Division of Endocrinology, Departments of 1 Medicine, Diabetes, and Medical Genetics and 2 Biochemistry/ Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425; Division of Endocrinology and Metabolism, Departments of 3 Pediatrics and 4 Cell Biology and Physiology, School of Medicine, Washington University, St. Louis, Missouri 63110
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ABSTRACT |
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O-linked glycosylation on Ser/Thr with single N-acetylglucosamine (O-GlcNAcylation) is a reversible modification of many cytosolic/nuclear proteins, regulated in part by UDP-GlcNAc levels. Transgenic (T) mice that overexpress GLUT1 in muscle show increased basal muscle glucose transport that is resistant to insulin stimulation. Muscle UDP-GlcNAc levels are increased. To assess whether GLUT4 is a substrate for O-GlcNAcylation, we translated GLUT4 mRNA (mutated at the N-glycosylation site) in rabbit reticulocyte lysates supplemented with [35S]methionine. O-GlcNAcylated proteins were galactosylated and separated by lectin affinity chromatography; >20% of the translated GLUT4 appeared to be O-GlcNAcylated. To assess whether GLUT4 or GLUT4-associated proteins were O-GlcNAcylated in muscles, muscle membranes were prepared from T and control (C) mice labeled with UDP-[3H]galactose and immunoprecipitated with anti-GLUT4 IgG (or nonimmune serum), and N-glycosyl side chains were removed enzymatically. Upon SDS-PAGE, several bands showed consistently two- to threefold increased labeling in T vs. C. Separating galactosylated products by lectin chromatography similarly revealed approximately threefold more O-GlcNAc-modified proteins in T vs. C muscle membranes. RL-2 immunoblots confirmed these results. In conclusion, chronically increased glucose flux, which raises UDP-GlcNAc in muscle, results in enhanced O-GlcNAcylation of membrane proteins in vivo. These may include GLUT4 and/or GLUT4-associated proteins and may contribute to insulin resistance in this model.
glucose transporter 4; glucose transporter 4-associated proteins; O-linked glycosylation on serine/threonine with single N-acetylglucosamine of membrane proteins; transgenic mice overexpressing glucose transporter 1 in muscle; rabbit reticulocyte lysate
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INTRODUCTION |
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POSTTRANSLATIONAL
O-GlcNac modification of proteins
(O-GlcNAcylation) is defined as the O-linked
attachment of single -N-acetylglucosamine moieties to
specific Ser/Thr hydroxyl groups. It was first described in 1984 and
differs in many respects from the "classical" forms of protein
glycosylation, i.e., Asn-linked N-glycosylation or O-GalNAc-Ser-(Thr)-linked O-glycosylation.
Whereas the latter processes occur in the endoplasmic reticulum and/or
Golgi apparatus, the major sites of O-GlcNAcylation are the
nucleus and the cytosol. While proteins bearing classical glycosyl side
chains are typically cell surface or secreted proteins,
O-GlcNAcylated proteins are ubiquitous and most abundant in
the nucleus and the cytoplasm. Known O-GlcNAcylated proteins
include nucleoporins, RNA polymerase II catalytic subunit, many
transcription factors, and numerous cytoskeletal proteins. All
O-GlcNAc-modified proteins identified to date are subject to
phosphorylation, and in many instances the sites of
O-phosphorylation and O-GlcNAcylation are
identical or adjacent, suggesting a regulatory role in cell
signaling (reviewed in Refs. 6 and 12).
O-GlcNAcylation is a dynamic and reversible process, and
enzymes that catalyze the addition (22, 27) and removal
(9) of O-GlcNAc to and from polypeptides have
been identified and cloned.
Insulin resistance is a hallmark of type 2 diabetes and is associated with uncontrolled type 1 diabetes. Sustained exposure to high glucose also causes insulin resistance, and "glucose toxicity" accounts for the insulin resistance in uncontrolled type 1 diabetes (39, 44). Insulin accelerates glucose utilization by responsive cells (skeletal muscle, heart muscle, and adipocytes) by stimulating glucose transport. The above cells express the insulin-responsive glucose transporter GLUT4, which is mainly segregated intracellularly in the basal state and translocates to the plasma membrane (PM) in response to insulin. GLUT1 is constitutively expressed at the PM and is mainly responsible for basal glucose transport (reviewed in Ref. 2).
Transgenic mice, which overexpress GLUT1 in skeletal muscle, exhibit mild fasting hypoglycemia, without significant changes in circulating insulin or glucagon. Basal glucose transport and glycogen stores are markedly increased in GLUT1 transgenic muscles (28, 36). A remarkable characteristic of these muscles is that, in vitro, insulin fails to stimulate glucose transport, although GLUT4 expression is unchanged. Other stimuli, e.g., insulin-like growth factor-I, hypoxia, and contractile activity, which normally stimulate muscle glucose transport and GLUT4 translocation, also fail to stimulate glucose transport (10).
UDP-GlcNAc is the major product of the hexosamine biosynthesis pathway (HBSP) and the obligatory substrate of polypeptide-O-GlcNAc transferase (OGT), the cytosolic/nuclear enzyme, which catalyzes protein O-GlcNAcylation (22, 27). Glucose entry into HBSP is regulated by glutamine-fructose-6-phosphate amidotransferase (GFAT). The products of the reaction are glucosamine 6-phosphate (GlcN-6-P) and glutamate (21). The concentration of UDP-N-acetylhexosamine(s) (UDP-GlcNAc + UDP-GalNAc, in an ~3:1 ratio) is increased two- to threefold in GLUT1-overexpressing muscles, and GFAT activity is increased ~60% (5).
Increased flux via HBSP has been implicated in glucose-induced insulin resistance (4, 5, 7, 13, 14, 16, 29, 37). In GLUT1-overexpressing muscles, glucose flux is chronically increased, and insulin-resistant glucose transport is associated with increased HBSP activity. We hypothesized that the increased intracellular concentrations of UDP-GlcNAc may promote O-GlcNAcylation of critical proteins involved in insulin-stimulated GLUT4 trafficking. In the experiments reported here, we investigated whether GLUT4 is a potential substrate of O-GlcNAcylation and assessed whether or not increased O-GlcNAcylation of GLUT4 or of proteins associated with GLUT4 are detectable in membrane preparations from GLUT1-overexpressing skeletal muscle.
