1 Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, and 2 Departments of Pharmacology, 3 Pathology, and 4 Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425
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ABSTRACT |
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Increased flux through the
hexosamine biosynthesis pathway has been implicated in the development
of glucose-induced insulin resistance and may promote the modification
of certain proteins with O-linked
N-acetylglucosamine (O-GlcNAc). L6 myotubes (a
model of skeletal muscle) were incubated for 18 h in 5 or 25 mM
glucose with or without 10 nM insulin. As assessed by immunoblotting
with an O-GlcNAc-specific antibody, high glucose and/or
insulin enhanced O-GlcNAcylation of numerous proteins,
including the transcription factor Sp1, a known substrate for this
modification. To identify novel proteins that may be
O-GlcNAc modified in a glucose
concentration/insulin-responsive manner, total cell membranes were
separated by one- or two-dimensional gel electrophoresis. Selected
O-GlcNAcylated proteins were identified by mass spectrometry
(MS) analysis. MS sequencing of tryptic peptides identified member(s)
of the heat shock protein 70 (HSP70) family and rat -tubulin.
Immunoprecipitation/immunoblot studies demonstrated several HSP70
isoforms and/or posttranslational modifications, some with selectively
enhanced O-GlcNAcylation following exposure to high glucose
plus insulin. In conclusion, in L6 myotubes, Sp1, membrane-associated
HSP70, and
-tubulin are O-GlcNAcylated; the modification
is markedly enhanced by sustained increased glucose flux.
insulin resistance; L6 myotubes; O-linked N-acetylglucosamine; heat shock protein 70
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INTRODUCTION |
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THE ADDITION OF A SINGLE N-acetylglucosamine (GlcNAc) moiety to serine or threonine residues in proteins (O-GlcNAcylation) is a common posttranslational modification. It is unlike other glycosylation events in that it occurs through an enzyme-catalyzed reaction in the cytosol and the nucleus rather than in the Golgi apparatus or the endoplasmic reticulum. O-GlcNAcylation is a dynamic and reversible process, regulated by the activities of O-GlcNAc transferase (37, 41) and N-acetylglucosaminidase (O-GlcNAcase) (18), which have been characterized and cloned. Although there is not a well-defined consensus sequence for O-GlcNAcylation, the modified regions generally contain a proline residue and usually multiple serines and threonines. These sites are often identical to the motifs used by serine and threonine kinases. In fact, in some cases, O-GlcNAcylation is reciprocal with phosphorylation (4, 6), which may have important consequences on protein activity and signaling. In addition to its potential role in regulating phosphorylation, O-GlcNAcylation also appears to play a role in protein transcription and translation, nuclear targeting and transport, and protein degradation (reviewed in Refs. 5 and 25). More information about the function of O-GlcNAcylation is being discovered as the proteins that undergo this modification are being identified.
O-GlcNAcylation may play a role in certain disease
states such as Parkinson's disease, Alzheimer's disease, and diabetes
(5, 64). In patients with diabetes, sustained
hyperglycemia leads to insulin resistance ("glucose toxicity")
(57). This glucose-induced insulin resistance may reflect,
at least in part, increased flux through the hexosamine
biosynthesis pathway (HBSP), a quantitatively minor
component of overall glucose disposal (42). HBSP is the obligatory source of essential building blocks for the glycosylation of
proteins and lipids. The major product is UDP-GlcNAc, the obligatory substrate for the transfer of O-GlcNAc to proteins. Studies
in rodents (27, 28) suggest a correlation between the
development of insulin resistance and increased UDP-GlcNAc
concentrations in muscle. The latter may enhance the
O-GlcNAc modification of certain proteins (37,
25), which may contribute to insulin resistance.
Insulin-sensitive tissues, such as skeletal muscle, respond to the
hormone with acceleration of glucose transport, which reflects, in
great part, increased expression of the insulin-responsive glucose
transporter isoform GLUT4 at the cell membrane. GLUT4 translocation and
vesicle fusion with the plasmalemma involve an intricate signaling
pathway linking the insulin receptor tyrosine kinase to numerous
adapter proteins, kinases, and vesicular and cytoskeletal proteins.
