Selective deletion of Pten in pancreatic beta cells leads to increased islet mass and resistance to STZ-induced diabetes

Abstract

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing beta cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of beta-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total beta-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in beta cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for beta-cell protection and regeneration therapies.

FIG. 1.

Generation of mouse model carrying Pten deletion in insulin-producing cells. (A) Tissue PCR confirms deletion of Pten in pancreas. Minor leakage in the brain is observed. (B) Immunofluorescent staining shows PTEN staining (red) in the duct (inset) and nuclei of β cells in the control pancreas. In the mutant animals, loss of PTEN immunoreactivity is associated with increased membrane P-AKT staining (green). Images (original magnification, ×60) were overlaid by confocal microscopy. (Right) Western blot analysis of protein lysates isolated from control and mutant islets. Blots were probed with PTEN, p27, phosphor-S6, and β-actin (as a loading control).

FIG. 2.

Pten deletion in insulin-producing cells resulted in increased islet mass. (A) Increased islet mass is observed in the Pten mutant pancreas from late embryonic stage through adulthood. Insets show lower-magnification images with more islets in mutants than in control. Histology images are shown at ×40 magnification. Inserts are at ×10 magnification. Bar, 25 μm. (B) Increases in both islet mass and islet numbers are observed with Pten mutant mice. (Left) Stereology-assisted mapping of selected pancreas sections showed increased islets throughout the pancreas; images are representative of five animals, each 3 months of age. (Right) Islet culture isolated and pooled from four animals per genotype group. Larger and more islets were also observed in these organ cultures of isolated islets. (C) Quantification of islets in control and mutant animals. Islet number and area were averaged from five animals, with three sections from each animal, 120 μm apart. The right-hand panel shows the percentage of islets in each size category.

FIG. 3.

Pten deletion led to increased proliferation and decreased apoptosis. (A) Increased BrdU-positive (green) cells were observed in mutant pancreas at E17.5. Double-positive cells for BrdU (green) and insulin (red) were quantified (bottom). Insets show higher-resolution images of BrdU and insulin staining. Arrows show proliferative activity adjacent to the insulin-positive cells in the mutant pancreas. (B) Apoptosis rate is measured in samples from P15 neonatal mice by TUNEL staining (green). TUNEL-positive cells were colocalized with insulin (red) and quantified (bottom). Inserts show higher-resolution images of TUNEL and insulin stainings. Immunofluorescent images were shown at ×40 magnification. Bar, 25 μm.

FIG. 4.

Pten mutant islets exhibit normal hormone profiles and cell surface markers. (A) Insulin immunohistochemistry staining of pancreas from different age cohorts (original magnification, ×40). Inset, lower-magnification image showing more insulin stained islets in mutants than in controls. Original magnification of insets, ×10. Bar, 25 μm. (B) Control and mutant pancreas stained with insulin (red) and a cocktail of non-insulin islet antibodies (glucagons plus somatostatin plus pancreatic polypeptide, green). (C, left) Insulin secretion from islet culture (pooled from four animals) with low (3 mM, right two bars) and high (20 mM, left two bars) glucose levels, as well as calculated changes (n-fold). The figure shows a representative sample from three independent tests. (C, right) Plasma insulin levels in control and mutant animals after 16-h fast. (D) Cell size measurement. (Left) GLUT2 staining of control and mutant islets. (Right) FACS analysis of control and mutant β cells. Original magnification, ×100. Bar, 25 μm.

FIG. 5.

Pten deletion resulted in fasting hyperglycemia of mutant animals. (A) Fasting plasma glucose levels. Animals fasted for 16 h, and plasma glucose levels were measured (10 animals). (B) Glucose tolerance test. Animals fasted overnight (16 h) before glucose tolerance test. Glucose (2 g/kg of body weight) was injected at time zero, and glucose levels were measured before injection and at 5, 15, 30, 60, and 120 min thereafter (10 animals). (C) Insulin tolerance test. Animals fasted overnight (16 h) before insulin tolerance test. Insulin (1 U/kg of body weight) was injected at time zero; glucose measurements were obtained before injection and at 5, 15, 30, 60, and 90 min thereafter (eight animals). Arrows indicate times at which most mutant animals needed to be rescued, due to hypoglycemic seizures. (D) Fasting plasma triglyceride and nonesterified fatty acid levels in samples from five animals. Data are presented as means ± standard error of the mean. *, statistical significance at P values of ≤0.05.

FIG. 6.

Pten deletion protected mutant animals from STZ-induced β-cell loss. Histology analysis of islets treated with STZ. Untreated pancreas (left); pancreas 7 days after STZ treatment (middle); pancreas 2 months after STZ treatment (right). (A to L) Hematoxylin and eosin (H&E)-stained sections, showing islet morphology. Magnification, ×20 (A to F); ×100 (G to L). Bar, 50 μm (A to F) or 25 μm (G to L). Arrows indicate lymphocyte infiltration. (M to R) Immunohistochemical staining of insulin-positive cells. Bar, 25 μm.

FIG. 7.

Pten deletion increases β-cell proliferation, decreases apoptosis, and prevents hyperglycemia in STZ-treated animals. (A) BrdU pulse-labeling of control and mutant islets 7 days after initial STZ treatment (magnifications, ×40 [left] and ×100 [middle]). Red, insulin; green, BrdU. Quantification is shown in the right-hand panel. (B) Colocalization of PTEN and BrdU⁺ proliferating cells (confocal image magnification, ×60). Red, PTEN; green, BrdU. Insets, higher magnification showing colocolization of BrdU and PTEN in control but not in mutant pancreas. (C) TUNEL staining of control and mutant islets 7 days after initial STZ treatment (magnification, ×100). Red, insulin; green, TUNEL. Quantification is shown in the righthand panel. (D) STZ-induced hyperglycemia. (Left) Glucose levels of six STZ-treated animals. (Right) Percentage of animals free of diabetes after STZ treatment.

FIG. 8.

Pten deletion protects mutant animals from oxidative stress. (A) Isolated islets from control and mutant animals were subjected to 2 mM STZ treatment for 30 min. Islets were allowed to recover overnight. Images were taken the next morning. Images are representative of three experiments. (B) TUNEL staining of pancreatic sections from animals treated with a single dose of 200 mg STZ/kg for 36 h. Experiment used two animals per genotype group.