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Originally published In Press as doi:10.1074/jbc.M312139200 on March 10, 2004

J. Biol. Chem., Vol. 279, Issue 20, 20636-20642, May 14, 2004
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Hyperglycemia and Inhibition of Glycogen Synthase in Streptozotocin-treated Mice

ROLE OF O-LINKED N-ACETYLGLUCOSAMINE*

Glendon Parker, Rodrick Taylor, Deborah Jones, and Donald McClain{ddagger}

From the Veterans Affairs Medical Center and Division of Endocrinology, University of Utah School of Medicine, Salt Lake City, Utah 84132

Received for publication, November 5, 2003 , and in revised form, February 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Glycogen synthase is post-translationally modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). In 3T3-L1 adipocytes exposed to high concentrations of glucose, O-GlcNAc contributes to insulin resistance of glycogen synthase. We sought to determine whether O-GlcNAc also regulates glycogen synthase in vivo. Glycogen synthase activity in fat pad extracts was inhibited in streptozotocin (STZ)-treated diabetic mice. The half-maximal activation concentration for glucose 6-phosphate (A0.5) was increased to 830 ± 120 µM compared with 240 ± 20 µM in control mice (C, p < 0.01), while the basal glycogen synthase activity (%I-form) was decreased to 2.4 ± 1.4% compared with 10.1 ± 1.8% in controls (p < 0.01). Glycogen synthase activity remained inhibited after compensatory insulin treatment. After insulin treatment kinetic parameters of glycogen synthase were more closely correlated with blood glucose (A0.5, r2 = 0.70; %I-form, r2 = 0.59) than insulin levels (A0.5, r2 = 0.04; %I-form, r2 = 0.09). Hyperglycemia also resulted in an increase in the level of O-GlcNAc on glycogen synthase (16.1 ± 1.8 compared with 7.0 ± 0.9 arbitrary intensity units for controls, p < 0.01), even though the level of phosphorylation was identical in diabetic and control mice either with (STZ: 2.9 ± 1.0 and C: 3.2 ± 0.8) or without (STZ: 12.2 ± 2.8 and C: 13.8 ± 3.0 arbitrary intensity units) insulin treatment. In all mice the percent activation of glycogen synthase that could be achieved in vitro by recombinant protein phosphatase 1 (230 ± 30%) was significantly greater in the presence of {beta}-D-N-acetylglucosaminidase (410 ± 60%, p < 0.01). This synergistic stimulation of glycogen synthase due to codigestion by protein phosphatase 1 and {beta}-D-N-acetylglucosaminidase was more pronounced in STZ-diabetic mice (310 ± 70%) compared with control mice (100 ± 10%, p < 0.05). The findings demonstrate that O-GlcNAc has a role in the regulation of glycogen synthase both in normoglycemia and diabetes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The rate-limiting enzyme in glycogen metabolism, glycogen synthase, is a major determinant of overall glucose metabolism (1, 2). Because of its central role in glucose metabolism glycogen synthase is responsive to endocrine factors, including insulin, glucagon, and catecholamines, as well as to metabolic status, such as the concentration of the allosteric activator glucose 6-phosphate (G6P)1 (3, 4). Glycogen synthase activity is modulated by phosphorylation that directly inhibits the enzyme and renders it less sensitive to allosteric activation by G6P (5). Insulin stimulation leads to removal of phosphate by protein phosphatase 1 (PP1), resulting in increased sensitivity to activation by G6P and a higher level of G6P-independent activity (6, 7). Glycogen levels, glycogen synthase activity, and responsiveness to insulin signaling are all reduced in diabetes (8–11). Both endogenous and exogenous phosphatases are also less able to fully activate glycogen synthase in streptozotocin (STZ)-diabetic rats (12).