Enhanced O-GlcNAc modification of participating proteins may
affect the insulin response. For instance, impaired insulin signaling
concomitant with increased O-GlcNAcylation of insulin
receptor substrate (IRS)-1 and IRS-2 was reported in skeletal muscle of
rats infused with glucosamine plus insulin (52), and enhanced O-GlcNAc modification of IRS-1 and -catenin was
observed in 3T3-L1 adipocytes rendered insulin resistant by treatment
with an O-GlcNAcase inhibitor (63).
Coinfusion of insulin and glucosamine for 6 h increases UDP-GlcNAc in skeletal muscle and enhances the O-GlcNAc modification of numerous unidentified proteins (66). Transgenic mice overexpressing GLUT1 in skeletal muscle exhibit chronically increased glucose flux and increased UDP-GlcNAc concentrations in muscle (3). Although basal glucose transport is markedly increased, it is resistant to further stimulation by insulin, although GLUT4 expression is normal (20). Muscles of these transgenic mice show two- to threefold enhancement of the O-GlcNAc modification of numerous membrane-associated proteins, including proteins that appear to be associated with GLUT4 (3). In the present study, we used L6 myotubes as a model system for skeletal muscle, aiming to identify proteins with enhanced O-GlcNAc modification in response to sustained increased glucose flux.
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MATERIALS AND METHODS |
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Materials.
Unless otherwise noted, materials were purchased from Sigma Chemical
(St. Louis, MO) and were of the highest quality available. -Minimal
essential growth medium (MEM) and Dulbecco's modified Eagle's medium
(DMEM) were purchased from GIBCO Invitrogen (Grand Island, NY). Human
recombinant insulin was a gift from Lilly Research Laboratories
(Indianapolis, IN). Rabbit polyclonal anti-Sp1 (PEP-2), anti-
-tubulin antibodies, and monoclonal anti-HSP70 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Rabbit polyclonal anti-HSP70 was obtained from Upstate Biotechnologies (Lake Placid, NY).
Monoclonal RL-2 anti-O-GlcNAc antibody was purchased from ABR (Golden, CO). Anti-O-GlcNAc (110.6) antibody was a kind
gift of Dr. G. W. Hart (7). Rabbit polyclonal
anti-Sp1 (3517) was a kind gift of Dr. J. E. Kudlow
(23). Nitrocellulose membranes were obtained from Osmonics
(Westborough, MA). Ampholytes, immobilized pH gradient (IPG)
isoelectric focusing strips, IPG strip Criterion gels, and
Sypro orange stain were purchased from Bio-Rad (Hercules, CA). E-zinc
stain was obtained from Pierce (Rockford, IL). Sequencing-grade acetonitrile, formic acid, and trifluoroacetic acid (TFA) were purchased from Fisher Chemical (Pittsburgh, PA). Sequencing-grade trypsin was obtained from Promega (Madison, WI), and C-18 ZipTips were
from Millipore (Bedford, MA).
L6 cell culture.
Cryopreserved L6 myocytes/myoblasts were a kind gift of Dr. Amira Klip.
The cells were passaged according to Mitsumoto and Klip
(44) in -MEM growth medium containing 100 U/ml
penicillin, 100 µg/ml streptomycin, 250 µg/ml amphotericin, and
10% fetal calf serum. To obtain differentiated myotube cultures, cells
were seeded at 2 × 104 cells/ml into
-MEM
containing 2% fetal calf serum and maintained in culture for
1 wk
postconfluence before experimentation.
L6 cell treatment and fractionation. Once differentiated, cells were incubated for 18 h in DMEM containing 1% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 250 µg/ml amphotericin, 4 mM glutamine, 1 mM sodium pyruvate, and either 5 or 25 mM glucose in the presence or absence of 10 nM insulin. Glucosamine-treated cells were incubated in medium containing 5 mM glucose and 2.5 mM glucosamine in the presence or absence of 10 nM insulin.
