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Originally published In Press as doi:10.1074/jbc.M802240200 on April 17, 2008

J. Biol. Chem., Vol. 283, Issue 24, 16283-16292, June 13, 2008
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O-GlcNAc Regulates FoxO Activation in Response to Glucose*Formula

Michael P. Housley{ddagger}, Joseph T. Rodgers§, Namrata D. Udeshi, Timothy J. Kelly§, Jeffrey Shabanowitz, Donald F. Hunt||, Pere Puigserver§, and Gerald W. Hart{ddagger}1

From the {ddagger}Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, the §Department of Cell Biology, Harvard Medical School and the Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, and the Departments of Chemistry and ||Pathology, University of Virginia, Charlottesville, Virginia 22904

Received for publication, March 20, 2008 , and in revised form, April 17, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
FoxO proteins are key transcriptional regulators of nutrient homeostasis and stress response. The transcription factor FoxO1 activates expression of gluconeogenic, including phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, and also activates the expression of the oxidative stress response enzymes catalase and manganese superoxide dismutase. Hormonal and stress-dependent regulation of FoxO1 via acetylation, ubiquitination, and phosphorylation, are well established, but FoxOs have not been studied in the context of the glucose-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification. Here we show that O-GlcNAc on hepatic FoxO1 is increased in diabetes. Furthermore, O-GlcNAc regulates FoxO1 activation in response to glucose, resulting in the paradoxically increased expression of gluconeogenic genes while concomitantly inducing expression of genes encoding enzymes that detoxify reactive oxygen species. GlcNAcylation of FoxO provides a new mechanism for direct nutrient control of transcription to regulate metabolism and stress response through control of FoxO1 activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nutrient homeostasis is tightly regulated to ensure the health of sensitive cells and tissues. Molecular mechanisms to counter the adverse effects of nutrient excess are overwhelmed in diabetes and result in diseases of the nervous system, heart, vasculature, and kidneys (1). Key molecules that respond to, and regulate, nutrient and stress levels include the forkhead transcription factor FoxO1 (FKHR)2 and the post-translational modification O-GlcNAc.

The FoxO transcription factors are important regulators of broad gene expression programs, which include metabolism (e.g. gluconeogenesis, amino acid catabolism, glycolysis, pentose phosphate shunt, and fatty acid/triglyceride/sterol synthesis) (2), cell cycle (3), stress response (4, 5), and longevity in Caenorhabditis elegans (6). Ubiquinitation and acetylation (7) are post-translational regulators of FoxO; however, phosphorylation by the insulin signaling pathway is the most well characterized signal transduction pathway that impinges upon FoxO (8). In the presence of insulin, this highly conserved pathway results in the nuclear exclusion of FoxO1 upon protein kinase B (PKB)/AKT phosphorylation. Three key sites of AKT phosphorylation have been identified for FoxO1: threonine 24, serine 256, and serine 319. Homologs of FoxO1, including FoxO3 and FoxO4, contain conserved AKT phosphorylation sites and are regulated by insulin signaling in a similar manner (9). Nuclear exclusion of FoxO eventually leads to its ubiquitination and degradation (10). Stress stimuli also alter FoxO3 activation and turnover via deacetylation by SIRT1 (7).

The highly abundant and dynamic post-translational modification, O-GlcNAc, is implicated in stress responses, insulin signaling, nutrient sensing, cell cycle progression, protein turnover, translation, and transcription, in addition to other biological processes (11, 12). O-GlcNAc rapidly cycles on serine and threonine residues of nuclear and cytoplasmic proteins in a fashion analogous to phosphorylation. Unlike phosphorylation, however, the addition of O-GlcNAc is performed by a single catalytic subunit (O-GlcNAc transferase; OGT) as opposed to hundreds of kinases and requires interactions with adaptor molecules for specificity to the hundreds of target proteins (11). O-GlcNAc functions as a nutrient sensor because the intracellular concentration of the donor sugar for GlcNAcylation, UDP-GlcNAc, rapidly responds to flux through multiple metabolic pathways, and OGT activity is highly dependent upon the concentration of this donor substrate (13). Elevated flux through the UDP-GlcNAc synthetic pathway results in insulin resistance (the hallmark of type II diabetes) of peripheral tissues (14). In murine adipocytes, elevated O-GlcNAc directly causes insulin resistance as measured by reduced 2-deoxyglucose uptake (15). Overexpression of OGT in muscle or adipose tissues of transgenic mice results in diabetes (16). However, elevated O-GlcNAc appears to be protective against heat shock (17). In the heart, O-GlcNAc is protective against ischemic stress (18), highlighting the importance of this post-translational modification to the stress response. In mammals and plants, deletion of OGT is lethal (19, 20). However, in C. elegans, an OGT knock-out was found to have altered glycogen and trehalose storage and dauer formation induced by a temperature-sensitive daf-2 allele, indicating a role for OGT in insulin signaling (21).