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EXPERIMENTAL PROCEDURES |
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Materials. Except where otherwise noted, all reagents were purchased from Sigma Chemical (St. Louis, MO) and were of the highest quality available. Site-specific, COOH-terminal polyclonal antibodies against GLUT1 and GLUT4 were raised in rabbits and purified before use by protein A affinity chromatography. Preimmune serum was purified identically. A monoclonal mouse antibody (RL-2) that recognizes O-GlcNAcylated proteins (17, 40) was purchased from Affinity Bioreagents (Golden, CO), and the mouse monoclonal site-specific anti-GLUT4 antibody was from Genzyme (Cambridge, MA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgGs were purchased from Jackson Immunoresearch Laboratories (West Grove, PA), and enhanced chemiluminescence (ECL) reagents were from Pierce (Rockford, IL).
Animals. The transgenic mouse line that overexpresses GLUT1 in skeletal muscle has been described previously (5, 10, 28, 36). Transcription of the human GLUT1 gene was controlled by the rat myosin light chain promoter (28). For experiments, mice expressing a single GLUT1 gene were mated with wild-type B65JLF1/J mice (Jackson Laboratories, Bar Harbor, ME). The offspring of these matings consisted of a 50:50 mixture of heterozygous transgenic mice and wild-type controls (5). Male transgenic mice and controls from the same litter were used in experiments. At the time of study, mice were between 2 and 6 mo old.
Mice were housed in a facility equipped with a 12:12-h light-dark cycle and were fed ad libitum (Rodent Blox; Ralston Purina, St. Louis, MO) until food was removed 2 h before death. Mice were killed at 10:00 AM under halothane anesthesia. Hindlimb muscles, including calf, thigh, and hip muscles, were rapidly removed and frozen in liquid nitrogen.Preparation of total muscle membranes. Frozen muscles (~2 g) were powdered in a mortar cooled with liquid nitrogen and homogenized immediately in 5 vol of ice-cold 10 mM Tris, pH 7.4, 1 mM EDTA, 250 mM sucrose, 10 µg/ml leupeptin and aprotinin, and 2 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged for 5 min at 1,000 g, and the pellet was reextracted with another 5 vol of homogenization buffer and centrifuged for 5 min at 1,000 g. Pooled supernatants were centrifuged for 5 min at 10,000 g. The supernatant was adjusted to 0.8 M KCl, mixed for 30 min at 4°C, and centrifuged for 2 h at 174,000 g. The pellet was resuspended in 50 mM HEPES, pH 7.4, 150 mM NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 10 µg/ml leupeptin and aprotinin and solubilized for 1 h; the resultant sample was centrifuged for 5 min at 10,000 g to remove insoluble material. Protein content was assessed by the Bradford assay using Coomassie Protein Assay Reagent (Pierce). The protein concentration was adjusted to 2 mg/ml for the galactosylation procedure.
Galactosylation of membrane proteins.
Terminally O- GlcNAc-modified membrane proteins were
identified by the galactosyltransferase probe method using purified
UDP-Gal-GlcNAc Gal-(1-4) galactosyltransferase
(Gal-transferase) and UDP-[3H]galactose, as described by
Roquemore et al. (38). Briefly, 0.5 or 1 mg of total crude
membrane proteins in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 0.5% CHAPS
was incubated for 1 h at 37°C with the addition of 10 mM HEPES,
pH 7.3, 10 mM galactose, 5 mM MnCl2, 1 mM dithiothreitol
(DTT), 25 mM 5'-AMP, 20 µCi/ml UDP-[6-3H]galactose (sp
act 40 Ci/mmol; American Radiolabeled Chemicals, St. Louis, MO), and 1 U/ml galactosyltransferase (previously autogalactosylated for 30 min at 37°C in 50 mM Tris, pH 7.3, 5 mM MgCl2, 1 mM
mercaptoethanol, 0.4 mM UDP-galactose, and 0.1 mg/ml aprotinin). Enzyme
reactions were terminated by the addition of 10 mM EDTA.
Immunoprecipitation of galactosylated membranes is described below.
Purification of galactosylated membrane proteins by ricinus communis lectin chromatography. Galactosylation of 0.5 mg muscle membrane proteins was performed as described above except that the reactions were stopped by adding 10 mM EDTA and 1% SDS. Reaction products were separated from unincorporated label on a Sephadex G-50 column equilibrated with 50 mM ammonium formate and 0.1% SDS. The macromolecular peak was lyophilized to remove the ammonium formate, reconstituted in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 10 mM EDTA, and the SDS concentration was reduced to 0.1% by spin filtration. The sample was reconstituted in the same buffer containing 1% Triton X-100. Average label incorporated per 0.5 mg protein was 1.6 × 106 disintegrations/min (dpm) for the control and 1.9 × 106 dpm for the transgenic mice. N-linked glycosyl side chains were removed by digesting the samples overnight at 37°C with 4 U/ml endoglycosidase F (Boerhinger Mannheim, Indianapolis, IN). Ricinus communis agglutinin I agarose (Vector Laboratories, Burlingame, CA) chromatography was performed on a 0.7 × 18-cm (7 ml) column preequilibrated with 50 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% Triton X-100 (buffer A). Sixty 0.4-ml fractions were collected before the addition of 0.2 M lactose in buffer A, after which an additional 20 fractions were collected. The radioactivity in the fractions was monitored by scintillation counting of 40-µl aliquots and the protein content by ultraviolet absorption of the undiluted fractions at 280 nm. Peak 1, which eluted just after the void volume, and peak 2, which eluted after the addition of lactose, were concentrated and analyzed by 10% SDS-PAGE. The only visible proteins using silver stain or Coomassie blue stain were from peak 1. The majority of the protein-associated radioactivity, however, was in peak 2. Proteins were transferred to nitrocellulose, and 0.5-cm slices were digested in dimethyl sulfoxide (DMSO) before scintillation counting.
Immunoprecipitation of galactosylated membrane proteins with GLUT1 and GLUT4 antibodies and endoglycosidase F digestion. Membrane preparations galactosylated as described above were preabsorbed with Protein G-Sepharose for 2 h at 4°C. Beads were pelleted by centrifugation, and supernatants were incubated overnight at 4°C with rabbit polyclonal anti-GLUT1 IgG (10 µg/ml) and Protein G-Sepharose. The supernatants were incubated overnight at 4°C with rabbit polyclonal anti-GLUT4 IgG or mouse monoclonal anti-GLUT4 IgG or nonimmune IgG (10 µg/ml) and Protein G-Sepharose. Beads were washed three times with 50 mM HEPES, pH 7.4, 150 mM NaCl, and 0.1% CHAPS, and bound proteins were eluted with 10 mM Tris, pH 8.3, 1% SDS, 10 mM DTT, and 25 mM iodoacetic acid at room temperature for 40 min. The elution buffer was exchanged by spin filtration with 50 mM HEPES, pH 7.4, 150 mM NaCl, and 10 mM EDTA. The detergent concentration was adjusted to 1% Triton X-100, and 10 µg/ml leupeptin and aprotinin were added. Samples were incubated overnight at 37°C with or without 4 U/ml endoglycosidase F. Digestions were terminated by the addition of Laemmli sample buffer, and proteins were separated by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane, and sample lanes were cut into 0.5-cm slices, which were dissolved in DMSO. Radioactivity was quantified by scintillation counting.