Glycogen synthase activity is also affected by the hexosamine biosynthetic pathway, which produces UDP-N-acetylhexosamines (13–17). UDP-N-acetylglucosamine is a substrate for O-linked N-acetylglucosaminyltransferase, which transfers the monosaccharide onto serine and threonine residues of cytosolic and nuclear proteins. Data recently published by our laboratory showed that glycogen synthase from extracts of 3T3-L1 adipocytes was modified by O-GlcNAc in a glucose-dependent manner (18). This modification inhibited the enzyme in a manner analogous to phosphate, and only after enzymatic removal of O-GlcNAc could the enzyme be fully activated by exogenous PP1 (18). This illustrated a direct link between increased glucose uptake, modification by O-GlcNAc, glycogen synthase inhibition, and resistance of the synthase to activation by insulin signaling. We therefore investigated the relative roles of O-GlcNAc and phosphate in regulating glycogen synthase in vivo in mice made diabetic by low dose STZ treatment. We show that hyperglycemia results in elevated O-GlcNAc on glycogen synthase and that removal of O-GlcNAc facilitates activation of the enzyme by PP1 especially in diabetes. This confirms that O-GlcNAc has an important regulatory function in vivo and plays a role in mediating the inhibitory effect of hyperglycemia on glycogen synthase in a diabetic animal model.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—The following primary antibodies were used in the current study: anti-glycogen synthase (Chemicon International, Inc., Temecula, CA), anti-phosphoglycogen synthase (Cell Signaling Technology, Inc., Beverly, MA), anti-O-GlcNAc monoclonal IgM antibody (CTD 110.6; a gift of Dr. Gerald Hart, The Johns Hopkins University, Baltimore, MD) (19). Secondary antibodies used were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (Amersham Biosciences) as well as goat anti-mouse IgM (Calbiochem-Novabiochem). Succinylated wheat germ agglutinin-agarose was obtained from EY Laboratories (San Mateo, CA). UDP-[6-3H]glucose was obtained from Amersham Biosciences. The insulin used in this study was recombinant human insulin (NovolinR, NovoNordisk, Bagsvaerd, Denmark). 6-Acetamido-6-deoxycastanospermine was obtained from Industrial Research Ltd. (Wellington, New Zealand). The protease inhibitors used were the Mini-Complete tablets from Roche Applied Science. Glucose was measured using a Glucometer Elite glucose meter and test strips from Bayer Corp. (Mishawaka, IN). The saline used was 0.9% sodium chloride (Baxter Health Products, Deerfield, IL). All other enzymes and chemicals were obtained from Sigma.

Animals and Treatments—Animals used in this study were male C57BL/6 mice. Mice were maintained in a 12-h light/dark cycle with ad libitum access to food. At 10–12 weeks of age mice were injected intraperitoneally with either saline or on sequential days with 85, 75, and 50 mg of STZ/kg of body weight to induce hyperglycemia. Blood glucose concentrations were measured daily in random fed animals. The STZ treatment resulted in 72% of the mice developing hyperglycemia (resting blood glucose, >13.9 mM) after 8.7 ± 0.5 days. After at least 3 days of hyperglycemia, the mice were weighed, resting blood glucose levels were measured, and STZ-treated and control mice were treated with 0.75 units of insulin/kg of body weight or the equivalent volume of saline. The mice were then left without food for 60 min at which time the blood glucose levels were measured, serum was collected, and both groups of animals were sacrificed by cervical dislocation. The animals were immediately dissected, and organs were frozen in liquid nitrogen and stored at –80 °C. Insulin levels were measured in serum using the Sensitive Rat Insulin radioimmunoassay kit (Linco Research, Inc., St. Charles, MO). The Institutional Animal Care and Use Committee of the University of Utah and Salt Lake City Veterans Affairs approved all procedures.

Preparation of Epididymal Fat Pad Extract—Frozen epididymal fat pads were finely diced with a razor and placed in 1 ml of ice-cold buffer containing 25 mM HEPES, pH 7.4, 100 mM NaCl, 5% glycerol (v/v), and protease inhibitors. They were then immediately homogenized with a Polytron PT 2100 homogenizer and PT-DA 2107/2EC probe (setting 26 for 15 s; Kinematica AG, Littau, Switzerland) and centrifuged at 20,000 x g for 2 min at 4 °C. The infranatant was aspirated, frozen as aliquots in liquid nitrogen, and stored at –80 °C.

Analysis of Glycogen Synthase—Glycogen synthase activity was measured as reported previously (18). Basal glycogen synthase activity (%I-form) was defined as the percentage of enzyme activity in the absence of G6P relative to activity in 10 mM G6P. The glycosylated form of glycogen synthase was quantified by binding the glycosylated proteins to immobilized succinylated wheat germ agglutinin followed by SDS-PAGE and staining the blot with anti-glycogen synthase as described previously (18). Phosphorylated glycogen synthase was measured by staining an immunoblot of fat pad extracts with an antibody specific for phosphorylation at the Ser-640 residue of glycogen synthase. Densitometry was conducted as described previously (18).

