The flavones apigenin and luteolin induce FOXO1 translocation but inhibit gluconeogenic and lipogenic gene expression in human cells.

The flavones apigenin (4',5,7,-trihydroxyflavone) and luteolin (3',4',5,7,-tetrahydroxyflavone) are plant secondary metabolites with antioxidant, antiinflammatory, and anticancer activities. We evaluated their impact on cell signaling pathways related to insulin-resistance and type 2 diabetes. Apigenin and luteolin were identified in our U-2 OS (human osteosarcoma) cell screening assay for micronutrients triggering rapid intracellular translocation of the forkhead box transcription factor O1 (FOXO1), an important mediator of insulin signal transduction. Insulin reversed the translocation of FOXO1 as shown by live cell imaging. The impact on the expression of target genes was evaluated in HepG2 (human hepatoma) cells. The mRNA-expression of the gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pc), the lipogenic enzymes fatty-acid synthase (FASN) and acetyl-CoA-carboxylase (ACC) were down-regulated by both flavones with smaller effective dosages of apigenin than for luteolin. PKB/AKT-, PRAS40-, p70S6K-, and S6-phosphorylation was reduced by apigenin and luteolin but not that of the insulin-like growth factor receptor IGF-1R by apigenin indicating a direct inhibition of the PKB/AKT-signaling pathway distal to the IGF-1 receptor. N-acetyl-L-cysteine did not prevent FOXO1 nuclear translocation induced by apigenin and luteolin, suggesting that these flavones do not act via oxidative stress. The roles of FOXO1, FOXO3a, AKT, sirtuin1 (SIRT1), and nuclear factor (erythroid-derived2)-like2 (NRF2), investigated by siRNA knockdown, showed differential patterns of signal pathways involved and a role of NRF2 in the inhibition of gluconeogenic enzyme expression. We conclude that these flavones show an antidiabetic potential due to reduction of gluconeogenic and lipogenic capacity despite inhibition of the PKB/AKT pathway which justifies detailed investigation in vivo

Dose-dependent induction of FOXO-GFP translocation by apigenin and luteolin, competition by insulin.

<p>Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) treated with apigenin (A), luteolin (B) 1–100 µM for 2 h −/+ addition of insulin 100 nM after 30 minutes. Cells were fixed and stained with DAPI. Fluorescence microscopic detection of nuclei, segmentation of cells, quantification of GFP intensities measured in nuclear and cytoplasmic areas and calculation of the GFP-ratio nucleus/cytoplasm were performed for all FOXO-GFP expressing cells by BD Image Data Explorer. Nonlinear regression was performed with Graph Pad Prism. Results are presented as mean ± SEM of quadruplets with *(p<0.05) significant differences vs. control (ANOVA + Post Hoc Tests). (A) Dose-dependent accumulation of FOXO1 in nuclei induced by apigenin 1–100 µM shown as mean ratio of FOXO1 nucleus/cytoplasm + SEM (n = 4) *p<0.05 (Dunnett T3) and EC50 = 13 µM calculated by nonlinear regression from sigmoidal dose response. Reversion by insulin 100 nM induced FOXO1 translocation from nuclei into the cytoplasm. (B) Luteolin induced FOXO1 nuclear accumulation shown as mean ratio of FOXO1 nucleus/cytoplasm + SEM (n = 4) *p<0.05 (Bonferroni) with EC50 = 12 µM and competing insulin effect.</p

Time- and dose-dependent modulation of gene expression in HepG2 cells induced by apigenin and luteolin.

<p>A–D: Human hepatoma cells (HepG2) were cultivated in EMEM + 10% FBS and starved without FBS 16 h before stimulation. Apigenin and luteolin were applied in the range of 1–100 µM diluted in EMEM. Incubation of HepG2 was performed for 2 h and 24 h respectively. Total RNA was extracted with Nucleospin RNA II isolation kit and reverse transcribed with the High capacity cDNA reverse transcription kit for quantitative realtime PCR (qRT-PCR) in triplicates using the Power SYBR green PCR master mix with primers pairs described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104321#pone-0104321-t001" target="_blank">Table 1</a>. qRT-PCR was run in triplicates using cDNA from control cells treated with DMSO 0.5% for standard dilutions. Levels of mRNA were normalized to the houskeeping gene ribosomal protein (RPL32). Three independent experiments were performed with different passages of HepG2. Results are presented as fold mRNA expression normalized to control expression as means ± SEM and significances versus control *(p<0.05). Gluconeogenic (A) phosphoenolpyruvate carboxykinase (PEPCK) and (B) glucose-6-phosphatase (G6Pc), lipogenic (C) fatty-acid synthase (FASN) and (D) acetyl-CoA-carboxylase (ACC).</p

FOXO1-GFP translocation induced by apigenin and luteolin in the presence of N-acetyl-L-cysteine, reduced reversion by insulin.

