Glucose Intolerance and Reduced Proliferation of Pancreatic β-Cells in Transgenic Pigs With Impaired Glucose-Dependent Insulinotropic Polypeptide Function
Article Figures & Tables
Figures
- FIG. 1.
Lentiviral vector, Southern blot analyses, and transgene expression. A: The lentiviral vector (LV-GIPRdn) carrying the cDNA of the dominant-negative GIPR (GIPRdn) under the control of the rat Ins2 gene promoter (RIP II). ApaI, restriction site of ApaI; LTR, long terminal repeat; ppt, polypurine tract; SIN, self-inactivating mutation; W, woodchuck hepatitis posttranscriptional regulatory element; probe, probe used for Southern blot analyses; wavy lines, pig genome. B: Southern blot analyses of ApaI-digested genomic DNA isolated from EDTA blood of piglets generated by subzonal injection of LV-GIPRdn (transgenic [tg]) and two nontransgenic littermates (wild type [wt]). Pigs of the F0 generation show either one or two single-copy integration sites of the transgene. Sires 50 and 51 (S 50/S 51) were selected to establish two transgenic lines. Note that pigs of the F1 generation show segregation of the integrants according to the Mendelian rules. C: Analysis of transgene expression (GIPRdn) in isolated porcine islets of Langerhans of transgenic (tg) and nontransgenic littermates (wt) by RT-PCR. β-Actin RT-PCR used for confirmation of reverse transcription efficiency. Due to the use of intron-spanning primers to detect β-actin, two different-sized bands are visible differentiating cDNA and genomic DNA. M: pUC Mix Marker; −RT wt: minus RT wild-type pigs; −RT tg: minus RT GIPRdn transgenic pigs (no signals were obtained from islets of transgenic offspring after omission of the RT step, demonstrating that expressed rather than integrated sequences were detected); +, genomic DNA of GIPRdn transgenic pig; −, aqua bidest.
- FIG. 2.
Body weight gain of GIPRdn transgenic pigs (tg) compared with control pigs (wt). Data are means ± SE. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 3.
Functional analysis of GIPRdn expression by GIP/exendin-4 stimulation test. Reduced insulinotropic action of GIP but enhanced insulinotropic action of exendin-4 in 11-week-old GIPRdn transgenic pigs (tg) compared with nontransgenic control animals (wt). A: Serum insulin levels of GIPRdn transgenic (tg) and control (wt) pigs after intravenous administration of glucose (Glc) ± GIP. B: Serum insulin levels of GIPRdn transgenic (tg) and control (wt) pigs after intravenous administration of glucose (Glc) ± exendin-4 (Exe-4). C and D: Corresponding serum glucose levels for the GIP (C) and exendin-4 (D) stimulation test. 0 min = point of Glc/GIP/exendin-4 administration. Data are means ± SE. *P < 0.05 vs. control; **P < 0.01 vs. control. E and F: Immunohistochemical staining of GIPR (E) and GLP-1R (F) in pancreas sections from 11-week-old GIPRdn transgenic pigs (tg) and nontransgenic control animals (wt) does not provide evidence for differences in receptor abundance. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 4.
Oral glucose tolerance in 11-week-old and 5-month-old GIPRdn transgenic pigs (tg) compared with nontransgenic littermates (wt). A and C: Serum glucose levels; 0 min = point of glucose administration. B and D: Serum insulin levels. AUC glucose/insulin for transgenic pigs (red) and wild-type pigs (blue). Data are means ± SE. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control. Note that in 11-week-old transgenic pigs, there is a delay in insulin secretion leading to a significant reduction of insulin secretion during the first 45 min following oral glucose load, although the total amount of insulin secreted over 120 min is not different from controls. In contrast, 5-month-old transgenic pigs display not only delayed but also reduced insulin secretion. A high-quality digital representation of this figure is available in the online issue.
- FIG. 5.
Intravenous glucose tolerance in GIPRdn transgenic pigs (tg) compared with nontransgenic controls (wt). A, C, and E: Serum glucose levels; 0 min = point of glucose administration. B, D, and F: Serum insulin levels. AUC glucose/insulin for transgenic pigs (red) and wild-type pigs (blue). Data are means ± SE. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control. Note that intravenous glucose tolerance (A) and insulin secretion (B) are not altered in 11-week-old transgenic pigs. In 5-month-old transgenic pigs, a tendency of reduced insulin secretion (D) is observed, while 11-month-old transgenic pigs display a significantly reduced intravenous glucose tolerance (E) due to a significantly reduced insulin secretion (F). (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 6.
