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J. Biol. Chem., Vol. 280, Issue 21, 20589-20595, May 27, 2005
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From the Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032
Received for publication, November 1, 2004 , and in revised form, March 18, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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FoxO activity is subject to complex regulation by growth factors and cellular stress. The former inhibit FoxO via serine-threonine phosphorylation and nuclear exclusion (10). The latter causes FoxO acetylation, thus promoting the interaction between FoxO and Sirt1, the mammalian ortholog of Sir2.1 (4, 5, 11, 12). However, the effect of Sirt1-dependent deacetylation on FoxO function remains somewhat controversial, with most (4, 5, 12), but not all (11), studies suggesting that deacetylation increases FoxO-dependent transcription.
In this study, we sought to uncover the mechanism by which stress-induced deacetylation affects FoxO activity. To this end, we studied FoxO translocation using live cell imaging, coupled to measurements of protein kinetics with fluorescence recovery after photobleaching (FRAP)1 and fluorescence loss in photobleaching (FLIP) experiments (13). We also measured expression of FoxO1 target genes and glucose production in hepatocyte cultures. Our findings are consistent with a model in which deacetylation promotes FoxO nuclear retention and increases FoxO-dependent glucose production, thus providing evidence for a mechanism to regulate FoxO activity via subnuclear targeting.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Culture—We cultured Sv40 hepatocytes or H4IIE rat hepatoma cells in 
-minimal essential medium supplemented with 4% fetal calf serum, 1% Pen-Strep, and 200 nM dexamethasone at 37 °C in a 5% CO2 incubator. We lysed cells in a buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% Na deoxycholate, 5 mM Na orthovanadate, 10 mM NaF, 1 mM dithiothreitol, 2 mg/ml pepstatin, 20 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 10 mM nicotinamide, and 2 µM trichostatin A (TsA). Immunoprecipitation was carried out using anti-HA (12CA5; Roche Applied Science) and anti-GFP monoclonal antibodies (A-11120; Molecular Probes). We processed a 50-µg aliquot of immunoprecipitated proteins for electrophoresis and Western blotting using antisera against FoxO1 (H-128; Santa Cruz), Sirt1 (Upstate Biotechnology), phospho-Ser253 FoxO1, Akt, phospho-Ser473-Akt, or acetyl lysine (Cell Signaling). We employed the following concentrations of reagents: H2O2 0.5 mM, insulin 100 nM, resveratrol 10 µM, nicotinamide 10 mM, and Trichostatin A 2 µM. All were from Sigma. We used Leptomycin B (LC Laboratories) at 20 nM. We cultured HEK 293 cells in minimal essential medium, 10% fetal calf serum, 1% Pen-Strep and transduced them with an adenoviral vector containing HA-tagged wild type FoxO1 24 h before the experiment (14).
Image Acquisition and Analysis—We plated and observed cells in LabTek chambers (Nalgene). We captured confocal microscope images on a LSM 510 META microscope (Zeiss) using 488 nm laser excitation for GFP and 568 for rhodamine. Images were captured with a x63 1.2 water objective and an open pinhole to collect fluorescence from the entire depth of the cell. We analyzed images with the Image J software (NIH). We measured fluorescence separately within the nucleus and the cytoplasm. To calculate relative nuclear fluorescence, we divided nuclear fluorescence by the total amount of cellular fluorescence. Time lapse sequences of images of FoxO1-GFP nuclear translocation were captured with a x63 1.2 water objective at 10-s intervals (15). All quantitative values represent averages from at least 20 cells from five independent experiments.
Immunofluorescence Microscopy—We prepared cells for immunofluorescence microscopy by transfecting FoxO1-GFP by FuGENE 6 (Roche Applied Science) and staining for Sirt1 in LabTek chambered slides (Nalgene). We fixed cells in 4% paraformaldehyde in PBS for 15 min, washed using PBS, and treated them with 0.1 M Tris, pH 8.1, for 10 min. Thereafter, we permeabilized cell membranes with PBS and 0.2% Triton X-100, washed in PBS, and incubated in blocking buffer containing 1% bovine serum albumin for 10 min. Subsequently, we incubated cells with Sirt1 antiserum (Cell Signaling) in PBS containing 1% bovine serum albumin for 1 h at room temperature, washed in PBS, and incubated with a rhodamine-conjugated secondary antibody for 1 h at room temperature. Slides were mounted using Fluoromount-G (Southern Biotech).