Digestion with Jack Bean {beta}-D-N-Acetylglucosaminidase and Protein Phosphatase 1—Epididymal fat pad extract (13.5 µg) was digested at 30 °C in a total volume of 50 µl in 50 mM HEPES, pH 7.4, 5% glycerol, 20 mM sodium chloride, 3.6 mM manganese chloride, and protease inhibitors with or without 1 unit of jack bean {beta}-D-N-acetylglucosaminidase and with or without 0.2 units of rabbit recombinant PP1 (Sigma). Incubations lacking {beta}-D-N-acetylglucosaminidase included 2 mM 6-acetamido-6-deoxycastanospermine, an inhibitor of O-linked {beta}-D-N-acetylglucosaminidase. Both {beta}-D-N-acetylglucosaminidase and phosphatase were either desalted into or diluted in desalting buffer (50 mM HEPES, pH 7.4, 5% glycerol, and protease inhibitors). After 30 min 103 µl of 20 mg of glycogen/ml, 50 mM sodium fluoride, 2 mM 2-acetamido-1-amino-1,2-dideoxy-{beta}-D-glucopyranose, 1 mg/ml bovine serum albumin fraction V, and 1% (v/v) protein phosphatase 1 inhibitor mixture (Sigma) were added to stop the digestion. Duplicate glycogen synthase assays, with and without 10 mM G6P, were then conducted for 30 min at 37 °C as described previously (18). A stock of PP1 was prepared by resuspension at a concentration of 7 units/µl in a 50% (v/v) mixture of the desalting buffer and glycerol and stored as aliquots at –80 °C.

To confirm that the conditions described above resulted in deglycosylation and dephosphorylation of glycogen synthase 400 µg of protein were treated under the same conditions with 30 units of {beta}-D-N-acetylglucosaminidase or 6 units of protein phosphatase 1. The incubation was terminated by addition of 750 µl of radioimmune precipitation assay buffer. Endogenous {beta}-D-N-acetylglucosaminidase activity was inhibited by prior addition of 1 mM 2-acetamido-1-amino-1,2-dideoxy-{beta}-D-glucopyranose unless a {beta}-D-N-acetylglucosaminidase digestion was conducted in which case it was added after the digestion. The glycosylation of glycogen synthase and phosphorylation were measured as described previously (18).

Statistical Analysis—Significance was determined by using the Student's t test (Microsoft Excel Version X, Microsoft Corp., Redwood, WA). The lines of best fit were determined using linear regression and significance (p < 0.05) determined from the degree of freedom and correlation coefficient. Data are presented as the means ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of STZ-diabetic Mice—We followed a protocol of low dose STZ treatment to induce moderate hyperglycemia with only mild hypoinsulinemia. STZ-treated mice (Fig. 1A) had an elevation in blood glucose to 20.5 ± 1.2 mM compared with 7.9 ± 0.6 mM in control mice (p < 0.001). Serum insulin levels of the STZ-treated mice (0.8 ± 0.2 ng/ml) tended to be lower than control mice (1.1 ± 0.3 ng/ml), but this difference was not significant (Fig. 1B). When treated with an intraperitoneal injection of 0.75 units of insulin/kg of body weight (20), serum insulin values increased after 60 min to similar levels of 1.8 ± 0.6 ng/ml in STZ-treated animals and 1.5 ± 0.3 ng/ml in control animals (Fig. 1B; p = 0.55). Glucose levels were reduced with insulin treatment to 2.4 ± 0.3 mM in controls (Fig. 1A; p < 0.001). STZ-treated mice were hyperglycemic even after insulin treatment with glucose decreasing only to 14.9 ± 4.3 mM (p = 0.07), 6.0-fold more than that in control mice (p < 0.01).



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  		FIG. 1.
Blood glucose and serum insulin concentrations in control, STZ-treated, and insulin-treated mice. A, blood glucose concentrations were measured in control mice (CON, white columns) and mice treated with a low dose STZ protocol (STZ, light gray columns). Animals were either left untreated or treated with 0.75 units of insulin/kg of body weight and sacrificed after 60 min (CON + I and STZ + I, dark gray and black columns, respectively). B, serum insulin concentrations were measured in the same mice. Significant relationships (p < 0.05) are indicated.