In Sp1 studies, total cell extracts were prepared as follows. L6 cells were rinsed with PBS, scraped into 1 ml of lysis buffer containing 10 mM Tris (pH 7.5), 600 mM NaCl, 1 mM dithiothreitol (DTT), 50 mM GlcNAc, 330 µM calpain inhibitor 1 (N-acetyl-leu-leu-norleu-al, LLnL), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/ml each of leupeptin, aprotinin, and pepstatin A, frozen and thawed three times, and centrifuged at 1,000 g for 1 min. The supernatant was homogenized with a Dounce homogenizer, mixed for 30 min at 4°C, and centrifuged at 10,000 g for 10 min. The supernatant was analyzed for protein content and used for SDS-PAGE or immunoprecipitation. For studies examining O-GlcNAcylation of membrane-associated proteins, plates were rinsed with cold PBS, and cells were scraped into 200 µl of preparation buffer containing 10 mM Tris (pH 7.4), 250 mM sucrose, 1 mM EDTA, 330 µM LLnL, 1 mM PMSF, and 10 µg/ml leupeptin and aprotinin. Cells were homogenized with a Dounce homogenizer, and cell debris was pelleted at 14,000 rpm in a microcentrifuge. The supernatant was centrifuged at 174,000 g for 2 h at 4°C. The resulting supernatant was designated the cytosol. The pellet was resuspended in 125 µl of reconstitution buffer containing 50 mM HEPES (pH 7.3), 150 mM NaCl, 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 330 µM LLnL, and 10 µg/ml leupeptin and aprotinin, and was designated the membrane fraction. Membrane fractions were mixed at 4°C forImmunoprecipitations.
Immunoprecipitation of Sp1 was carried out overnight in 1 ml of lysis
buffer diluted to normal salt (150 mM NaCl) concentration containing
500-600 µg of total cell extract protein, 25 µl of packed
protein A-Sepharose beads, and 2 µg/ml rabbit polyclonal anti-Sp1 antibody (Santa Cruz Biotechnologies). Immunoprecipitation of other proteins of interest from membrane fractions was carried out
overnight in 0.5-1 ml of membrane reconstitution buffer diluted to
0.5% CHAPS containing 300-500 µg of membrane protein,
25-40 µl of packed protein A-Sepharose (Sigma) or
anti-IgM-agarose (Sigma) beads, and 1-2 µg of primary antibody.
Rabbit polyclonal antibodies were used for the immunoprecipitation of
HSP70 (Upstate Biotechnologies) and -tubulin (Santa Cruz
Biotechnologies). An anti-O-GlcNAc (IgM) antibody (110.6)
(7) was used to immunoprecipitate
O-GlcNAcylated proteins.
SDS-PAGE and 2-DE. For Sp1 experiments, proteins were separated by SDS-PAGE (7% polyacrylamide) and were transferred to nitrocellulose. Blots were developed using the mouse monoclonal RL-2 anti-O-GlcNAc antibody (ABR) or a rabbit Sp1 antibody (23).
For 2-DE analysis, samples were cup loaded onto Bio-Rad IPG isoelectric focusing strips (various pH gradients depending on protein of interest) at 300 V for 3 h. Voltage was gradually increased to 3,500 V over the next hour, and strips were focused for an additional 18 h at 3,500 V. After being focused, strips were incubated for 15 min in 2-DE equilibration buffer containing 50 mM Tris · HCl (pH 6.8), 6 M urea, 30% glycerol, 2% SDS, a trace of bromphenol blue dye, and 15 mg/ml DTT. This buffer was removed, and the strips were incubated in the same buffer containing 18 mg/ml iodoacetamide. After this equilibration, the proteins in the strips were separated on either 10-20% gradient or 10% Bio-Rad Criterion IPG strip Tris gels (preequilibrated with SDS). Gels were then either stained immediately with E-zinc stain or Sypro orange or transferred to nitrocellulose for Western blot analysis. Bands of interest were excised from protein-stained gels, destained, and either digested immediately with trypsin or frozen atMass spectrometry: in-gel trypsin digestion. Excised gel bands were incubated in 100 mM ammonium bicarbonate for 1 h. This was removed, and 50 µl of a solution containing 50% 100 mM ammonium bicarbonate and 50% acetonitrile were placed on each sample and then removed. Gel pieces were then cut into 1 × 1-mm fragments and placed into tubes with 100 µl of acetonitrile. After 15 min of incubation, acetonitrile was removed, and samples were dried in a Speed-Vac. Ten microliters of 10 ng/ml sequencing-grade trypsin were placed on each of the samples and allowed to soak into the gel pieces for 10 min. Enough 100 mM ammonium bicarbonate solution to cover the gel pieces was placed into each tube, and the samples were incubated overnight at 37°C. The following morning, the solution from each tube was removed, saved, and replaced with a similar volume of 50% acetonitrile and 5% formic acid, and the samples were sonicated for 20 min. The solution was removed from the gel pieces and saved, and the procedure was repeated. The three extracts were combined and dried for 2-3 h in a Speed-Vac.
