Insulin Resistance of Glycogen Synthase Mediated by
O-Linked N-Acetylglucosamine*
Glendon J.
Parker,
Kelli C.
Lund,
Rodrick
P.
Taylor, and
Donald A.
McClain
From the Veterans Affairs Medical Center and Division of
Endocrinology, University of Utah School of Medicine,
Salt Lake City, Utah 84132
Received for publication, August 1, 2002, and in revised form, November 27, 2002
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ABSTRACT |
We have investigated the mechanism by which high
concentrations of glucose inhibit insulin stimulation of glycogen
synthase. In NIH-3T3-L1 adipocytes cultured in low glucose (LG; 2.5 mM), the half-maximal activation concentration
(A0.5) of glucose 6-phosphate was 162 ± 15 µM. Exposure to either high glucose (HG; 20 mM) or glucosamine (GlcN; 10 mM) increased the
A0.5 to 558 ± 61 or 612 ± 34 µM.
Insulin treatment with LG reduced the A0.5 to 96 ± 10 µM, but cells cultured with HG or GlcN were
insulin-resistant (A0.5 = 287 ± 27 or
561 ± 77 µM). Insulin resistance was not explained by increased phosphorylation of synthase. In fact, culture with GlcN
decreased phosphorylation to 61% of the levels seen in cells cultured
in LG. Hexosamine flux and subsequent enzymatic protein O-glycosylation have been postulated to mediate nutrient
sensing and insulin resistance. Glycogen synthase is modified by
O-linked N-acetylglucosamine, and the level of
glycosylation increased in cells treated with HG or GlcN. Treatment of
synthase in vitro with protein phosphatase 1 increased
basal synthase activity from cells cultured in LG to 54% of total
activity but was less effective with synthase from cells cultured in HG
or GlcN, increasing basal activity to only 13 or 16%. After enzymatic
removal of O-GlcNAc, however, subsequent digestion with
phosphatase increased basal activity to over 73% for LG, HG, and GlcN.
We conclude that O-GlcNAc modification of glycogen synthase
results in the retention of the enzyme in a glucose
6-phosphate-dependent state and contributes to the reduced
activation of the enzyme in insulin resistance.
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INTRODUCTION |
Glycogen synthase, which incorporates activated glucose into
glycogen, is a major gatekeeper of carbohydrate metabolism. The enzyme
is highly regulated, both allosterically and by several kinases and
phosphatases (1, 2). Phosphorylation inactivates the enzyme. The
process is complex, since there are nine phosphorylation sites,
targeted by several kinases, each with different effects on
enzyme activity (3-5). The inactivation of glycogen synthase can be
overcome by allosteric interaction with glucose 6-phosphate (G6P)1 (6). Insulin
stimulates glycogen synthase primarily through the phosphatidylinositol
3-kinase/Akt pathway, resulting in inhibition of glycogen synthase
kinase-3 and dephosphorylation of the enzyme by protein phosphatase 1 (1, 7, 8). Protein phosphatase 1 is itself regulated by stimulation and
by specific targeting to glycogen (9-11). With activation by insulin,
glycogen synthase becomes more sensitive to G6P, and basal,
G6P-independent activity is increased.
In type 2 diabetes mellitus there is resistance to the stimulation of
glycogen synthase by insulin and a reduction of glycogen synthase
activity (12-14). How this insulin resistance is triggered is not
known. Work from Marshall's laboratory originally suggested that
insulin resistance could be mediated by an increase in carbohydrate flux through the hexosamine biosynthesis pathway (HBP) (15). Consistent
with that hypothesis, acute infusions of glucosamine or transgenic
overexpression in muscle and fat of the rate-limiting enzyme in the
HBP, glutamine:fructose-6-phosphate amidotransferase, result in insulin
resistance (16, 17).
The terminal metabolites of the hexosamine pathway are UDP-hexosamines.
UDP-N-acetylglucosamine is a substrate for the cytosolic UDP-N-acetylglucosamine:peptide glycosyltransferase (OGT),
which glycosylates nuclear and cytosolic proteins with a single
N-acetylglucosamine moiety on serine and threonine residues
(O-GlcNAc) (18, 19). This recently described protein
modification is in many ways analogous to phosphorylation; it is
dynamic and has been shown to occur exclusively on phosphoproteins.
Additionally, it has been shown to often have a reciprocal relationship
with the degree of phosphorylation of a protein (20, 21). Recent
studies have suggested possible links between the O-GlcNAc
modification on proteins and the pathogenesis of diabetes and insulin
resistance. For example, insulin resistance of endothelial nitric-oxide
synthase stimulation results when the Akt phosphorylation site of
endothelial nitric-oxide synthase is modified by O-GlcNAc
(22). Transgenic overexpression of OGT in skeletal muscle and fat
results in the development of insulin resistance in mice, mimicking the
effects of increased hexosamine flux (23).
