Diabetes and exocrine pancreatic insufficiency in E2F1/E2F2 double-mutant mice

Abstract

E2F transcription factors are thought to be key regulators of cell growth control. Here we use mutant mouse strains to investigate the function of E2F1 and E2F2 in vivo. E2F1/E2F2 compound-mutant mice develop nonautoimmune insulin-deficient diabetes and exocrine pancreatic dysfunction characterized by endocrine and exocrine cell dysplasia, a reduction in the number and size of acini and islets, and their replacement by ductal structures and adipose tissue. Mutant pancreatic cells exhibit increased rates of DNA replication but also of apoptosis, resulting in severe pancreatic atrophy. The expression of genes involved in DNA replication and cell cycle control was upregulated in the E2F1/E2F2 compound-mutant pancreas, suggesting that their expression is repressed by E2F1/E2F2 activities and that the inappropriate cell cycle found in the mutant pancreas is likely the result of the deregulated expression of these genes. Interestingly, the expression of ductal cell and adipocyte differentiation marker genes was also upregulated, whereas expression of pancreatic cell marker genes were downregulated. These results suggest that E2F1/E2F2 activity negatively controls growth of mature pancreatic cells and is necessary for the maintenance of differentiated pancreatic phenotypes in the adult.

Figure 1

Hyperglycemia and pancreatic atrophy in E2F1/E2F2 compound-mutant mice. (A) Life span data obtained from WT (n = 40), DKO male (DKO M; n = 25), and DKO female (DKO F; n = 25) mice were analyzed using a log-rank nonparametric test (P < 0.01) and expressed as Kaplan-Meier survival curves. (B) Spot blood glucose levels in WT, E2F1^_/_, E2F2^_/_, and DKO mice were determined at the indicated times. Results are the means ± SEM for five to ten animals per sex and genotype. Data were analyzed using the two-tailed t test (***P < 0.001). (C) Insulin and glucagon levels in serum of 2-month-old and 6-month-old WT and DKO mice (n ≥ 7 per genotype; *P < 0.05; **P < 0.01). (D) Insulin-tolerance test was performed in starved animals following intraperitoneal injection of insulin into 5- to 6-month-old hyperglycemic female DKO mice (n = 8) or normoglycemic female WT controls (n = 7). Results are expressed as percentage of initial blood glucose concentration (*P < 0.05). Similar results were obtained for males (not shown). (E) RT-PCR analysis of RNA samples from 2-week-old WT, E2F1^_/_, E2F2^_/_, and DKO (n = 2 per genotype) mice showing expression of E2F1, E2F2, E2F5, and E2F6 in the pancreas of WT mice (E2F3 and E2F4 expression was absent in pancreas). As shown, E2F5 and E2F6 were similarly expressed in the WT and DKO pancreas. The last row shows expression of an internal standard (GAPDH) amplified in a reaction parallel to the test genes. (F) Pancreas weight expressed as fraction of total body weight. Shown are means ± SEM for four to six animals per genotype and sex at each time point analyzed. The nonparametric Mann-Whitney U test was used for comparative analysis (*P < 0.05).

Figure 2

Pancreatic histopathology of E2F1/E2F2 double homozygotes. Pancreas sections obtained from E2F-deficient and WT animals stained with H&E. (A and B) Representative pancreas section of 7-day-old (A) and 2-month-old (B) WT male mice. Magnification, ∞600. (C and D) Representative pancreas section of 2-month-old (C) and 6-month-old (D) E2F1-deficient male mice showing aberrantly large nuclei (marked with arrowheads). Magnification, ∞600. (E_G) Representative pancreas section of 7-day-old (E) and 2-month-old (F and G) DKO male mice. (E and F) Magnification, ∞600. (G) Magnification, ∞400. The acini and islets (marked with an asterisk) in the DKO pancreas are abnormal and have lost their typical tubular organization, which has been replaced by ductal structures. Note the dysplasic changes in acinar cells, characterized by hypertrophy and karyomegaly. (H) Representative pancreas section of a 2-month-old E2F2-deficient male mouse. Magnification, ∞600. The morphology of the pancreas in female E2F-deficient mice was essentially the same as the morphology of the pancreas in male counterparts (data not shown). The sections shown are representative examples from histology for six WT (n = 3 male, n = 3 female), 15 DKO (n = 8 male, n = 7 female), six E2F1^_/_ (n = 3 male, n = 3 female), and six E2F2^_/_ (n = 3 male, n = 3 female) mice.

Figure 3

Ultrastructural analysis of E2F1/E2F2 double-homozygote pancreas sections. Exocrine (A) and endocrine (B) cells in pancreata from WT mice. Insets show the aspect of normal acinar and β cell granules. Pancreas from a 3-month-old male DKO mouse (C_F). Ductal structures composed of transitional cells with ductal cell features, but containing zymogen granules (C), or both zymogen and endocrine granules, can be observed in DKO mice (D). Cells containing both types of granules (pointed arrows, acinar granules; round arrows, endocrine granules) (E and F). Original magnification: (A, B, E, F) ∞3,400; (C) ∞1,100; (D) ∞2600; insets in A and B, ∞10,500.

Figure 4

Immunohistochemical analysis of expression of pancreas-specific protein markers. Shown are representative pancreas sections of 7-day-old and 2-month-old WT and DKO male mice immunostained with Ab’s to insulin, glucagon, and α-amylase. A light hematoxylin counterstaining was performed in all sections (insulin and glucagon, ∞400; α-amylase, ∞200). Similar results were obtained when DKO female mice were analyzed (data not shown).

Figure 5

Immunohistochemical analysis of transcription factor expression in the pancreas. Shown are representative pancreatic islets of 7-day-old WT and DKO male mice immunostained with polyclonal Ab’s to Pdx1, Pax6, and Isl1 (∞400). The expression of all three transcription factors in DKO mice was indistinguishable from age-matched WT mice.

Figure 6

Increased proliferation and apoptosis in pancreas of E2F1/E2F2 compound-mutant mice. In vivo BrdU labeling (as described in Methods) identified pancreatic cells in S phase (appearing brown) in pancreata from 15-day-old mice. As shown, young DKO mice have an increased number of cells in S phase in the pancreas in vivo. Arrowheads in lower left panel indicate BrdU-positive nuclei. The presence of the expected labeling of intestinal crypt cells with BrdU in both genotypes (inset) serves as an internal control for the injection and incorporation of the tracer. Cell death was detected by TUNEL labeling in 15-day-old mice. Arrowheads in lower right panel denote apoptotic nuclei (appearing brown) detected by the TUNEL assay. Virtually no BrdU-positive or TUNEL-positive cells were detected in 15-day-old WT pancreata. The presence of the expected staining of thymocytes with TUNEL in both genotypes (inset) serves as an internal control for the experiment.