Matrix-assisted laser desorption ionization (MALDI) analysis was performed on a Voyager-DE Biospectrometry Workstation (Applied Biosystems). Samples were resuspended in 10% acetonitrile and 0.1% TFA and desalted using a C-18 Zip-Tip. After a wash with 0.1% TFA, peptides were eluted in 2 µl of 50% acetonitrile and 0.1% TFA for MALDI analysis and 50% acetonitrile and 2% acetic acid for nanospray analysis. Samples were mixed 1:3 (vol/vol) with the matrixStatistical analyses. Means ± SE are shown. The significance of differences between means was evaluated by two-tailed, unpaired Student's t-test and by one-way analysis of variance (ANOVA; Microsoft Excel 2000; Redmond, WA). P < 0.05 was considered significant.
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RESULTS |
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O-GlcNAcylation of Sp1.
The ability of L6 myotubes to O-GlcNAcylate the
transcription factor Sp1 was examined using
immunoprecipitation/immunoblotting. Sp1 was immunoprecipitated from
total cellular proteins using the anti-Sp1 rabbit polyclonal antibody
(PEP-2). O-GlcNAcylation was determined by immunoblotting
with RL-2 antibody. We found that Sp1 is O-GlcNAcylated in
L6 myotubes. When incubated in 5 mM glucose, the addition of 10 nM
insulin enhanced Sp1 O-GlcNAcylation approximately threefold
(Fig. 1, A and C).
Incubation in 25 mM glucose (without insulin) further enhanced
Sp1 O-GlcNAcylation (~4-fold increase compared with 5 mM
glucose without insulin). Concurrent treatment with insulin and 25 mM
glucose did not result in additional enhancement of the modification.
The observed changes in O-GlcNAcylation were not due to
differences in Sp1 protein levels (Fig. 1, B and
D).
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Identification of O-GlcNAcylated proteins in L6 membrane fractions.
In view of previous studies that indicated that chronically increased
glucose flux promoted the O-GlcNAc modification of membrane proteins in skeletal muscle (2), we next examined the
O-GlcNAcylation of membrane-associated proteins resolved on
7% SDS-PAGE and immunoblotted with RL-2 (Fig.
2A). Numerous proteins
appeared to be O-GlcNAcylated in L6 membrane
fractions. Incubation in media containing 25 mM glucose or 2.5 mM
glucosamine added to 5 mM glucose enhanced the O-GlcNAc
modification of several protein bands (compared with 5 mM glucose) in
the absence of insulin. Insulin markedly enhanced O-GlcNAcylation in cells incubated in 5 mM glucose and,
unlike Sp1, further increased the signal from several proteins in cells treated with either 25 mM glucose or 2.5 mM glucosamine.
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HSP70 is O-GlcNAcylated in L6 myotubes.
Protein samples that corresponded to immunostained proteins from L6
membrane fractions were excised from two-dimensional gels and digested
with trypsin. Proteins were extracted and analyzed by MALDI. A
representative spectrum is shown in Fig.
3A. The ProFound software
program (PROWL, Rockefeller University) was used to analyze the tryptic
fragments. Mining of the database revealed a match with the HSP70
family of proteins.
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-Tubulin is O-GlcNAcylated in L6 myotubes.
Protein samples that corresponded to immunostained proteins from L6
membrane fractions were excised from 10% SDS-PAGE gels and digested
with trypsin. Proteins were extracted and analyzed by MALDI. A
representative spectrum is shown in Fig.