FRAP Analysis—We plated, transfected, and observed cells in LabTek II chambers (Nalgene). We performed selective photobleaching of the nucleus on a LSM 510 META microscope (Zeiss) using 488 nm laser excitation for GFP and a x63 1.2 water objective. We photobleached a defined region (outlined in the figures) at full laser power (100% power for 15 s). Thereafter, we monitored fluorescence recovery by scanning the whole cell at low laser power (4% power every 7.5 s). We observed no photobleaching during recovery. For FLIP experiments, we repeatedly imaged and bleached cells at 20-s intervals. Bleaching and imaging settings were identical to the ones used in FRAP. We generated FRAP recovery curves from background-subtracted images. We determined the total cell fluorescence for each image using Image J software (NIH) and compared the percentage of nuclear fluorescence to the initial value to account for the amount lost during the bleach pulse (15). All quantitative values represent averages from at least 10 cells from five independent experiments.
Gene Expression Studies—We isolated RNA using RNeasy mini-kit (Qiagen), followed by DNase (Qiagen) digestion to remove contaminating DNA. We carried out first strand synthesis with the Superscript system (Invitrogen) according to the manufacturer's instructions. We carried out real-time quantitative PCR using a DNA Engine Opticon 2 System instrument (MJ Research) and DyNAmo SYBR Green quantitative PCR kit (Finnzymes). Reactions were carried out in triplicate, using 
-actin or Hprt to normalize mRNA content (16). Primer sequences are available upon request. We employed standard PCR conditions.
Measurement of Glucose Release from H4IIE Hepatoma Cells—To measure glucose production, we removed the growth medium, washed cells with PBS three times, and incubated with glucose- and serum-free -minimal essential medium for 1 h. We then washed with PBS and incubated cells in glucose- and serum-free -minimal essential medium supplemented with 200 nM dexamethasone, 20 µM forskolin, 2 mM glutamine, 16 mM lactate, and 4 mM pyruvate (Sigma) (17). We measured glucose concentration in triplicate using the Amplex red glucose assay (Invitrogen). Results are the mean of two independent experiments performed in triplicate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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45 min in H2O2-treated cells and achieved steady-state levels (nuclear percentage levels above 70%) at 120 min, with a t
= 80 min (Fig. 1C). We obtained identical results in Cos7 cells (Fig. 1D). The changes in FoxO1 distribution could not be accounted for by perturbations to the fluorophor, as the localization of GFP expressed in the same cells was unaffected by oxidative stress (Fig. 1E). The delayed nuclear translocation of FoxO1-GFP during peroxide treatment may be caused by a transient increase in Akt activity (data not shown). This may account for the apparent discrepancy between reports of FoxO phosphorylation (18) and nuclear translocation (5) in response to H2O2. To further clarify this point, we measured whether insulin treatment could override the effect of H2O2. Addition of insulin promoted FoxO1-GFP localization to the cytoplasm (Fig. 1, A and B) with a rapid time course (t = 13 min, data not shown) but was unable to do so in the presence of H2O2 (Fig. 1, A and B). Consistent with these results, we observed that insulin-induced FoxO phosphorylation was abolished in H2O2-treated cells (Fig. 1F).
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= 15 min (data not shown), suggesting that the interaction of FoxO1 with Sirt1 contributes to its nuclear distribution. Indeed, co-immunoprecipitation experiments indicated that Sirt1 could be detected in FoxO1 immunopellets following H2O2 treatment (Fig. 2C, middle lane). Conversely, treatment with the Sirtuin inhibitor nicotinamide (NAM) and the class I/II histone deacetylase (HDAC) inhibitor TsA prevented Sirt1 binding to FoxO1 (Fig. 2C, right lane). Furthermore, immunohistochemical studies showed that FoxO1-GFP colocalized with Sirt1 in the nucleus during H2O2 treatment (Fig. 2D). Combined treatment with resveratrol and H2O2 also caused nuclear translocation (Fig. 2, A and B). To determine whether the effect of oxidative stress on FoxO1 is mediated solely through Sirtuins or also through class I/II HDACs, we compared the effects of NAM and TsA on FoxO1-GFP translocation. Treatment with either TsA or NAM failed to affect FoxO1-GFP localization. TsA had no effect on H2O2-dependent nuclear accumulation of FoxO1, whereas NAM completely reversed the effect of H2O2 (Fig. 2, A and B). In addition, prior NAM treatment prevented FoxO1-GFP nuclear translocation in response to H2O2 (data not shown). Moreover, NAM was able to prevent resveratrol-induced nuclear localization of FoxO1-GFP, whereas TsA treatment did not affect the response to resveratrol (Fig. 2, A and B). As Sirtuins have been shown to deacetylate FoxO (4, 5, 11), we sought to determine the acetylation status of FoxO1 under different conditions. Treatment with NAM alone for 2 h increased FoxO1 acetylation, whereas incubation with insulin, H2O2, or resveratrol had no effect (Fig. 2E). Unlike previous data showing promiscuous FoxO deacetylation by Sirtuins and HDACs (4, 5, 11), these findings are consistent with the possibility that Sirtuins are the specific FoxO deacetylases in hepatocytes.