 
Glycogen Synthase from Adipose Tissue Is Inhibited in Hyperglycemic Conditions Even after Exogenous Insulin Treatment—We next sought to determine the relative roles of glycemia and insulin levels on regulation of glycogen synthase in this mouse model of diabetes. Extracts were made from the epididymal fat pads of control and STZ-treated mice and control and STZ-treated mice that had been treated with insulin 60 min prior to sacrifice. Glycogen synthase activity was measured over a range of 0–10 mM G6P to measure the half-maximal activation concentration for G6P (A0.5) and %I-form. Glycogen synthase from the epididymal fat pads of STZ-treated mice was inhibited with the A0.5 value increasing from 240 ± 20 µM G6P in fat pad extracts of control mice to 830 ± 120 µM G6P in fat pads of STZ-treated mice (Fig. 2A; p < 0.01). A similar result was observed with basal glycogen synthase activity: STZ treatment resulted in a decrease of basal glycogen synthase activity from 10.1 ± 1.8% in control mice to 2.4 ± 1.4% in STZ-treated mice (Fig. 2B; p < 0.01). The inactivation seen with STZ treatment is expected as a result of both diminished insulin signaling and hyperglycemia. To separate the two factors, we compensated for insulin deficiency by additional insulin treatment at levels typically used in insulin tolerance tests (21). The treatment significantly activated glycogen synthase from both STZ-treated and control mice (p < 0.01). However, even with compensatory insulin treatment there was still a relative inhibition of A0.5 values and basal glycogen synthase activity in STZ-treated mice. Control mice treated with insulin had an A0.5 value of 150 ± 20 µM G6P, whereas in STZ-treated mice the A0.5 value was 290 ± 40 µM G6P (Fig. 2A; p < 0.01). The basal glycogen synthase activity showed the same pattern of resistance to stimulation by insulin with 15.1 ± 1.4% for control mice and 9.1 ± 2.2% for STZ-treated mice (Fig. 2B; p < 0.05).



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  		FIG. 2.
Glycogen synthase activity in epididymal fat pad extracts from STZ- and insulin-treated mice. Epididymal fat pad extracts (7.5 µg of protein) were obtained from control mice (CON), STZ-treated mice (STZ), and control and STZ-treated mice also treated with 0.75 units of insulin/kg of body weight (CON ± I and STZ ± I, respectively). Glycogen synthase activity was assayed over a range of 0-10 mM G6P to determine both the A0.5 (A) and %I-form (B). Data are represented as the mean ± S.E. Significant relationships are indicated (p < 0.05).

 
The Phosphorylation of Glycogen Synthase Is the Same in Both Hyperglycemic and Control Mice—We next determined whether the inhibition of glycogen synthase in the hyperglycemic mice could be explained by increased phosphorylation of the enzyme. Epididymal fat pad extracts from control and diabetic mice, treated with and without insulin, were resolved by SDS-PAGE, immunostained with an antibody specific for a key regulatory phosphorylation site (Ser-640) on glycogen synthase, and quantified with densitometry (Fig. 3, A and B). The level of phosphoglycogen synthase was similar in the fat pads of control and STZ-treated mice (13.8 ± 3.0 arbitrary intensity units (AIU) compared with 12.2 ± 2.8 AIU, respectively). With insulin treatment both control and diabetic mice underwent dephosphorylation at the Ser-640 site equally well with levels of phosphorylation of 3.2 ± 0.8 AIU for control and 2.9 ± 1.0 AIU for STZ-treated mice, reductions of 76 and 77%, respectively. Thus the inhibition of glycogen synthase and its resistance to activation by insulin in STZ-treated mice could not be explained solely by the phosphorylation state of the enzyme at Ser-640.



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  		FIG. 3.
Degree of phosphorylation of glycogen synthase in epididymal fat pad extracts from STZ- and insulin-treated mice. Extracts were obtained from the epididymal fat pads of control mice (CON), streptozotocin-treated mice (STZ), and mice treated additionally with 0.75 units of insulin/kg of body weight (CON + I and STZ + I). A, immunoblots of the extracts were developed with an antibody specific for phosphorylation at the Ser-640 site. The blot shown is a single representative exposure in which lanes were arranged to maintain consistency of presentation. B, the relative levels of phosphorylated glycogen synthase (pGS) were quantified with densitometry. Significant relationships (p < 0.05) are indicated. Data are represented as the mean ± S.E.

