Permanent neonatal diabetes in INS(C94Y) transgenic pigs

Abstract

Mutations in the insulin (INS) gene may cause permanent neonatal diabetes mellitus (PNDM). Ins2 mutant mouse models provided important insights into the disease mechanisms of PNDM but have limitations for translational research. To establish a large animal model of PNDM, we generated INS(C94Y) transgenic pigs. A line expressing high levels of INS(C94Y) mRNA (70-86% of wild-type INS transcripts) exhibited elevated blood glucose soon after birth but unaltered β-cell mass at the age of 8 days. At 4.5 months, INS(C94Y) transgenic pigs exhibited 41% reduced body weight, 72% decreased β-cell mass (-53% relative to body weight), and 60% lower fasting insulin levels compared with littermate controls. β-cells of INS(C94Y) transgenic pigs showed a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum. Cataract development was already visible in 8-day-old INS(C94Y) transgenic pigs and became more severe with increasing age. Diabetes-associated pathological alterations of kidney and nervous tissue were not detected during the observation period of 1 year. The stable diabetic phenotype and its rescue by insulin treatment make the INS(C94Y) transgenic pig an attractive model for insulin supplementation and islet transplantation trials, and for studying developmental consequences of maternal diabetes mellitus.

FIG. 1.

Generation of diabetic INS^C94Y transgenic (tg) pigs (expression construct, Southern blot, glucose levels, transgene expression). A: Expression cassette including the porcine INS gene with the Cys→Tyr exchange at amino acid position 94, essential regulatory elements, and a neomycin selection cassette (neo). Restriction site of BamHI and binding site of the probe (bar) used for Southern blot analysis are indicated. B: Southern blot analysis of BamHI digested genomic DNA from INS^C94Y tg pigs and littermate control animals (wt) showing different integration sites in founders 9725, 9726, 9727, 9728, 9745, 9746, and 9747. Tg offspring (1334, 1336, 1340, 1341) of founder 9747 show the same integration pattern as founder 9747, demonstrating a single integration site. C: Fasting blood glucose levels of INS^C94Y tg founder boars showing hyperglycemia in founder 9747 progressively deteriorating over time, whereas blood glucose levels of the six other founders remain within the reference range (70–115 mg/dL, depending on the laboratory); arrow indicates start of insulin therapy. D: Quantification of INS^C94Y and wt INS transcripts in pancreatic tissue of INS^C94Y tg pigs by next- generation sequencing of RT-PCR amplicons. Founder 9747 shows at least five-fold higher expression of the mutant INS^C94Y compared with the other six founders (F0) and similar expression compared with its F1 offspring (F1; n = 3). Inset: RT-PCR products of INS^C94Y/INS transcripts in pancreatic tissue of all INS^C94Y tg founders (left) and founder 9747, as well as its offspring (right). M, pUC Mix Marker; gDNA, genomic DNA; H₂O, Aqua bidest.

FIG. 2.

Glucose control and insulin secretion in INS^C94Y transgenic (tg) pigs. A: Random blood glucose levels (left) of INS^C94Y tg pigs and littermate controls (wt) up to age 8 days. Glucose levels of tg pigs are significantly elevated 24 h after birth. Random and fasting blood glucose levels (right) of tg and wt pigs show deterioration of glucose control in tg pigs with increasing age. B: Fasting insulin levels of 8-day-old and 4.5-month-old INS^C94Y tg pigs and littermate controls reveal hypoinsulinemia in 4.5-month-old INS^C94Y tg pigs vs. littermate controls. C: The 42-h continuous glucose monitoring of an insulin-treated INS^C94Y tg pig and a nontransgenic control (wt) pig. Insulin dose per day: 0.5 to 1 units per kilogram of body weight. IGC, interstitial glucose concentration. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control.

FIG. 3.

Reduced β-cell mass and ultrastructural changes in INS^C94Y transgenic (tg) pigs. A: Immunohistochemical staining for insulin in 8-day-old pigs and double immunohistochemical staining for insulin (pink) and glucagon, somatostatin, and pancreatic polypeptide (brown) in 4.5-month-old pigs. Representative histological sections of pancreatic tissue from a control (wt) and an INS^C94Y tg pig. Scale bar = 50 µm. B: Quantitative stereological analyses of the pancreas. Unaltered total β-cell volume (V_{(β-cell, Pan)}) in 8-day-old INS^C94Y tg pigs (n = 4 per group) and significant reduction of V_{(β-cell, Pan)} in 4.5-month-old INS^C94Y tg pigs (n = 7 per group) compared with littermate controls. Data are means ± SEM. ***P < 0.001 vs. control. C: Transmission electron microscopy of pancreatic tissue from a representative 8-day-old and 4.5-month-old INS^C94Y tg and a littermate control (wt) pig. In 4.5-month-old INS^C94Y tg pigs, only a few insulin granules (arrows) and severe dilation of endoplasmic reticulum (*) are visible, whereas in 8-day-old tg pigs insulin granules are present and dilation of the endoplasmic reticulum is less severe.

FIG. 4.

Diabetes-related alterations of INS^C94Y transgenic (tg) pigs. A: Body weight gain in INS^C94Y tg pigs (red circles; n = 9) and controls (blue circles; n = 7) up to age 4.5 months. B: Increased relative (rel.) kidney weight (percent body weight) and ratio of mean glomerular volume to body weight (BW) (v[glom] / BW [µm³/kg] × 10³) in 4.5-month-old INS^C94Y tg pigs vs. littermate controls (wt; n = 7 per group). C: Unaltered true harmonic mean thickness of the GBM in 4.5-month-old INS^C94Y tg pigs compared with littermate controls (n = 3 per group) and representative transmission electron microscopic sections. Scale bar, 500 nm. D: Representative histological sections (left) of a tibial nerve fascicle of a 4.5-month-old INS^C94Y tg pig and a littermate control demonstrating unchanged fiber density without structural changes in axons and myelin. Azur II-methylene blue-safranin staining. Scale bar, 100 µm. Single fiber teasings (right) showing unchanged fiber myelinisation and morphology of nodes of Ranvier. Scale bar, 100 µm. E: Eye lenses of 8-day-old and 4.5-month-old INS^C94Y tg pigs and littermate controls (wt) show a progressive cataract with increasing opacity in tg pigs, whereas lenses of wt pigs remain clear. Data are means ± SEM. *P < 0.05 vs. control; ***P < 0.001 vs. control. US, urinary space; CL, glomerular capillary lumen; p, podocyte foot process.