Physiological and morphological characterization of trangenic pigs expressing a dominant-negative glucose-dependent insulinotropic polypeptide receptor (GIPRdn)

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.