Figure 1
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  		FIGURE 1.
FoxO1 is O-GlcNAcylated. A, Fao cells were infected with Ad-FLAG-FoxO1. FLAG-FoxO1 was immunoprecipitated (IP) and blotted (IB) with the anti-O-GlcNAc antibody CTD110.6 or the terminal GlcNAc-binding lectin succinylated wheat germ agglutinin. Specificity was confirmed by GlcNAc competition and increased antibody or lectin reactivity following treatment with the O-GlcNAcase inhibitor PUGNAc. B, autoradiograph showing endogenous FoxO1 immunoprecipitated from rat liver and labeled with [3H]UDP-galactose using a GlcNAc-specific galactosyltransferase. BSA, bovine serum albumin.

 
Preliminary studies have shown FoxO1 to be GlcNAcylated (22, 23); however, detailed mechanistic insights and their relevance to diabetes have not been elucidated. Here, we show that FoxO1 is GlcNAcylated in liver and that the modification is increased in diabetic rats. Furthermore, the hexosamine biosynthetic pathway serves to sense glucose levels and regulate FoxO1 transcriptional activation through GlcNAcylation of key amino acids in a complex interplay with FoxO phosphorylation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—Human FoxO1 expression plasmids were obtained from Addgene. pGEX-4T3-GST-FKHR (Addgene plasmid 10706) was used for in vitro assays. pcDNA-FLAG-FKHR (Addgene plasmid 13507) (24) and pcDNA3 FLAG FKHR AAA mutant (Addgene plasmid 13508) (24) were used for cell culture experiments, luciferase assays, and site-directed mutagenesis (QuikChange, Stratagene; PAGE-purified primers, Invitrogen). pGEX-4T3-GST-FoxO3 wild type (Addgene plasmid 1790), pGEX-4T3-GST-FoxO3 1–525aa (Addgene plasmid 10826), and pGEX-4T3-GST-FoxO3 1–525aa TM 8351 (Addgene plasmid 8351) (25) were used for in vitro assays.

Adenoviral Infections—Adeno-OGT (26) was infected at an multiplicity of infection of 100 in serum-free DMEM for 24 h. Cyclization recombinase and GFP adenovirus were purchased from the Baylor College of Medicine Vector Development Laboratories (vector.bcm.tmc.edu). Adeno-FLAG-FoxO1, adeno-FLAG-3A FoxO1 (T24A/S256A/S319A), and adeno-FLAG-HA-PGC-1 were used as described (27). OGT–/flox mouse embryonic fibroblasts (MEFs) (19, 28) were infected with adeno-CRE or adeno-GFP for 3 days prior to harvesting.

Cell Culture and Treatments—Fao rat hepatoma cells were cultured in Coon's modification of Ham's F-12 medium (2 g/liter glucose; Sigma) supplemented with 5% fetal bovine serum. MEFs were cultured in DMEM (1 g/L glucose) supplemented with 10% fetal bovine serum. The cells were treated with 100 µM O-(2-acetamido-2-deoxy-D-glusopyranoslidene) amino-N-phenylcarbamate (PUGNAc; Toronto Research Chemicals) overnight. The cells were treated with 1, 5, or 25 mM glucose or 25 mM glucose +10 mM glucosamine in serum-free DMEM overnight. Insulin was used at indicated concentrations for 1 h.

Transcription Activation Assays—FoxO1 transcriptional activation was measured using a luciferase reporter construct pGL3 promoter, Promega) containing three copies of the FoxO1-binding site from the IGFBP promoter (29). 10 ng/well of the FoxO1 reporter construct as well as 10 ng/well of a β-galactosidase control construct were transfected into HEK293 cells in 24-well dishes using Lipofectamine 2000 (Invitrogen). pShuttle-OGT was used at 100 ng/well. The cells were transfected for 4 h in serum-free DMEM (Invitrogen). The medium was then replaced with serum-free DMEM containing varying amounts of glucose and incubated overnight. Luminescence was normalized to β-galactosidase activity.

Gene Expression Analysis—RNA was collected using TRIzol (Invitrogen) and cDNAs were reverse transcribed using Super Script II (Invitrogen). Real time PCR was performed on a Stratagene MX3000 using SYBR mix (Invitrogen). The data were normalized to 18 S rRNA.

