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Fehlings, Christiane (2010): Physiological and morphological characterization of trangenic pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn): a large animal model of diabetes research. Dissertation, LMU München: Tierärztliche Fakultät



The incretin hormones GIP (glucose-dependent insulinotropic polypeptide) and glucagon-like peptide-1 (GLP-1) mediate the so-called incretin effect, which describes the phenomenon that glucose given orally causes a higher insulin response compared to an isoglycemic intravenous glucose load. The insulinotropic action of GIP is reduced to almost absent in type 2 diabetes patients, while the action of GLP-1 is vastly preserved. GIPRdn transgenic pigs were generated by lentiviral genetransfer to establish a large animal model to investigate the effects of an impaired insulinotropic action of GIP on glucose homeostasis. At the age of 5 months GIPRdn transgenic pigs showed a disturbed oral glucose tolerance going along with reduced insulin secretion. Eleven-month-old GIPRdn transgenic pigs exhibited an impaired intravenous glucose tolerance and reduced insulin secretion as well as a significantly reduced total β-cell volume compared to controls. In this work different age classes (11 weeks, 5 months, 1-1.4 years) of GIPRdn transgenic were investigated to obtain detailed data about physiological and morphological characteristics. To proof specifity of the GIPRdn GIP/Exendin 4 stimulation tests were carried out in 11 week-old GIPRdn transgenic pigs. The insulinotropic action of intravenously injected porcine GIP was impaired, while this of Exendin-4, a GLP-1 mimetic, was enhanced in GIPRdn transgenic pigs compared to controls. Marked alterations in the expression profile of the GIPR and the GLP-1R were excluded as no apparent differences of immunohistochemically stained pancreas sections for GIPR and GLP-1R were detectable between GIPRdn transgenic pigs and controls at any age group. The effects of the impaired insulinotropic action of GIP on glucose metabolism were investigated by oral and intravenous glucose tolerance tests. Eleven-week-old GIPRdn transgenic pigs exhibited significantly reduced oral glucose tolerance with a delay in insulin secretion compared to controls. The area under the insulin curve (AUC insulin) during the first 45 minutes following glucose load was 31% smaller in transgenic pigs compared to controls. The total amount of insulin secretion was not different between the two 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 exhibited a tendency towards reduced intravenous glucose tolerance and reduced insulin secretion in response to an intravenous glucose challenge. 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. However, pancreatic -cell mass was reduced by almost 40% in 5-month-old and by 60% in adult (1 1.4 years) GIPRdn transgenic pigs compared to controls. Furthermore, the cellular composition of the islets was analyzed by quantitative stereological investigations. The relative volumes of α- and -cells in the islets were increased in 1-1.4-year-old GIPRdn transgenic pigs but the absolute volumes of these non-β-cell populations were not different from those of age-matched controls. To investigate the reason for the reduced pancreatic β-cell mass in GIPRdn transgenic pigs, β-cell proliferation and apoptosis rate was determined performing a double-immunohistochemistry for insulin and the proliferation marker Ki67 and cleaved caspase-3, respectively. Eleven-week-old GIPRdn transgenic pigs showed significantly less Ki67 positive cell nuclei compared to controls, whereas proliferation rates in 5 month-old and 1-1.4-year-old GIPRdn transgenic pigs reached no statistical significance. No differences were shown in the apoptosis rates of GIPRdn transgenic pigs compared to controls at any age, although a trend of higher numbers of cleaved caspase-3 positive β-cells was visible in 1-1.4-year-old GIPRdn transgenic pigs. In conclusion, GIPRdn transgenic pigs exhibit a comparable situation as in type 2 diabetes mellitus patients like impaired insulinotropic action of GIP, disturbed glucose tolerance and reduced β-cell mass. Moreover, the results of this work demonstrate an essential role of GIP for the physiological expansion of β-cell mass. In this context GIPRdn transgenic pigs represent a valuable model for further investigations on type 2 diabetes mellitus including diet studies and therapeutic trials.