Preparation of muscle extracts for Western blotting with RL-2 antibody. Frozen, powdered hindlimb muscle was homogenized in 10 vol of 50 mM HEPES, pH 7.4, 1 mM EDTA, 10% glycerol, 150 mM sodium chloride, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.2 mM sodium vanadate, 100 µg/ml aprotinin, 10 µg/ml leupeptin, 1.5 mg/ml benzamidine, and 1 mM PMSF (homogenization buffer). Samples were centrifuged at 2,000 g for 2 min, and supernatants were saved. Pellets were reextracted in 8 vol of homogenization buffer and centrifuged as before. Pooled supernatants were solubilized for 1 h in 1% Igepal CA-630 (Sigma) at 4°C. After centrifugation at 10,000 g for 10 min, supernatant protein concentrations were normalized, samples were diluted with Laemmli sample buffer, and 40 µg of protein were analyzed by 7% SDS-PAGE. Proteins were transferred to nitrocellulose membranes, incubated overnight with RL-2 antibody (1:1,000), and developed by the ECL procedure.
In vitro expression and galactosylation of GLUT4. GLUT4(Thr57) was transcribed, translated, and O-GlcNAcylated by incubation for 90 min at 37°C using the Promega TNT rabbit reticulocyte lysate coupled transcription/translation system (Promega, Madison, WI) in the presence of canine microsomal membranes, in a methionine-free amino acid mixture supplemented with [35S]methionine (2 mCi/ml; ICN, Costa Mesa, CA) in a final volume of 50 µl. Asn57 is the only N-glycosylation site in GLUT4. Mutated aglyco-GLUT4 was translated to avoid galactosylating terminal GlcNAc moieties of the N-glycosyl side chain. The template was the rat GLUT4 cDNA in the oocyte expression vector pSP64T (24). Site-directed mutagenesis was carried out using the Clontech Transformer site-directed mutagenesis kit (Clontech Laboratories, Palo Alto, CA). Pancreatic microsomes were prepared as described (42). After 90 min of translation at 37°C, samples were pelleted by centrifugation at 10,000 g for 30 min at 4°C. Pellets were rinsed with five reaction volumes of 10 mM Na2HPO4, pH 7.4, and 150 mM NaCl (buffer B) and resuspended in 1 volume of 1% SDS in buffer B. Samples were diluted with 2.5 vol of 3% Nonidet P-40 (NP-40) in buffer B and galactosylated as described for total membrane proteins from muscle, except that UDP-galactose (0.3 mM) was not labeled. Galactosylated products were diluted to 1 ml with 0.2% NP-40 in buffer B (buffer C) and mixed at room temperature for 30 min with 400 µl of ricinus communis agglutinin I agarose prewashed with 30 ml of buffer C. The lectin beads were transferred to a column for washing and elution. The flow through and a 1-ml wash were collected, and the beads were washed with an additional 20 ml of buffer C. Beads were mixed with 1 ml of 0.5 M GlcNAc in buffer C (buffer D) for 30 min, the eluate was collected, and the column was washed with 2 ml of buffer D and 2 ml of buffer C. The beads were mixed for 30 min with 1 ml of 0.5 M galactose in buffer C (buffer E), the eluate was collected, and the beads were washed with two 1-ml aliquots of buffer E. All fractions were precipitated with TCA or immunoprecipitated with GLUT4 antibody and Protein G-Sepharose as described for membrane proteins from muscle, except that the immunoprecipitates were washed with buffer C. Washed TCA precipitates and immunoprecipitates were solubilized in Laemmli sample buffer, separated by 10% SDS-PAGE, and analyzed by fluorography using En3Hance reagent (NEN Life Science, Boston, MA).
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RESULTS |
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To assess whether or not GLUT4 is a potential substrate for
O-GlcNAcylation, we analyzed the products generated by the
rabbit reticulocyte lysate system during translation of GLUT4 mRNA,
mutated at the N-glycosylation site Asn57 to
Thr57. The commercially available rabbit reticulocyte
lysate preparation is known to contain sufficient substrate
(UDP-GlcNAc) and OGT enzyme activity for efficient
O-GlcNAcylation of susceptible proteins (38).
Indeed, translation in this system has been recommended for the
identification of low-abundance proteins, which are subject to
O-GlcNAcylation (38). After galactosylation,
O-GlcNAc-bearing proteins can be separated from complex
mixtures by lectin affinity chromatography on a ricin column. Because
Gal-transferase does not distinguish between O-GlcNAc
attached to Ser/Thr residues and terminal GlcNAc moieties in glycosyl
side chains, mutated GLUT4 mRNA, which is not subject to
N-glycosylation, was translated (Fig.
1).
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The data in Fig. 1 are consistent with the concept that GLUT4 is a substrate for O-GlcNAcylation. In the presence of GLUT4 mRNA, the major labeled translation product was an ~50-kDa protein, consistent with GLUT4 (Fig. 1A, lanes 2, 5, and 7). In samples that were not treated with galactosyltransferase, essentially all of the translated GLUT4 was recovered in the flow-through fraction of the ricin column (Fig. 1A, lane 2), and elution of the column with 0.5 M GlcNAc (to test for nonspecific binding,) or with 0.5 M galactose (specific binding) yielded essentially no labeled products (Fig. 1A, lanes 3 and 4). In the Gal-transferase-treated samples (Fig. 1A, lanes 5-7), a major portion of translated GLUT4 was recovered in the flow-through fraction of the ricin column (Fig. 1A, lane 5). Eluting the column with GlcNAc yielded essentially no GLUT4 (lane 6), but, upon elution with 0.5 M galactose, a prominent GLUT4 band appeared (Fig. 1A, lane 7). In the experiments shown in Fig. 1A, the labeled proteins were precipitated with TCA before SDS-PAGE. Essentially identical results were obtained when the column fractions (corresponding to lanes 5 and 7 in Fig. 1A) were immunoprecipitated with mouse or rabbit anti-GLUT4 antibodies before SDS-PAGE (Fig. 1B), confirming the identity of the labeled bands. Quantitative analysis of GLUT4 recovery in the different fractions indicated that at least 20% of the translated GLUT4 was galactosylated, presumably on O-GlcNAc. This calculation may underestimate the extent of O- GlcNAcylation in view of the known inefficiency of the galactosyltransferase reaction, which was likely incomplete (38), and because proteins bearing only one O-GlcNAc may be retarded but not firmly bound to the lectin column (15). Qualitatively similar results were also obtained when wild-type GLUT4 was translated in the reticulocyte lysate system, adsorbed to a wheat germ agarose column, which after extensive washing was eluted with 0.5 M GlcNAc, and the eluate was digested with endoglycosidase F (data not shown). However, the yield of O-GlcNAcylated GLUT4 was greater using the method shown in Fig. 1.