Immunoblot Analysis—O-GlcNAc levels were determined by immunoprecipitation and measuring relative reactivity to the anti-O-GlcNAc antibody CTD110.6 (30) (Covance) or the GlcNAc-binding lectin succinylated wheat germ agglutinin (31) (EY Labs). FoxO1 was immunoprecipitated using the FKHR antibody (Cell Signaling number 9462). FoxO1 blots were done with anti-FKHR (Santa Cruz sc-11350) and anti-FoxO1 phosphoserine 256 (Santa Cruz sc-22158).

Protein Interaction Analysis—Co-immunoprecipitation assays were performed on lysates from Fao cells infected with Ad-FLAG-HA-PGC-1a. Anti-FLAG agarose (Sigma) or anti-OGT (AL28) was applied to cells lysed in Tris-buffered saline with 1%Nonidet P-40. The immunoprecipitates were washed three times and separated by SDS-PAGE gel electrophoresis and blotted using anti-HA or anti-OGT (DM-17; Sigma).

UDP-GlcNAc Determination—UDP-GlcNAc levels were measured by capillary zone electrophoresis (32).


Figure 2
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  		FIGURE 2.
O-GlcNAcylation of FoxO1 is elevated in diabetes. A, FoxO1 was immunoprecipitated from control and STZ-induced diabetic rat livers and blotted with anti-O-GlcNAc antibodies (CTD110.6). Specificity was confirmed by GlcNAc competition. B, UDP-GlcNAc levels were determined from control and STZ-induced diabetic rat livers using capillary electrophoresis (y axis) and plotted against the densitometry analysis from C (x axis). C, RT-PCR analysis of Pepck and G6pc expression show an elevated mRNA expression following STZ-induced diabetes. The dash represents the mean. D, FoxO1 was immunoprecipitated from control and high fat-fed mice and blotted with anti-O-GlcNAc antibodies (CTD110.6). Densitometry analysis reveals elevated GlcNAcylation of FoxO1. IB, immunoblotting.

 
OGT and Kinase Assays—Recombinant AKT was obtained from Cell Signaling (7500). GST-FoxO1 was labeled on beads for 1 h at room temperature in the presence of 200 µM ATP as indicated by the manufacturer. Plasmid expressing recombinant OGT was a kind gift from Suzanne Walker (33). OGT assays following AKT labeling were performed on bead-bound FoxO1 for 2 h at room temperature in 50 mM Tris, pH 7.5. with 1 µM UDP-GlcNAc. 0.25 µCi of [3H]UDP-GlcNAc (American Radiolabeled Chemicals) was added to the reaction and incubated for 1 h at room temperature. The beads were then washed, and incorporated label was measured by scintillation counting or SDS-PAGE and autoradiography.

ETD MS/MS—GST-FoxO1 bound to glutathione-conjugated beads (Amersham Biosciences) was carbamidomethylated with dithiothreitol (Amersham Biosciences) and iodoacetamide (Sigma-Aldrich) at room temperature as previously described (34). The protein-bound beads were enzymatically digested with either endoproteinase LysC (Roche Applied Science) or endoproteinase AspN (Roche Applied Science) at an enzyme-to-substrate ratio of 1:20 in 100 mM ammonium bicarbonate, pH 8, at room temperature while shaking for 6.5 h. Supernatant containing the resulting proteolytic peptides was removed from the spun-down beads and acidified to pH 3.5 with glacial acetic acid (Sigma-Aldrich).

An aliquot of the supernatant from the digested sample was loaded onto a polyimide-coated, fused silica capillary reverse phase precolumn (360-µm outer diameter x 75-µm inner diameter; Polymicro Technologies) packed with C18 resin (5–20-µm diameter, 120-Å pore size; YMC Inc.) and desalted with 0.1% acetic acid. The precolumn was connected to a capillary analytical column (360-µm outer diameter x 50-µm inner diameter) packed with C18 resin (5-µm diameter, 120-Å pore size; YMC Inc.) and equipped with an integrated, electrospray emitter as described in Ref. 35. The peptides were eluted into the mass spectrometer at a flow rate of 60 nl/min with a gradient of 0–60% B in 60 min and 60–100% B in 5 min (A = 0.1 M acetic acid, B = 70% acetonitrile, 0.1 M acetic acid) using a 1100 series binary high pressure liquid chromatography (Agilent Technologies) (35).

Aliquots of samples were analyzed on a Thermo Electron LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific). Full mass spectra were acquired with the Orbitrap analyzer operated at a resolving power of 30,000 (at m/z 400). Collision-activated dissociation tandem mass spectra were acquired data-dependently with the quadrupole linear ion trap analyzer. ETD MS/MS spectra were acquired on a Finnigan LTQ (Thermo Fisher Scientific) modified in house with a chemical ionization source to generate fluoranthene radical anions for fragmentation via ETD (45 msec ETD reaction) (36). Targeted ETD MS/MS were acquired on a second Finnigan LTQ (Thermo Fisher Scientific) equipped with a prototype Thermo Fisher ETD upgrade (100-ms ETD reaction, AGC target for ETD reagent ions 1E5). All of the data were interpreted manually.

