The forkhead transcription factor Foxo1 (Fkhr) confers insulin sensitivity onto glucose-6-phosphatase expression

Abstract

Type 2 diabetes is characterized by the inability of insulin to suppress glucose production in the liver and kidney. Insulin inhibits glucose production by indirect and direct mechanisms. The latter result in transcriptional suppression of key gluconeogenetic and glycogenolytic enzymes, phosphoenolpyruvate carboxykinase (Pepck) and glucose-6-phosphatase (G6p). The transcription factors required for this effect are incompletely characterized. We report that in glucogenetic kidney epithelial cells, Pepck and G6p expression are induced by dexamethasone (dex) and cAMP, but fail to be inhibited by insulin. The inability to respond to insulin is associated with reduced expression of the forkhead transcription factor Foxo1, a substrate of the Akt kinase that is inhibited by insulin through phosphorylation. Transduction of kidney cells with recombinant adenovirus encoding Foxo1 results in insulin inhibition of dex/cAMP-induced G6p expression. Moreover, expression of dominant negative Foxo1 mutant results in partial inhibition of dex/cAMP-induced G6p and Pepck expression in primary cultures of mouse hepatocyes and kidney LLC-PK1-FBPase(+) cells. These findings are consistent with the possibility that Foxo1 is involved in insulin regulation of glucose production by mediating the ability of insulin to decrease the glucocorticoid/cAMP response of G6p.

Figure 1

Northern blot analysis of G6p and Pepck expression in LLC. (a) Time-course analysis of dex/cAMP–induced gene expression. Cell monolayers were incubated with dex/cAMP for the indicated periods of time. Thereafter, mRNA was extracted and size-fractionated on denaturing formaldehyde/agarose gels prior to membrane transfer and Northern hybridization, with the cDNA probes indicated to the left of the autoradiograms. (b) Lack of insulin effect on G6p and Pepck expression. LLC cells were incubated in serum-free medium in the absence (lanes 1 and 4) or presence of dex/cAMP for 4 hours (lanes 2 and 3) or 16 hours (lanes 5 and 6), followed by the addition of insulin (lanes 3 and 6) for 6 hours. mRNA isolation and Northern blot analysis were performed as described in Methods. (c) Mean ± SEM of the percentage of insulin inhibition of dex/cAMP–induced expression at 16 hours was calculated from three independent experiments using densitometric scanning of the autoradiograms. mRNA loading was normalized by subsequent hybridization with a β-actin probe.

Figure 2

Immunodetection of insulin and IGF-1 receptors in LLC cells. Detergent extracts were prepared from SV40-transformed hepatocytes (lanes 1 and 3) and LLC cells (lanes 2 and 4) and immunoprecipitated with antisera against IR (lanes 1 and 2) and IGF-1 receptor (lanes 3 and 4). Equal amounts of protein extracts were used for both experiments.

Figure 3

Expression of Foxo isoforms in hepatocytes and LLC cells. (a) mRNA was isolated from SV40-transformed hepatocytes and LLC cells as indicated in Methods and hybridized with the probes indicated to the left of the autoradiogram. Exposure time was 12 hours for all probes, except Gapdh, which was exposed for 1 hour. (b) Quantification of the data in a. The signal obtained with each probe was quantitated by scanning densitometry of the autoradiogram. To evaluate the relative expression of the three Foxo isoforms, the densitometric data were corrected for the specific activity of each Foxo probe. The highest level of Foxo expression (Foxo1 in hepatocytes) was set to 100%. Data were normalized by hybridization of the blots with a Gapdh probe.

Figure 4

(a) Subcellular localization of Foxo1 and Foxo3 in LLC cells. Cells at approximately 50% confluence were transiently transfected with c-Myc–tagged Foxo1 or c-Myc–tagged Foxo3. After transfection, cells were seeded into four-well slide culture chambers, cultured overnight in serum-free medium, and incubated in the absence (upper panels) or presence (lower panels) of dex/cAMP for 16 hours. Thereafter, they were treated with insulin (100 nM) for the indicated periods of time. Epitope-tagged Foxo1 and Foxo3 were visualized with anti–c-Myc mAb and FITC-conjugated anti–mouse IgG. At least 200 transfected cells were visually scored for localization of transfected proteins in each experiment. Data represent mean ± SEM from three independent transfection experiments. *P < 0.01 between the number of cells with nuclear staining in Foxo1- and Foxo3-expressing cells by one-factor ANOVA. (b) Insulin-induced Foxo1 phosphorylation. LLC cells were transduced with adenovirus encoding WT Foxo1. After 24 hours, cells were stimulated with insulin (100 nM) for the indicated lengths of time. At the end of the incubation, cells were harvested and detergent extracts were subjected to immunoprecipitation with anti-HA Ab, followed by sequential immunoblotting with anti–phospho-specific Ab’s or anti-Foxo1 Ab, as indicated next to each panel. A representative experiment is shown.

