The expression and function of glucose-dependent insulinotropic polypeptide in the embryonic mouse pancreas

Abstract

Objective: Glucose-dependent insulinotropic polypeptide (GIP) is a member of a structurally related group of hormones that also includes glucagon, glucagon-like peptides, and secretin. GIP is an incretin, known to modulate glucose-induced insulin secretion. Recent studies have shown that glucagon is necessary for early insulin-positive differentiation, and a similar role for incretins in regulating embryonic insulin-positive differentiation seems probable. Here we studied the role of GIP signaling in insulin-positive differentiation in the embryonic mouse pancreas.

Research design and methods: The ontogeny of the GIP ligand and GIP receptor in the embryonic pancreas was investigated by immunohistochemistry and RT-PCR. GIP signaling was inhibited in cultured embryonic pancreata using morpholine-ring antisense against GIP ligand and receptor, or small interfering RNA (siRNA) for GIP ligand and receptor. Markers of endocrine cells and their progenitors were studied by immunohistochemistry and RT-PCR.

Results: GIP and GIP receptor mRNA were both detected in the embryonic pancreas by embryonic day 9.5 and then persisted throughout gestation. GIP was generally coexpressed with glucagon by immunostaining. The GIP receptor was typically coexpressed with insulin. Morpholine-ring antisense or siRNA against either GIP ligand or GIP receptor both inhibited the differentiation of insulin-positive cells. Inhibition of GIP or its receptor also led to a decrease in the number of Pdx-1-positive and sox9-positive cells in the cultured embryonic pancreas. The number of Pax6- and Nkx2.2-positive cells, representative of developing pancreatic endocrine cells and β-cells, respectively, was also decreased.

Conclusions: GIP signaling may play a role in early embryonic pancreas differentiation to form insulin-positive cells or β-cells.

FIG. 1.

Immunohistochemistry showing the ontogeny of GIP ligand (A, C, and E) and GIPR (B, D, and F). GIP ligand is present in the early embryonic pancreas, and the expression increases during late gestational ages. GIPR expression was weak at early ages, but strong expression was found at later gestational ages (magnification size bars: A and B = 20 μmol/L; C and D = 50 μmol/L; E and F = 100 μmol/L).

FIG. 2.

RT-PCR for GIP ligand showed that mRNA expression of GIP was present as early as E9.5 and continued through later gestational stages and into postnatal stages (A). Semiquantitative RT-PCR showed increasing GIP expression after E9.5 (C). B: RT-PCR of GIPR (GIP Rec) showed that there was consistent expression of GIPR in early and late developmental and postnatal stages. D: Semiquantitative RT-PCR expression pattern for GIPR showed low relative values at days 9–13, followed by a progressive increase in expression over increasing gestational age. Error bars represent SEM.

FIG. 3.

Immunohistochemical staining for GIPR at serial gestational ages E11.5 (A–D), E13.5 (E–H), E15.5 (I–L), and E18.5 (M–P). Definite GIPR-positive staining was first identified at E13.5 and continued through E18.5 (A, E, I, and M). Insulin (B, F, J, and N), glucagon (C, G, K, and O), and merged images (D, H, L, and P) are shown. GIPR expression overlapped primarily with insulin. At E13.5, a few glucagon cells also expressed GIPR (compare 3D and 3H) in addition to the insulin-positive cells that expressed GIPR (magnification size bars: D and H = 20 μmol/L; L and P = 50 μmol/L). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Immunohistochemistry of GIP siRNA-treated embryonic pancreas showed decreased insulin and Pdx-1–positive staining (A, B, E, and F). There was no apparent change in amylase staining (C and G). Area of insulin staining was calculated by using image analysis software, and the positive proportion of the entire pancreas was then calculated. The insulin-staining area of GIP siRNA-treated cultured pancreas was significantly less than the GAPDH siRNA-treated pancreas (n = 3, P = 0.003) (I). Pdx-1–positive cells were also significantly decreased in GIP siRNA-treated pancreas (J). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Semiquantitative RT-PCR was performed for insulin and glucagon (A and B, respectively) and showed significant decreases of mRNA with GIP siRNA. Similarly, the Pax6- and Nkx2.2-positive staining areas were decreased significantly in GIP siRNA-treated pancreas (C and D). Error bars represent SEM.

FIG. 6.

E11 pancreas cultured for 6 days in the presence of morpholine-ring antisense against GIP, GIPR, or control. Immunohistochemistry was performed for insulin and Pdx-1 (A, B, and C), Nkx2.2 and caspase (D, E, and F), BrdU (G, H, and I), and sox9 (J, K, and L). Both ligand and receptor antisense treatment significantly affected endocrine differentiation, as shown by decreased insulin and Nkx2.2 staining (B, C, E, and F). We also saw a decrease in an undifferentiated progenitor cell population suggested by decreased Pdx-1 and sox9 staining. However, the antisense treatment did not induce apoptosis (D, E, and F) or change the proliferation rate (G, H, and I) (magnification size bars: 20 μmol/L). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

GIPR siRNA. Immunohistochemistry of GIPR siRNA-treated cultured pancreas for insulin and Pdx-1 (A and B) is shown. In GIPR siRNA-treated cultured pancreas, there was a significant decrease in the number of insulin-positive staining (C). Pdx-1–positive cells were counted, and there was a significant decrease in GIPR siRNA-treated tissues (D) (magnification size bars: 100 μmol/L). Semiquantitative RT-PCR for GIPR was performed to confirm effective gene knockdown (E). (A high-quality digital representation of this figure is available in the online issue.)