Original Article

Glucose Intolerance and Reduced Proliferation of Pancreatic β-Cells in Transgenic Pigs With Impaired Glucose-Dependent Insulinotropic Polypeptide Function

  1. Simone Renner1,
  2. Christiane Fehlings1,
  3. Nadja Herbach2,
  4. Andreas Hofmann3,
  5. Dagmar C. von Waldthausen1,
  6. Barbara Kessler1,
  7. Karin Ulrichs4,
  8. Irina Chodnevskaja4,
  9. Vasiliy Moskalenko4,
  10. Werner Amselgruber5,
  11. Burkhard Göke6,
  12. Alexander Pfeifer3,7,
  13. Rüdiger Wanke2 and
  14. Eckhard Wolf1
  1. 1Chair for Molecular Animal Breeding and Biotechnology and Laboratory for Functional Genome Analysis, Gene Center, Ludwig Maximilians University (LMU) Munich, Munich, Germany;
  2. 2Institute of Veterinary Pathology, Faculty of Veterinary Medicine, LMU Munich, Munich, Germany;
  3. 3Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany;
  4. 4Department of Experimental Transplantation Immunology, Surgical Clinic I, University Hospital of Würzburg, Würzburg, Germany;
  5. 5Institute of Anatomy and Physiology, University of Stuttgart-Hohenheim, Stuttgart, Germany;
  6. 6Medical Clinic II, Klinikum Grosshadern, LMU Munich, Munich, Germany;
  7. 7Pharma Center Bonn, University of Bonn, Bonn, Germany.
  1. Corresponding author: Eckhard Wolf, ewolf{at}lmb.uni-muenchen.de.

  1. C.F. and N.H. contributed equally to this article.

Diabetes 2010 May; 59(5): 1228-1238. https://doi.org/10.2337/db09-0519

Article Figures & Tables

Figures

  • FIG. 1.
    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.
    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.
    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.
    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.
    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.
    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.
    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)

    Parameter11 weeks (n = 5 wt, 5 tg)5 months (n = 4 wt, 4 tg)1–1.4 years (n = 5 wt, 5 tg)ANOVA
    Means ± SEMeans ± SEMeans ± SEGroupAgeGroup × age
    Pancreas weight (g)
        Wild type34.5 ± 4.2115.3 ± 5.6183.4 ± 13.8NS<0.0001NS
        Transgenic32.7 ± 3.1125.7 ± 6.1206.1 ± 4.9
    VV(β-cell/islet) (%)
        Wild type69.8 ± 2.289.0 ± 1.590.2 ± 1.20.0066<0.00010.0024
        Transgenic70.8 ± 1.387.0 ± 1.276.4 ± 3.2*
    VV(α-cell/islet) (%)
        Wild type14.1 ± 1.25.0 ± 0.85.0 ± 0.70.0122<0.00010.0008
        Transgenic12.2 ± 0.76.5 ± 0.713.8 ± 2.3*
    VV(δ-cell/islet) (%)
        Wild type13.5 ± 2.84.3 ± 0.82.2 ± 0.6NS<0.0001NS
        Transgenic13.0 ± 1.05.5 ± 0.95.8 ± 0.9†
    VV(pp-cell/islet) (%)
        Wild type2.7 ± 0.71.8 ± 0.32.8 ± 0.8NS0.0355NS
        Transgenic3.9 ± 0.51.3 ± 0.33.8 ± 1.1
    V(β-cell/islet) (mm3)
        Wild type168.7 ± 29.51,088.2 ± 82.01,694.6 ± 251.70.0002<0.00010.0024
        Transgenic152.1 ± 17.1664.1 ± 74.5†663.7 ± 130.5*
    V(α-cell/islet) (mm3)
        Wild type32.6 ± 8.758.4 ± 6.395.7 ± 17.4NS<0.0001NS
        Transgenic24.5 ± 1.747.7 ± 4.5112.8 ± 14.5
    V(δ-cell/islet) (mm3)
        Wild type26.6 ± 3.949.5 ± 6.136.8 ± 6.3NS0.0014NS
        Transgenic25.9 ± 1.839.6 ± 3.547.5 ± 5.2
    V(pp-cell/islet) (mm3)
        Wild type6.0 ± 1.820.7 ± 2.952.4 ± 12.6NS<0.0001NS
        Transgenic8.2 ± 1.79.3 ± 2.130.3 ± 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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May 2010, 59(5)
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Glucose Intolerance and Reduced Proliferation of Pancreatic β-Cells in Transgenic Pigs With Impaired Glucose-Dependent Insulinotropic Polypeptide Function
Simone Renner, Christiane Fehlings, Nadja Herbach, Andreas Hofmann, Dagmar C. von Waldthausen, Barbara Kessler, Karin Ulrichs, Irina Chodnevskaja, Vasiliy Moskalenko, Werner Amselgruber, Burkhard Göke, Alexander Pfeifer, Rüdiger Wanke, Eckhard Wolf
Diabetes May 2010, 59 (5) 1228-1238; DOI: 10.2337/db09-0519
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