4A. The ProFound software
program was used to analyze the tryptic fragments. Mining of the
database revealed a match with
-tubulin.
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O-GlcNAcylation of HSP70 and -tubulin is enhanced in high
glucose-treated L6 myotubes.
The ability of L6 cells to O-GlcNAcylate HSP70 and
-tubulin was further confirmed using
immunoprecipitation/immunoblotting. Total membranes were isolated from
L6 cells treated with either normal glucose (5 mM) alone or high
glucose (25 mM) plus 10 nM insulin. Proteins were separated on 7%
SDS-PAGE gels and transferred to nitrocellulose. Resulting immunoblots
using either the RL-2 anti-O-GlcNAc antibody or monoclonal
anti-HSP70 revealed that O-GlcNAcylation of proteins in
the molecular weight range of HSP70 increased following exposure
of the cells to high glucose, whereas protein levels of HSP70 did not
change significantly (Fig.
5A). We also
immunoprecipitated HSP70 with a polyclonal antibody, and separated
these immunoprecipitates on pH 5-8 two-dimensional gels. After
transferring these immunoprecipitated proteins to nitrocellulose, we
immunoblotted again with either monoclonal anti-HSP70 or RL-2. The
immunoblots resolved several proteins that reacted with the anti-HSP70
antibody. There was very little change in protein levels between the 5 and 25 mM glucose treatment groups, except for a small decrease in
immunostaining in the proteins with lower isoelectric point (pI).
However, when these blots were probed with RL-2, there was a
substantial increase in immunostaining of several protein spots with a
greater pI in the high-glucose treatment group (Fig. 5B).
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DISCUSSION |
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Increased UDP-GlcNAc in skeletal muscle is associated with insulin resistance in numerous experimental models (3, 27, 28). It may promote the O-GlcNAc modification of proteins in part by mass action and in part as an allosteric regulator of O-GlcNAc transferase, enhancing the affinity of the enzyme for certain protein substrates (38). Indeed, enhanced O-GlcNAc modification of numerous proteins has been demonstrated in skeletal muscle in vivo following sustained infusions of glucosamine and insulin (52, 66), a condition that causes insulin resistance and large increases in UDP-GlcNAc. In transgenic mice overexpressing GLUT1 in muscle, chronically increased glucose flux into muscle is associated with insulin resistance of muscle glucose transport, approximately twofold increases in UDP-GlcNAc, and markedly increased O-GlcNAc modification of numerous membrane-associated muscle proteins. These proteins coimmunoprecipitated with GLUT4 (the insulin-responsive glucose transporter), suggesting that they may be involved in the translocation to or fusion with the cell membrane of GLUT4-containing vesicles (2). A major objective of the present work was to identify some of the membrane-associated proteins that responded to increased glucose flux with enhanced O-GlcNAc modification with the use of L6 myotubes as a model for skeletal muscle.
Preincubation in 25 mM glucose plus 100 nM insulin for 24 h impairs subsequent acute insulin-stimulated GLUT4 translocation in L6 myotubes. The proximal insulin-signaling cascade is downregulated in terms of both protein expression of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) and phosphorylation (activation) of IR, IRS-1, and phosphatidylinositol 3-kinase (PI 3 kinase) and Akt. Concomitantly, the intrinsic activity of GLUT4 appears to be upregulated, which in part compensates for the translocation defect and may reflect enhanced p38 mitogen-activated protein kinase activity (29). Insulin-stimulated GLUT4 translocation is also impaired in muscles of insulin-resistant transgenic mice, which overexpress glutamine fructose-6-phosphate amidotransferase (the rate-limiting enzyme of HBSP) in skeletal muscle and adipocytes (9). On the other hand, in GLUT1-overexpressing muscles, insulin-stimulated GLUT4 translocation to the plasma membrane appears to be unimpaired (as assessed by exofacial photolabeling and by electron microscopy) (24), and the impaired insulin-stimulated glucose transport is consistent with inappropriate insertion of GLUT4 into the plasma membrane and/or decreased GLUT4 intrinsic activity. The O-GlcNAc modification of numerous proteins is increased both in L6 cells incubated in high glucose plus insulin and in GLUT1-overexpressing muscles. There are many differences between the two models; the difference in the chronic ambient insulin concentration, which is <0.1 nM in serum of GLUT1-transgenic mice, deserves comment. In 3T3-L1 adipocytes, sustained