Disruption of growth factor receptor-binding protein 10 in the pancreas enhances β-cell proliferation and protects mice from streptozotocin-induced β-cell apoptosis

Abstract

Defects in insulin secretion and reduction in β-cell mass are associated with type 2 diabetes in humans, and understanding the basis for these dysfunctions may reveal strategies for diabetes therapy. In this study, we show that pancreas-specific knockout of growth factor receptor-binding protein 10 (Grb10), which is highly expressed in pancreas and islets, leads to elevated insulin/IGF-1 signaling in islets, enhanced β-cell mass and insulin content, and increased insulin secretion in mice. Pancreas-specific disruption of Grb10 expression also improved glucose tolerance in mice fed with a high-fat diet and protected mice from streptozotocin-induced β-cell apoptosis and body weight loss. Our study has identified Grb10 as an important regulator of β-cell proliferation and demonstrated that reducing the expression level of Grb10 could provide a novel means to increase β-cell mass and reduce β-cell apoptosis. This is critical for effective therapeutic treatment of both type 1 and 2 diabetes.

FIG. 1.

Generation of pGrb10KO mice. A: Schematic diagram of the strategy used to target the mouse Grb10 locus. The targeting vector is shown. B: Southern blot analysis of WT, homozygous (Ho), and heterozygous (Het) Grb10 floxed mice. Genomic DNA extracted from the mice was digested with EcoRV and gel-fractionated. The blot was hybridized with 5′- or 3′-probe as indicated. C: Western blot analysis of tissues from pGrb10KO and control mice to confirm the successful knockout of Grb10 in the pancreas. ERK1/2 is shown as a loading control. D: The pancreases of pGrb10KO (KO) and WT littermates were sectioned at 10 µm by using a cryostat. Protein expression in pancreas sections was determined using goat anti-insulin (C-12) antibody (Santa Cruz Biotechnology) and a homemade, affinity-purified rabbit polyclonal antibody to Grb10 (8). The slide images were obtained using a laser confocal fluorescence microscope. Scale bars, 40 µm. B, brain; F, fat; H, heart; K, kidney; L, liver; Lu, lung; M, muscle; P, pancreas; S, spleen. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Pancreatic knockout of Grb10 increases pancreas weight, β-cell mass, and BrdU incorporation rate in β-cells. A: Mouse tissues were isolated from 5-month-old male WT (n = 6) and pGrb10KO mice (n = 5) fed with normal diet and weighed by electronic balance. B: β-Cell size between male pGrb10KO mice and WT littermates (n = 4/group; >500 cells were counted per slide) was measured using Image-Pro Plus software (Version 5.0; Media Cybernetics, Inc). C: Average β-cell mass between 5-month-old male pGrb10KO mice and WT littermates fed with normal diet (WT: n = 7; pGrb10 KO: n = 4). D: Knockout of Grb10 increased β-cell proliferation as determined by BrdU cell proliferation assay. BrdU was injected into 2-month-old male mice (100 mg/kg), and mice were killed 16 h after injection. More than 2,500 insulin-positive cell nuclei were counted per mouse under a Nikon TE2000 microscope (Nikon; WT: n = 3; pGrb10KO: n = 4). Black bars, Grb10 flox^+/−; white bars, pGrb10 KO. All data represent mean ± SEM. *P < 0.05; **P < 0.01 (t test).

FIG. 3.

Pancreatic knockout of Grb10 increases glucose tolerance and insulin secretion. A: GTT was performed in 5-month-old male pGrb10KO mice (n = 9) and age-matched WT littermates (n = 8) fed with normal diet. Blood glucose was withdrawn from the tail vein at 0, 15, 30, 60, and 120 min after intraperitoneal injection of glucose (2 g/kg body weight) and measured using a glucometer. B: GTT was performed on overnight-fasted male pGrb10KO mice and WT littermates (WT: n = 11; pGrb10KO: n = 9) fed with 60% HFD for 16 weeks. Blood glucose after intraperitoneal injection of glucose (2 g/kg body weight) was withdrawn from the tail vein at 0, 15, 30, 60, and 120 min. C: The glucose infusion rates in 3-month-old normal chow diet male pGrb10KO and WT control mice were determined by hyperglycemic clamp experiments (n = 4/group). D: Insulin levels of male pGrb10KO and WT littermates during hyperglycemic clamp experiments (n = 4/group). The data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way repeated-measure ANOVA).