Galactose Labeling Assays—O-GlcNAc levels were measured by [3H]UDP-galactose labeling of GlcNAc (37). FoxO1 was immunoprecipitated using cold-labeled antibodies (Cell Signaling Technology) from 0.1 g of liver lysed in 10 ml of radio-immune precipitation assay buffer. The immunoprecipitates were then labeled overnight, separated by SDS-PAGE, and stained with Coomassie G-250. Following destaining, the gels were treated with En3Hance (PerkinElmer Life Sciences) and exposed to autoradiography film.

Animal Experiments—Male Sprague-Dawley rats (~16 weeks old) were given a single injection of streptozotocin (50 mg of STZ/kg of body weight) to induce hyperglycemia. After 2 weeks the animals were sacrificed, and the livers were harvested. Control animals received vehicle alone.

For OGT overexpression studies, eight 9-week-old male balb/c mice (Taconic) were injected with 8.0 x 108 adeno-OGT or adeno-GFP virus particles. Gene expression was corrected by 36B4 and normalized to GFP.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
FoxO1 GlcNAcylation Is Elevated in Diabetes—Given that FoxO1 is a transcription factor that controls many aspects of metabolism and stress responses in the liver and O-GlcNAc functions as a nutrient sensor, we tested whether FoxO1 is O-GlcNAc modified in the liver. Fig. 1 shows that FLAG-tagged FoxO1 expressed in Fao rat hepatoma cells is GlcNAcylated using the anti-O-GlcNAc antibody, CTD110.6, and the terminal GlcNAc-specific lectin, succinylated wheat germ agglutinin (Fig. 1A). Specificity was confirmed by GlcNAc competition and increased O-GlcNAc levels following treatment of the cells with the O-GlcNAcase inhibitor, PUGNAc. Endogenous FoxO1 immunoprecipitated from rat liver was shown to be O-GlcNAc modified by using a terminal GlcNAc-specific galactosyltransferase probe (Fig. 1B) (37). We next asked whether GlcNAcylation of FoxO1 is dysregulated by hyperglycemia in an model for diabetes. Using an O-GlcNAc-specific antibody (CTD110.6), we found that liver FoxO1 from STZ-induced diabetic rats was more heavily GlcNAcylated (Fig. 2A and supplemental Fig. S1). The reduction in antibody reactivity by free GlcNAc further demonstrates antibody specificity. The donor sugar for GlcNAcylation is UDP-GlcNAc, a product of the hexosamine biosynthetic pathway (HBP). Elevated glucose can increase flux through the HBP resulting in increased UDP-GlcNAc (14), upon which OGT activity is dependent (13). We therefore measured UDP-GlcNAc levels in the control and STZ diabetic rat livers and found an increase in the sugar nucleotide (Fig. 2B; 55.5 ± 2.5 versus 88.6 ± 12.3 pmol/mg liver, p < 0.05 by Student's t test). These data suggest that the HBP may act as a nutrient sensor by regulating OGT activity toward transcription factors, such as FoxO1. The STZ diabetic rats also had elevated mRNA expression of the FoxO1 targets Pepck and G6pc, consistent with previous reports (Fig. 2C) (38). Additionally, we measured the GlcNAcylation of FoxO1a from high fat fed diabetic mice and found an increase similar to the STZ-induced diabetic rats (Fig. 2D).


Figure 3
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  		FIGURE 3.
Insulin reduces FoxO1 GlcNAcylation elevated by high glucose. A, FLAG-FoxO1 was immunoprecipitated (IP) from Fao cells that were treated with insulin for 1 h. GlcNAcylation was detected by Western blotting using the CTD110.6 antibody. Glucose increases, whereas insulin decreases, FoxO1 GlcNAcylation. B, insulin treatment does not reduce total cellular GlcNAcylation in Fao cells. C, insulin treatment does not alter O-GlcNAcase activity in Fao cells. IB, immunoblotting.