incubation with insulin 100 nM or higher downregulates the proximal insulin-signaling cascade (53, 54), whereas this pathway is not affected by sustained exposure to low or high glucose in the presence of 0.6 nM insulin (47, 48). The insulin resistance of glucose transport that develops in the presence of 25 mM glucose and 0.6 nM insulin in 3T3-L1 adipocytes appears to be consistent with modest impairment of GLUT4 translocation (49) and, in addition, possible dysregulation of GLUT4 insertion into the cell membrane and/or decreased GLUT4 intrinsic activity (47, 49). Insulin-mediated Akt activation is impaired distally to PI 3-kinase activation in these insulin-resistant 3T3-L1 adipocytes (48) as well as in rat skeletal muscle after incubation in high glucose (39). Which of these mechanisms, including protein modifications such as O-GlcNAcylation, is primarily responsible for glucose-induced insulin resistance of glucose transport in skeletal muscle, in vivo, in rodents or humans (57) is yet to be determined.
Sp1 is a transcription factor, which, along with several other RNA
polymerase II transcription factors, is known to be O-GlcNAc modified at multiple sites (12). To characterize the
regulation of the O-GlcNAc modification in L6 myotubes, in
initial experiments we examined whether these cells responded to
insulin and/or exposure to high glucose by increasing the
O-GlcNAc modification of Sp1. In addition to regulating the
expression of housekeeping genes, hormones, and metabolic enzymes such
as acetyl-CoA carboxylase (10), leptin (8,
15), fatty acid synthase, and ATP citrate-lyase (15,
16), enhanced transcriptional activity of Sp1 has been associated with glucose-induced secondary diabetic complications through enhancement of transforming growth factor (TGF)-
(58), TGF-
, and plasminogen activator inhibitor (PAI)-1
transcription (12, 33, 36). Functional effects of
O-GlcNAcylation on Sp1 include protection from protein
degradation (22) and disruption of the interaction of Sp1
with TATA binding-associated factor 110 and holoSp1 (55).
In the present study, O-GlcNAcylation of Sp1 in L6 cells was
apparent and was enhanced when glucose uptake was increased either through addition of insulin in normal (5 mM) glucose medium or after
exposure of the cells to high glucose (25 mM). Total expression of Sp1
protein was unchanged under all conditions (Fig. 1B). In a
previous report (22), O-GlcNAcylation enhanced
Sp1 protein expression by slowing Sp1 degradation. However, the
experimental conditions in Ref. 22 were markedly different
from ours. Normal rat kidney-derived cells were incubated for 39 h
in glucose-free medium, and forskolin was added during the last 24 h to elevate intracellular cAMP. The latter promoted Sp1
deglycosylation and Sp1 degradation only in glucose-starved cells; it
was ineffective in cells incubated in 5 mM glucose or in 5 mM
glucosamine. In our experiments, control L6 cells were incubated in 5 mM glucose. Possibly, the site that stabilizes Sp1 protein is already
O-GlcNAc modified under physiological conditions, and the
enhanced O-GlcNAcylation in response to increased glucose
flux may involve additional Sp1 sites. Similarly, in other studies
where incubation in high glucose (compared to physiological glucose)
promoted Sp1 O-GlcNAcylation and the induction of TGF-1
and PAI-1, no effect on Sp1 protein expression was observed
(12). Hyperglycemia-mediated activation of the PAI-1
promoter was blocked by mutating its two Sp1-binding sites or by
decreasing HBSP activity, which reduced UDP-GlcNAc availability
(12). DNase I protection studies failed to demonstrate increased DNA binding of O-GlcNacylated Sp1, although its
transcriptional efficiency appeared to be markedly enhanced
(32). O-GlcNAcylation of Sp1 decreases its
O-phosphorylation (12, 21). Because
O-GlcNAc modification of Ser484 in the
glutamine-rich transactivation domain of Sp1 inhibits known Sp1 protein
interactions (55), it has been proposed that enhanced
O-GlcNAcylation may increase Sp1-mediated transactivation by
inhibiting the binding of nuclear repressor proteins, such as p74 and
the retinoblastoma-related protein p107 (11, 46), to the
amino terminus of the Sp1 transactivation domain (12). However, O-GlcNAcylation of the Sp1 activation domain
peptide or of native Sp1 decreases its transcriptional efficiency in an in vitro assay system (65). Thus, although there is
apparent consensus that O-GlcNAcylation modulates Sp1
transcriptional activity, the mechanisms of regulation are
controversial. As with Sp1, enhanced O-GlcNAc modification
of HSP70 or of
-tubulin had no significant effect on the
concentration of these proteins in our study (Figs. 5 and 6).