FIG. 4.

Pancreatic-specific knockout of Grb10 increased β-cell mass, insulin granule numbers, and insulin/IGF-1 signaling in islets. pGrb10KO mice and WT littermates were fed with 60% HFD for 16 weeks. A: Average β-cell mass between male pGrb10KO (n = 4) and WT littermates (n = 3). B: Insulin granule numbers in islets of male pGrb10KO (n = 4) and WT littermates (n = 3) were detected. The numbers of docked granules were measured in a cell-surface area of 100 μm². Scale bars, 2 μm. The data represent mean ± SEM. C: The average β-cell size was analyzed from male pGrb10KO and WT littermates (n = 4/group; >500 cells were counted per slide) using Image-Pro Plus software (Version 5.0; Media Cybernetics, Inc). D: Insulin signaling and IGF-1 signaling in β-cell mass. Islets were isolated from HFD-fed male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) or IGF-1 (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and ERK1/2 Thr²⁰²/Tyr²⁰⁴ were determined by Western blot using specific antibodies as indicated. E: mTOR signaling in β-cell mass. Islets were isolated from male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) with or without wortmannin (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and Ser⁴⁷³, ERK1/2 Thr²⁰²/Tyr²⁰⁴, p70S6K Thr³⁸⁹, and 4E-BP1 Thr^37/46 were determined by Western blot using specific antibodies as indicated. Data were quantified by using the Scion Image program (Scion Corporation, Frederick, MD). Black bars, Grb10 flox^+/−; white bars, pGrb10 KO. The data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (t test).

FIG. 4.

Pancreatic-specific knockout of Grb10 increased β-cell mass, insulin granule numbers, and insulin/IGF-1 signaling in islets. pGrb10KO mice and WT littermates were fed with 60% HFD for 16 weeks. A: Average β-cell mass between male pGrb10KO (n = 4) and WT littermates (n = 3). B: Insulin granule numbers in islets of male pGrb10KO (n = 4) and WT littermates (n = 3) were detected. The numbers of docked granules were measured in a cell-surface area of 100 μm². Scale bars, 2 μm. The data represent mean ± SEM. C: The average β-cell size was analyzed from male pGrb10KO and WT littermates (n = 4/group; >500 cells were counted per slide) using Image-Pro Plus software (Version 5.0; Media Cybernetics, Inc). D: Insulin signaling and IGF-1 signaling in β-cell mass. Islets were isolated from HFD-fed male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) or IGF-1 (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and ERK1/2 Thr²⁰²/Tyr²⁰⁴ were determined by Western blot using specific antibodies as indicated. E: mTOR signaling in β-cell mass. Islets were isolated from male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) with or without wortmannin (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and Ser⁴⁷³, ERK1/2 Thr²⁰²/Tyr²⁰⁴, p70S6K Thr³⁸⁹, and 4E-BP1 Thr^37/46 were determined by Western blot using specific antibodies as indicated. Data were quantified by using the Scion Image program (Scion Corporation, Frederick, MD). Black bars, Grb10 flox^+/−; white bars, pGrb10 KO. The data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (t test).

FIG. 4.

