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• W2058876100 abstract "O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. Here we report on regulation of O-GlcNAcylation over a broad range of glucose concentrations. We have discovered a significant induction of O-GlcNAc modification of a limited number of proteins under conditions of glucose deprivation. Beginning 12 h after treatment, glucose-deprived human hepatocellular carcinoma (HepG2) cells demonstrate a 7.8-fold increase in total O-GlcNAc modification compared with cells cultured in normal glucose (5 mm; p = 0.008). Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (20 mm) or glucosamine (10 mm) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity. Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation (p = 0.006). Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels (p = 0.03). We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and that these changes affect cell metabolism, thus inactivating glycogen synthase. O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. Here we report on regulation of O-GlcNAcylation over a broad range of glucose concentrations. We have discovered a significant induction of O-GlcNAc modification of a limited number of proteins under conditions of glucose deprivation. Beginning 12 h after treatment, glucose-deprived human hepatocellular carcinoma (HepG2) cells demonstrate a 7.8-fold increase in total O-GlcNAc modification compared with cells cultured in normal glucose (5 mm; p = 0.008). Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (20 mm) or glucosamine (10 mm) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity. Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation (p = 0.006). Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels (p = 0.03). We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and that these changes affect cell metabolism, thus inactivating glycogen synthase. Dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification is a critical modulator of the fate and function of diverse nuclear and cytoplasmic proteins. O-GlcNAcylation of target proteins is dependent upon substrate synthesis in the hexosamine biosynthetic pathway (HBP) 2The abbreviations used are:HBPhexosamine biosynthetic pathwayO-GlcNAcO-linked N-acetylglucosamineOGTO-linked N-acetylglucosaminyltransferasencnucleocytoplasmicmmitochondrialGSglycogen synthaseHPLChigh pressure liquid chromatographysWGAsuccinylated wheat germ agglutinin-agaroseNONOnon-POU domain-containing octamer-binding proteinPUGNAcO-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate.2The abbreviations used are:HBPhexosamine biosynthetic pathwayO-GlcNAcO-linked N-acetylglucosamineOGTO-linked N-acetylglucosaminyltransferasencnucleocytoplasmicmmitochondrialGSglycogen synthaseHPLChigh pressure liquid chromatographysWGAsuccinylated wheat germ agglutinin-agaroseNONOnon-POU domain-containing octamer-binding proteinPUGNAcO-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate. coupled with O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein modification. In many eukaryotic systems, HBP flux has been shown to parallel substrate (glucose) availability, making the HBP a nutrient sensor (1Hebert Jr., L.F. Daniels M.C. Zhou J. Crook E.D. Turner R.L. Simmons S.T. Neidigh J.L. Zhu J.S. Baron A.D. McClain D.A. J. Clin. Investig. 1996; 98: 930-936Crossref PubMed Scopus (279) Google Scholar, 2Yki-Jarvinen H. Virkamaki A. Daniels M.C. McClain D. Gottschalk W.K. Metab. Clin. Exp. 1998; 47: 449-455Abstract Full Text PDF PubMed Scopus (93) Google Scholar, 3Nelson B.A. Robinson K.A. Koning J.S. Buse M.G. Am. J. Physiol. 1997; 272: E848-E855PubMed Google Scholar, 4Gazdag A.C. Wetter T.J. Davidson R.T. Robinson K.A. Buse M.G. Yee A.J. Turcotte L.P. Cartee G.D. Am. J. Physiol. 2000; 278: R504-R512Google Scholar). In the cell, the HBP converts imported glucose and glucosamine to UDP-GlcNAc. Glutamine:fructose-6-phosphate amidotransferase is the rate-limiting enzyme in this pathway. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-GlcNAcase catalyzes O-GlcNAc removal.O-GlcNAc is known to affect multiple metabolic pathways and has been implicated specifically as a contributor to insulin resistance and type 2 diabetes (5McClain D.A. Crook E.D. Diabetes. 1996; 45: 1003-1009Crossref PubMed Google Scholar, 6Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar, 7Buse M.G. Am. J. Physiol. 2006; 290: E1-E8Crossref PubMed Scopus (361) Google Scholar). Chronically elevated HBP flux, a result of hyperglycemia, is known to exacerbate metabolic dysregulation in part by targeting metabolic enzymes. For example, in diabetic mice, glycogen synthase (GS) becomes resistant to insulin stimulation as its level of O-GlcNAc modification increases (8Parker G.J. Lund K.C. Taylor R.P. McClain D.A. J. Biol. Chem. 2003; 278: 10022-10027Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 9Parker G. Taylor R. Jones D. McClain D. J. Biol. Chem. 2004; 279: 20636-20642Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). AMP-activated protein kinase is activated in adipocytes with elevated HBP flux, resulting in O-GlcNAc-mediated elevation of fatty acid oxidation (10Luo B. Parker G.J. Cooksey R.C. Soesanto Y. Evans M. Jones D. McClain D.A. J. Biol. Chem. 2007; 282: 7172-7180Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). To date, the majority of reports of O-GlcNAc-mediated metabolic changes attribute increased O-GlcNAc modification to increased HBP flux. We report a novel and significant induction of O-GlcNAc modification in glucose-deprived HepG2 cells that is independent of increased HBP flux and appears distinct from previously reported stress-induced O-GlcNAc induction. Rather, increased O-GlcNAc with glucose deprivation is mediated by induction of OGT and down-regulation of O-GlcNAcase. Increased O-GlcNAcylation of GS in these conditions contributes to decreased GS activity.EXPERIMENTAL PROCEDURESAntibodies and Reagents—The following antibodies were used in the current study: anti-O-GlcNAc monoclonal IgM (CTD110.6, a gift of Dr. G. Hart, Johns Hopkins University, Baltimore, MD), anti-O-GlcNAcase (a gift of Dr. S. W. Whiteheart, University of Kentucky, Lexington, KY), anti-glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology), anti-β-actin (Cell Signaling), anti-OGT (DM-17, Sigma-Aldrich), anti-glycogen synthase (Cell Signaling; catalog no. 3886), anti-phosphoglycogen synthase (Cell Signaling), and horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (GE Healthcare) and anti-mouse IgM (Calbiochem). Succinylated wheat germ agglutinin-agarose (sWGA; EY Laboratories, San Mateo, CA), the HepG2 cell line (ATCC, Manassas, VA), and Protein A/G PLUS-agarose (Santa Cruz Biotechnology) were obtained from the indicated sources. All enzymes and chemicals were obtained from Sigma with the exception of the following: UDP-[6-3H]glucose (GE Healthcare); Dulbecco's modified Eagle's medium and fetal calf serum (Invitrogen); O-GlcNAcase inhibitor, O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate (PUGNAc; Toronto Research Chemicals, Ontario, Canada); Complete tablet protease inhibitors (Roche Applied Science); and TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). All chromatography media and columns were obtained from GE Healthcare. The Beckman Glucose Analyzer II (Beckman Coulter) was used for media glucose determination.Growth, Treatment, and Extraction of HepG2 Cells—HepG2 cells were grown in 10 ml of Dulbecco's modified Eagle's medium containing 20 mm glucose, 10% fetal calf serum, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate in 10-cm plates (Corning Glass) at 37 °C in 5% CO2. The medium was replaced 1 day prior to experimental treatment initiation; media glucose concentrations at treatment initiation averaged 10 mm. Experimental treatments were initiated once cells reached 70% confluence. We found 70% confluence to be optimal for promoting the