 
FoxO Transcription Factors Integrate Nutrient and Hormone Information—To confirm that hyperglycemia elevates GlcNAcylation of FoxO1, we employed a hepatoma cell culture model. Fig. 3A shows that culturing Fao hepatocytes in increasing glucose concentrations resulted in a dose-dependent increase in O-GlcNAc levels on FLAG-FoxO1 as detected by the CTD110.6 antibody. Interestingly, the addition of 100 nM insulin for 1 h down-regulated O-GlcNAc specifically on FoxO1 but did not reduce total cellular O-GlcNAc levels (Fig. 3, A and B) or alter O-GlcNAcase activity (Fig. 3C). We next assayed the O-GlcNAc levels of the insulin-insensitive mutant FoxO1 (T24A,S256A,S319A, termed "3A"). The lack of AKT phosphorylation sites results in much higher O-GlcNAc levels (Fig. 4A). To further address the interplay between O-GlcNAc and O-phosphate, we employed in vitro OGT and AKT assays. In vitro GlcNAcylation of GST-FoxO1 had no effect on subsequent in vitro AKT phosphorylation at Ser256 (Fig. 4B). Additionally, in vitro AKT phosphorylation of GST-FoxO1 did not affect subsequent in vitro GlcNAcylation (Fig. 4C), suggesting that the sites are not directly reciprocal. To test whether GlcNAcylation and Phosphorylation are functionally reciprocal, we assayed wether high glucose or PUGNAc (the O-GlcNAcase inhibitor) alters FoxO localization. Fao cells with a basal AKT activity, despite serum starvation, have most of their FoxO1 localized in the cytoplasm. FoxO does not translocate to the nucleus upon the addition of 25 mM glucose or PUGNAc treatment, whereas 10 µM LY294002 (PI3K inhibitor) does cause translocation. Consistent with Fig. 3A, it appears that AKT-mediated phosphorylation is dominant to GlcNAcylation with regards to FoxO. Another GST-tagged FoxO isoform, FoxO3 (FKHRL1), was also an in vitro target of OGT (Fig. 2E). A truncated FoxO3 (amino acids 1–525) incorporated less O-GlcNAc, showing that some sites are found between amino acids 526 and 673. A mutant lacking three AKT phosphorylation sites (T32A,S253A,S315A, termed "TM") of the truncated form labeled to the same extent as the truncation containing the hydroxyamino acids, consistent with the model that phosphorylation by AKT is not directly reciprocal with GlcNAcylation.


Figure 4
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  		FIGURE 4.
There is a complex interplay between phosphorylation and GlcNAcylation of FoxO1. A, the AKT-insensitive and constitutively nuclear FoxO1 mutant (T24A,S256A,S319A, called 3A) is hyper-GlcNAcylated in Fao cells. B, recombinant FoxO1 was in vitro GlcNAcylated then subsequently in vitro AKT labeled, subjected to SDS-PAGE, and blotted with anti-FoxO1 Ser(P)256, stripped, and then blotted with anti-O-GlcNAc antibodies (CTD110.6). Specificity of the Ser(P)256 antibody was confirmed by alkaline phosphatase treatment. C, recombinant FoxO1 was in vitro AKT-phosphorylated, then subsequently in vitro O-GlcNAc labeled, subjected to SDS-PAGE, and blotted with anti-O-GlcNAc antibodies (CTD110.6), stripped, and then blotted with anti-FoxO1 Ser(P)256 antibodies. Specificity of the Ser(P)256 antibody was confirmed by alkaline phosphatase treatment. In vitro AKT phosphorylation does not block in vitro OGT labeling of FoxO1. D, Fao cells were infected with adenovirus expressing FLAG-tagged FoxO1. 24 h after infection cells were serum-starved in RPMI + 0.5% bovine serum albumin for 4 h and then treated as indicated for an additional 16 h in RPMI + 0.5% bovine serum albumin. FoxO1 was visualized using anti-FoxO1 antibody. PUGNAc (an O-GlcNAcase inhibitor) or 25 mM glucose did not result increased nuclear translocation of FoxO1 under conditions tested. E, the FoxO3 isoform is an in vitro substrate for OGT. [3H]UDP-GlcNAc incorporation was detected by autoradiography following Coomassie G250 staining. The truncated (amino acids 1–525) form incorporated significantly less label, indicating a significant number of sites are in the C-terminal region. The mutant FoxO3 lacking AKT phosphorylation sites (T24A,S215A,S316A, or "triple mutant" or TM) incorporates [3H]UDP-GlcNAc similar to the truncated form, indicating that AKT phosphorylation sites are not significantly in vitro O-GlcNAc labeled. IB, immunoblotting; wt, wild type.

