gradual loss of graft function over time and permanent need for immunosuppression, only a small subgroup of patients can benefit from this treatment option so far. To test alternative strategies, e.g. islet macroencapsulation, diabetic large animal models showing close physiological and anatomical similarities to humans, e.g. pigs, are needed. To date, diabetes in the pig can be induced either by pancreatectomy or chemical agents, e.g. streptozotocin. Due to substantial drawbacks of these existing models, our aim was to generate a transgenic diabetic pig model, expressing a mutant porcine insulin gene (INSC94Y) resembling the human and murine INSC96Y mutation which leads to permanent neonatal diabetes. Materials and methods:INSC94Y transgenic (tg) pigs were generated by somatic cell nuclear transfer using porcine fetal fibroblasts stably transfected with a porcine INSC94Y expression vector (provided by Dr. H. Flaswinkel). The ratio of INSC94Y and INS transcripts in pancreatic tissue was determined by next generation sequencing technology. The total β-cell volume was determined by quantitative-stereological analyses. Results: In total, 7 INSC94Y tg founder boars were born. One founder showed elevated fasting blood glucose levels at the age of 85 days, further increasing over time. The ratio of INSC94Y to INS transcripts (0.751) in pancreas of the diabetic founder was at least 5-fold higher than in the 6 non-diabetic founders. Under insulin treatment, the diabetic boar developed nearly normal and was successfully mated to non-transgenic sows. According to mendelian rules the transgene was inherited to 50% of the offspring and INSC94Y to INS transcript ratio in pancreas of F1-offspring was similar to the founder boar (0.775 ± 0.05; n = 3). Transgenic F1-/F2-offspring exhibited significantly elevated random blood glucose levels within 24 hours after birth vs. controls (194 ± 12 vs. 146 ± 9 mg/dl; p=0.03). Fasting insulin levels were unaltered in 8-day-old INSC94Y tg pigs, but were significantly reduced at the age of 4.5 months vs. controls (2.0 ± 0.42 vs. 5.1 ± 0.77 μU/ml; p< 0.01). Also, 4.5-month-old INSC94Y tg pigs revealed a significantly elevated HOMA-IR index (2.4 ± 0.48 vs. 1.2 ± 0.24; p< 0.05) indicating insulin resistance. Despite the early onset of hyperglycemia, total β-cell volume of 8-day-old tg pigs was not different compared to controls (136 ± 37 vs. 137 ± 15 mm3; p=0.68). In contrast, 4.5-month-old INSC94Y tg pigs revealed a 77% reduced total β-cell volume related to BW (4 ± 0.45 vs. 17.2 ± 2.4 mm3/kg; p< 0.0001). Electron microscopy of β-cells from 4.5-month-old INSC94Y tg pigs showed a very low number of insulin secretory granules and severe dilation of the endoplasmic reticulum (ER) while in 8-day-old tg piglets insulin granules were present and the ER-dilation less severe. The evaluation for the development of diabetic complications is currently ongoing. Conclusion: INSC94Y tg pigs exhibit a stable diabetic phenotype and survive under insulin treatment and are therefore a valuable animal model for numerous applications.