Having shown that GLUT4 is susceptible to
O- GlcNAcylation, we addressed the question whether GLUT4
or proteins associated with it are subject to the O-GlcNAc
modification in skeletal muscles, in vivo, and whether the process is
augmented in GLUT1-overexpressing, insulin-resistant muscles. The
graphs in Fig. 2A represent
[3H]galactose incorporation into total membrane
preparations from muscles of heterozygote transgenic mice
overexpressing GLUT1 in skeletal muscle and their wild-type
littermates. The galactosylation reaction serves as a probe for the
presence of protein-associated terminal GlcNAc moieties. The transgenic
mice express 10-fold more GLUT1 in muscle than the controls but have
equal amounts of GLUT4 (28). To enrich the preparation in
the proteins of interest and minimize nonspecific background
radioactivity, the galactosylated muscle membranes were submitted to
two-step immunoprecipitation, first with an anti-GLUT1 antibody
followed by immunoprecipitation of the supernatant with either
anti-GLUT4 or nonimmune -globulin. [3H]galactose
associated with the anti-GLUT4 immunoprecipitates from 0.5-mg muscle
membranes prepared from control rats was very low and similar to the
radioactivity associated with membranes from control or transgenic
muscles that were treated with nonimmune
-globulin. However, there
was significantly more radioactivity associated with membrane proteins
of GLUT1-overexpressing muscles that had been immunoprecipitated with
anti-GLUT4 IgG. As assessed by their migration on SDS-PAGE, there were
three major peaks between ~100 and ~40 kDa and three minor peaks
ranging from 23 to 35 kDa. Comparing the heights of the major peaks,
the [3H]galactose associated with these fractions in
anti-GLUT4 immunoprecipitates prepared from transgenic muscles was
consistently two to three times higher than in identical preparations
from control rats.
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To assess whether the [3H]galactose was incorporated in
O-GlcNac-modified sites rather than into N-linked
glycosyl side chains, a parallel sample of the immunoprecipitates was
digested with endoglycosidase F before SDS-PAGE (Fig. 2B).
The results are essentially identical to those shown in Fig.
2A. In each experiment, an aliquot of the immunoprecipitated
membranes was saved for immunoblotting before and after endoglycosidase
F digestion (Fig. 3). The change in the
apparent molecular mass of GLUT4 after digestion indicates that the
N-glycosyl side chain was removed by the endoglycosidase. Therefore, the enhanced galactosylation of membrane proteins from the
transgenic mice likely represents enhanced
O- GlcNAcylation of these proteins. It is noteworthy that,
after treatment with endoglycosidase F, GLUT4 still migrated as a broad
band, albeit with increased mobility, suggesting that GLUT4
heterogeneity on SDS-PAGE is not determined solely by the heterogeneity
of the N-glycosyl side chain and that additional
posttranslational modifications are also likely involved.
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In Fig. 2, A and B, membrane proteins were immunoprecipitated with a polyclonal anti-GLUT4 antibody raised in rabbits. To ensure that the immunoprecipitation of O-GlcNAcylated proteins from transgenic mice was not an artifact peculiar to the antibody used, we repeated the experiments using a monoclonal mouse anti-GLUT4 antibody for immunoprecipitation (Fig. 2C). The data shown represent the distribution of [3H]galactose among the immunoprecipitated proteins after endoglycosidase F digestion. Although the overall recovery of labeled proteins was less with the mouse antibody than with the polyclonal rabbit antibody, the pattern of protein recovery and distribution was similar.
For statistical analyses of the data in Fig. 2, the areas under the
curves were integrated using National Institutes of Health Image
software. Data from three independent experiments were analyzed for
each parameter shown in Fig. 2, A-C. The graphic analysis eliminates variations in labeling between experiments but allows for
comparison of controls and trangenics in each experiment because they
were always processed in parallel. Data were analyzed as uncorrected
and corrected (specific) anti-GLUT4 immunoprecipitates. The latter
represent the area under the immunoprecipitate curve minus the area
under the curve generated from a corresponding sample treated with
nonimmune -globulin. The significance of differences between
means was analyzed by two-tailed Student's t-test or by
paired Student's t-test where indicated in the text. Values
represent means (in arbitrary units) ± SE. For Fig.
2A, uncorrected mean values were 7,061 ± 793 for
controls and 11,441 ± 332 for transgenics (n = 3, P < 0.01), and corrected values were 2,394 ± 651 for controls and 5,896 ± 1,196 for transgenics (P < 0.07). The mean ratio of specific immunoprecipitates
(transgenic/control) was 2.66 ± 0.4 (P < 0.025).
In Fig. 2B, means for uncorrected immunoprecipitates were 7,969 ± 1,280 and 10,980 + 892 for controls and trangenics, respectively (n = 3, P < 0.03, paired Student's t-test), and the corrected values were 2,141 ± 614 for controls and 4,960 ± 940 for trangenics (n = 3, P < 0.03, paired Student's t-test). The mean ratio of specific immunoprecipitates (transgenic/control) was 2.53 ± 0.398 (P < 0.03). In Fig. 2C, the corresponding values were 7,710 ± 1,199 for controls and 14,687 ± 1,375 for transgenics (uncorrected immunoprecipitates, n = 3, P < 0.02), and the specific immunoprecipitates were 700 ± 296 for controls and 7,049 ± 967 for trangenics (P < 0.005, n = 3).