 
Blood Glucose Levels Are a Better Predictor of Glycogen Synthase Kinetics than Serum Insulin Levels—Glycogen synthase activities in fat pad extracts were next correlated with the blood glucose levels and serum insulin levels at the time of harvesting. Basal glycogen synthase activity in mice that were not treated with insulin (open circles) correlated equally well with either blood glucose (Fig. 4A; r2 = 0.42, p = 0.013) or serum insulin levels (Fig. 4B; r2 = 0.38, p = 0.020). However, treatment with exogenous insulin (closed circles) eliminated the correlation with insulin levels (Fig. 4B; r2 = 0.09, p = 0.28), while the correlation with blood glucose levels was even more significant (Fig. 4A; r2 = 0.59, p < 0.005). A0.5 values were also better correlated with glycemia than insulinemia. Blood glucose concentrations showed significant correlations with the A0.5 value of insulin-untreated and -treated mice (Fig. 4C; r2 = 0.70, p < 0.005; r2 = 0.61, p < 0.005). However, serum insulin concentrations showed no significant correlation with the A0.5 value in animals treated with or without insulin (Fig. 4D; r2 = 0.23, p = 0.08; r2 = 0.04, p = 0.51). Both A0.5 values and basal activities were also significantly correlated with the blood glucose levels that were measured prior to insulin treatment (A0.5, r2 = 0.49, p < 0.005; %I-form, r2 = 0.27, p < 0.05; data not shown).



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  		FIG. 4.
Correlation of glycogen synthase kinetic parameters with glucose and insulin concentrations in serum. %I-form (A and B) and A0.5 (C and D) were correlated with blood glucose (A and C) and blood insulin levels (B and D). Data points from epididymal fat pad extracts of insulin-treated (closed circles) and untreated mice (open circles) are shown. The lines of best fit, achieved using linear regression, are shown for both insulin-treated (solid line) and untreated mice (dashed line). Significant (p < 0.05 and p < 0.005) correlations are indicted by the plus sign and asterisk, respectively. The blood glucose concentrations represent those obtained after insulin treatment at the time of sacrifice and tissue harvesting.

 
Glycogen Synthase Is Differentially Modified by O-GlcNAc in STZ-treated Mice—The lack of relationship of phosphorylation of glycogen synthase to the basal activities and A0.5 values combined with the better correlation of both parameters with blood glucose compared with insulin levels suggests that hyperglycemia may inhibit glycogen synthase by mechanisms that are independent of insulin action. In cultured 3T3-L1 adipocytes increased glucose flux results in inhibition of glycogen synthase due to modification by O-GlcNAc (18). We therefore next examined the potential role of O-GlcNAc on glycogen synthase regulation in vivo in control and diabetic mice.

To determine whether STZ-induced diabetes increased the level of O-GlcNAc on cytosolic and nuclear proteins, we resolved proteins from epididymal fat pad extracts of STZ-treated and untreated mice and stained the resulting immunoblot with an anti-O-GlcNAc antibody to examine global changes in protein glycosylation (Fig. 5A). As indicated by the asterisk a select, and at this stage uncharacterized, population of proteins in STZ-treated mice exhibited an increase in glycosylation. To determine whether glycogen synthase (84 kDa) was one of this group, we incubated extracts with immobilized succinylated wheat germ agglutinin to isolate proteins that were hyperglycosylated with O-GlcNAc. Resolution of these proteins and subsequent probing with an anti-glycogen synthase antibody indicated that the relative amount of glycogen synthase modified by O-GlcNAc was elevated in hyperglycemic mice (Fig. 5B). Densitometry of this population of glycogen synthase showed a glycosylation level of 16.1 ± 0.9 AIU relative to 7.0 ± 1.8 AIU in control mice (Fig. 5C; p < 0.005). Both bands shown in Fig. 5B represent differently modified populations of glycogen synthase that were resolved by SDS-PAGE.



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  		FIG. 5.
Modification of glycogen synthase with O-GlcNAc in epididymal fat pad extracts from STZ-treated mice. A, extracts from the epididymal fat pads of STZ-treated (+) and control (–) mice were resolved by SDS-PAGE, and the resulting immunoblot was stained with the anti-O-GlcNAc antibody CTD110.6. An example of an unidentified protein differentially glycosylated in STZ-treated mice is indicated by an asterisk. B, extracts from the fat pads of STZ-treated (+) and control (–) mice were incubated with immobilized succinylated wheat germ agglutinin. An immunoblot of the isolated O-GlcNAc-modified proteins was stained with an anti-glycogen synthase antibody. The two bands present in the immunoblot indicate differences in the post-translational modification and migration of glycogen synthase. C, levels of glycosylated glycogen synthase (gGS) were quantified by densitometry. Data are represented as the mean ± S.E., and significance (p < 0.05) is indicated by a line. CON, control.

