Up-regulation of O-GlcNAc Transferase with Glucose Deprivation in HepG2 Cells Is Mediated by Decreased Hexosamine Pathway Flux

O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. We have previously shown a significant induction of O-GlcNAc modification under conditions of glucose deprivation. Increased O-GlcNAc modification was mediated by increased mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (ncOGT). We have investigated the mechanism mediating ncOGT induction with glucose deprivation. The signal does not appear to be general energy depletion because no differences in AMP-dependent kinase protein levels or phosphorylation were observed between glucose-deprived and normal glucose-treated cells. However, treatment of glucose-deprived cells with a small dose (1 mm) of glucosamine blocked the induction of ncOGT mRNA and subsequent increase in O-GlcNAc protein modification, suggesting that decreased hexosamine flux is the signal for ncOGT up-regulation. Consistent with this, treatment of glucose-deprived cells with an inhibitor of O-GlcNAcase (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamat) completely prevented the subsequent up-regulation of ncOGT. Glucosamine treatment also resulted in a 40% rescue of the down-regulation of glycogen synthase activity normally seen after glucose deprivation. We conclude that deglycosylation of proteins within the first few hours of glucose deprivation promotes ncOGT induction. These findings suggest a novel negative feedback regulatory loop for OGT and O-GlcNAc regulation. O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. We have previously shown a significant induction of O-GlcNAc modification under conditions of glucose deprivation. Increased O-GlcNAc modification was mediated by increased mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (ncOGT). We have investigated the mechanism mediating ncOGT induction with glucose deprivation. The signal does not appear to be general energy depletion because no differences in AMP-dependent kinase protein levels or phosphorylation were observed between glucose-deprived and normal glucose-treated cells. However, treatment of glucose-deprived cells with a small dose (1 mm) of glucosamine blocked the induction of ncOGT mRNA and subsequent increase in O-GlcNAc protein modification, suggesting that decreased hexosamine flux is the signal for ncOGT up-regulation. Consistent with this, treatment of glucose-deprived cells with an inhibitor of O-GlcNAcase (O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamat) completely prevented the subsequent up-regulation of ncOGT. Glucosamine treatment also resulted in a 40% rescue of the down-regulation of glycogen synthase activity normally seen after glucose deprivation. We conclude that deglycosylation of proteins within the first few hours of glucose deprivation promotes ncOGT induction. These findings suggest a novel negative feedback regulatory loop for OGT and O-GlcNAc regulation. Dynamic O-linked N-acetylglucosamine (O-GlcNAc) 2The abbreviations used are: O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-linked N-acetylglucosaminyltransferase; nc, nucleocytoplasmic; AMPK, AMP-dependent kinase; PUGNAc, O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamate; HBP, hexosamine biosynthetic pathway; UDP, uridine diphospho; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high pressure liquid chromatography. 2The abbreviations used are: O-GlcNAc, O-linked N-acetylglucosamine; OGT, O-linked N-acetylglucosaminyltransferase; nc, nucleocytoplasmic; AMPK, AMP-dependent kinase; PUGNAc, O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino N-phenyl carbamate; HBP, hexosamine biosynthetic pathway; UDP, uridine diphospho; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high pressure liquid chromatography. 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) coupled with O-linked N-acetylglucosaminyltransferase (OGT)-mediated protein modification. The HBP converts a portion of imported glucose to uridine 5′-diphospho (UDP)-GlcNAc. OGT catalyzes GlcNAc transfer to serine and threonine residues of target proteins, whereas O-GlcNAcase catalyzes O-GlcNAc removal (1Hart G.W. Housley M.P. Slawson C. Nature.. 2007; 446: 1017-1022Google Scholar). HBP flux is known to parallel substrate (glucose) availability, making the HBP a nutrient sensor (2Hebert 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.1.. 1996; 98: 930-936Google Scholar, 3Yki-Jarvinen H. Virkamaki A. Daniels M.C. McClain D. Gottschalk W.K. Metab. Clin. Exp.. 1998; 47: 449-455Google Scholar, 4Nelson B.A. Robinson K.A. Koning J.S. Buse M.G. Am. J. Physiol.. 