We identified HSP70 as one of the membrane-associated,
high-glucose/insulin-responsive O-GlcNAcylated proteins in
L6 myotubes. The HSP70 family is one of several ubiquitous molecular
chaperone protein families. HSP70s are generally cytosolic but can also be associated with cytoskeletal and membrane proteins
(61). HSP70s have been found to be important during
protein translation, folding, and transport in the cell, as well as in
regulating protein aggregation and degradation (26, 59).
In renal tubular epithelial cells, HSP70 associates with the
sodium-dependent glucose transporter, increasing its expression at the
apical membrane, resulting in enhanced glucose transport into the cell
(30). Whether or not HSP70 interacts with facilitative
glucose transporters or plays a role in the genesis and/or trafficking
of GLUT4-containing vesicles is unknown. Alternatively, HSPs may serve
as a sink for modified (e.g., O-GlcNAcylated) proteins that
occur under various "stress" conditions (e.g., hyperglycemia).
Interestingly, in rat liver extracts, members of the HSP70 family,
including heat shock cognate protein 70 (HSC70), were found not only to
be O-GlcNAc modified, but also to bind to, or serve as
"lectins" for, other O-GlcNAc-modified proteins in both
cytosolic and nuclear fractions (40). This observation may
explain how the O-GlcNAc modification protects some
proteins, e.g., Sp1, from degradation. A recent study implicates HSP70
in the regulation of protein kinase C (PKC) activity. HSP70 specifically binds to the unphosphorylated turn motif of the carboxy terminus of mature PKC (Thr642 in PKCII), thus
stabilizing the protein, allowing rephosphorylation of the enzyme, and
prolonging its half-life. This role of HSP70 appears to apply to all
PKC isoforms [including PKC
and the other members of the AGC
superfamily, e.g., Akt/PKB and PKA (17)]. We are
currently testing the hypothesis that enhanced HSP70
O-GlcNAcylation may modify these interactions.
In the present study, we observed immunostaining with anti-O-GlcNAc of a "doublet" band at ~70 kDa from one-dimensional SDS-PAGE. This result, along with the observation that, on two-dimensional Western blots of HSP70 immunoprecipitates it appeared to migrate as several protein "spots," led us to speculate that we were detecting either more than one isoform of HSP70 or that the HSP70 that we isolated contained several modifications, including O-GlcNAcylation. In skeletal muscle, four HSP70s are known to be present, including the glucose-regulated proteins GRP75 and GRP78, HSP72, and HSC70. After several MALDI spectra from tryptic digests were obtained and some of these tryptic peptides sequenced, our data indicate that the major HSP70 in our fractions is not a GRP (based on both tryptic fragment size and sequence) and matches most closely in all characteristics to rat HSP70.1/2 (SswissProt accession no. Q07439). The sequence AIAYGLDR that we sequenced from the 1,687.9-MW tryptic fragment is unique to HSP70.1/2; the similar fragment from HSC70 (accession no. NP077327) contains a COOH-terminal lysine rather than an arginine (AIAYGLDK). Therefore, it appears that the major HSP70 that we detected in our samples was HSP70.1/2, although we cannot rule out the presence of HSC70 or other unidentified HSP70s on the basis of the collective physical characteristics of the protein or the antibodies used in the study, as these are shared by or are reactive to all members of the HSP70 family.