Immunohistochemistry for insulin and total β-cell volume in the pancreas [V(β-cell, Pan)] determined with quantitative stereological methods. A–C: Representative histological sections of pancreatic tissue from a control (wt) and a GIPRdn transgenic pig (tg); scale bar = 200 μm. Unaltered total β-cell volume in 11-week-old GIPRdn transgenic pigs (n = 5 per group) (A) but reduction of the total β-cell volume in 5-month-old (n = 4 per group) (B) and young adult (1–1.4 years old) (n = 5 per group) GIPRdn transgenic pigs (C) compared with controls. Data are means ± SE. *P < 0.05 vs. control; **P < 0.01 vs. control. A high-quality digital representation of this figure is available in the online issue.
- FIG. 7.
β-Cell proliferation and apoptosis. A and C: Representative histological sections doublestained for insulin (blue) and Ki67 (brown) (A) or for insulin (light brown) plus cleaved caspase-3 (dark blue; see arrow) (C). B and D: Determination of the number of Ki67− (B) and cleaved caspase-3–positive β-cells (D). Wild type: blue bars, transgenic: red bars. Wild type: n = 5, transgenic: n = 5 for 11-week-old and 1- to 1.4-year-old pigs; wild type: n = 4, transgenic: n = 4 for 5-month-old pigs. Data are means ± SE. *P < 0.05 vs. control; scale bar = 20 μm. Note the significantly (P < 0.05) reduced β-cell proliferation rate in 11-week-old GIPRdn transgenic pigs. (A high-quality digital representation of this figure is available in the online issue.)
Tables
- TABLE 1
Quantitative stereological analyses of the endocrine pancreas of GIPRdn transgenic pigs (tg) and wild-type control pigs (wt)
Parameter 11 weeks (n = 5 wt, 5 tg) 5 months (n = 4 wt, 4 tg) 1–1.4 years (n = 5 wt, 5 tg) ANOVA Means ± SE Means ± SE Means ± SE Group Age Group × age Pancreas weight (g) Wild type 34.5 ± 4.2 115.3 ± 5.6 183.4 ± 13.8 NS <0.0001 NS Transgenic 32.7 ± 3.1 125.7 ± 6.1 206.1 ± 4.9 VV(β-cell/islet) (%) Wild type 69.8 ± 2.2 89.0 ± 1.5 90.2 ± 1.2 0.0066 <0.0001 0.0024 Transgenic 70.8 ± 1.3 87.0 ± 1.2 76.4 ± 3.2* VV(α-cell/islet) (%) Wild type 14.1 ± 1.2 5.0 ± 0.8 5.0 ± 0.7 0.0122 <0.0001 0.0008 Transgenic 12.2 ± 0.7 6.5 ± 0.7 13.8 ± 2.3* VV(δ-cell/islet) (%) Wild type 13.5 ± 2.8 4.3 ± 0.8 2.2 ± 0.6 NS <0.0001 NS Transgenic 13.0 ± 1.0 5.5 ± 0.9 5.8 ± 0.9† VV(pp-cell/islet) (%) Wild type 2.7 ± 0.7 1.8 ± 0.3 2.8 ± 0.8 NS 0.0355 NS Transgenic 3.9 ± 0.5 1.3 ± 0.3 3.8 ± 1.1 V(β-cell/islet) (mm3) Wild type 168.7 ± 29.5 1,088.2 ± 82.0 1,694.6 ± 251.7 0.0002 <0.0001 0.0024 Transgenic 152.1 ± 17.1 664.1 ± 74.5† 663.7 ± 130.5* V(α-cell/islet) (mm3) Wild type 32.6 ± 8.7 58.4 ± 6.3 95.7 ± 17.4 NS <0.0001 NS Transgenic 24.5 ± 1.7 47.7 ± 4.5 112.8 ± 14.5 V(δ-cell/islet) (mm3) Wild type 26.6 ± 3.9 49.5 ± 6.1 36.8 ± 6.3 NS 0.0014 NS Transgenic 25.9 ± 1.8 39.6 ± 3.5 47.5 ± 5.2 V(pp-cell/islet) (mm3) Wild type 6.0 ± 1.8 20.7 ± 2.9 52.4 ± 12.6 NS <0.0001 NS Transgenic 8.2 ± 1.7 9.3 ± 2.1 30.3 ± 7.9 Data were analyzed by the general linear models procedure (SAS Institute) taking the effects of group (wild type, transgenic), age (11 weeks, 5 months, 1–1.4 years), and the interaction group × age into account. For significant effects of these factors P values are indicated in the last three columns. In addition, significant differences between groups within age classes are marked by:
*P < 0.01;
†P < 0.05;
NS, not significant.
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