glucose deprivation effect; under- and over-confluent cells demonstrated a diminished glucose deprivation effect. Experimental treatment of each plate comprised 10 ml of glucose-free Dulbecco's modified Eagle's medium, 1% fetal calf serum, 1 mm sodium pyruvate, 4 mm l-glutamine, and 0–40 mm glucose. Glucosamine treatments included glucose-free Dulbecco's modified Eagle's medium, 1% fetal calf serum, 1 mm sodium pyruvate, 4 mm l-glutamine, 2.5 mm glucose, and 10 mm d-glucosamine. Because media glucose concentrations depleted significantly over time in pilot experiments, media glucose concentrations were assayed every 3 h (using the Beckman Glucose Analyzer II), and glucose was replenished to achieve consistent glucose concentrations throughout treatment. Experimental treatment lasted 0–24 h. No cell death was observed for any of the treatment durations. For protein extracts, plates were placed on ice and washed twice with ice-cold Krebs-Ringer bicarbonate HEPES buffer (25 mm HEPES, pH 7.4, 150 mm sodium chloride, 4.4 mm potassium chloride, 1.2 mm sodium phosphate, pH 7.4, 1 mm magnesium chloride, and 1.9 mm calcium chloride), and then the cells were harvested in 0.75 ml of extraction buffer (50 mm HEPES, pH 7.4, 100 mm sodium chloride, 5% (v/v) glycerol, 50 μm PUGNAc, and protease inhibitors). The resulting cell suspension was sonicated with a Sonic Dismembrator F60 for 15 s at setting 6 (Thermo) and centrifuged at 20,000 × g for 2 min at 4 °C. Supernatant aliquots were immediately frozen in liquid nitrogen. Cells whose lysates were subsequently digested with hexosaminidase were harvested as above, but in a modified extraction buffer lacking PUGNAc and EDTA to prevent hexosaminidase inhibition. For cells used for RNA determination, the medium was aspirated/discarded, and 1 ml of TRI Reagent was immediately applied to the cells. Cells were scraped, disrupted by repeated pipetting, and immediately frozen in liquid nitrogen.Western Blotting—Protein concentrations of HepG2 lysates were determined using Bio-Rad protein reagent. Lysates were prepared for gel electrophoresis by dilution with extraction buffer and 5× Laemmli buffer. 10 μg of protein was added to each lane. SDS-PAGE was conducted using the Bio-Rad Mini-PROTEAN 3 electrophoresis cell, and resolved proteins were transferred to an Immobilon-PSQ transfer membrane (Millipore Corp., Bedford, MA). Resulting blots were blocked with TBST (20 mm Tris, pH 7.4, 150 mm sodium chloride, and 0.5% Tween 20) containing 4% (w/v) nonfat dried milk for 1 h at room temperature or overnight at 4 °C. 4% (w/v) bovine serum albumin was used in lieu of dried milk for detection with the anti-O-GlcNAc antibody. Blots were incubated with primary antibodies for 1 h at room temperature or overnight at 4 °C, washed three times in TBST, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were washed five times in TBST and imaged by treatment with Super Signal West Dura reagents (Pierce) and by exposure to Classic Blue BX autoradiography film (Molecular Technologies, St. Louis, MO). Densitometry measurements were obtained using an Epson Perfection 3200 photo scanner and NIH Image version 1.62 software (rsb.info.nih.gov/ij). In all experiments, glyceraldehyde-3-phosphate dehydrogenase protein levels were used to normalize changes in protein/modification. Glyceraldehyde-3-phosphate dehydrogenase protein levels were not affected by the various cell treatments of these studies (see Fig. 1, A and C; and Fig. 2C).FIGURE 2GS O-GlcNAcylation is increased with glucose deprivation, and this increase contributes to a decrease in GS activity. A, glucose-deprived cells exhibit a 60% decrease in maximal glycogen synthase activity compared with 5 mm glucose treatment (p = 0.004; n = four independent determinations). B, glucose-deprived cells exhibit a 6.75-fold increase in O-GlcNAc modification of glycogen synthase (p = 0.05; n = six independent determinations). Glycogen synthase was immunoprecipitated (IP) from cell lysates and immunoblotted (IB) with α-GS to demonstrate equivalent GS pulldown and with α-O-GlcNAc (CTD110.6) to demonstrate changes in O-GlcNAc modification of GS. Failure to precipitate GS with rabbit IgG confirmed specific GS precipitation (not shown). C, glucose-deprived cells exhibit a 3.3-fold increase in sWGA-bound/O-GlcNAc-modified glycogen synthase (p = 0.002; n = five independent determinations). Inset, sWGA-precipitated proteins immunoblotted with α-GS are shown. D, immunoblots of total GS and phospho-GS (pGS) levels demonstrate no change among treatments, suggesting that changes in glycogen synthase activity are not due to changes in GS protein or phospho-GS levels (representative blots of at least three independent determinations per treatment). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was probed as a loading control (n = at least three independent determinations per treatment). E, glucose-deprived lysates digested with hexosaminidase demonstrate a 40% rescue of glycogen synthase total activity (0–versus 0+; p = 0.003; n = six independent determinations). **, p ≤ 0.001. A.I.U., arbitrary intensity units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Immunoprecipitation of Glycogen Synthase—We followed the glycogen synthase antibody manufacturer's protocol for immunoprecipitation. Briefly, 300 μl of cell lysate (600 μg of protein) combined with glycogen synthase antibody (1:25) was rotated overnight at 4 °C. Incubation with rabbit IgG served as the negative control. 20 μl of Protein A/G PLUS-agarose beads (50% bead slurry) was added, and mixtures were rotated for 3 h at 4 °C. Beads were pelleted by centrifugation (20,000 × g for 30 s at 4 °C), and the supernatant was discarded. Some supernatant was retained and run as a positive control. Beads were washed five times with 500 μl of 1× lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, and 1 μg/ml leupeptin). Pellet was resuspended in 20 μl of 3× SDS sample buffer. Samples were boiled at 95 °C for 5 min and loaded onto SDS-polyacrylamide gel (4–15%). Western-blotted membranes were probed with α-O-GlcNAc (CTD110.6) and α-GS.Immobilization of O-GlcNAc-modified Proteins with Wheat Germ Agglutinin—400 μl of lysate (1 mg of protein/ml) was incubated with 25 μl of sWGA and 400 μl of radioimmune precipitation buffer (11 mm sodium phosphate, pH 7.4, 150 mm sodium chloride, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 40 mm sodium fluoride, 0.5 mm 2-acetamido-1-amino-1,2-dideoxyglucopyranose, and protease inhibitors). The preparation was rotated for 16 h at 4 °C and washed two times with extraction buffer/radioimmune precipitation buffer (1:1). Immobilized proteins were eluted by boiling in Laemmli buffer, resolved by SDS-PAGE, and transferred to an Immobilon-PSQ membrane. Levels of O-GlcNAc modification were determined by probing for sWGA-bound/O-GlcNAc-modified target proteins with the appropriate antibodies.Quantitation of mRNA by Reverse Transcription-PCR—RNA was prepared from –70 °C frozen TRI Reagent/cell suspensions according to the manufacturer's protocol (Molecular Research Center, Inc.) and dissolved in water. RNA concentrations were measured spectrophotometrically. First-strand cDNA synthesis was carried out using 3 μg of RNA, oligo(dT) primers (Invitrogen), and SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol. Realtime PCR was performed with a rapid thermal cycler (Light-Cycler, Roche Diagnostics). Reactions (10 μl) were performed using ∼16 ng of cDNA as a template with 0.5 μm each primer, 200 μm each deoxynucleotide triphosphate, 50 mm Tris, pH 8.3, 500 μg/ml nonacetylated bovine serum albumin (Sigma), 3.0 mm MgCl2, 0.04 units/μl Platinum Taq DNA polymerase (Invitrogen), and a 1:30,000 dilution of SYBR Green I fluorescent dye (Molecular Probes, Eugene, OR). Primers based on human sequences were chosen using the Primer3 program: nucleocytoplasmic OGT, 5′-CTTTAGCACTCTGGCAATTAAACAG-3′ and 