1997; 272: E848-E855Google Scholar, 5Gazdag 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). O-GlcNAcylation is regulated principally by substrate availability. Previous work has indicated that protein O-GlcNAcylation is proportional to substrate (glucose) availability (8Robinson K.A. Weinstein M.L. Lindenmayer G.E. Buse M.G. Diabetes.. 1995; 44: 1438-1446Google Scholar). However, we have shown that human hepatocellular carcinoma (HepG2) cells demonstrate a robust O-GlcNAc increase when deprived of glucose, and this O-GlcNAc induction is mediated not by substrate-driven HBP flux increase but instead by increased OGT expression and O-GlcNAcase down-regulation (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar). It has subsequently been shown that glucose deprivation of Neuro-2a neuroblastoma cells also results in OGT and O-GlcNAc induction (7Cheung W.D. Hart G.W. J. Biol. Chem.. 2008; 283: 13009-13020Google Scholar). We have therefore investigated the mechanism for regulation of OGT in HepG2 cells and determined that the signal responsible for the induction of OGT mRNA in glucose deprivation is an early decrease in HBP flux and O-GlcNAc modification of proteins. Thus, the levels of O-GlcNAc in these cells are maintained through a feedback mechanism responsive to the degree of protein O-GlcNAc modification. Antibodies and Reagents—The following antibodies were used in the current study: anti-O-GlcNAc monoclonal IgM (CTD 110.6, a gift of Dr. Gerald Hart, Johns Hopkins University, Baltimore, MD), anti-GAPDH (Santa Cruz), anti-AMP-dependent kinase α (Cell Signaling), anti-phospho-AMPK (Cell Signaling), anti-acetyl-CoA carboxylase (Cell Signaling), and horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (GE Healthcare) and anti-mouse IgM (Calbiochem). HepG2 cell line (ATCC, Manassas, VA). All of the 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-phenyl carbamate (PUGNAc; Toronto Research Chemicals, Toronto, Canada), complete tablet protease inhibitors (Roche Applied Science), and TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH). 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, 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 (measured 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 0–20 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, and 0–10 mm d-glucosamine for 0–12 h. For PUGNAc experiments, treatment medium was augmented with PUGNAc, resulting in a final PUGNAc concentration of 50 μm. Because media glucose concentrations deplete significantly over time, 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. No cell death was observed for any of the treatment durations. For protein extracts, the 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 harvested in 0.75 ml of extraction buffer (50 mm HEPES, pH 7.4, 100 mm sodium chloride, 5% glycerol (v/v), 50 μm PUGNAc, and protease inhibitors). The resulting cell suspension was sonicated with a Sonic Dismembrator F60 for 6 s twice at setting 6 (Thermo) and centrifuged at 20,000 × g for 2 min at 4 °C. Supernatant aliquots were immediately frozen in liquid nitrogen. For cells used for RNA determination, the medium was aspirated/discarded, and 1 ml of TRI reagent was immediately applied to the cells. The cells were scraped, then disrupted by repeated pipetting, and immediately frozen in liquid nitrogen. UDP-N-acetylhexosamine Assay—The levels of UDP-N-acetylhexosamines (consisting of UDP-GlcNAc and UDP-Gal-NAc), products of the hexosamine biosynthesis pathway, were measured in cell extracts as previously described (11Kreppel L.K. Hart G.W. J. Biol. Chem.. 1999; 274: 32015-32022Google Scholar). The 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), and HPLC was performed on a Partisil 10.5Ax column (25 cm x 4.6 mm; Waters Corp., Taunton, MA), 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-HexNAc levels were quantified by UV absorption at 254 nm, compared with external standards. Western Blotting—Protein concentrations of HepG2 lysates were determined using Bio-Rad protein reagent. The lysates were prepared for gel electrophoresis by dilution with extraction buffer and 5× Laemmli buffer. 10 μg of protein were added to each lane. SDS-PAGE was conducted using the Bio-Rad Mini-PROTEAN 3 electrophoresis cell, and resolved proteins were transferred to Immobilon-PSQ transfer membrane (Millipore Corp., Bedford, MA). The 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. The 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 exposure to Classic Blue Autoradiography Film BX (Molecular Technologies, St. Louis, MO). Densitometry measurements were obtained from non-saturated developed film using an EPSON Perfection 3200 Photo scanner (EPSON, Long Beach, CA) and National Institutes of Health Image version 1.62 software. In all of the experiments, the GAPDH protein levels were used to normalize changes in protein/modification. GAPDH protein levels are not affected by the various cell treatments of these