We have previously demonstrated that treatment of fibroblasts with high
concentrations of glucose or glucosamine results in decreased basal
activity of glycogen synthase and decreased stimulation by insulin
(24-26). To explore whether these effects might be due to direct
modification of glycogen synthase by O-GlcNAc, we have examined glycogen synthase in differentiated NIH3T3-L1 adipocytes, treated with either low glucose, high glucose, or glucosamine. We show
that treatment with high glucose or glucosamine results in insulin
resistance, a reduction of basal glycogen synthase activity, and
decreased activation by G6P. These effects are associated with
increased levels of O-GlcNAc modification of glycogen
synthase and can be reversed by enzymatic removal of
O-GlcNAc.
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EXPERIMENTAL PROCEDURES |
Antibodies and Reagents--
The following antibodies were used
in the current study: anti-glycogen synthase (Chemicon International,
Inc., Temecula, CA), anti-phospho-glycogen synthase (Oncogene, Boston,
MA), anti-O-GlcNAc monoclonal IgM antibody (CTD 110.6, a gift of Dr.
Gerald Hart, Johns Hopkins University, Baltimore, MD) (27), and
horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG
(Amersham Biosciences). Succinylated wheat germ agglutinin-agarose
(sWGA) was obtained from EY laboratories (San Mateo, CA). NIH 3T3-L1 cells were obtained from the ATCC (Manassas, VA).
UDP-[6-3H]glucose was obtained from Amersham Biosciences;
Dulbecco's modified Eagle's medium and fetal calf serum were from
Invitrogen. The insulin used in this study was recombinant human
insulin (NovolinR; NovoNordisk, Bagsvaerd, Denmark).
6-Acetaminido-6-deoxy-castanospermine (CaspNAc) was obtained from
Industrial Research Ltd. (Wellington, New Zealand). The protease
inhibitors used were the Complete tablets from Roche Molecular
Biochemicals. All chromatography media and columns were obtained from
Amersham Biosciences, with the exception of UDP-hexanolamine-agarose,
which was obtained from Sigma. All other enzymes and chemicals were
obtained from Sigma.
Differentiation, Treatment, and Extraction of NIH 3T3-L1
Adipocytes--
NIH 3T3-L1 cells were grown in 10 ml of Dulbecco's
modified Eagle's medium containing 20 mM glucose and 10%
fetal calf serum in 10-cm plates (Corning Glass) and with 10%
CO2. Two days after confluence, 1 µM
dexamethasone, 1 µg/ml insulin, and 0.5 mM
isobutylmethylxanthine were added for 3 days, followed by 3 days
with insulin alone. The cells were additionally cultured in Dulbecco's
modified Eagle's medium with 20 mM glucose and 10% fetal
calf serum for 5-10 days, with the medium being changed every 3 days.
Cells were passaged every 3 days with care taken to ensure that
confluence was not reached before passaging (28, 29). Experimental
protocols were then begun. The differentiated adipocytes were placed in Dulbecco's modified Eagle's medium containing 1% fetal calf serum and either 2.5 mM glucose (low glucose (LG)), 20 mM glucose (high glucose (HG)), or 2.5 mM
glucose plus 10 mM glucosamine (glucosamine (GlcN)) for
24 h and again 30 min prior to harvesting. For insulin-treated dishes, insulin (10 nM) was added 30 min prior to
harvesting. The cells were harvested by placing on ice and washing
twice with ice-cold KRBH (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 extracted
with 0.75 ml of extraction buffer (50 mM HEPES, pH 7.4, 100 mM sodium chloride, 5% glycerol (v/v), 1 mM
2-acetamido-1-amino-1,2-dideoxyglucopyranoside, 40 mM
sodium fluoride, and protease inhibitors). If
-D-hexosaminidase or phosphatase digests were to be
conducted, then the extraction buffer consisted of 25 mM
HEPES, pH 7.4, 100 mM sodium chloride, and 5% glycerol
with protease inhibitors. The resulting extracts were sonicated with a
Sonic Dismembrator F60 for 15 s at setting 4 (Fisher) and
centrifuged at 20,000 × g for 2 min at 4 °C. The supernatant was aspirated, and aliquots were immediately frozen in
liquid nitrogen.
Glycogen Synthase Assay--
The assay for glycogen synthase was
based on that published by Thomas et al. (30). Extracts from
differentiated adipocytes (7.5 µg of protein) were mixed in a volume
of 100 µl with 10.0 µmol of HEPES, pH 7.4, 0.5 µmol of EDTA, pH
7.4, 0.8 mg of glycogen (type III from rabbit liver), 0.2 µmol of
UDP-glucose, 10 µl of glycerol, 0-0.3 µmol of G6P, and 0.4 µCi
of UDP-[6-3H]glucose and incubated for 45 min at
37 °C. The G6P concentrations used (0-3 mM) reflect the
physiological range in vivo (31). The incubation was
terminated by application to Whatman qualitative filter paper number 3 (Maidstone, UK) and immersion in 60% (v/v) ethanol. After five washes
in 400 ml of 60% ethanol, the paper squares were washed once in
acetone, dried, and assayed for tritium. All assays were done in
duplicate. The incorporation of tritium was found to be optimal at
37 °C and linear for 120 min. The resulting data were analyzed by
the least squares algorithm for a single rectangular hyperbola, using
SigmaPlot for Windows version 4.0 (SSPS Science, Chicago, IL).
