Emut Foxa2 is nuclear in hyperinsulinemic mice. Ob/ob mice were injected with the indicated adenovirus, and livers were analyzed 10 days post-injection by cellular fractionation and immunoblotting (A) or immunofluorescence microscopy (B). γ-Tubulin was used as a general loading control, and LSD1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the nuclear and cytoplasmic extraction controls, respectively. Recombinant Foxa2 is shown in green, and red staining indicates the nuclear dye 4,6-diamidino-2-phenylindole (DAPI).

Nuclear/cytoplasmic shuttling of Foxa2 requires a functional NES. A, immunoblots showing cellular fractionation of HepG2 cells stably expressing HA-Emut Foxa2, serum-starved overnight, and stimulated with 500 nm insulin. B, immunoblots showing cellular fractionation of livers from fasted or fed C57Bl/6 mice infected with adenoviruses expressing GFP, Foxa2, T156A, or Emut. Livers of mice that were either fasted for 18 h or fed ad libitum were harvested 5 days post-infection. γ-Tubulin was used as a loading control, and LSD1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels were used as the nuclear and cytosolic markers, respectively.

Emut Foxa2 is inactive in livers of ob/ob mice. Transcript levels of Foxa2 and target genes and metabolic parameters were measured in ob/ob mice injected with Ad-GFP (green), Ad-Foxa2 (black), Ad-T156A (blue), Ad-Emut (red), or Ad-TAE (magenta) after a 6-h fast. A, mean mRNA levels of Foxa2 target genes from livers, n ≥ 3. MCAD, medium chain acyl-CoA dehydrogenase; VLCAD, very long chain acyl-CoA dehydrogenase; CPT1α, carnitine palmitoyltransferase 1α; HMGCS, HMG-CoA synthase. B, mean mRNA levels of Foxa2. Primers were designed to recognize both rat and mouse isoforms. β-Oxidation (C) and ketone body generation (D) by liver mitochondria was measured by the formation of ¹⁴CO₂ and ¹⁴C-labeled acid-soluble products, respectively, from ¹⁴C-palmitic acid. E, blood glucose levels. Time point zero indicates the time of adenovirus injection. F, plasma insulin levels. Values shown represent the mean ± S.E. *, p < 0.05; **; p < 0.01; ***, p < 0.001 by unpaired t test compared with Ad-GFP.

Emut Foxa2 is phosphorylated and inactivated by insulin. A, primary hepatocytes isolated from C57Bl/6 mice were infected with recombinant adenovirus expressing the indicated HA-tagged Foxa2 prior to overnight serum starvation and stimulation with 100 nm insulin. HA-Foxa2 and variants were immunoprecipitated using a mouse anti-HA antibody; immunoblots of whole cell lysates (In) and precipitates (IP) were probed with rabbit antibodies against HA- and phospho-Foxa2 (P-Foxa2). B, HepG2 cells were transfected with expression vectors containing Foxa2 constructs alone or in combination with AKT2. Vector p6xCdx-TkLuc was used as a reporter gene. Luciferase activity, normalized to Renilla luciferase, is shown relative to vector only controls. All experiments were performed in triplicate, and values shown represent the mean of three independent experiments ± S.E. *, p < 0.05; **, p < 0.01 by unpaired t test. C, chromatin immunoprecipitations from serum-starved or insulin-stimulated primary hepatocytes that were infected with the indicated adenoviruses. Chromatin was immunoprecipitated with HA or IgG (control) antibodies. Binding of Foxa2 and mutants to promoter sites in target genes was assayed by PCR. CPT1, carnitine palmitoyltransferase 1; HMGCS, HMG-CoA synthase; l-PK, liver pyruvate kinase.

Foxa2 contains an LMB-sensitive nuclear export signal. A, cellular fractionation of HepG2 cells stably expressing HA-tagged Foxa2. Cells were serum-starved overnight and stimulated with 500 nm insulin with or without pretreatment with 2.5 ng/ml LMB. Foxa2 localization was determined by Western blotting. B, partial sequence alignment of Foxa2 from six different species and the HIV-REV NES (32, 33). The putative NES (red) and AKT phosphorylation site (yellow) are highlighted, with an asterisk indicating the phosphorylated Thr-156. The heat map illustrates the degree of conservation, and numbering is based on the rat Foxa2 amino acid sequence. C, schematic depiction of Foxa2 showing the putative NES in relation to other known domains along with mutant constructs. D, partial sequence alignment of FoxA family members with regions corresponding to the putative NES (red) and AKT phosphorylation site (yellow) highlighted. H, human; R, rat; M, mouse; C, chicken; Xl, Xenopus laevis; Dm, Drosophila melanogaster; II–V, transactivation domains; p, phosphorylation site; NLS, nuclear localization signal (N or C terminus of the DNA binding domain); DBD, DNA binding domain; PGC1β, PGC1β interaction domain; Foxa2, rat wild-type Foxa2; T156A, mutated at residue T156A; Emut, mutated at residues L110A and L113A; TAE, mutated at residues T156A, L110A, and L113A.