 
O-GlcNAc Regulates FoxO1 Activity—To investigate the role of O-GlcNAc on FoxO1, we first asked whether glucose could activate FoxO1-dependent transcription. Luciferase reporter assays in HEK293 cells (Fig. 5A) demonstrate that overnight incubation in 25 mM glucose resulted in increased FoxO1 transactivation. Immunoblotting showed that FoxO1 protein levels did not increase with increasing glucose. The AKT-insensitive FoxO1 mutant is also activated by glucose independent of insulin signaling. To further demonstrate that the hexosamine biosynthetic pathway can regulate FoxO1 in a mechanism that is distinct from nuclear localization, we tested FoxO1 3A activation while blocking the rate-limiting step in UDP-GlcNAc synthesis (Fig. 5B) with the glutamine:fructose-6-phosphate amidotransferase inhibitor, 6-diazo-5-oxonorleucine (DON). DON attenuated FoxO1 activation by 25 mM glucose (Fig. 5C), whereas the addition of glucosamine, which enters the UDP-GlcNAc synthesis pathway after glutamine: fructose-6-phosphate amidotransferase, restored FoxO1 activation, indicating that constitutively nuclear FoxO1 is activated by O-GlcNAc. The addition of glucosamine to the culture medium of Fao cells was confirmed to increase FoxO1 O-GlcNAc levels, whereas treatment with DON reduced FoxO1 glycosylation (Fig. 5D). Overexpressing OGT enhanced glucose-induced FoxO1 transcriptional activation (Fig. 5E) and GlcNAcylation (Fig. 5F).


Figure 5
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  		FIGURE 5.
O-GlcNAc increases FoxO-dependent luciferase reporter transcription in HEK293 cells via the HBP. A, luciferase reporter activity is increased by high glucose for both wild type (wt) and 3A FoxO1 (all panels are plotted as relative luciferase activity normalized to β-galactosidase activity; error bars indicate standard errors; *, p < 0.05 by Student's t test). FoxO1 protein levels are unaffected. B, schematic of the UDP-GlcNAc synthesis pathway in which 2–5% of glucose that enters the cells is used for production of the donor sugar nucleotide. C, addition of 50 µM DON, which inhibits the rate-limiting enzyme in UDP-GlcNAc synthesis (glutamine:fructose-6-phosphate amidotransferase, GFAT), reduces high glucose activation of luciferase activity. 10 mM glucosamine (GlcN) rescues activation of FoxO1. D, O-GlcNAc levels on FLAG-FoxO1 in Fao cells following treatment with 10 mM glucosamine and 50 µM DON. E, overexpression of OGT increases FoxO-dependent luciferase activity. F, overexpression of OGT increases FoxO GlcNAcylation. CTD110.6 antibodies were used to detect FLAG-precipitated FoxO1. IP, immunoprecipitation; IB, immunoblotting.

 
To examine the effects of glucose sensing via O-GlcNAc on FoxO1 target genes, we performed RT-PCR analysis on mRNA isolated from Fao hepatoma cells cultured overnight in 1, 5, or 25 mM glucose. mRNA levels of Pepck and G6pc were elevated in a dose-dependent manner following overnight glucose treatment (Fig. 6A). We next asked whether other FoxO1 target genes are also elevated, specifically those involved in the oxidative stress responses, and found both catalase and MnSOD expression to be increased (Fig. 6B). This RT-PCR analysis was performed with either Ad-GFP or an adenovirus expressing a dominant negative FoxO1 mutant ({Delta}256) (39). The Ad-FoxO1 {Delta}256-infected cells did not respond to glucose, demonstrating that the glucose sensing is specifically enhancing FoxO1-dependent transcription of gluconeogenesis and stress response genes. To confirm that the increase in catalase and MnSOD expression requires OGT, we employed MEFs that have loxP recombination sites flanking the OGT gene (19). mRNA expression of the stress response genes was not increased by culturing in high glucose conditions when the cells had been infected with an adenovirus expressing Cre recombinase causing a reduction in OGT compared with adeno-GFP controls (Fig. 6C). The reduced mRNA expression in the 25 mM glucose samples may be due to an effect on basal transcriptional machinery (40, 41). Additionally, OGT overexpression (via adenovirus) in Fao cells was found to increase Pepck and G6pc expression as compared with GFP controls (Fig. 6D).


Figure 6
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  		FIGURE 6.
Glucose activates expression of FoxO1 target genes in an OGT-dependent manner. A, RT-PCR analysis shows gluconeogenic enzyme expression (G6pc and Pepck) is elevated by glucose in Fao hepatoma cells (all panels are relative expression normalized to 18 S rRNA; the error bars indicate standard errors; *, p < 0.05 and **, p < 0.005 by Student's t test). B, RT-PCR analysis shows reactive oxygen species detoxification enzyme expression (Catalase, MnSOD) is elevated by glucose. Infection with an adenovirus expressing a dominant negative FoxO1 ({Delta}256) prevents activation of target genes by high glucose. C, RT-PCR analysis in OGT–/flox MEFs shows that OGT is required for high glucose activation of Catalase and MnSOD. D, RT-PCR analysis in Fao cells following OGT overexpression shows elevated Pepck and G6pc. E, RT-PCR analysis of mouse livers 7 days post-injection of adeno-OGT or GFP viruses. F, electrophoretic mobility shift assay with recombinant GlcNAcylated or naked GST-FoxO1 and a 300-base pair fragment of the IRE-luciferase reporter plasmid (used in Fig. 5) and visualized with ethidum bromide. G, electrophoretic mobility shift assay as in F with either 2 µg of GST + 6 µg of OGT, 2 µg of FoxO1, or 2 µg of FoxO1 + 6 µg of OGT.