In the course of the experiments shown in Fig. 2, we also routinely assessed the recovery of [3H]galactosylated proteins in the immunoprecipitates generated with the anti-GLUT1 rabbit antibody, after separating the immunoprecipitated proteins by SDS-PAGE. We were unable to detect a consistent pattern or reproducible differences between the anti-GLUT1 immunoprecipitates prepared from muscle membranes of control and transgenic mice (data not shown).
In the experiments described above, the yield of
O-GlcNacylated labeled proteins was too low to attempt
identification. In an effort to improve the yield, we performed lectin
affinity chromatography of the galactosylated membrane proteins from
muscles of control and transgenic mice. After labeling with
[3H]galactose, the membranes were digested with
endoglycosidase F before affinity chromatography on a ricine column.
Most of the radiolabeled proteins were retained on the column and were
recovered upon elution with 0.2 M lactose. The distribution of the
radiolabeled proteins after separation by SDS-PAGE is shown in Fig.
4. Although the yield of radiolabeled
proteins had increased compared with the immunoprecipitation
experiments, it was insufficient for visual detection of labeled bands
by fluorography after 1 wk of exposure. Therefore, the proteins were
transferred to nitrocellulose, cut into 0.5-cm segments, solubilized,
and counted. Clearly, the yield of galactosylated proteins was greater
from muscle membranes of transgenic mice compared with controls over
the entire range of apparent molecular masses. The predominant activity
migrated as a broad peak between 50 and 120 kDa, with an apparent
maximum between 55 and 70 kDa. As in the previous experiments, there
appeared to be smaller peaks of lower-approximate-molecular mass
(~15-35 kDa) GlcNAcylated proteins, which again were enriched in
muscle membranes of transgenic mice.
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The integrated areas under the curves were determined by graphic analysis of four independent experiments, as described in Fig. 2. Means (arbitrary units ± SE) were 2,913 ± 378 and 6,275 ± 663 for controls and transgenics, respectively (n = 4, P < 0.005). Unfortunately, the protein concentrations in the concentrated lactose eluates were insufficient for detection after SDS-PAGE by silver or by Coomassie blue staining. We also attempted to ascertain whether GLUT4 was among the proteins that were retained on the column and eluted with 0.2 M lactose. Although we were able to identify GLUT4 in peak 1 (which eluted after the void volume) by immunoblotting, we did not detect it in the lactose eluate (data not shown).
The RL-2 mouse monoclonal antibody was originally raised against
nuclear pore proteins, which are O-GlcNAcylated (17,
40); it recognizes the O-GlcNAc modification on
numerous proteins. To confirm our finding that
O-GlcNAcylation was increased in muscles of mice that
overexpress GLUT1, proteins separated by SDS-PAGE from postnuclear
muscle extracts of transgenic and control mice were immunoblotted with
the RL-2 antibody (Fig. 5). As expected, the antibody recognized numerous proteins, and the signal was markedly
increased in muscles from transgenic mice vs. the controls. Inclusion
of 0.1 M N-acetylglucosamine with the RL-2 antibody, as a
competitive inhibitor of specific binding, diminished the signal in
both controls and transgenics, although it did not abolish it. GlcNAc
competes relatively poorly with protein-bound O-GlcNAc for
binding to the antibody.
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DISCUSSION |
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In recent years, there has been increasing awareness of the potential role of protein O-GlcNAclyation in cellular regulation. Studies from several laboratories suggest potential roles in 1) signaling (as an alternative to or modulator of phosphorylation); 2) protein-protein interactions; 3) modulation of protein degradation; and 4) regulation of DNA transcription and mRNA translation, as well as other processes (6, 12, 38). Despite this interest, there are very few studies addressing the metabolic regulation of protein O- GlcNAcylation in vivo. Yki-Jarvinen et al. (45) reported increased O-GlcNAcylation of proteins in rat skeletal muscles after 6 h of coinfusion of insulin and glucosamine (GlcN). GlcN infusions caused insulin resistance, impaired activation of early insulin-signaling intermediates in muscle, and, based on RL-2 reactivity, enhanced the O-GlcNAcylation of insulin receptor substrate-1 (32). Using in vivo metabolic radiolabeling, Hawkins and colleagues (13, 14) reported markedly enhanced glycosylation of skeletal muscle proteins associated with GLUT4-containing vesicles (GCV) in rats rendered insulin resistant by 7 h of infusion of GlcN plus insulin. However, >90% of the label was incorporated into N-linked glycosyl side chains (13). Thirty minutes of sustained hyperglycemia markedly increased protein O-GlcNAcylation in the islets of Langerhans, based on RL-2 immunocytochemistry. Pancreatic islets appear to be exceptionally rich in O-OGT (26).
GlcN-6-P is the product of the transamidase reaction catalyzed by GFAT, the first and rate-limiting step in HBSP. The entry of extracellular GlcN into cells is facilitated by glucose transporters (in the case of muscle, GLUT1 and GLUT4). GlcN is phosphorylated to GlcN-6-P by hexokinase and enters the HBSP, bypassing GFAT. GFAT activity is subject to allosteric, negative feedback regulation by the major product of the HBSP, UDP-GlcNAc (21, 29). The circulating concentration of GlcN is normally very low (13). Increasing the ambient GlcN concentration can cause metabolic changes in the intact animal (41) and in cell culture systems (18, 30, 31), which do not mimic the effects of increased glucose flux via HBSP. Thus, although both GlcN and sustained hyperglycemia cause insulin resistance, the cellular mechanisms may not always be identical (18, 30, 31, 41).
