(a) Tissue survey of Foxo isoform expression in mice. We hybridized multiple tissue Northern blots (filter membranes; Clontech Laboratories Inc.) with probes encoding the three Foxo isoforms, labeled with ³²P at similar specific activities to obtain readily comparable hybridization signals. (b) Foxo1 expression in cultured β cells. We isolated mRNA from SV40-transformed mouse hepatocytes (lane 1), βTC-3 (lane 2), and HEK293 cells (lane 3), and performed Northern blot analysis using specific probes for Foxo1, Foxo3, and Foxo4. (c) Time course analysis of Foxo1 expression in islets. We isolated mRNA from islets of wild-type mice at 2, 4, 8, and 24 weeks of age. We performed RT-PCR using primers for Foxo1, Foxo3, Foxo4 (not shown, because no amplification could be seen), Irs2, Pdx1, insulin, glucagon, pancreatic polypeptide, and β-actin as indicated in Methods. (d) Islet immunohistochemistry. We performed double immunohistochemistry with anti-Foxo1 and anti-insulin antisera on the same section to localize Foxo1 expression. We visualized the anti-insulin antibody with CY3-conjugated anti–guinea pig IgG, and the anti-Foxo1 with FITC-conjugated anti-rabbit IgG.

(a) Pancreatic histology in mice with targeted null alleles of Irs2 and Foxo1. We stained pancreatic sections from 8-week-old mice of the indicated genotypes with anti-insulin and anti-glucagon antibodies. (b) Foxo1 phosphorylation in Irs2^–/– islets. We transduced islets from wild-type and Irs2^–/– mice with adenovirus encoding hemagglutinin-tagged wild-type Foxo1. Following immunoprecipitation with anti-hemagglutinin antibody, we carried out immunoblotting with anti–phospho-Foxo1^S253. We then stripped and reprobed the blots with anti-Foxo1 antibody. One of three experiments is shown, and mean phosphorylation ± SEM is summarized in the graph above (WT, white bar; Irs2^–/–, black bar), following scanning densitometry and normalization for total Foxo1 protein levels. (c) Islet morphometry. We quantitated β and α cell area in mice of the indicated genotypes using NIH Image 1.60 analysis software. Results are expressed as the percentage of total surveyed area containing insulin- or glucagon-immunoreactive cells. (d) Measurements of mitotic indices in pancreatic cells. We labeled cells traversing S phase of the replication cycle with BrdU in vivo and visualized them in pancreatic sections using double immunohistochemistry with anti-BrdU and anti-insulin antibodies. We calculated labeling indices by counting BrdU-positive cells as percentage of total number of cells within each microscopic field. We scored exocrine acinar cells based on their morphological appearance. Results represent the mean ± SEM of 12 sections from at least four mice for each genotype. For each genotype, we scored at least 50 microscopic fields.

Foxo1 haploinsufficiency restores Pdx1 expression in β cells of Irs2^–/– mice. Real time RT-PCR analysis of gene expression. We isolated mRNA from islets and amplified it with primers encoding the genes indicated above each graph. An asterisk indicates a statistically significant difference (P < 0.05 by ANOVA) between genotypes.

(a) Immunohistochemical analysis of Pdx1 expression in β cells of wild-type, Irs2^–/–, and Irs2^–/–Foxo1^+/– mice. We costained pancreatic sections with anti-Pdx1 (green) and anti-insulin antibodies (red). We show a representative section. (b) Subcellular localization of Foxo1 in Pdx1-positive and Pdx1-negative β cells from wild-type mice. We double-stained adjacent pancreatic sections from 2-week-old mice with antisera against insulin (red) and Pdx1 (mauve pseudocolor), or insulin (red) and Foxo1 (green). We acquired images using a SPOT-RT digital camera and overlaid them using the insulin-stained sections to match the position of the same nucleus in the two sections. Thereafter, we analyzed the colocalization of Pdx1 and Foxo1. We show a ×60 magnification. In the schematic diagram, an empty circle indicates an overlapping nucleus in the two sections. These nuclei have been numbered to facilitate identification of matching cells in the two sections. A mauve nucleus indicates exclusive Pdx1 immunoreactivity; a green nucleus, exclusive Foxo1 immunoreactivity.

(a) Pdx1 expression in βTC-3 cells transfected with c-Myc–tagged wild-type (left column) or ADA-Foxo1 (right column). We performed immunocytochemistry with anti–c-Myc (top row) and anti-Pdx1 (middle row). We visualized anti–c-Myc immunoreactivity with CY3-conjugated anti-mouse IgG (red) and anti-Pdx1 immunoreactivity with FITC-conjugated anti-rabbit IgG (green). We show double staining in the bottom row. (b) Foxo1 localization in pancreatic duct–associated cells. We treated frozen sections for immunohistochemistry with anti-insulin and anti-Pdx1 antisera, followed by incubation in X-gal to detect β-gal expression from the Foxo1 locus. In the top row, we show a representative endocrine islet, demonstrating that X-gal reactivity colocalizes with islets. Occasional X-gal–positive cells can also be visualized in ducts and acini. In the middle and bottom rows, we show that X-gal reactivity in ducts can be detected in cells that do not express insulin and/or Pdx1 (red arrows), as well as cells with insulin and Pdx1 immunoreactivity (black arrows).

Foxo1 binds to the Pdx1 promoter and inhibits Foxa2-induced Pdx1 transcription. (a and b) Gel shift assays. (a) We used nuclear extracts from kidney epithelial LLC cells transduced with Foxo1 adenovirus to test the specificity of binding to the Pdx1 probe. We chose this cell type because it does not express Foxa2 (13), which is abundant in βTC-3 cells. The concentrations of cold competitor used are indicated by the triangle and are 0, 100-, 300-, and 900-fold excess for both wild-type and mutant probes. (b) Antibody-induced supershift. We incubated βTC-3 nuclear extracts with antisera against Foxo1 and Foxa2 or with nonimmune serum (control). I and II correspond to the two main complexes detected in the absence of antiserum, III and IV to the supershifted complexes. (c) Foxa2, but not Foxo1, induces Pdx1 promoter activity. We cotransfected βTC-3 cells with the plasmids indicated at the bottom of the graphs. After 48 hours, we measured luciferase activity and normalized it by SEAP activity relative to the basic pCMV5 vector. (d) Foxo1 inhibits Foxa2-dependent Pdx1 transcription. We cotransfected βTC-3 cells with the Pdx1-luciferase reporter gene and pCMV5 vector or pCMV5-Foxa2. Five hours after transfection, cells were transduced with control adenovirus or with adenovirus encoding ADA-Foxo1 at the indicated moi. After 36 hours, we measured relative luciferase activity. (e) We determined immunoreactive Foxo1 and Foxa2 levels by Western blotting in cells transduced with Foxo1 at different moi’s. Foxo1 was not detectable by this approach in untransduced cells because of its low expression levels.