 
To test how OGT affects gene expression in animals, we infected mice with adeno-OGT virus and preformed RT-PCR analysis from mRNA isolated from the livers. Fig. 6E shows that Pepck and G6pc expression are elevated by ~50% compared with adeno-GFP infected control mice, whereas Cox7a1 did not change significantly. These data, consistent with previous reports (42), suggest that, in response to glucose, FoxO1 and O-GlcNAc are involved in the ability of the cell to regulate transcription of gluconeogenic and stress response genes.

To better understand how O-GlcNAc regulates FoxO1, we investigated the DNA binding properties of this transcription factor. Using the FoxO-binding portion of the luciferase reporter vector used in Fig. 5, we performed electrophoretic mobility shift assays on recombinant FoxO1. No difference in DNA binding was found between naked or GlcNAc-FoxO1 nor between FoxO and FoxO incubated with OGT (Fig. 6, F and G). These results suggest that mechanisms other than nuclear localization or DNA binding, such as recruitment of transcription machinery, mediate the activation of FoxO1 by glucose.

To identify which of the 131 serine and threonine residues in human FoxO1 might be O-GlcNAcylated, we employed a relatively new technique, ETD MS/MS. The O-GlcNAc modification is lost under conventional, collision-activated dissociation mass spectrometry but retained with ETD (43). As a result, we were able to determine that FoxO1 residues Ser550, Thr648, Ser654, and either Thr317 or Ser318 were GlcNAcylated (Fig. 7A and supplemental Fig. S3). We then mutated several of these sites and tested whether they were activated by glucose in a luciferase reporter assay. Mutation of threonine 317 to alanine reduced transcriptional activation by high glucose, whereas mutation of serine 318 had no effect (Fig. 7B). Under the conditions tested, mutation of the other identified sites had no effect in the FoxO1 luciferase reporter assay (supplemental Fig. S4). Western blot analysis showed reduced O-GlcNAc on the T317A mutant versus wild type after overnight culture in 25 mM glucose (Fig. 6C).


Figure 7
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  		FIGURE 7.
FoxO1 is GlcNAcylated at multiple sites. A, schematic indicating four O-GlcNAc sites mapped using ETD MS/MS on human FoxO1 (Thr317, Ser550, Thr648, and Ser656). B, luciferase reporter assays of point mutants of FoxO1, indicating that mutation of Thr317 to alanine reduces activation by 25 mM glucose (expressed as relative activity normalized to β-galactosidase; error bars represent standard errors; *, p < 0.05 by Student's t test). C, HEK293 cells were transfected with FLAG-FoxO1 vectors containing the indicated site mutations, subjected to SDS-PAGE, and blotted using anti-O-GlcNAc antibodies (CTD110.6). IP, immunoprecipitation; IB, immunoblotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The highly abundant post-translational modification O-GlcNAc has been proposed to be a nutrient sensor as the donor sugar, UDP-GlcNAc, receives input from multiple metabolic pathways and that OGT activity depends upon UDP-GlcNAc concentration (11). We have shown that hyperglycemia elevates UDP-GlcNAc levels in rat liver, resulting in augmented FoxO1 GlcNAcylation and activation of this transcription factor. Early work showed that O-GlcNAc is enriched in chromatin (40) and at active sites of gene transcription in polytene chromosomes. Many transcription factors have been shown to be GlcNAcylated (for review see Ref. 12), and the RNA polymerase II C-terminal domain is extensively GlcNAcylated in a competitive manner with phosphorylation (41). The modification has been shown to regulate transactivation by potentially altering stability, nuclear localization, recruitment of transcriptional machinery, or DNA binding. In the case of Sp1, activation is regulated by O-GlcNAc and depending on cell type or promoter via DNA binding or other mechanisms (44, 45). Another mechanism by which O-GlcNAc regulates transcription factors is through nuclear localization. Pancreatic NeuroD1 translocates to the nucleus under high glucose or inhibition of O-GlcNAcase (46). However, we have shown that a constitutively nuclear mutant FoxO1 (3A) is also regulated by glucose via the hexosamine biosynthetic pathway (Fig. 5), indicating that additional, intranuclear mechanisms of transactivation exist. It is possible that this mechanism involves GlcNAcylation of upstream regulators of FoxO because AKT and insulin receptor substrate 1 are also GlcNAcylated (47). This mechanism is unlikely, however, because the mutation of a very specific hydroxyamino acid (Thr317 but not Ser318), which we have shown to be GlcNAcylated, reduces FoxO1 stimulation by hyperglycemia. The fact that steady-state levels of FoxO1 protein did not vary in high glucose conditions speaks against FoxO regulation by the proteasome, a possibility given that FoxO is ubiquitinated (48) and proteasome function is inhibited by O-GlcNAc (49). Another possible mechanism for O-GlcNAc activation of FoxO1 is through mediating the recruitment of basal transcription factors, many of which are also GlcNAcylated (50). GlcNAcylation of Sp1 in its transactivation domain inhibits the interaction with TATA-binding-associated factor (TAF110) (51).