Skeletal muscle is a major site of insulin resistance in diabetic patients and in animal models of type 1 and type 2 diabetes. The major defect is impaired insulin stimulation of glucose transport (reviewed in Ref. 33). The transgenic mouse overexpressing GLUT1 in skeletal muscle is an interesting model, because the only underlying abnormality is chronically increased glucose flux into muscle. Circulating levels of nutrients, e.g., glucose and free fatty acids, and hormones, e.g., insulin and glucagon, are essentially normal. GLUT4 expression is similar to that of controls, and GLUT4 translocates to the PM in response to insulin (11). However, glucose transport is not stimulated by insulin (10, 11, 28, 36), suggesting that either the translocated GLUT4 is inappropriately inserted in the PM or GLUT4 or proteins associated with GLUT4 are modified in a manner that impairs the intrinsic activity of GLUT4 (11). The chronically increased glucose flux into muscle cells increases flux via HBSP, resulting in increased concentrations of UDP-GlcNAc (5). The data presented here demonstrate concommitant marked increases in the O-GlcNAcylation of muscle membrane proteins (Fig. 4), a subset of membrane proteins that coimmunoprecipitate with GLUT4 (Fig. 2), and numerous proteins in postnuclear total muscle extracts (Fig. 5). To our knowledge, this is the first demonstration of increased protein O-GlcNAcylation without high glucose or GlcN in the extracellular milieu, indicating that increased glucose flux via HBSP, resulting in increased UDP-GlcNAc, is sufficient to promote the O-GlcNAcylation of certain proteins in vivo. The insulin resistance that develops in rats infused with lipid emulsions, resulting in increased plasma free fatty acids, is also associated with increased UDP-GlcNAc in skeletal muscle, presumably reflecting impaired glycolytic flux distal to fructose 6-phosphate (F-6-P), resulting in increased F-6-P flux via HBSP (14). Because increased circulating free fatty acids and their enhanced utilization by muscle are clearly associated with the insulin resistance of obesity and type 2 diabetes, the question arises whether these conditions also promote O- GlcNAcylation of certain proteins in skeletal muscle. Relatively modest elevations in UDP-GlcNAc may promote protein O-GlcNAcylation in part by mass action and by positively regulating OGT activity (23). The concentration of UDP-GlcNAc appears to regulate not only the velocity of the enzyme, but also its Michaelis constant (Km) for different peptide substrates, i.e., substrate selectivity. Furthermore, OGT itself is subject to posttranslational modification by tyrosine phosphorylation and O-GlcNAcylation (23).
Our data obtained in the reticulocyte lysate system (Fig. 1) support the concept that GLUT4 can serve as a substrate for OGT. Although the majority of O-GlcNAc-modified proteins are nuclear or cytosolic, several membrane-associated O-GlcNAcylated proteins have been identified (6, 12, 32). As reported for other O-GlcNAc-modified proteins, GLUT4 is subject to reversible O-phosphorylation (25). The COOH-terminal portion of GLUT4 contains two sequences, which may represent potential O-GlcNAcylation sites, i.e., Val and/or Pro in close proximity to Ser/Thr (38): Thr-Pro-Ser-Leu-Leu-Glu-Gln-Glu-Val-Lys-Pro-Ser-Thr (amino acids 486-498) and Val-Pro-Glu-Thr-Ser (amino acids 469-472). Ser488 is an identified phosphorylation site (25). However, we were not able to conclusively determine that GLUT4 was O-GlcNAcylated in muscle in vivo. Clearly, the muscle membranes, which were precipitated with anti-GLUT4 antibody, yielded GLUT4 in the immunoprecipitates (Fig. 3), and the labeled protein peaks, which represent the O-GlcNAcylated membrane proteins prepared from GLUT1-overexpressing muscles, may contain the O-GlcNAcylated GLUT4 (consisting of 509 amino acids, ~51 kDa without the NH2-linked glycosyl side chain; see, e.g., peak 3 in Fig. 2B). Unfortunately, the low yield precluded the characterization and identification of O-GlcNAcylated proteins. We chose the Gal-transferase method to probe for O-GlcNAcylated proteins because it has been extensively used and validated (38). Provided that N-glycosyl side chains are removed, the probe is specific for proteins modified by O-GlcNAc. However, galactosylation is not stoichiometric, and the yield is low. This reflects, in part, the lability of the O-GlcNAc modification, which may be lost during sample preparation, the relatively high Km of Gal-transferase for UDP-galactose, and the accessibility of the peptide substrate to the enzyme (23, 38).
The proteins that are highly O-GlcNAcylated in
GLUT1-overexpressing muscle membranes and coimmunoprecipitate with
GLUT4 (Fig. 2) have not been identified. The question arises whether
they represent proteins and/or protein complexes, which interact with GLUT4 or nonspecific coimmunoprecipitation of
O- GlcNAcylated proteins, which are more abundant in
membrane preparations from transgenic muscles. The following support
the former interpretation. 1) The O-GlcNAcylated
protein peaks were not observed in "sham immunoprecipitates"
treated with nonimmune -globulin. 2) There was no
difference in the distribution or abundance of
O-GlcNAcylated proteins between membranes prepared from
control and transgenic muscles after immunoprecipitation with
anti-GLUT1
-globulin (GLUT1 and GLUT4 are largely segregated in
separate vesicles; reviewed in Ref. 34). 3)
Similar results were obtained using two different anti-GLUT4 antibodies
(rabbit polyclonal and mouse monoclonal). The source of the putative
GLUT4-associated O- GlcNAcylated membrane proteins is not
clear. GLUT4 is associated with the endoplasmic reticulum and the Golgi
apparatus; it is stored in GCV, which translocate to the PM. The GCV
interact with cytoskeletal components in the process of translocation,
with the docking/fusion machinery at the PM, and GLUT4 recycles in
endocytotic vesicles (34). The COOH-terminal portion of
GLUT4 appears to be associated with unidentified proteins in skeletal
muscle, which mask the antibody recognition site, an association that
is released by insulin (43).
To galactosylate the O-GlcNAcylated membrane proteins, membranes had to be resuspended in detergent (1% CHAPS, a zwitterionic detergent)-containing buffer. It seems highly unlikely that this treatment preserved the integrity of GCV. However, GCV share many characteristics with small synaptic vesicles (SSV) in neurons (34, 35). Solubilization of SSV membranes in CHAPS preserved multimeric protein complexes (including SNARE proteins), but not solubilization in Triton X-100 or octylglucoside (3), and CHAPS-stabilized complexes were resistant to treatment with high salt or DTT, suggesting lypophilic interactions (3). Numerous cytoskeletal and bridging proteins, which are subject to O- GlcNAcylation, have been identified, including the microtubule-associated proteins-1, -2, and -4, tau, vincullin, talin, clathrin assembly protein-3 (AP-3) and synapsin 1, which is thought to anchor synaptic vesicles to the cytoskeleton and mediate their release upon phosphorylation (reviewed in Refs. 6 and 12). O-GlcNAcylation may modulate protein-protein interactions involved in numerous cellular processes (6), which may include GLUT4 trafficking, its appropriate docking/fusion at the PM, and/or its intrinsic activity.