 
Removal of O-GlcNAc Improves Activation of Glycogen Synthase by Protein Phosphatase 1—To provide direct evidence for a role of O-GlcNAc in the regulation of glycogen synthase we conducted a series of digestions of epididymal fat pad extracts with jack bean {beta}-D-N-acetylglucosaminidase and submaximal levels of recombinant rabbit PP1 or a combination of both treatments (Fig. 6). Averaged across the combined populations of all mice, phosphatase digestion alone resulted in a 230 ± 30% increase (p < 0.005), and hexosaminidase digestion alone resulted in an activation of 30 ± 10% (p = 0.67) (Fig. 6A). The combination of both hexosaminidase and phosphatase treatment resulted in a 410 ± 60% increase (p < 0.005), a significant increase compared with phosphatase alone (p < 0.005). This synergistic effect on activation of glycogen synthase by removal of both phosphate and O-GlcNAc is more pronounced in diabetic mice. In control mice, the increase seen with both treatments is 100 ± 10% greater than with phosphatase alone compared with 310 ± 70% greater in STZ-treated hyperglycemic mice (p < 0.05).



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  		FIG. 6.
Activation of glycogen synthase with {beta}-D-N-acetylglucosaminidase and protein phosphatase 1. A, epididymal fat pad extracts (13.5 µg of protein) from control and STZ-treated mice were digested with 1 unit of {beta}-D-N-acetylglucosaminidase (open columns), 0.2 units of protein phosphatase 1 (shaded columns), or both together for 30 min at 30 °C (hatched columns). Glycogen synthase activity was assayed with 0 and 10 mM glucose 6-phosphate in duplicate. The percentage of increase in %I-form (average ± S.E.) was determined for all mice (COMBINED), control mice only (CONTROL), and STZ-treated diabetic mice (STZ-TREATED). The {beta}-D-N-acetylglucosaminidase-dependent component of the glycogen synthase activation observed in the combined digests is represented as black columns. Significant increases (p < 0.05) in STZ-diabetic compared with control mice are indicated by asterisks. B, epididymal fat pad extracts (400 µg of protein) from control mice were digested with either 30 units of {beta}-D-N-acetylglucosaminidase or 6 units of protein phosphatase 1 under the same conditions used in A and C. The resulting mixtures were incubated overnight with O-GlcNAc-specific succinylated wheat germ agglutinin-agarose, and immobilized proteins were resolved on an immunoblot. O-GlcNAc-modified glycogen synthase (gGS) was measured by staining with an anti-glycogen synthase antibody. The degree of phosphorylation at the regulatory Ser-640 site (pGS) was measured by staining an immunoblot of each digest with a phosphospecific glycogen synthase antibody. C, epididymal fat pad extracts from insulin-untreated control and STZ-treated mice were digested with 1 unit of {beta}-D-N-acetylglucosaminidase and 0.2 units of protein phosphatase 1 compared with protein phosphatase 1 alone. The resulting difference in %I-form that is attributable to {beta}-D-N-acetylglucosaminidase is plotted against blood glucose concentrations from each animal (control mice, n = 2; STZ-treated mice, n = 3). The line of best fit was achieved using linear regression.

 
The glycosylation and phosphorylation of glycogen synthase was changed as a result of digestion with either {beta}-D-N-acetylglucosaminidase or protein phosphatase 1. Digestion with {beta}-D-N-acetylglucosaminidase resulted in a 45 ± 4% (p < 0.01) decrease in binding to succinylated wheat germ agglutinin. Digestion with recombinant protein phosphatase 1 decreased phosphorylation by 82 ± 7% (p < 0.01). Interestingly {beta}-D-N-acetylglucosaminidase digestion increased the level of glycogen synthase phosphorylation by 165 ± 7% (p < 0.01). This indicates that removal of O-GlcNAc facilitates rephosphorylation of the enzyme. This would help explain the lower degree of glycogen synthase activation with only {beta}-D-N-acetylglucosaminidase (Fig. 6A, open bars; control: 40 ± 10% and STZ-treated: 30 ± 10%) compared with {beta}-D-N-acetylglucosaminidase-dependent activation in combined digests (Fig. 6A, closed bars; control: 100 ± 10% and STZ-treated: 310 ± 70%). Phosphatase digestion did not change the glycosylation status of the enzyme.