5′-TCAAATAACATGCCTTGGCTTC-3′; mitochondrial OGT, 5′-TTTACCTCCTTTCCCTCCCATC-3′ and 5′-CTGTCAAAAATGCGTGCCTCT-3′; all OGT isoforms, 5′-CTGCCCCAGAACCGTATCA-3′ and 5′-TTCCAGACTTTGCCACGAACT-3′; O-GlcNAcase, 5′-AGCCTTGAGTGGTGAGCCTA-3′ and 5′-TCTGGGGATTTTGATTCAGC-3′; and NONO (non-POU domain-containing octamer-binding protein), 5′-CAAGTGGACCGCAACATCA-3′ and 5′-CGCCGCATCTCTTCTTCAC-3′. We assayed the expression of six different potential normalizer genes and found that NONO expression was consistent across all cell treatments. Amplification occurred over 26–45 four-step cycles, with a rate of temperature change between steps of 20 °C/s. The steps were 95 °C with a 0-s hold, 60 °C with a 0-s hold, 72 °C with an 11-s hold, and 80 °C with a 1-s hold. Fluorescence was detected during the fourth step at a temperature determined previously to be below the melting temperature of the PCR products. After amplification, a melting curve was generated by slowly heating the double-stranded DNA product. Analysis of the postamplification melting curves confirmed the absence of nonspecific DNA products. For each amplification's fluorescence versus cycle line, the LightCycler software determined the second derivative maximum (the threshold cycle at which fluorescence clearly increased above background). Standard curves of log cDNA versus second derivative maximum (fractional cycle number) were constructed for each quantitated transcript and for the NONO normalization transcript, from cDNA mixtures comprising equal amounts of all cell treatment condition cDNAs. Standard curve points of 0, 6, 10, 16, 26, and 32 ng of combined cell cDNA were always included with the same PCR run with the entire set of individual cDNA amplifications of the same transcript. Results for each individual cDNA were normalized by dividing the relative amount of each transcript by the relative amount of NONO transcript from the same experiment. Within each experiment the same mixture was used, containing everything but the specific primers.UDP-N-Acetylhexosamine Assay—Levels of UDP-N-acetylhexosamines (consisting of UDP-GlcNAc and UDP-GalNAc), products of the hexosamine biosynthesis pathway, were measured in cell extracts as described previously (11Robinson K.A. Weinstein M.L. Lindenmayer G.E. Buse M.G. Diabetes. 1995; 44: 1438-1446Crossref PubMed Scopus (133) Google Scholar). Cell extracts were homogenized at 4 °C in 4 volumes of perchloric acid (300 mm). The precipitates were centrifuged (10,000 × g for 15 min at 4 °C), and the lipid was extracted from the supernatants with 2 volumes of tri-n-octylamine:1,1,2-trichlorofluoroethane (1:4). The aqueous phase was stored at –80 °C until analysis by HPLC. The extracts were filtered (0.45 μm); HPLC was performed on a Partisil 10.5Ax column (25 cm x 4.6 mm, Waters Corp., Taunton, MA); and the extracts were eluted with a concave gradient from 5 mm potassium phosphate (pH 7.2) to 750 mm potassium phosphate (pH 7.2) over 48 min at a flow rate of 1 ml/min. UDP-N-acetylhexosamine levels were quantified by UV absorption at 254 nm and compared with external standards.Glycogen Synthase Assay—The assay for glycogen synthase was performed as described previously (8Parker G.J. Lund K.C. Taylor R.P. McClain D.A. J. Biol. Chem. 2003; 278: 10022-10027Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). HepG2 lysate (7.5 μg of protein) was incubated in a 100-μl final volume with 100 mm HEPES, pH 7.4, 5 mm EDTA, pH 7.4, 0.8 mg of glycogen (type III from rabbit liver), 2 mm UDP-glucose, 10 μl of glycerol, 0 or 10 mm glucose 6-phosphate, and 0.4 μCi of UDP-[6-3H]glucose for 45 min at 37 °C. The incubation was terminated by application to Whatman qualitative filter paper (No. 3MM, Maidstone, UK) and immersion in 60% (v/v) ethanol. After five washes in 400 ml of 60% ethanol, the paper squares were washed once in acetone, dried, and assayed for tritium. All assays were done in duplicate. The incorporation of tritium was found to be optimal at 37 °C and