studies. Quantitation of mRNA by Reverse Transcription-PCR—RNA was prepared from –70 °C-frozen TRI reagent/cell suspensions according to the manufacturer's (TRI reagent; Molecular Research Center, Inc) protocol 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. Real time PCR was performed with a rapid thermal cycler (LightCycler; Roche Applied Science). The reactions (10 μl) were performed using ∼16 ng of cDNA as 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 unit/μl of Platinum Taq DNA polymerase (Invitrogen), and 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. For nucleocytoplasmic OGT, 5′-CTTTAGCACTCTGGCAATTAAACAG-3′ and 5′-TCAAATAACATGCCTTGGCTTC-3′. For NONO (Non-POU domain-containing octamer-binding protein), 5′-CAAGTGGACCGCAACATCA-3′ and 5′-CGCCGCATCTCTTCTTCAC-3′. We assayed 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 previously determined 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 post-amplification 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 and for the NONO from cDNA of all cell treatment Standard curve of and ng of cell cDNA were included with the PCR with the of cDNA of the for each cDNA were by the of each by the of NONO from the each the was containing the Assay—The for glycogen synthase was performed as previously described G.J. R.P. McClain D.A. J. Biol. Chem.. 278: Scholar). HepG2 μg of was incubated in 100 of final with pH 7.4, pH 7.4, glycogen III from 10 glucose or 10 and of UDP-[6-3H]glucose for min at 37 °C. The was by to 3 and in After five in ml of the were washed once in and assayed for All of the were in The of was found to be optimal at 37 °C and for glycogen synthase activity was as the activity at glucose (10 mm). are as the of the from at three The was used to differences between between ncOGT mRNA and levels negative feedback regulation of We have previously a robust increase in protein O-GlcNAcylation in HepG2 human cells deprived of glucose (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar). the mechanism for this we assayed changes in ncOGT mRNA levels after 6 h of of HepG2 cells in medium containing or 5 mm glucose previously we observed a increase in ncOGT mRNA levels with glucose deprivation compared with cells in normal mm) glucose (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar). In this we measured ncOGT mRNA levels over a of glucose concentrations and with glucosamine treatment. We measured ncOGT levels at 6 the time at which we previously observed ncOGT induction with glucose deprivation. We that ncOGT levels are with mm glucose and decrease with glucose under conditions of HBP flux by 10 mm glucosamine ncOGT levels are The levels of ncOGT are to the levels of the of the HBP, We previously changes in UDP-GlcNAc levels with glucose concentrations (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar). In cells deprived of glucose demonstrate ncOGT UDP-GlcNAc levels are the UDP-GlcNAc levels at h is because this is the time at which O-GlcNAc induction first UDP-GlcNAc levels increase with glucose concentration a maximum with glucosamine treatment. Glucosamine is a of the HBP because the HBP of the in hexosamine J. Biol. Chem.. Scholar). O-GlcNAc over 6 h after Glucose observed between ncOGT mRNA and UDP-GlcNAc levels a negative feedback mechanism. We therefore that ncOGT mRNA induction in glucose-deprived cells a to a in HBP flux in the first 6 h following glucose deprivation. Consistent with this cells deprived of glucose demonstrate a of O-GlcNAcylation between 1 and 6 the induction of ncOGT and cells in normal glucose no change in O-GlcNAcylation over the time Glucosamine the in UDP-GlcNAc and O-GlcNAc with Glucose and of OGT and O-GlcNAcylation and of the induction of OGT mRNA with glucose deprivation is by decreased HBP we that we prevented this in HBP flux with glucosamine in glucose we OGT induction. was the The induction of O-GlcNAc modification by glucose deprivation is when the first and treatment with high concentrations of glucosamine and 10 mm) to an increase in O-GlcNAc modification through increased HBP flux also concentrations of glucosamine and 1 mm) decrease O-GlcNAc modification in glucose-deprived cells to levels observed in cells with normal glucose and 1 mm glucosamine treatment also the in UDP-GlcNAc levels and O-GlcNAcylation at and 6 h and previously demonstrated to with glucose deprivation and 1 mm glucosamine treatment ncOGT induction by in glucose-deprived cells and therefore the subsequent increase in O-GlcNAcylation seen after h in glucose-deprived cells We previously demonstrated increased O-GlcNAc modification of glycogen synthase with glucose which to a decrease in glycogen synthase activity (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar). 