G6P-independent glycogen synthase activity (I-form) is defined as basal
activity. The half-maximal activation value
(A0.5) was defined as the concentration of G6P to achieve 50% activation of the G6P dependent activity
(D-form). p values for the
A0.5 value and basal activity were determined using the values from each separate experiment and conducting a T-Test
on Microsoft Excel 98 (Microsoft Corp.). The total activity (I-form + D-form) was defined as the activity at maximal G6P (3 mM).
Western Blotting--
Protein concentrations in adipocyte
extracts were determined using the Bio-Rad protein reagent. Extracts
were prepared for gel electrophoresis by dilution with extraction
buffer and 5× Laemmli buffer (32). 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). Resulting blots were blocked with TBST (20 mM Tris, pH
7.4, 150 mM sodium chloride, and 0.5% Tween 20) containing
4% (w/v) nonfat dried milk for 1 h at room temperature or
overnight at 4 °C. 4% (w/v) bovine serum albumin was used in lieu
of dried milk for detection with the anti-O-GlcNAc antibody.
Blots were incubated with primary antibodies for 1 h at room
temperature, washed three times in TBST, and then incubated with the
appropriate horseradish peroxidase-conjugated secondary antibody for
1 h. The blots were washed five times in TBST and imaged by
treating with Super Signal West Femto reagents (Pierce) and exposure to
Hyperfilm (Amersham Biosciences). Densitometry measurements were
obtained using a UMAX Astra 3450 scanner (UMAX Technologies, Fremont,
CA) and NIH Image version 1.62 software. All densitometric values were
normalized to the signal obtained from the low glucose cultured extract.
Immobilization of Glycogen Synthase with Wheat Germ
Agglutinin--
400 µl of extracts (2 mg of protein/ml) were
incubated with 50 µg of sWGA and 600 µl of radioimmune
precipitation buffer (11 mM sodium phosphate, pH 7.4, 150 mM sodium chloride, 1% IGEPAL, 0.5% sodium deoxycholate,
0.1% SDS, 40 mM sodium fluoride, 0.5 mM
2-acetamido-1-amino-1,2-dideoxyglucopyranose, and protease inhibitors).
The preparation was incubated on a rotator for 16 h at 4 °C and
washed three times with extraction buffer. Bound proteins were eluted
by boiling in Laemmli buffer and resolved by SDS-PAGE.
Purification of Glycogen Synthase from Rabbit Muscle--
Rabbit
muscle (500 g; Pel-Freez, Rogers, AR) was homogenized in 500 ml of
2× standard buffer to give a final concentration of 100 mM
HEPES, pH 7.4, 10 mM EDTA, 4 mM dithiothreitol,
20% glycerol, 200 mM GlcNAc (to inhibit hexosaminidases),
and protease inhibitors. The homogenate was centrifuged at 13,300 × g for 1 h at 4 °C, and the resulting supernatant
was precipitated with 313 g/liter ammonium sulfate. The pellet was
resuspended in standard buffer and applied to a phenyl-Sepharose column
(2.5 × 40 cm) equilibrated in standard buffer containing 1 M sodium chloride and 0.5 M ammonium sulfate.
The flow-through fraction was dialyzed against 10 volumes of standard
buffer using Spectro/Por dialysis tubing (Spectrum Laboratories Inc.,
Rancho Dominguez, CA) and applied to a concanavalin A-Sepharose column
(1 × 30 cm) equilibrated in standard buffer. The filtrate from
the concanavalin A column was then applied to a HiPrep SephQ XL column
(10/10) equilibrated in standard buffer and eluted with a gradient of
50-500 mM sodium chloride. The active fractions were
pooled and concentrated to 5 ml in Vivaspin-15 centrifugal
concentrators (molecular weight cut-off of 10,000; Millipore). The
preparation was then applied to a UDP-hexanolamine column HR(5/15)
(Sigma) and eluted with a gradient of 0-1 M sodium
chloride. The active fractions were desalted into standard buffer and
concentrated to 250 µl in Vivaspin-0.5 centrifugal concentrators
(molecular weight cut-off of 10,000). More than 95% of the protein in
the active fractions reacted on Western blotting with the anti-glycogen
synthase antibody.
Labeling of Glycogen Synthase with
[6-3H]Galactose--
Purified glycogen synthase (25 µg) from rabbit muscle was incubated for 16 h on ice with 75 milliunits of autogalactosylated
-1,4-galactosyltransferase, 50 nmol
of MnCl2, 10 nmol of AMP, 0.1 µmol of HEPES, pH 8.0, 0.1 µmol of galactose, and 40 µCi of UDP-[6-3H]galactose
in 100 µl (33). The mixture was concentrated to 15 µl using a
Vivaspin-0.5 centrifugal concentrator (molecular weight cut-off of
30,000; Millipore), boiled with 5 µl of 5× Laemmli buffer, and
analyzed by SDS-PAGE and autoradiography.