linear for 120 min. Total GS activity was defined as the activity at maximal glucose 6-phosphate (10 mm).Digestion of HepG2 Extracts with Hexosaminidase—Hexosaminidase digestions were conducted by treating cell lysates (50 μg of protein) with 1 unit of N-acetylglucosaminidase from jack beans (Sigma) and 25 μl of hexosaminidase buffer (20 mm sodium citrate, pH 4.5, 10% glycerol, 100 mm sodium chloride, and protease inhibitors) and incubating the preparation for 1 h at 30 °C. Lysates not digested with hexosaminidase were protected from deglycosylation by the addition of PUGNAc (50 μm); PUGNAc inhibits endogenous O-GlcNAcase. Immediately following digestion, glycogen synthase activation was measured as described above.Statistics—Descriptive statistics are represented as mean ± S.E. Each mean represents data from at least three independent experiments. Student's t test (two-tail) was used to compare differences between groups.RESULTSGlucose Deprivation of HepG2 Cells Stimulates O-GlcNAc Protein Modification—We assayed the HepG2 human liver cell line for changes in O-GlcNAcylation of proteins in response to changes in ambient glucose concentration. We chose to study O-GlcNAc changes in liver cells because of the liver's central role in carbohydrate metabolism and its glucose responsiveness. HepG2 cells were cultured for 12 h with 0–40 mm glucose. O-GlcNAc levels were determined by Western blot analysis of HepG2 cell lysates using the O-GlcNAc-specific antibody CTD110.6. Media glucose concentration had a significant effect on O-GlcNAc modification of a number of cellular proteins. Cells cultured in 0 mm glucose showed a total increase in O-GlcNAc of 7.8-fold (p = 0.008) and 4.1-fold (p = 0.05) compared with cells cultured in normal and high glucose, respectively (Fig. 1). Appreciable increases of O-GlcNAc with glucose deprivation first appear at 12 h and continue to increase through 18 and 24 h (Fig. 1C). Glucosamine treatment has been shown to stimulate O-GlcNAc modification even more potently than high glucose in a variety of systems (12Marshall S. Nadeau O. Yamasaki K. J. Biol. Chem. 2004; 279: 35313-35319Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). However, we observed 3-fold greater O-glycosylation with glucose deprivation than with glucosamine treatment (p = 0.02; Fig. 1, A and B).Although some protein targets demonstrate increased O-GlcNAc modification with both glucose deprivation and high glucose/glucosamine conditions, others are highly glycosylated exclusively with glucose deprivation. For two protein bands (Fig. 1A, bands b and c, ∼250 and ∼150 kDa, respectively), O-glycosylation is maximal with glucose deprivation and also increases with high glucose/GlcN treatment, with the lowest glycosylation levels at 1 mm glucose. Other proteins (Fig. 1A, band a, >250 kDa) fail to show glycosylation with high glucose/glucosamine treatment, but demonstrate robust glycosylation exclusively with glucose deprivation. Yet other targets (Fig. 1A, band d, ∼75 kDa) demonstrate bimodal modification in which glycosylation is lowest at normal (5 mm) glucose and increases with both lower and higher glucose concentrations, suggesting that some targets are regulated by O-GlcNAc modification throughout the entire range of physiologic glucose concentrations.Increased O-GlcNAcylation of GS in Glucose-deprived Cells Contributes to a Decrease in Activity—The O-GlcNAc modification induced by glucose deprivation is functionally significant. We have shown previously in adipocytes that O-GlcNAc modification of GS in conditions of high glucose decreases GS activity (8Parker G.J. Lund K.C. Taylor R.P. McClain D.A. J. Biol. Chem. 2003; 278: 10022-10027Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). This is also the case with glucose deprivation of HepG2 cells. HepG2 cells cultured in 0 mm glucose for 12 h exhibit a 60% decrease in maximal glycogen synthase activity compared with 5 mm glucose treatment (p = 0.004; Fig. 2A). We