The third protein that we identified as O-GlcNAcylated in L6
cells is the cytoskeletal protein -tubulin. In adipocytes, integrity of the cytoskeletal network and motor proteins such as kinesin appear
to be crucial for proper translocation of GLUT4-containing vesicles
following insulin stimulation (13, 14, 19, 51). Microtubules are a critical component of the cytoskeletal network for cell motility and organelle and vesicle transport and are composed
of tubulin
/
-heterodimers (50, 62).
-Tubulin
was, in fact, found in purified preparations of GLUT4 vesicle membranes (19). A microtubule-associated protein, tau, found in
neurons, is also modified by O-GlcNAc (1). The
discovery that
-tubulin is O-GlcNAcylated and that this
modification is enhanced under hyperglycemic conditions may be of
significance, particularly if this modification affects microtubule
dynamics and/or interactions with ligands. Tubulin is subject to
numerous posttranslational modifications, including Ser/Thr
phosphorylation (50), but the O-GlcNAc
modification has not been previously reported. One of these
modifications, enzymatic detyrosination, affects the interaction of
tubulin with kinesin motors (56). In an in vitro
microtubule-binding assay using 3T3-L1 adipocytes, Olson et al.
(51) observed that GLUT4-containing vesicles and IRS-1
specifically bind to microtubules. Interactions between tubulin
isoforms, including
-tubulin and PI 3-kinase, have been reported
(31, 34, 35). Thus tubulin may play a role in regulating
the intracellular trafficking of GLUT4 (13, 14, 19, 51)
and of important mediators in the proximal insulin-signaling cascade
(31, 34, 35, 51). Note, however, that the requirement for
an intact microtubule network for insulin's stimulation of glucose
transport and GLUT4 translocation has been questioned (45,
60).
Two recent articles strongly support a role for enhanced protein
O-GlcNAcylation in glucose-induced insulin resistance
(43, 63). When treated with a specific competitive
inhibitor of O-GlcNAcase (21), 3T3-L1
adipocytes manifest enhanced protein O-GlcNAc modification, insulin resistance of glucose transport, and Akt activation in the
presence of normal IR/IRS-1/IRS-2 activation (63), thus mimicking the characteristics of glucose-induced insulin resistance in
these cells (48). Transgenic mice with modest
overexpression of O-GlcNAc transferase in skeletal muscle
and adipocytes develop hyperinsulinemia, insulin resistance (assessed
by euglycemic insulin clamp studies), and hyperleptinemia without
changes in body weight, glycemia, or GLUT4 expression in muscle
(43). We found that Sp1, HSP70, and -tubulin are
O-GlcNAcylated in L6 myotubes and that the
O-GlcNAc modification on each of these proteins is markedly enhanced by exposure to high glucose and insulin. Further
identification of glucose flux-responsive O-GlcNAcylated
proteins may contribute to our understanding of glucose-induced insulin
resistance in muscle. Whether or not the O-GlcNAc
modification affects HSP70 and/or microtubular function and is
implicated in insulin resistance of glucose transport in muscle
warrants further investigation.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the excellent technical assistance of K. Robinson and A. DiNovo, gifts of antibodies from Dr. J. E. Kudlow (University of Alabama) and Dr. G. W. Hart (Johns Hopkins University), the gift of L6 myocytes from Dr. Amira Klip (University of Toronto), and the gift of human recombinant insulin from Lilly Research Laboratories.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health (NIH) Research Grant DK-02001 (to M. G. Buse), NIH Postdoctoral Training Grant T32-HL-07260 (to J. L. E. Walgren), and the Medical University of South Carolina Mass Spectrometry Facility.
Parts of this paper were presented at the 62nd Annual Meeting of the American Diabetes Association, June 2002, San Francisco, CA (Diabetes 51, Suppl 2: A205, 2002).
Address for reprint requests and other correspondence: M. G. Buse, Division of Endocrinology, MUSC Dept. of Medicine, 96 Jonathan Lucas St., CSB 323, P.O. Box 250624, 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.
First published October 22, 2002;10.1152/ajpendo.00382.2002
Received 28 August 2002; accepted in final form 16 October 2002.
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