The discovery that GlcNAcylation of serine and threonine residues also occurs at phosphorylation sites led to the "Yin-Yang" hypothesis where O-GlcNAc moieties could directly oppose O-phosphate. We now know that this model is overly simple. When the three AKT phosphorylation sites are mutated in FoxO1, the O-GlcNAc levels become amplified. FoxO1 O-GlcNAc levels are elevated by high glucose and can subsequently be reduced by treating cells with insulin. These findings indicate that there is a Yin-Yang effect with FoxO. However, in vitro labeling experiments indicate that the sites of phosphorylation and GlcNAcylation do not overlap. ETD MS/MS allowed for identification several O-GlcNAc sites, one of which is adjacent to an AKT phosphorylation site. Mutation of threonine 317 reduces hyperglycemic activation in the FoxO luciferase assay, but determining the function of the other sites will require different assays and/or experimental conditions. If a GlcNAc site is also phosphorylated, mutation of a hydroxyamino acid to alanine will not be able to determine whether a phenotype for this mutant is due to the lack of phosphorylation or GlcNAcylation. What is clear is that multiple post-translational modifications at either shared or distinct sites give the cell greatly expanded molecular diversity.

This complex molecular diversity may allow a liver cell to sense and respond to multiple stimuli and respond with changes in transcription target activation and specificity. Here we show that glucose up-regulates mRNA expression of Pepck and G6pc via increasing GlcNAcylation of FoxO1 in the absence of insulin. This potentially establishes a dangerous positive feedback loop of gluconeogenesis and demonstrates that inappropriate gluconeogenesis (52) is not merely an effect of lack of insulin but a pathologically activated pathway (53). It has been asked why, from an evolutionary perspective, would an organism up-regulate glucosenegenesis under conditions of hyperglycemia (54)? The answer may lie in the finding that GlcNAcylation of FoxO1 also activates reactive oxygen species detoxification enzyme expression. Given that glucose metabolism leads to reactive oxygen species production, direct glucose control of this stress response pathway would be advantageous to the cell. O-GlcNAc senses and protects cells from stress (17, 18, 55), perhaps by activating expression of protective genes. Therefore, investigation into the pathological activation of gluconeogenesis as a drug target must also consider increased oxidative damage as a potential side effect.

In addition to gluconeogenesis, FoxO transcription factors regulate cell cycle, apoptosis, and longevity in C. elegans in addition to their roles in liver metabolism. O-GlcNAc is also implicated in cell cycle progression (56), apoptosis, and dauer formation in C. elegans, suggesting there may be other processes controlled by GlcNAcylation of FoxO1. Altogether, these data suggest a novel form of FoxO1 regulation relevant not only to diabetes, but to a wide array of cellular processes.


   FOOTNOTES
 
* This work was supported, in whole or in part, by National Institutes of Health Grants DK61671 and HD13563 (to G. W. H.) and GM37537 (to D. F. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 Recipient of a share of royalties on university sales of CTD110.6 antibody. To whom correspondence should be addressed. E-mail: gwhart{at}jhmi.edu.

2 The abbreviations used are: FKHR, forkhead transcription factor FoxO1; O-GlcNAc, O-linked β-N-acetylglucosamine; Pepck, phosphoenolpyruvate carboxykinase; G6pc, glucose-6-phosphatase; MnSOD, manganese superoxide dismutase; OGT, O-GlcNAc transferase; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate; HA, hemagglutinin; RT, reverse transcription; ETD, electron transfer dissociation; MS/MS, tandem mass spectrometryl; HBP, hexosamine biosynthetic pathway; DON, 6-diazo-5-oxonorleucine; STZ, streptozotocin. Back


   ACKNOWLEDGMENTS
 
We thank Kaoru Sakabe, Win Cheung, Kyoungsook Park, Chad Slawson, and other members of the Hart Laboratory for helpful discussion and technical assistance. Dawn Chen and Robert Cole at the Johns Hopkins University Mass Spectrometery core confirmed immunoprecipitations by mass spectrometery analysis. We thank William Sellers for the use of pGEX-4T3-GST-FKHR.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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