In mice that were not treated with insulin the absolute increase in basal glycogen synthase activity seen after digestion with PP1 plus {beta}-D-N-acetylglucosaminidase, compared with PP1 treatment alone, correlated significantly with blood glucose concentrations (r2 = 0.98, p < 0.005; Fig. 6C). The correlation, however, was eliminated with insulin treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Metabolism of glycogen is dependent on the metabolic demands of the cell and the total organism (3, 22–24). Glycogen synthase is dynamically regulated by multiple mechanisms, including substrate availability, hormone signaling, subcellular localization, targeting of phosphatase, and allosteric activation (4, 22, 23, 25). In addition we recently reported that glycogen synthase is regulated by modification with O-GlcNAc (18). Treatment of 3T3-L1 adipocytes with high concentrations of glucose resulted in an increase in glycosylation of glycogen synthase, which inactivated the enzyme and contributed to a reduced response to insulin signaling. Removal of O-GlcNAc was able to correct the resistance to PP1-mediated activation of glycogen synthase (18).

We therefore sought to extend the findings in cultured cells and determine whether O-GlcNAc exerts a biologically significant effect on glycogen synthase activity in the intact organism where glycogen synthase is subject to the full range of physiologic regulation by hormones and metabolic factors. Results reported here verify that O-GlcNAc modification does occur and does affect glycogen synthase activity in vivo. Glycogen synthase from STZ-diabetic mice was inhibited even after insulin treatment. This inhibition of glycogen synthase was more tightly correlated with blood glucose than insulin levels. Additionally the inhibition of glycogen synthase in these diabetic animals could not be explained by increased phosphorylation at the key regulatory 3a site (Ser-640) either before or after insulin treatment. This normal dephosphorylation after insulin treatment in vivo demonstrates that dephosphorylation may be necessary but not sufficient for complete activation of glycogen synthase. Full activation of glycogen synthase in vitro required both {beta}-D-N-acetylglucosaminidase and PP1. The increase in the efficiency of activation by {beta}-D-N-acetylglucosaminidase was much greater in extracts from STZ-treated mice, which also have increased levels of glycosylation. This illustrates a relationship between hyperglycemia, glycosylation of glycogen synthase, and resistance to activation by PP1. In this model of moderate and short term hyperglycemia the ability of insulin to lead to dephosphorylation was unimpeded, implying no critical effects on insulin signal transduction upstream of glycogen synthase. However, other studies using different model systems have demonstrated inhibition of insulin signal transduction pathways mediated by the hexosamine biosynthetic pathway (26–30).

The level of O-GlcNAc on nuclear and cytosolic proteins is a function of cellular nutritional status. Flux through the hexosamine biosynthetic pathway is rate-limited by the concentration of fructose 6-phosphate, and hexosamine flux increases with increased glucose or fatty acid uptake (14, 15, 31). The terminal metabolite of the hexosamine biosynthetic pathway, UDP-N-acetylglucosamine, is a substrate for O-linked N-acetylglucosaminyltransferase, which covalently modifies serine and threonine residues with O-GlcNAc (32). The Km value of O-linked N-acetylglucosaminyltransferase for UDP-N-acetylglucosamine is relatively high (33). Thus, high levels of O-GlcNAc on nuclear and cytoplasmic proteins reflect a high level of nutrient uptake (32, 34). This makes O-GlcNAc an attractive candidate for a nutrient sensing mechanism and for mediating the cellular consequences of excess nutrient flux (34–37).

Consistent with the proposed nutrient sensing role of O-GlcNAc, insulin resistance develops in mice with transgenic expression of O-linked N-acetylglucosaminyltransferase or the rate-limiting enzyme for the hexosamine biosynthetic pathway: glutamine:fructose-6-phosphate amidotransferase (38, 39). In addition to modulation of glycogen synthase activity, a direct role for O-GlcNAc in metabolic signaling has been demonstrated by several studies. Glycosylation of the Akt site on endothelial nitric-oxide synthase prevents activation by insulin signaling (40). Modification by O-GlcNAc of transcription factors involved in metabolism, such as Sp1, YY1, and c-myc, affects protein-protein interactions, transcriptional activation, and protein stability (41–47). Pharmacological inhibition of the enzyme that removes O-GlcNAc from proteins results in down-regulation of elements of the insulin signaling pathway and reduced glucose uptake by glucose transporter 4 (30).