The development of glucose-induced insulin resistance is time
dependent. It requires several hours of hyperglycemia in animal models
and in cell culture systems or, as in the model presented here,
chronically increased glucose flux without hyperglycemia. Numerous
O-GlcNAcylated proteins are transcription factors. Altered regulation of gene expression may contribute to the insulin resistance associated with chronically increased glucose flux into muscle and the
resulting increase in the UDP-GlcNAc pool. Work from several laboratories links the deleterious effects of chronic hyperglycemia on
the vascular system to increased HBSP activity. Chronic exposure to
high glucose of cultured vascular smooth muscle cells increases OGT
protein expression and activity and induces qualitative and quantitative changes in nuclear O-GlcNAcylated proteins
(1). In cultured mesangial cells, the high glucose
induction of transforming growth factor (TGF)-1
(20) and of plasminogen inhibitor-1 (19) is
mediated at least in part by products of HBSP. The
transcription factor Sp-1 has several O-GlcNAcylation
sites, and the O-GlcNAc modification competes with Ser/Thr
phosphorylation (reviewed in Refs. 6, 8, and
12). In bovine aortic endothelial cells, culture in high
glucose induces TGF-
1 and PAI-1 expression, which is
dependent on GFAT activity. O-GlcNAcylation of Sp-1 is
enhanced in high glucose, and mutation of Sp-1 binding sites on the
PAI-1 promoter prevents its induction by glucose. Thus induction of genes known to be involved in the vascular complications of diabetes reflect, at least in part, the enhanced
O-GlcNAcylation of Sp-1 (8).
In conclusion, our data indicate that chronically increased glucose flux into skeletal muscle is associated with insulin resistance, increased intracellular UDP-GlcNAc, and enhanced O-GlcNAc modification of membrane proteins in vivo. They support the notion that the UDP-GlcNAc concentration in muscle may serve as an indicator of and a contributor to the development of insulin resistance (5, 13) by promoting the O-GlcNAcylation of critical proteins. These may include GLUT4 and/or GLUT4-associated proteins.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02001 (to M. G. Buse) and DK-38495, by the Juvenile Diabetes Foundation International, and by the Diabetes Research and Training Center at Washington University (to M. M. Mueckler).
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FOOTNOTES |
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This paper was presented in part at the 60th Annual Meeting of the American Diabetes Association (San Antonio, June 2000) and was published in abstract form (Diabetes 49, Suppl 1: A58, 2000).
Address for reprint requests and other correspondence: M. G. Buse, Medical Univ. of South Carolina, Div. of Endocrinology, Dept. of Medicine, 96 Jonathan Lucas St., Rm. 323 CSB, Charleston, SC 29425 (E-mail: busemg{at}musc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 27, 2002;10.1152/ajpendo.00060.2002
Received 11 February 2002; accepted in final form 18 March 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Akimoto, Y,
Kreppel LK,
Hirano H,
and
Hart GW.
Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation.
Arch Biochem Biophys
389:
166-175,
2001[ISI][Medline].
2.
Bell, GI,
Burant CF,
Takeda J,
and
Gould GW.
Structure and function of mammalian sugar transporters.
J Biol Chem
268:
19161-19164,
1993
3.
Bennett, MK,
Calakos N,
Kreiner T,
and
Scheller RH.
Synaptic vesicle membrane proteins interact to form a multimeric complex.
J Biol Chem
116:
761-775,
1992.
4.
Buse, MG,
Robinson KA,
Gettys T,
McMahon E,
and
Gulve E.
Increased activity of the hexosamine biosynthesis pathway in muscles of insulin resistant ob/ob mice.
Am J Physiol Endocrinol Metab
272:
E1080-E1088,
1997
5.
Buse, MG,
Robinson KA,
Marshall BA,
and
Mueckler M.
Differential effects of GLUT1 or GLUT4 overexpression on hexosamine biosynthesis by muscles of transgenic mice.
J Biol Chem
271:
23197-23202,
1996
6.
Comer, FI,
and
Hart GW.
O-glycosylation of nuclear and cytosolic proteins.
J Biol Chem
275:
29179-29182,
2000
7.
Cooksey, RC,
Herbert LF,
Zhou JH,
Wofford P,
Garvey WT,
and
McClain DA.
Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione.
Endocrinology
140:
1151-1157,
1999
8.
Du, XL,
Edelstein D,
Rossetti L,
Fantus IG,
Goldberg H,
Ziyadeh F,
Wu J,
and
Brownlee M.
Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing SP-1 glycosylation.
Proc Natl Acad Sci USA
97:
12222-12226,
2000
9.
Gao, Y,
Wells L,
Comer FI,
Parker GJ,
and
Hart GW.
Dynamic O-glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a neutral, cytosolic -N-acetylglucosaminidase from human brain.
J Biol Chem
276:
9838-9845,
2001
10.
Gulve, EA,
Ren J-M,
Marshall BA,
Gao J,
Hansen PA,
Holloszy JO,
and
Mueckler M.
Glucose transport activity in skeletal muscles of transgenic mice overexpressing GLUT1 increased basal transport is associated with a defective response to diverse stimuli that activate GLUT4.
J Biol Chem
259:
18366-18370,
1994.
11.
Hansen, PA,
Wang W,
Marshall BA,
Holloszy JO,
and
Mueckler M.
Dissociation of GLUT4 translocation and insulin-stimulated glucose transport in transgenic mice overexpressing GLUT1 in skeletal muscle.
J Biol Chem
273:
18173-18179,
1998
12.
Hart, GW.
Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins.
Annu Rev Biochem
66:
315-335,
1997[ISI][Medline].
13.
Hawkins, M,
Angelov I,
Lui R,
Barzilai N,
and
Rossetti L.
The tissue concentration of UDP-N-acetylglucosamine modulates the stimulatory effect of insulin on skeletal muscle glucose uptake.
J Biol Chem
272:
4889-4895,
1997
14.
Hawkins, M,
Barzilai N,
Lui R,
Hu M,
Chen W,
and
Rossetti L.
Role of the hexosamine synthesis pathway in fat-induced insulin resistance.
J Clin Invest
99:
2173-2182,
1997
15.
Hayes, BK,
Greis KD,
and
Hart GW.
Specific isolation of O-linked N-acetylglucosamine glycopeptides from complex mixtures.
Anal Biochem
228:
115-122,
1995[ISI][Medline].
16.
Herbert, LF,
Daniels M,
Zhou J,
Crook E,
Simmons S,
Neidigh J,
Baron A,
and
McClain D.
Overexpression of glutamine-fructose-6-phosphate: amidotransferase in transgenic mice leads to insulin resistance.
J Clin Invest
98:
930-936,
1996
17.
Holt, GD,
Snow CM,
Senior A,
Haltiwanger RS,
Gerace L,
and
Hart GW.
Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.
J Cell Biol
104:
1157-1164,
1987[Abstract].
18.
Hresko, RC,
Heimberg H,
Chi MM,
and
Mueckler M.
Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP.
J Biol Chem
273:
20658-20668,
1998
19.
James, LR,
Fantus G,
Goldberg H,
Ly H,
and
Scholey JW.
Overexpression of GFAT activates PAI-1 promoter in mesangial cells.
Am J Physiol Renal Physiol
279:
F718-F727,
2000
20.
Kolm-Litty, V,
Sauer U,
Nerlich A,
Lehmann R,
and
Schleicher ED.
High glucose-induced transforming growth factor -1 production is mediated by the hexosamine pathway in porcine mesangial cells.
J Clin Invest
101:
160-169,
1998
21.
Kornfeld, S,
Kornfeld R,
Neufeld EF,
and
OBrien PJ.
The feedback control of sugar nucleotide biosynthesis in liver.
Proc Natl Acad Sci USA
52:
371-379,
1964[ISI][Medline].
22.
Kreppel, LK,
Bloomberg MA,
and
Hart GW.
Dynamic glycosylation of nuclear and cytosolic proteins: cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.
J Biol Chem
272:
9308-9315,
1997
23.
Kreppel, LK,
and
Hart GW.
Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats.
J Biol Chem
274:
32015-32022,
1999
24.
Krieg, PA,
and
Melton DA.
Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.
Nucleic Acids Res
12:
7057-7070,
1984[Abstract].
25.
Lawrence, JC, Jr,
Hiken JF,
and
James DE.
Phosphorylation of the glucose transporter in rat adipocytes. Identification of the intracellular domain at the carboxyl terminus as a target for phosphorylation in intact cells and in vitro.
J Biol Chem
265:
2324-2332,
1990
26.
Liu, K,
Paterson AJ,
Chin E,
and
Kudlow JE.
Glucose stimulates protein modification by O-linked GlcNAc in pancreatic beta cells: linkage of O-linked GlcNAc to beta cell death.
Proc Natl Acad Sci USA
97:
2820-2825,
1984
27.
Lubas, WA,
Frank DW,
Krause M,
and
Hanover JA.
O-linked GlcNAc transferase is a conserved nucleoplasmic protein containing tetratricopeptide repeats.
J Biol Chem
272:
9316-9324,
1997
28.
Marshall, BA,
Ren JM,
Johnson DW,
Gibbs EM,
Lillquist JS,
Soeller WC,
Holloszy JO,
and
Mueckler M.
Germline manipulation of glucose homeostasis via alteration of glucose transporter levels in skeletal muscle.
J Biol Chem
268:
18442-18445,
1993
29.
Marshall, S,
Bacote V,
and
Traxinger R.
Discovery of a metabolic pathway mediating glucose-induced densitization of the glucose transport system.
J Biol Chem
266:
4706-4712,
1991
30.
Nelson, BA,
Robinson KA,
and
Buse MG.
High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes.
Diabetes
49:
981-991,
2000[Abstract].
31.
Nelson, BA,
Robinson KA,
and
Buse MG.
Defective Akt activation is associated with glucose- but not glucosamine-induced insulin resistance.
Am J Physiol Endocrinol Metab
282:
E497-E506,
2002
32.
Patti, ME,
Virkamaki A,
Landaker EJ,
Kahn CR,
and
Yki-Jarvinen H.
Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early post-receptor signaling events in skeletal muscle.
Diabetes
48:
1562-1571,
1999[Abstract].
33.
Pessin, JE,
and
Saltiel AR.
Signaling pathways in insulin action: molecular targets of insulin resistance.
J Clin Invest
106:
165-169,
2000
34.
Pessin, JE,
Thurmond DC,
Elmendorf JS,
Coker KJ,
and
Okada S.
Molecular basis of insulin-stimulated GLUT4 vesicle trafficking.
J Biol Chem
274:
2593-2596,
1999
35.
Rea, S,
and
James DE.
Moving GLUT4. The biogenesis and trafficking of GLUT4 storage vesicles.
Diabetes
46:
1667-1677,
1997[Abstract].
36.
Ren, JM,
Marshall BA,
Gulve EA,
Gao J,
Johnson DW,
Holloszy JO,
and
Mueckler M.
Evidence from transgenic mice that glucose transport is rate-limiting for glycogen deposition and glycolysis in skeletal muscle.
J Biol Chem
268:
16113-16115,
1993
37.
Robinson, KA,
Weinstein M,
Lindenmayer G,
and
Buse MG.
Effects of diabetes and hyperglycemia on the hexosamine synthesis pathway in rat muscle and liver.
Diabetes
44:
1438-1446,
1995[Abstract].
38.
Roquemore, EP,
Chou T,
and
Hart GW.
Detection of O-linked n-acetylglucosamine (O-GlcNAc) on cytoplasmic and nuclear proteins.
Methods Enzymol
230:
443-460,
1994[ISI][Medline].
39.
Rossetti, L,
Giaccari A,
and
DeFronzo R.
Glucose toxicity.
Diabetes Care
13:
610-630,
1990[Abstract].
40.
Snow, CM,
Senior A,
and
Gerace L.
Monoclonal antibodies identify a group of nuclear pore complex glycoproteins.
J Cell Biol
104:
1143-1156,
1987[Abstract].
41.
Virkamaki, A,
and
Yki-Jarvinen H.
Allosteric regulation of glycogen synthetase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart.
Diabetes
48:
1101-1107,
1999[Abstract].
42.
Walter, P,
and
Blobel G.
Preparation of microsomal membranes for cotranslational protein translocation.
Methods Enzymol
96:
84-93,
1983[ISI][Medline].
43.
Wang, W,
Hansen PA,
Marshall BA,
Holloszy JO,
and
Mueckler M.
Insulin unmasks a COOH-terminal GLUT4 epitope and increases glucose transport across T-tubules in skeletal muscle.
J Cell Biol
135:
415-430,
1996[Abstract].
44.
Yki-Jarvinen, H,
Helve E,
and
Koivisto V.
Hyperglycemia decreases glucose uptake in type 1 diabetes.
Diabetes
36:
892-896,
1987[Abstract].
45.
Yki-Jarvinen, H,
Virkamaki A,
Daniels MC,
McClain D,
and
Gottschalk WK.
Insulin and glucosamine infusions increase O-linked N-acetylglucosamine in skeletal muscle proteins in vivo.
Metabolism
47:
449-455,
1998[ISI][Medline].