Digestion of Adipocyte Extracts with Hexosaminidase and Protein
Phosphatase 1--
Phosphatase digests were conducted by treating 120 µg of protein at 37 °C for 30 min in 100 µl of a buffer
consisting of 50 mM HEPES, pH 7.4, 10% glycerol, 1.2 mM manganese chloride, protease inhibitors (Roche Molecular
Biochemicals), and 10 units of the recombinant active subunit of
protein phosphatase 1
(Sigma catalog no. P-7937) (34). When extracts
were to be treated only with phosphatases, the treatments were done in
the presence of 100 µM CaspNAc. Complete digestion had
occurred by 15 min (data not shown). For digestion with
-D-hexosaminidase, the pH of the extract (100 µg of
protein in 25 µl) was adjusted to 5.5 by the addition of 25 µl of
hexosaminidase buffer (20 mM sodium citrate, pH 4.5, 10%
glycerol, 100 mM sodium chloride, and protease inhibitors) with or without 2 units of
-N-acetylglucosaminidase from
jack beans (Sigma) in the same buffer. After the addition of 5 µl of 1 mg/ml bovine serum albumin (Roche Molecular Biochemicals) and 5 µl
of 20 mg/ml glycogen, the preparation was incubated for 2 h at
30 °C. The reaction was stopped by the addition of 20 µl of 100 mM HEPES, pH 8.8, and 1 µl of 100 mM CaspNAc
(35). When CaspNAc was added before hexosaminidase, no change in
glycogen synthase was observed (data not shown). After subsequent
phosphatase digestion, the glycogen synthase activation was measured by
duplicate assays at 0 and 3 mM G6P.
 |
RESULTS |
Glycogen Synthase from Cells Cultured with High Glucose or
Glucosamine Exhibits Reduced Sensitivity to Glucose
6-Phosphate--
Differentiated NIH 3T3-L1 adipocytes were cultured
for 24 h with 2.5 mM glucose (low glucose), 20 mM glucose (high glucose), or 10 mM glucosamine
plus 2.5 mM glucose (glucosamine). These treatments had a
significant effect on the sensitivity of glycogen synthase to G6P. In
cells cultured in low glucose, glycogen synthase was sensitive to G6P
(A0.5 = 162 ± 15 µM) and
became more sensitive when cells were treated with insulin
(A0.5 = 96 ± 10 µM,
p < 0.05, Fig. 1,
a and d). Glycogen synthase from cells cultured
in high glucose was less sensitive to G6P (A0.5 = 558 ± 61 µM, p < 0.02), and
insulin did not increase the sensitivity to G6P to the same degree as
was observed with low glucose (A0.5 = 287 ± 27 µM, p = 0.1, Fig. 1, b
and d). Culture of cells in glucosamine led to a similar
degree of insensitivity to G6P (A0.5 = 612 ± 35 µM, p < 0.005) and resulted in
complete insulin resistance (A0.5 = 561 ± 77 µM, p = 0.86, Fig. 1, c and
d). Similar results were obtained from cells cultured in 5 mM glucosamine (A0.5 = 728 ± 132 µM, data not shown). Total activities
(Vmax) of glycogen synthase did not differ among
experimental treatments (see legend to Fig. 1).

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Fig. 1.
Sensitivity of glycogen synthase to G6P in
3T3-L1 adipocytes cultured in low glucose, high glucose, or
glucosamine, with or without insulin. NIH 3T3-L1 differentiated
adipocytes were incubated for 24 h in either 2.5 mM
glucose (LG; and ) (a); 20 mM glucose
(HG; and ) (b); or 2.5 mM glucose plus 10 mM glucosamine (GlcN; and ) (c).
Adipocytes were also treated in the absence ( , , ,
open bar) or presence ( , , ,
closed bar) of 10 nM insulin for 30 min prior to extraction. Duplicate glycogen synthase assays were
conducted in the presence of 0-3 mM G6P. The half-maximal
activation concentration of G6P (A0.5 ± S.E.)
was determined using the least squared algorithm from four independent
cultures and is shown with each curve. A summary of all of the
A0.5 values ± S.E. is presented in
d. Significant difference relative to the low glucose
culture (*, p 0.05) and to the non-insulin-treated
cells (+, p 0.05) is also indicated. Absolute values
for basal and maximal synthase activity, given as nmol of glucose
transferred/min/mg of protein ± S.E. in extracts from the
differently treated cultures were as follows: low glucose, basal = 0.29 ± 0.06, maximal = 3.38 ± 0.76; low glucose with
insulin treatment, basal = 0.51 ± 0.13, maximal = 2.87 ± 0.69; high glucose, basal = 0.04 ± 0.01, maximal = 3.70 ± 0.87; high glucose with insulin treatment,
basal = 0.17 ± 0.07, maximal = 3.68 ± 1.02;
glucosamine, basal = 0.08 ± 0.01, maximal = 2.74 ± 0.67; glucosamine with insulin treatment, basal = 0.24 ± 0.12, maximal = 2.94 ± 0.88.