Pancreatic-specific knockout of Grb10 increased β-cell mass, insulin granule numbers, and insulin/IGF-1 signaling in islets. pGrb10KO mice and WT littermates were fed with 60% HFD for 16 weeks. A: Average β-cell mass between male pGrb10KO (n = 4) and WT littermates (n = 3). B: Insulin granule numbers in islets of male pGrb10KO (n = 4) and WT littermates (n = 3) were detected. The numbers of docked granules were measured in a cell-surface area of 100 μm². Scale bars, 2 μm. The data represent mean ± SEM. C: The average β-cell size was analyzed from male pGrb10KO and WT littermates (n = 4/group; >500 cells were counted per slide) using Image-Pro Plus software (Version 5.0; Media Cybernetics, Inc). D: Insulin signaling and IGF-1 signaling in β-cell mass. Islets were isolated from HFD-fed male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) or IGF-1 (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and ERK1/2 Thr²⁰²/Tyr²⁰⁴ were determined by Western blot using specific antibodies as indicated. E: mTOR signaling in β-cell mass. Islets were isolated from male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) with or without wortmannin (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and Ser⁴⁷³, ERK1/2 Thr²⁰²/Tyr²⁰⁴, p70S6K Thr³⁸⁹, and 4E-BP1 Thr^37/46 were determined by Western blot using specific antibodies as indicated. Data were quantified by using the Scion Image program (Scion Corporation, Frederick, MD). Black bars, Grb10 flox^+/−; white bars, pGrb10 KO. The data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (t test).

FIG. 4.

Pancreatic-specific knockout of Grb10 increased β-cell mass, insulin granule numbers, and insulin/IGF-1 signaling in islets. pGrb10KO mice and WT littermates were fed with 60% HFD for 16 weeks. A: Average β-cell mass between male pGrb10KO (n = 4) and WT littermates (n = 3). B: Insulin granule numbers in islets of male pGrb10KO (n = 4) and WT littermates (n = 3) were detected. The numbers of docked granules were measured in a cell-surface area of 100 μm². Scale bars, 2 μm. The data represent mean ± SEM. C: The average β-cell size was analyzed from male pGrb10KO and WT littermates (n = 4/group; >500 cells were counted per slide) using Image-Pro Plus software (Version 5.0; Media Cybernetics, Inc). D: Insulin signaling and IGF-1 signaling in β-cell mass. Islets were isolated from HFD-fed male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) or IGF-1 (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and ERK1/2 Thr²⁰²/Tyr²⁰⁴ were determined by Western blot using specific antibodies as indicated. E: mTOR signaling in β-cell mass. Islets were isolated from male pGrb10KO and WT control mice (n = 5/group), serum starved for 2 h, and treated with insulin (100 nmol/L) with or without wortmannin (100 nmol/L) for 30 min. Phosphorylation and the protein levels of Akt Thr³⁰⁸ and Ser⁴⁷³, ERK1/2 Thr²⁰²/Tyr²⁰⁴, p70S6K Thr³⁸⁹, and 4E-BP1 Thr^37/46 were determined by Western blot using specific antibodies as indicated. Data were quantified by using the Scion Image program (Scion Corporation, Frederick, MD). Black bars, Grb10 flox^+/−; white bars, pGrb10 KO. The data represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (t test).

FIG. 5.

Pancreatic-specific knockout of Grb10 enhances resistance to STZ-induced diabetes in mice. Male pGrb10KO and WT control mice (2 months old) were treated with STZ (75 mg/kg) for 5 days. Blood glucose levels (A) and body weight (B) were measured in WT (n = 8) and pGrb10KO (n = 11) mice every 3 days at the same time point (12 p.m. each day) after STZ treatment. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way repeated-measure ANOVA). C: pGrb10KO (n = 6) and WT (n = 8) mice were treated with STZ for 22 days and killed. Pancreases were isolated and weighed. D: pGrb10KO mice (n = 4) and WT littermates (n = 3) were treated with STZ for 22 days and then killed. Serum was collected for insulin measurement. E: Average β-cell mass between pGrb10KO (n = 4) and WT littermates (n = 3). F: Cell apoptosis rate was analyzed by TUNEL assay between pGrb10KO mice (n = 4) and WT littermates (n = 3). Mice were injected with STZ twice daily (0 and 24 h) and killed 24 h later. More than 2,500 insulin-positive cells were counted per mice. Black bars, Grb10 flox^+/−; white bars, pGrb10 KO. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01 (t test).