Figure 5

Foxo1 expression confers insulin inhibition on G6p, but not on Pepck. (a) Time-course analysis of dex/cAMP effect in cells transduced with Foxo1. LLC cells were transduced with adenovirus encoding WT Foxo1 and incubated with dex/cAMP for the indicated periods of time. At the end of the incubation, mRNA was extracted and analyzed by Northern blot analysis using the relevant cDNA probes. (b) Time-course analysis of insulin effect. LLC cells were transduced with Foxo1 adenovirus. After 48 hours, the medium was replaced with serum-free medium, and incubation was continued overnight. Thereafter, dex/cAMP was added for 16 hours, followed by 6 hours of treatment in the presence (lanes 2–5) or in the absence of insulin (lanes 6–9) for various lengths of time, as indicated. Northern blot analysis was performed as described above. (c) Northern blot of G6p and Pepck mRNAs following transduction of LLC cells with adenoviral vector encoding Foxo1. LLC cells were incubated in serum-free medium overnight before addition of dex/cAMP for 4 hours (lanes 2 and 3) or 16 hours (lanes 5 and 6), followed by insulin stimulation for 6 hours (lanes 3 and 6). Thereafter, mRNA was isolated and Northern blot analysis was performed with cDNA probes encoding G6p (upper panel), Pepck (middle panel), and β-actin (lower panel). A representative experiment is shown, and data from three independent adenoviral transductions are summarized in (d). Mean ± SEM of the percentage of insulin inhibition of dex/cAMP–induced expression at 16 hours was calculated from three independent experiments using densitometric scanning of the autoradiograms. The mRNA loading was normalized by subsequent hybridization with a β-actin probe. *P < 0.05 ANOVA.

Figure 6

Effects of constitutively active and dominant negative Foxo1 mutants on G6p and Pepck in LLC cells and in primary hepatocytes. (a) Expression of HA-tagged Δ256- and ADA-Foxo1 mutants in LLC cells was measured as indicated in Methods. Lane 1, untransduced LLC cells; lane 2, LLC cells transduced with the Δ256 mutant; lane 3, LLC cells transduced with the ADA mutant; lane 4, LLC cells cotransduced with both Δ256 and ADA mutants at a 1:10 moi. (b) Insulin inhibits G6p and Pepck in primary cultures of mouse hepatocytes. Hepatocytes were isolated as described in Methods. Cell monolayers were incubated in serum-free medium for 4 hours before the addition of dex/cAMP for 8 hours. Thereafter, cells were incubated in the absence (lanes 1 and 2) or in the presence of insulin (lane 3) for 6 hours. The data are representative of three separate hepatocyte preparations. (c) Effects of Foxo1 mutants on dex/cAMP–induced G6p and Pepck expression in LLC cells. Cells were transduced with the Δ256 mutant at increasing moi, in the absence (lanes 1–3) or presence (lanes 4–6) of a fixed amount of ADA-Foxo1 mutant. After overnight incubation in serum-free medium, cells were incubated in the presence of dex/cAMP for 8 hours. A representative experiment is shown. (d) Effects of Foxo1 mutants on dex/cAMP–induced G6p and Pepck expression in primary cultures of mouse hepatocytes. After transduction with Δ256- and/or ADA-Foxo1 mutant adenoviruses, hepatocyte cultures were incubated in serum-free medium for 4 hours before the addition of dex/cAMP for 8 hours. A representative experiment is shown, and data from three separate experiments for each cell type are summarized in Figure 7.

Figure 7

Summary of the effects of the constitutively active and dominant negative Foxo1 mutants on dex/cAMP–induced gene expression. Data from three independent experiments in LLC cells and three different hepatocyte preparations are expressed as mean ± SEM of the percentage of inhibition of dex/cAMP–induced G6p (a and c) and Pepck (b and d) expression by the dominant negative Δ256 Foxo1 mutant in the absence (filled bars) or presence (open bars) of the constitutively active ADA Foxo1 mutant. (a and b) Summary of the results in LLC cells. (c and d) Summary of the results in primary hepatocyte cultures. *P < 0.01 by ANOVA.