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Culture with high glucose or glucosamine also affected the
G6P-independent, or basal activity of glycogen synthase. When
adipocytes cultured with low glucose were treated with insulin the
basal activity increased from 8.7 ± 1.0 to 0.18 ± 1.0% of
total activity (p < 0.002). Both high glucose and
glucosamine treatments resulted in lower basal synthase activities and
less insulin stimulation. When cultured with high glucose, insulin
treatment increased G6P-independent activity from 1.2 ± 0.1 to
only 4.7 ± 0.7% (p < 0.005 and
p < 0.001, with and without insulin, compared with low
glucose). Cells cultured with glucosamine showed a change of basal
activity from 3.0 ± 0.5 to 7.4 ± 1.8% with insulin
stimulation (p = 0.075 and p < 0.005, with and without insulin, compared with low glucose).
Changes in Glycogen Synthase Activity Induced by Treatment in High
Glucose or Glucosamine Do Not Correlate with the Phosphorylation State
of the Enzyme--
Normally an increase in the
A0.5 value for G6P and concomitant decrease in
basal activity of glycogen synthase would be associated with increased
phosphorylation. We therefore examined the phosphorylation state of the
enzyme, using a polyclonal antibody against a phosphopeptide of the
GSK-3 recognition region of the enzyme (hGS 642-662) (Fig. 2). Synthase from cells cultured with
high glucose showed a slight increase in phosphorylation relative to
low glucose treatment that was not statistically significant (39 ± 29%; p = 0.17). Paradoxically, cells cultured with
glucosamine showed a decrease in phosphorylation to 61 ± 9% of
the level seen in low glucose (p < 0.001), despite the
high A0.5 value for G6P and lower basal
activity.

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Fig. 2.
Phosphorylation of glycogen synthase in
different metabolic conditions. a, differentiated NIH
3T3-L1 adipocytes were cultured with LG (lanes 1 and 2), HG (lanes 3 and 4),
and GlcN (lanes 5 and 6), in the
absence (lanes 1, 3, and 5)
or the presence (lanes 2, 4, and
6) of 10 nM insulin, as described under
"Experimental Procedures." Total glycogen synthase (GS)
and phosphoglycogen synthase (pGS) were detected by
immunoblotting, the latter by using an antibody against a
phosphopeptide from the GSK3 recognition domain of the enzyme (hGS
642-662). b, densitometric data (arbitrary
intensity units; AIU ± S.E.) from three independent experiments, each
assayed in duplicate blots, were averaged and displayed in Fig.
2b.
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The phosphorylation of the enzyme was responsive to insulin (Fig. 2).
Glycogen synthase from adipocytes cultured with low glucose showed a
70 ± 7% decrease in phosphorylation when treated with 10 nM insulin (p < 0.001). However,
adipocytes treated with high glucose or glucosamine exhibited insulin
resistance. In these cases, phosphorylation decreased by 44 ± 11% (p < 0.05) and 30 ± 13% (p = 0.10) after insulin treatment, less than that observed for cells in
low glucose (p < 0.05).
High Glucose and Glucosamine Treatments Increase Global Levels of
Protein Modification by Terminal O-GlcNAc--
The changes in glycogen
synthase seen in cells cultured in high glucose or glucosamine were not
paralleled by predicted changes in phosphorylation. For example, the
A0.5 value for G6P was high in
glucosamine-treated cells despite lower levels of phosphorylation (Figs. 1 and 2). Increased flux in the hexosamine pathway, resulting in
increased levels of protein modification by O-linked GlcNAc, has been proposed as a general mechanism for regulation of metabolism by carbohydrate levels (21, 36, 37). The global level of protein
O-glycosylation, as determined from immunoblots stained with
the CTD 110.6 monoclonal antibody, was increased 190 ± 30% (p < 0.05) in adipocytes cultured with high glucose
(Fig. 3, lanes 3 and 4) relative to low glucose (lanes
1 and 2) (27). Cells treated with glucosamine
(lanes 5 and 6) increased levels of
O-GlcNAc to 610 ± 70% (p < 0.001).
Treatment with insulin did not significantly affect the levels of
terminal O-GlcNAc (Fig. 2, lanes 2,
4, and 6).

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Fig. 3.
Effect of different metabolic conditions on
the level of cytosolic O-GlcNAc. NIH3T3-L1
adipocytes were cultured for 24 h in 2.5 mM glucose
(LG, lanes 1 and 2), 20 mM glucose (HG, lanes 3 and 4), or in 2.5 mM glucose plus 10 mM glucosamine (GlcN, lanes
5 and 6), in the absence (lanes
1, 3, and 5) or in the presence
(lanes 2, 4, and 6) of 10 nM insulin. 10 µg of each extract was resolved on
SDS-PAGE and stained with an antibody specific for O-GlcNAc.
The blot shown is a single representative exposure of five separate
experiments whose lanes were arranged to maintain consistency of the
order of the presentation.
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Glycogen Synthase Is an O-GlcNAc Protein That Is Dynamically
Glycosylated--
To determine whether glycogen synthase itself is
modified by O-GlcNAc, highly purified glycogen synthase from
rabbit muscle was resolved on SDS-PAGE. The protein, stained with
Coomassie Blue, is shown in Fig.