Sirtuin-mediated Deacetylation Perturbs FoxO1 Nucleocytoplasmic Shuttling—We measured the shuttling properties of FoxO1 using FRAP. In these experiments, an area of the cell is photobleached with a high intensity laser pulse and the movement of unbleached molecules from neighboring areas into the bleached area is recorded by time lapse microscopy (13). Fluorescence recovery depends on the amount of time required for total cell fluorescence to achieve steady-state distribution between different cellular compartments following the photobleach. We selectively photobleached the nuclear FoxO1-GFP pool and monitored the recovery from the cytoplasmic pool. In the presence of serum, nuclear FoxO1-GFP fluorescence recovered its prebleaching levels within 200 s (Fig. 3, A and B, squares). Importantly, after selective bleaching of the nuclear pool, cytoplasmic FoxO1-GFP fluorescence decreased as nuclear fluorescence increased. We then performed FRAP experiments in serum-deprived cells. Within 350 s of selectively photobleaching the nuclear FoxO1-GFP pool, the nucleus completely recovered the prebleaching fluorescence levels (Fig. 3, A and B, triangles), whereas the cytoplasmic pool of FoxO1-GFP fluorescence decreased. These data show that FoxO1-GFP is mobile between the two compartments and shuttles constitutively between the nucleus and the cytoplasm independently of serum. The main difference between the two conditions is that the rate of nucleocytoplasmic shuttling is slower in serum-deprived cells (t = 350 versus 200 s), probably reflecting reduced phosphorylation-dependent nuclear export.
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Because NAM prevented nuclear accumulation of FoxO1-GFP in H2O2-treated cells, we tested whether it could restore FoxO1 shuttling in the presence of H2O2 or resveratrol. FRAP analysis of cells treated with H2O2 or resveratrol showed that NAM, but not TsA (Fig. 4A), was able to restore nuclear fluorescence to prebleaching levels. This indicated that FoxO1-GFP is mobile between the nucleus and cytoplasm when Sirtuins, but not when class I/II HDACs, are inhibited. As a control to rule out interference from the fluorophor during oxidative stress on FoxO1-GFP kinetics, we performed FRAP experiments in cells transfected with GFP and treated with H2O2. Within 80 s of selectively photobleaching the nuclear pool of GFP, the nucleus recovered the prebleaching fluorescence levels (data not shown). These data indicate that GFP is freely mobile between the nucleus and the cytoplasm and that H2O2 does not obstruct GFP kinetics.