observed no differences in GS activity among cells treated with 5 and 20 mm glucose or glucosamine (Fig. 2A). The decrease in activity observed with glucose deprivation correlates with a 675% increase in O-GlcNAc-modified GS levels (p = 0.05; Fig. 2B) and a 330% increase in the amount of GS precipitated by sWGA (p = 0.002; Fig. 2C), a lectin that specifically binds terminal GlcNAc. We observed no difference in glycogen synthase protein amounts across glucose treatments or phosphoglycogen synthase levels among treatments (Fig. 2, B and D). We also detected no change in the Km of GS from glucose-deprived cells for its substrate UDP-glucose or its allosteric activator glucose-6-phosphate (data not shown).To demonstrate directly that O-GlcNAc modification contributes to the observed decrease in GS activity, we treated cell lysates with hexosaminidase to reduce the levels of O-GlcNAc modification. This treatment resulted in a 40% rescue of total GS activity after glucose deprivation (p = 0.003), whereas digestion of lysates from cells treated with normal glucose demonstrated no change in total activity (Fig. 2E). We observed a significant decrease in O-GlcNAc modification of proteins to background levels after hexosaminidase digestion, confirming deglycosylation (data not shown).Increased O-GlcNAc with Glucose Deprivation Does Not Result from Increased HBP Flux—Previous reports have mainly attributed increases of cellular O-GlcNAc to increased HBP flux and synthesis of the end product of the pathway, UDP-GlcNAc (1Hebert Jr., L.F. Daniels M.C. Zhou J. Crook E.D. Turner R.L. Simmons S.T. Neidigh J.L. Zhu J.S. Baron A.D. McClain D.A. J. Clin. Investig. 1996; 98: 930-936Crossref PubMed Scopus (279) Google Scholar, 13Cooksey R.C. Hebert Jr., L.F. Zhu J.H. Wofford P. Garvey W.T. McClain D.A. Endocrinology. 1999; 140: 1151-1157Crossref PubMed Google Scholar). This is not the case in glucose-deprived HepG2 cells. Cells cultured for 12 h in 0 mm glucose, which demonstrate a 7.8-fold increase in O-GlcNAc (Fig. 1, A and B), exhibit a 40% decrease in UDP-GlcNAc levels compared with normal glucose-treated cells (p = 0.01). UDP-GlcNAc levels increased 236% (p = 0.008) with high glucose and 616% (p = 0.01) with glucosamine treatment compared with 0 mm glucose treatment (Fig. 3).FIGURE 3UDP-GlcNAc and HBP flux are decreased with glucose deprivation. HepG2 cells were cultured for 12 h with 0–40 mm glucose or 10 mm GlcN. UDP-GlcNAc levels were measured, normalized to protein concentration, and calculated as proportions of the level for normal glucose treatments to allow for experiment-to-experiment averaging. Cells cultured in 0 mm glucose exhibited a 40% decrease in UDP-GlcNAc levels (1.8 μm/μg of protein/μl) compared with normal glucose (2.9 μm/μg of protein/μl; p = 0.01). UDP-GlcNAc levels increased 236% with high glucose (4.1μm/μg of protein/μl; p = 0.008) and 616% with glucosamine (12.2 μm/μg of protein/μl; p = 0.01) compared with 0 mm glucose treatment. Average UDP-GlcNAc levels are based on at least three independent determinations per treatment. **, p ≤ 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Nucleocytoplasmic OGT mRNA and OGT Protein Levels Are Increased in Glucose-deprived Cells—To explore the mechanisms for increased O-GlcNAc modification in the absence of increased HBP flux, we examined expression of OGT in conditions of glucose deprivation. Multiple OGT variants have been described (14Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar, 15Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus" @default.
• W2058876100 created "2016-06-24" @default.
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• W2058876100 date "2008-03-01" @default.
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• W2058876100 title "Glucose Deprivation Stimulates O-GlcNAc Modification of Proteins through Up-regulation of O-Linked N-Acetylglucosaminyltransferase" @default.
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