4a, lane
1. The stained bands are recognized by
anti-glycogen synthase antibody (lane 2) and are
consistent with the apparent molecular mass of glycogen synthase (84 kDa) and previously described degradation products (38). The purified glycogen synthase also reacted with an O-GlcNAc-specific
antibody (lane 3) and was radiolabeled after
treatment with UDP-[3H]galactose and
1,4-galactosyltransferase, a method that specifically probes for
terminal GlcNAc (lane 4) (33). The radiolabeling procedure was also performed using ovalbumin, a protein known to
contain terminal GlcNAc, and autogalactosylated galactosyltransferase as positive and negative controls (Fig. 4a, lanes
5 and 6).

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Fig. 4.
Glycogen synthase is modified by
O-GlcNAc and is dynamically glycosylated.
a, glycogen synthase was purified from rabbit muscle as
described under "Experimental Procedures," run on SDS-PAGE, and
stained with Coomassie Blue (lane 1). Immunoblots
of the purified protein were stained with anti-glycogen synthase
(lane 2) and anti-O-GlcNAc
(lane 3). Specific radiolabeling of
O-GlcNAc with [3H]galactose was conducted by
incubation of the protein with -1,4-galactosyltransferase and
UDP-[3H]galactose (lane 4).
Ovalbumin, a known GlcNAc protein, and -1,4-galactosyltransferase
alone were also incubated as positive and negative controls for
detection of O-GlcNAc (lanes 5 and
6). b, NIH3T3-L1 adipocytes were cultured for
24 h in 2.5 mM glucose (lanes 1 and 2), 20 mM glucose (lanes
3 and 4), or 2.5 mM glucose with 10 mM glucosamine (lanes 5 and
6), in the absence (lanes 1,
3, and 5) or the presence (lanes
2, 4, and 6) of 10 nM
insulin. Extracts were incubated with sWGA-agarose and thoroughly
washed. Immobilized proteins were resolved on SDS-PAGE. Glycogen
synthase (GSase) was detected using an anti-glycogen
synthase antibody. The presented immunoblot is representative of two
separate experiments conducted in duplicate.
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To demonstrate that glycogen synthase from adipocytes was also modified
by O-GlcNAc and that the level of the O-GlcNAc
modification was dynamic, extracts from cells treated in low glucose,
high glucose, and glucosamine were incubated with immobilized
sWGA-agarose to immobilize hyperglycosylated O-GlcNAc
proteins. After SDS-PAGE, the amount of glycogen synthase that bound to
sWGA was measured by immunoblotting. The level of glycogen synthase
binding to sWGA increased 190 ± 40% in cells cultured in high
glucose relative to low glucose (Fig. 4b). Glucosamine
treatment increased the level of glycogen synthase binding to sWGA by
630 ± 70%. Insulin treatment did not affect the level of binding
to sWGA. Although the blot shows an apparent increase in glycosylation
of glycogen synthase with insulin in low glucose, this trend was not
significant (42 ± 40% increase, p = 0.4, n = 4).
O-GlcNAc Blocks Phosphatase-mediated Activation of Glycogen
Synthase--
In order to gain direct evidence of the role of
O-linked GlcNAc in glycogen synthase regulation, cell
extracts were treated in vitro with
-hexosaminidase
and/or the active subunit of human protein phosphatase 1 to remove the
O-GlcNAc and/or the phosphate from the protein. Protein
phosphatase 1 targets glycogen synthase in vivo, mediating
insulin-stimulated activation (7, 9). When extracts from low glucose
cultures were digested with protein phosphatase 1 in vitro,
basal activity of glycogen synthase increased to 54 ± 7%
compared with 12 ± 2% in nondigested extracts (p < 0.001, Fig. 5a). When
extracts from high glucose cultures were digested with phosphatase, the
basal activity increased only to 13 ± 2%. Extracts from
glucosamine cultured cells were also resistant to phosphatase
treatment, with basal activity increasing to only 16 ± 2%.
Consistent with the change in basal activity, the
A0.5 concentration for G6P decreased with
phosphatase digestion of the low glucose cultured extract, from 78 ± 12 to 13 ± 5 µM G6P (p < 0.025). The decrease in the A0.5 value was less
in the high glucose and glucosamine extracts (51 ± 6 µM G6P for high glucose and 37 ± 4 µM
G6P for glucosamine treatment). The experiments above were performed in
the presence of CaspNAc, an inhibitor of the endogenous
O-GlcNAcase activity in the cell extracts.

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Fig. 5.
