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The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted in response to nutrients and enhance glucose-induced insulin secretion. The insulinotropic action of GIP is impaired in type 2 diabetes (T2D) whereas that of GLP-1 is preserved. To evaluate the role of an impaired GIP function in the pathogenesis of T2D in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPRdn) in the pancreatic islets. GIPRdn transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the pancreas were performed in 3 different age groups to investigate the effects of an impaired insulinotropic action of GIP on glucose metabolism and pancreas morphology. The insulinotropic action of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in 11-week-old GIPRdn transgenic pigs compared with control pigs. Eleven-week-old GIPRdn transgenic pigs (n = 5) exhibited significantly reduced oral glucose tolerance (P < 0.05) with a delay in insulin secretion compared with controls (n = 5). The area under the insulin curve (AUC) during the first 45 min following glucose load was 31% smaller (P < 0.05) in transgenic pigs compared with controls. The total insulin secretion capacity was not different between the 2 groups indicating that GIPRdn expression initially only interferes with the incretin effect. This was supported by the fact that intravenous glucose tolerance and insulin secretion in transgenic pigs were not different from controls. Five-month-old GIPRdn transgenic pigs revealed markedly reduced insulin secretion in response to oral glucose challenge (P < 0.01), resulting in significantly elevated glucose levels (P < 0.05). Also, intravenous glucose tolerance and insulin secretion were diminished in 11-month-old transgenic pigs. To determine the reason for the alterations in glucose metabolism, quantitative-stereological analyses of the pancreas were performed. In 11-week-old pigs, transgenic and control groups showed similar β-cell mass (n = 5 in each group). However, pancreatic β-cell mass was reduced by almost 40% (P < 0.05) in 5-month-old pigs and by 60% (P < 0.01) in adult (1 to 1.4 years) GIPRdn transgenic pigs compared with controls. To investigate the reason for the progressive reduction of pancreatic β-cell mass in GIPRdn transgenic pigs, β-cell proliferation rate was determined performing a double-immunohistochemistry for insulin and the proliferation marker Ki67. Eleven-week-old GIPRdn transgenic pigs showed significantly fewer Ki67-positive cell nuclei compared with controls (P < 0.05). In conclusion, GIPRdn transgenic pigs exhibit a comparable situation to T2D, such as impaired insulinotropic action of GIP, disturbed oral and intravenous glucose tolerance, and progressive reduction of β-cell mass. These alterations are at least partly attributable to diminished proliferation of β-cells. Grant support: Deutsche Forschungsgemeinschaft (GRK 1029), Bayerische Forschungsstiftung (492/02).

Among the candidate organs or tissues for pig-to-primate xenotransplantation, pancreatic islets are probably closest to clinical application. Rejection of islet xenografts occurs mainly by cellular mechanisms; that is, T cells. A candidate molecule to protect porcine islets against the attack by human T cells is CTLA-4Ig, which represents the T-cell-inactivating extracellular domain of the human CTLA-4 protein linked to a region of the human immunoglobulin (Ig). This recombinant soluble fusion protein binds to CD80 and CD86, blocking their interaction with CD28 and thereby inhibiting T-cell proliferation and T-cell-dependent antibody production. The survival of human, rabbit, and porcine islets after transplantation into streptozotocin-treated diabetic mice was found to be prolonged after treatment with CTLA-4Ig. In order to facilitate local protection of pig-to-primate islet xenografts, we generated transgenic pigs expressing LEA29Y, a modification of the original CTLA-4Ig with higher potency, specifically in the pancreatic islets. In LEA29Y, 2 amino acids in the binding region of CTLA-4 are altered. The LEA29Y coding sequence was placed under the control of the 1.3-kb core promoter from the porcine insulin gene (INS), and the polyadenylation signal from the bovine growth hormone gene (GH) was added. The construct was linked with a floxed neomycin resistance cassette and transfected into porcine fetal fibroblasts. The cells were selected and stable clones were pooled and used as donors for nuclear transfer. After electrofusion and activation, embryos were transferred to 2 synchronized gilts; 8 piglets survived to term with 7 of them carrying the transgene. Southern blot analysis suggested that the founder animals contain 1 or 2 independent integration sites. Four founders were autopsied at the age of 3 months to evaluate expression of LEA29Y in the pancreatic islets by immunohistochemistry. The ratio of immunohistochemically stained islet cell profiles to all islet cell profiles in the islet profiles visible in the sections was estimated. The staining intensity was also estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Although 2 founders exhibited single LEA29Y-positive islet cells in some pancreatic islet profiles, the other 2 founders showed a high percentage of strongly positive cells in all islet profiles examined, suggesting beta-cell specific expression. Fibroblasts from the latter 2 founders are currently being used for recloning to generate multiple pigs with constitutive expression of LEA29Y in the pancreatic islets. The protective effect of this strategy will be tested by transplanting LEA29Y-expressing porcine islets in diabetic mouse models with a humanized immune system and in diabetic nonhuman primate models. Supported by the Deutsche Forschungsgemeinschaft (FOR 535).

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by specific cell types in the intestine and are responsible for the so-called incretin effect, the phenomenon that an oral glucose load elicits a higher insulin response than does an intravenous glucose load. In patients with type 2 diabetes mellitus the overall incretin effect is reduced. This fact is mostly attributed to a lowered insulinotropic effect of GIP, while the effect of GLP-1 is preserved. In order to better understand the consequences of impaired function of GIP, knockout mice lacking a functional GIP receptor (GIPR–/–) as well as transgenic mice expressing a dominant negative GIPR (GIPRdn) were established. While GIPR–/– mice show only relatively mild changes in glucose homeostasis, GIPRdn mice display a distinct diabetic phenotype due to disturbed development of the endocrine pancreas (Herbach et al. 2005 Regul. Pept. 125, 103–117). To further clarify the underlying mechanisms, we used a novel, highly efficient gene transfer technology based on lentiviral vectors (Hofmann et al. 2003 EMBO Rep. 4, 1054–1060; Hofmann et al. 2006 Mol. Ther. 13, 59–66) to generate transgenic pigs expressing a GIPRdn under the control of the rat Ins2 promoter (RIP). RIP-GIPRdn transgenic pigs develop normally and do not display diabetes mellitus up to at least one year of age. Weekly measured fasting blood glucose levels in transgenic animals did not show a significant difference compared to control pigs. The same was true for monthly determined fructosamine levels. However, RIP-GIPRdn transgenic pigs exhibited reduced insulin release and higher glucose levels than non-transgenic littermate controls in an oral glucose tolerance test. The area under the curve (AUC) for insulin was 49% smaller (P < 0.01) and the AUC for glucose 26% larger (P < 0.05) in RIP-GIPRdn transgenic pigs (n = 5) than in their non-transgenic littermate controls (n = 5). These findings demonstrate that expression of a GIPRdn, which was shown by RT-PCR in isolated pancreatic islets, disturbs the function of GIP in transgenic pigs. Thus we have created a novel, clinically relevant animal model for studying the roles of the GIP/GIPR system. Quantitative morphological studies of the pancreas are being performed to clarify whether GIPR function is essential for pancreatic islet development and maintenance.