<p>Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) were incubated with the antioxidant N-acetyl-L-cysteine (NAC) 5 mM and 25 mM for 30 minutes before treatment with apigenin 30 µM and luteolin 30 µM −/+ insulin 100 nM for 2 h and 24 h respectively. Cells were fixed and stained with DAPI. Experiments were performed in quadruplets and fluorescence microscopic analyses performed with the BD Pathway 435 system, BD Attovision and BD Image Data Explorer. GFP-ratios nucleus/cytoplasm were normalized to untreated control cells. Results are presented as relative FOXO1 translocations resulting from means of ratios GFP Nuc/Cyt normalized to control ± SEM. Oneway ANOVA and Dunnett T3 significances are shown *(p<0.05), **(p<0.01), and ***(p<0.001) in blue for flavone induced FOXO1 nuclear accumulation vs control DMEM, in red for insulin induced FOXO1 export into cytoplasm, and in yellow for NAC effect on FOXO1 translocation.</p

FOXO1-GFP translocation in stably transfected U-2 OS (human osteosarcoma cells).

<p>Intracellular localization of the forkhead box transcription factor O1 labelled with green fluorescent protein FOXO1-GFP visualized by fluorescence microscopy after nuclear staining with DAPI (colour coded red). Merged images in left panel: Cytoplasm green from FOXO1-GFP, nuclei red→orange→yellow→green depending on GFP overlay from FOXO1-GFP accumulated in nuclei (A–C). Right panel GFP images (A′–C′). (A, A′) Transfected U-2 OS cells with pEGFP-FOXO1 (after 1 h starvation in DMEM without FBS) treated with DMSO 0.15% (control) 2 h with cytoplasmic and perinuclear localization of FOXO1-GFP. (B, B′) Apigenin 30 µM in 0.15% DMSO induced nuclear accumulation of FOXO1-GFP. (C, C′) Luteolin 30 µM in DMSO 0.15% induced translocation of FOXO1-GFP from cytoplasm into nuclei in nearly all transfected U-2 OS cells with stable expression of GFP tagged FOXO1.</p

Cell proliferation assay.

<p>Vitality of HepG2 cells treated with apigenin and luteolin 1 µM–100 µM for 24 h.</p><p>HepG2 survival was measured using the CellTiter96 Aqueous ONE Solution from Promega applied to cell-cultures after 24 h treatment with flavones apigenin and luteolin in the range from 1–100 µM. Following incubation with 20 µM (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS)/phenazine methosulfate (PMS - electron coupling reagent) for 4 h at 37°C, the optical density of the MTS bioreduction product formazan was measured at 490 nm. Means of OD-values were normalized to mock treated cells (100% survival) and analyzed by Oneway ANOVA for 0, 1, 2, 5, 10, 20, 50, and 100 µM apigenin and luteolin respectively with Levene statistics for analyses of variance. Significant reductions of vitality were found for 100 µM apigenin or luteolin vs 0 µM in mock treated control cells with DMSO 0.5% analyzed by Dunnett T3 (unequal variance for apigenin) or Bonferroni (equal variance for luteolin) respectively.</p

Time-dependent induction of FOXO-GFP translocation by apigenin A) and luteolin B) obtained by fluorescence-microscopic life cell imaging.

<p>Stably transfected human osteosarcoma cells with FOXO1-GFP (U2OS-FOXO1-GFP) were incubated with apigenin 10 µM (A) and transiently transfected human hepatic cells with FOXO1-GFP (HepG2-FOXO1-GFP) were incubated with luteolin 10 µM (B), respectively, for 1 h at 37°C in an incubation chamber connected to the inverted fluorescence microscope Axio Observer.Z1 from Zeiss. Images were taken every minute with the filter for GFP up to 60 minutes. FOXO1-GFP translocation with nuclear accumulation of FOXO1 is shown in a time-dependent course.</p

Effect of apigenin on the auto-phosphorylation of the IGF-1 receptor.

<p>Human embryonic kidney (HEK) cells overexpressing the insulin-like growth factor receptor (IGF-1R) were incubated with different concentrations of IGF-1 in the presence or absence of apigenin 20 µM for 16 minutes. Stimulated cells were lysed and transferred to a normal ELISA-sandwich assay using a mouse monoclonal IGF-1R antibody as a capture antibody and anti-mouse horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody as a detection antibody to quantify the phosphorylation of IGF-1 receptor. Bars show means ± SEM of two experiments performed in duplicate.</p

Posttranslational phosphorylation in the AKT-signaling cascade.

<p>A–E: Modifications of molecules at nodal points of AKT intracellular signaling were analysed in lysates of human hepatoma (HepG2) cells treated with apigenin 20 µM, luteolin 20 µM or DMSO 0.1% for 30′ ± pretreatment with insulin 100 nM for 15′. Cell lysis was performed in presence of phosphatase inhibitors and lysates analyzed for protein phosphorylation or cleavage using the PathScan Intracellular Signaling Array Kit (fluorescent readout) from Cell Signaling technology. Data are shown as means of integrated intensities of three independent experiments (n = 3) + SEM normalized to untreated control cells and significances shown as # p<0.05 and ## p<0.01 vs mock stimulated HepG2 with DMSO 0.1% (T-Test) or * p<0.05, ** p<0.01 and ***p<0.001 as indicated (Oneway ANOVA and posthoc Bonferroni or Dunnett T3 multiple comparisons). (A) protein kinase B PKB/AKT (Thr308), (B) AKT (Ser473), (C) proline-rich AKT/PKB substrate 40 kDa PRAS40 (Thr246), (D) mammalian target of rapamycin mTor (Ser2448), (E) p70S6 kinase p70S6K (Thr389), (F) ribosomal protein S6 (Ser235/236).</p

Primer sequences for qRT-PCR.

<p>Primer design by Primer Express for qRT-PCR based on human mRNA sequences.</p