Digestion of glycogen synthase preparations
with protein phosphatase 1 and
-N-acetylglucosaminidase.
a, extracts from differentiated NIH 3T3-L1 adipocytes
cultured in 2.5 mM glucose (LG), 20 mM glucose (HG), or 2.5 mM glucose
plus 10 mM glucosamine (GlcN) were incubated in
the absence or presence of 10 units of recombinant PP1 for 30 min and
assayed in duplicate with 0 and 3 mM G6P. Basal glycogen
synthase activity ± S.E. in the absence of G6P is expressed as
the percentage of maximal activity seen in 3 mM G6P.
b, combined -N-acetylglucosaminidase
(Hex) and phosphatase digestion of the adipocyte extracts
occurred by predigestion in the absence or presence of 2 units of
-N-acetylglucosaminidase, followed by digestion in the
absence or presence of 10 units protein phosphatase 1 . Basal
glycogen synthase activity ± S.E. was determined by duplicate
assays with 0 and 3 mM G6P. c, to confirm that
the phosphatase digestion was complete, 10 µg of protein from
extracts of low glucose cultured adipocytes was digested with
-N-acetylglucosaminidase, with or without subsequent
protein phosphatase digestion. The digested extracts were then resolved
on SDS-PAGE, and the resulting blot was stained with the
anti-phosphoglycogen synthase antibody. Absolute values for basal and
maximal synthase activity, given as nmol of glucose transferred/min/mg
of protein ± S.E., in extracts from the differently treated
cultures were as follows. a, LG, basal = 4.81 ± 0.02, maximal = 36.31 ± 1.88; LG + PP1, basal = 26.96 ± 0.71, maximal = 48.69 ± 2.80; HG, basal = 1.68 ± 0.30, maximal = 32.56 ± 1.74; HG + PP1,
basal = 2.36 ± 0.15, maximal = 17.91 ± 0.38;
GlcN, basal = 1.77 ± 0.25, maximal = 39.92 ± 1.33; GlcN + PP1, basal = 7.07 ± 0.42, maximal = 40.24 ± 1.00. b, LG, basal = 0.36 ± 0.14, maximal = 44.12 ± 8.70; LG + Hex, basal = 3.32 ± 0.51, maximal = 52.8 ± 8.25; LG + Hex and PP1, basal = 39.52 ± 6.99, maximal = 55.71 ± 8.84; HG, basal = 0.33 ± 0.15, maximal = 42.99 ± 6.60; HG + Hex,
basal = 2.26 ± 0.67, maximal = 52.79 ± 8.29; HG + Hex and PP1, basal = 40.07 ± 9.00, maximal = 54.92 ± 9.10; GlcN, basal = 0.40 ± 0.14, maximal = 35.35 ± 7.75; GlcN + Hex, basal = 2.22 ± 0.30, maximal = 48.84 ± 8.09; GlcN + Hex and PP1, basal = 39.54 ± 8.29, maximal = 55.11 ± 7.98.
|
|
We next determined whether the resistance to activation of glycogen
synthase by protein phosphatase 1 was due to modification by
O-GlcNAc. Adipocyte extracts without added CaspNAc were
predigested with
-D-N-acetylglucosaminidase
before phosphatase digestion. With the removal of O-GlcNAc,
glycogen synthase could be normally activated (Fig. 5b).
With combined hexosaminidase and phosphatase digestion, basal activity
increased to 74 ± 3, 77 ± 3, and 80 ± 0% of total
activity for low glucose, high glucose, and glucosamine cultured extracts, respectively. A similar increase also occurred in
samples from insulin-treated cells (data not shown). Digestion of the
extracts with hexosaminidase alone produced smaller increases in basal
activity to 7.3 ± 0.3, 15 ± 0.0, and 11 ± 1%,
respectively. To confirm that the phosphatase digestion was complete, a
Western blot of each digest condition was stained with the
anti-phosphoglycogen synthase antibody (Fig. 5c).
 |
DISCUSSION |
Acute regulation of net glycogen synthesis by carbohydrate occurs
through multiple mechanisms, including substrate availability, hormone
signaling, and allosteric activation. In addition, chronic hyperglycemia leads to changes in glycogen synthase activity by mechanisms that remain poorly understood. Our data demonstrate that one
mechanism affecting synthase activity is mediated by increases in
hexosamine flux and subsequent O-glycosylation of the
enzyme. A nutrient sensing and signaling role for the HBP was first
demonstrated by Marshall et al. (15), who showed that HBP
mediated the ability of high glucose to induce insulin resistance in
cultured adipocytes. Since then, studies in cultured cells and animals
have verified that the HBP plays a major role in the regulation of
metabolism and growth (16, 17, 24, 36, 39-46). The HBP has also been
shown to be responsible for glucose regulation of several proteins
including transforming growth factor-
, steroid response
element-binding protein 1, transforming growth factor-
1, plasminogen
activator inhibitor-1, leptin, NF-
B, and endothelial nitric-oxide
synthase (17, 22, 47-55). Flux through the HBP is responsive not only
to glucose but also to fatty acids and oxidative stress (56-58).
UDP-GlcNAc, the terminal metabolite of the HBP, is a substrate for
protein glycosylation, suggesting a possible mechanism for the
regulatory effects of the hexosamine pathway. Glycosylation of secreted
or plasma membrane proteins is not responsive to glucose flux (59).