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Sirtuin Activation and Hepatic Glucose Metabolism—Regulation of hepatic glucose production is an important physiologic function of FoxO1 (14, 24, 25). To correlate the changes in FoxO1 distribution with its biologic effects, we measured glucose production in H4IIE rat hepatoma cells following resveratrol or insulin treatment. Incubation of cells transduced with wild type FoxO1 adenovirus in glucose-free medium supplemented with the glucogenetic precursors lactate and pyruvate resulted in a time-dependent increase in the amount of glucose released in the culture medium. Insulin suppressed glucose production by 50%, whereas resveratrol increased it by 30% (Fig. 6A). To determine whether the effect of resveratrol was mediated by FoxO1, we transduced cells with constitutively nuclear (ADA) and dominant negative (
256) FoxO1 adenoviruses (14). Expression of ADA-FoxO1 completely blunted the effect of insulin without changing the response to resveratrol (Fig. 6B). In contrast, 256-FoxO1 decreased glucose release under all conditions tested (Fig. 6C). We next measured mRNA levels of the glucogenetic FoxO1 target genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (Pck1), as well as insulin-like growth factor-binding protein 1 (Igfbp1). Resveratrol increased G6pc expression in cells expressing wild type and ADA-FoxO1, but not in cells expressing 256-FoxO1 (Fig. 6D). Unlike G6pc, Pck1 levels did not increase in response to resveratrol treatment in cells expressing wild type FoxO1 but did so in cells expressing ADA-FoxO1. The dominant negative 256-FoxO1 suppressed both basal and resveratrol-induced Pck1 expression (Fig. 6E). Igfbp1 expression patterns in response to the various treatments mirrored those of G6pc (Fig. 6F). These data are consistent with a model in which resveratrol increases glucose production by increasing FoxO1-dependent transcription of glucogenetic genes. It is noteworthy that resveratrol increased gene expression in cells expressing a constitutively nuclear FoxO1. These findings indicate that nuclear expression per se does not increase FoxO1-dependent transcription. Rather, there appears to be an additional resveratrol-dependent step required for FoxO1 activation, consistent with a role for deacetylation to increase FoxO1-mediated transcription.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The effect of resveratrol could be mimicked in our experimental system by H2O2 treatment, a reagent that is commonly employed to induce oxidative stress by increasing intracellular concentrations of reactive oxygen species. It should be emphasized that the effects of H2O2 in cultured cells are complex. At early time points, H2O2 is known to have insulin-mimetic effects (31), which include increased Akt activity and FoxO1 nuclear exclusion (18). With prolonged incubation, there is down-regulation of insulin signaling and FoxO1 localization becomes predominantly nuclear (5).
Previous studies have dealt with the effects of deacetylation on FoxO1-dependent transcription. Most (4, 5, 32), but not all (11), authors have found that deacetylation promotes FoxO-dependent transcription. However, these studies focused primarily on the effects of FoxO proteins on the cell cycle. The present data lend further support to the idea that deacetylation is a positive modulator of FoxO1 function by providing evidence that FoxO1-dependent regulation of hepatic glucose production is also affected by resveratrol, a small molecule activator of Sirtuins. Our findings are at odds with those of Motta et al. (11), who found increased G6pc and Pck1 expression in mice lacking Sirt1. However, it is unclear whether their data were obtained in fasted or fed mice. Moreover, as the Sirt1–/– mice were hyperglycemic, it is possible that the increase in Pck1 and G6pc is secondary to hyperglycemia rather than Sirt1 ablation. Further work will be required to establish a consensus on this point. Moreover, it should be noted that Sirt1 has also been shown to promote Pgc1-dependent gluconeogenesis, a conclusion that is fully consistent with our findings (33). This is a key question if therapies based on Sirtuin modulation are to be pursued for the treatment of metabolic disorders. For example, if Sirtuin activation increases hepatic glucose production in vivo as it does in cultured cells, then hepatic Sirtuins should be inhibited to treat diabetes. On the other hand, in other tissues such as pancreatic cells Sirtuin activation may indeed prove beneficial.
Deacetylation of transcription factors is generally associated with reduced activity. For example, Sirt1-mediated deacetylation dampens p53-dependent transcription (34–36). This process involves changes in p53 degradation (37), but currently there is no information on its effect on p53 subnuclear kinetics. Conversely, it is unclear whether deacetylation affects FoxO-dependent transcription by altering its stability. The finding that deacetylation increases the nuclear residence time of FoxO seemingly supports the idea that deacetylation increases FoxO-dependent transcription, but it should be emphasized that the net outcome of this modification is likely to be rather complex as FoxO can be both an activator (27) and a suppressor of gene expression (16, 38). Further studies with site-directed mutants that prevent or mimic acetylation will be required to address the specific effects of this important post-translational modification on the pleiotropic actions of FoxO proteins.
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FOOTNOTES |
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Both authors contributed equally to this work.
To whom correspondence should be addressed: 1150 St. Nicholas Ave., New York, NY 10032. Tel.: 212-851-5332; Fax: 212-851-5331; E-mail: da230{at}columbia.edu.
1 The abbreviations used are: FRAP, fluorescence recovery after photobleaching; FLIP, fluorescence loss in photobleaching; GFP, green fluorescent protein; HA, hemagglutinin; PBS, phosphate-buffered saline; NAM, nicotinamide; HDAC, histone deacetylase; TsA, trichostatin A.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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