Glycogen synthase has been previously regarded as being primarily regulated by phosphorylation-dephosphorylation and allosteric activation by G6P. These factors are interdependent: increased phosphorylation reduces sensitivity to G6P activation, but G6P changes the conformation of the protein to simultaneously activate the enzyme and make it a better substrate for dephosphorylation (4, 23, 48, 49). In diabetes this regulation breaks down, and glycogen synthase is inhibited and less responsive to insulin activation even though G6P levels are unchanged (9, 10, 22, 50–52). In some situations this may be partly attributable to upstream effects of the hexosamine pathway on signal transduction elements such as phosphatidylinositol 3-kinase, insulin receptor substrate-1, or {beta}-catenin (26, 27, 30, 53–55). However, in studies on glycogen synthase from the livers of STZ-treated rats, diabetes also directly inhibited the ability of phosphatases to activate the enzyme (12). The current findings regarding glycosylation of the enzyme may explain these results.

The simplest model to explain these findings is that O-GlcNAc is directly inhibitory in a manner analogous to phosphate and that removal is required for full activation of the enzyme. In diabetic conditions modification of glycogen synthase by O-GlcNAc is increased. Removal of either O-GlcNAc or phosphate alone is not sufficient for full activation of the enzyme, but removal of O-GlcNAc facilitates the activation of the synthase by dephosphorylation. This hexosaminidase-dependent increase in activation of the synthase by PP1 is greater in diabetes, consistent with an increased level of O-GlcNAc on the enzyme. There are two sites of phosphorylation on glycogen synthase that are sufficient for inhibition of the enzyme: Ser-7 and Ser-640 (5, 56). If either of these were modified by O-GlcNAc, then dephosphorylation alone would not be sufficient to fully activate the total enzyme population, especially in diabetic conditions. Deglycosylation by hexosaminidase would lower the threshold for phosphatase-mediated activation by prior removal of an inhibitory group on the other serine. The inability of hexosaminidase digestion alone to activate glycogen synthase was explained in part by the phosphorylation status of glycogen synthase. In the digestion conditions used in this study, removal of O-GlcNAc by hexosaminidase was followed by subsequent rephosphorylation of the enzyme by endogenous kinases. This would have blunted the expected activation of glycogen synthase. Alternatively glycosylation may sterically hinder access by phosphatases or result in a conformation of the synthase that is a poorer substrate for phosphatase. G6P is well known to change the conformation of glycogen synthase and make it a better substrate for phosphatases (57). O-GlcNAc may block the conformational changes induced by G6P or even limit access of G6P itself, impeding the ability of phosphatase to activate the enzyme. The role of O-GlcNAc could also be an indirect one. O-GlcNAc is now documented to interfere with some protein-protein interactions (44). Thus, the association of PP1 with glycogen-targeting regulatory subunits and glycogen synthase may well be inhibited by glycosylation of the synthase or other components of the glycogen synthetic machinery.

The current results are significant in that they demonstrate a role and provide a mechanism by which excess nutrients lead directly to insulin resistance and altered protein function in diabetes. The identification of the sites of glycosylation on glycogen synthase and its partners is necessary for resolving the exact function of O-GlcNAc in modulating glycogen metabolism. Recently new methodologies have been developed to facilitate the identification of O-GlcNAc sites using mass spectroscopy (58), and applying these techniques to identify O-GlcNAc sites on glycogen synthase should further clarify the mechanisms of inhibition.


   FOOTNOTES
 
* This work was supported by the research service of the Veterans Administration, National Institutes of Health Grant R01 DK43526, 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} 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{at}hsc.utah.edu.

1 The abbreviations used are: G6P, glucose 6-phosphate; AIU, arbitrary intensity units; O-GlcNAc, O-linked N-acetylglucosamine; PP1, protein phosphatase type 1 ({alpha}-isoform); STZ, streptozotocin; %I-form, basal glycogen synthase activity; A0.5, half-maximal activation concentration for glucose 6-phosphate. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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