However, O-linked glycosylation of cytosolic proteins is
substrate-limited and proportional to glucose flux, making feasible a
nutrient-sensing function for that pathway (59-62). OGT, which
catalyzes the modification of cytosolic proteins by GlcNAc at serine
and threonine residues, has a high Km value,
allowing the production of UDP-GlcNAc to be reflected in levels of
protein glycosylation (19, 60, 63). Several lines of evidence now
support the hypothesis that the effects of the HBP on insulin
resistance are mediated by the modification of proteins by
O-GlcNAc. First, pharmacological inhibition of
O-GlcNAc removal from proteins results in insulin resistance
at the level of Akt activation and a reduction of glucose uptake (64).
Second, a transgenic mouse model that overexpresses OGT in fat and
muscle exhibits insulin resistance and hyperleptinemia independently of
UDP-GlcNAc levels (23). Direct evidence for a role of
O-GlcNAc in modifying protein behavior is the recent
demonstration that O-GlcNAc modification of endothelial
nitric-oxide synthase at the Akt phosphorylation site blocks normal
Akt-mediated insulin activation of endothelial nitric-oxide synthase
and lowers endothelial nitric-oxide synthase activity in vascular
endothelial cells (22).
The current study confirms the above findings that O-GlcNAc
modification of proteins can be responsible for the development of
insulin resistance and that O-GlcNAc can directly modify the kinetic properties of an enzyme. Activation of glycogen synthase by
insulin has been known to be decreased in cells exposed to high
concentrations of glucose or glucosamine (24-26, 43). We show here
that this insulin resistance of glycogen synthase stimulation can be
explained by its modification by O-GlcNAc. Namely, the enzyme becomes intrinsically resistant to activation by protein phosphatase 1, which normally mediates activation of the enzyme upon
insulin stimulation (7-9). The current studies do not exclude other
HBP-mediated events that might affect signal transduction upstream of
glycogen synthase. For example, there is evidence that
phosphatidylinositol 3-kinase and Akt activation are affected by
hexosamine flux, and many proteins including insulin receptor substrate
1 and GSK3 are known to be glycosylated (64-69). Thus, the HBP
may operate on several levels to coordinately alter hormone signaling
and metabolism in response to excess nutrient flux.
The simplest model for the effect of O-GlcNAc on glycogen
synthase is that O-GlcNAc is inhibitory in a manner
analogous to phosphate. Enzyme inhibition in this model would be
proportional to flux through the HBP and provide a parallel means of
regulation dependent on the nutritional state of the cell. Thus, in
situations of excess nutrient flux, synthase activity would be held in
a state that could not be activated by insulin because insulin does not
stimulate deglycosylation of proteins as it does dephosphorylation. This would be an adaptive response to excess feeding, aiding in the
limitation of glycogen accumulation and allowing excess calories to be
diverted for storage as fat. This model would predict that the same
sites used for inhibitory phosphorylation would also be used for
glycosylation and that loss of phosphorylation sites (e.g.
by mutagenesis) would parallel loss of glycosylation sites and loss of
inhibition. This model is generally consistent with our observations of
a reciprocal relationship between phosphorylation and
O-glycosylation (Fig. 2a and 4b). An
alternative model is that O-GlcNAc indirectly regulates
phosphorylation events on the enzyme, perhaps by changing the affinity
or specificity of glycogen synthase for one or more of its regulatory
phosphatases and kinases. It was noted that despite relatively low
levels of phosphorylation on glycogen synthase, hexosaminidase
treatment alone was insufficient to restore significant basal activity.
This implies that a complete understanding of the regulation of
synthase by these modifications will require a complete mapping of all
such sites. Further investigation of the role of the
O-GlcNAc modification on glycogen synthase promises to
complement our current understanding of glycogen metabolism and of
phosphate-based regulation in general. The model of regulation of
glycogen synthase by O-GlcNAc should also further elucidate the mechanisms by which excess nutrients contribute to insulin resistance and type 2 diabetes through physiologic and adaptive pathways that become maladaptive when the organism is faced with a
chronic excess of food.
 |
FOOTNOTES |
*
This work was supported by the Research Service of the
Veterans Administration, National Institutes of Health Grant R01
DK43526, the American Diabetes Association (mentor-based postdoctoral
award), and the Ben B. and Iris M. Margolis Foundation.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.
To whom correspondence should be addressed: Division of
Endocrinology, University of Utah, 30 North 2030 East, Salt Lake City, UT 84132. Tel.: 801-581-7755; Fax: 801-585-0956; E-mail:
donald.mcclain@hsc.utah.edu.
Published, JBC Papers in Press, January 1, 2003, DOI 10.1074/jbc.M207787200
 |
ABBREVIATIONS |
The abbreviations used are:
G6P, glucose
6-phosphate;
GSK3, glycogen synthase kinase-3;
HBP, hexosamine
bioynthesis pathway;
O-GlcNAc, O-linked
N-acetylglucosamine;
sWGA, succinyl wheat germ agglutinin;
LG, low (2.5 mM) glucose;
HG, high (20 mM)
glucose;
GlcN, glucosamine;
CaspNAc, 2-acetamido-2-deoxy-castanospermine;
PP1, protein phosphatase 1;
OGT, O-glycosyltransferase.
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