1 mm glucosamine treatment results in a rescue of glycogen synthase activity in glucose-deprived cells These demonstrate that of the in HBP flux in glucose-deprived cells by 1 mm glucosamine treatment the of glucose deprivation on ncOGT and glycogen synthase PUGNAc ncOGT with Glucose suggest that glucose deprivation results in HBP flux over the first 6 h. We that this in results in a in O-GlcNAcylation of a of proteins that the subsequent induction of We therefore a second to protein treatment of glucose-deprived cells with 50 μm PUGNAc, an inhibitor of PUGNAc treatment the in O-GlcNAc seen with glucose deprivation In O-GlcNAc levels increase over the treatment PUGNAc treatment completely induction of ncOGT mRNA Because of the of PUGNAc on we no the subsequent of PUGNAc on O-GlcNAc levels and glycogen synthase activity not The AMP-dependent Glucose of OGT and pathway is a pathway that is by in the to pathway in cells deprived of glucose and that this pathway to the observed in ncOGT mRNA and protein However, we observed no differences in phosphorylation or protein levels at or or phosphorylation of target carboxylase at h in glucose-deprived compared with normal glucose-treated cells. Thus, the pathway does not ncOGT and O-GlcNAc induction in HepG2 cells deprived of glucose from regulation by substrate availability, is known the regulation of O-GlcNAc modification of proteins. OGT modification, and regulation suggest regulation of For regulation of OGT activity and substrate are across a of UDP-GlcNAc concentrations (11Kreppel L.K. Hart G.W. J. Biol. Chem.. 1999; 274: 32015-32022Google Scholar). OGT is and this modification is on OGT is L.K. Hart G.W. J. Biol. Chem.. 1997; 272: Scholar). For phosphorylation and O-GlcNAcylation are and is that this of O-GlcNAc and is at by the of OGT and protein 1 L.K. Hart G.W. J. Biol. Chem.. Scholar). OGT have been that in the of and these differences are to OGT target L.K. Hart G.W. J. Biol. Chem.. 1997; 272: Scholar, J. Biol. Chem.. 1997; 272: Scholar, D. Scholar). is through feedback and current results demonstrate that regulation of O-GlcNAcylation is no The between UDP-GlcNAc and ncOGT mRNA levels and the of ncOGT induction by concentrations of glucosamine or PUGNAc this we and have O-GlcNAcylation in to glucose deprivation are mediated by changes in OGT expression (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar, W.D. Hart G.W. J. Biol. Chem.. 2008; 283: 13009-13020Google Scholar). mechanism for the of O-GlcNAc on protein function is regulation. OGT is known to with and O-GlcNAcase a C. A.J. J. Biol. Chem.. O-GlcNAcylation with and is Hart G.W. as are RNA polymerase II Hart G.W. J. Biol. Chem.. Scholar). that of O-GlcNAc are is that OGT expression are and that this be the mechanism changes in UDP-GlcNAc with ncOGT We previously that ncOGT induction results in a robust increase in O-GlcNAc modification of a of proteins, but ncOGT mRNA levels to normal by h (6Taylor R.P. Parker G.J. Hazel M.W. Soesanto Y. Fuller W. Yazzie M.J. McClain D.A. J. Biol. Chem.. 2008; 283: 6050-6057Google Scholar). In the the of ncOGT levels to normal after induction by glucose deprivation be consistent with of OGT that were early in resulting in of ncOGT. O-GlcNAcylation has been shown to protein DNA and Scholar, Hart G.W. Scholar). For the first a decreased rate 1997; and nuclear J. when modified by O-GlcNAc, and when modified by O-GlcNAc, demonstrate with proteins H. D. A. J. Biol. Chem.. 278: Scholar, C. Hart C. J. Biol. Chem.. or increased DNA Y. J. Hart G.W. Scholar). or regulation of OGT expression at the of O-GlcNAcylation of RNA polymerase II is known to Hart G.W. 1999; Scholar, 1997; Scholar). the of is also that O-GlcNAcylation of the up-regulation of ncOGT but not with glucose deprivation. OGT is suggesting potential negative feedback regulation of activity as as L.K. Hart G.W. J. Biol. Chem.. 1997; 272: Scholar). It was that increased O-GlcNAc modification of proteins mediated by increased OGT expression is also seen in glucose-deprived Neuro-2a cells (7Cheung W.D. Hart G.W. J. Biol. Chem.. 2008; 283: 13009-13020Google Scholar). In Neuro-2a the OGT induction is mediated by of the no increase in pathway with glucose deprivation. in glucose-deprived HepG2 or deprivation has no on O-GlcNAc induction not In HepG2 O-GlcNAcylation not in to a decrease in energy but to a decrease in UDP-GlcNAc and Thus, that of O-GlcNAc induction in to glucose deprivation according to suggesting an of to the regulation of It has previously been that increased O-GlcNAcylation in is by increased substrate availability. we that O-GlcNAcylation is also regulated by feedback across the and of glucose The results suggest that are for cell function in protein O-GlcNAc levels at and normal glucose We the of of the McClain and

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