The Effects of Glucose and the Hexosamine Biosynthesis Pathway on Glycogen Synthase Kinase-3 and Other Protein Kinases that Regulate Glycogen Synthase Activity

Abstract

Background

Glycogen synthase (GS) activity is determined by its phosphorylation state. We have previously demonstrated that high glucose (HG) downregulates both basal and insulin-stimulated GS activity in rat-1 fibroblasts and that the hexosamine biosynthesis pathway (HBP) may be involved in mediating some of the effects of glucose. In this study we investigate the influence of high glucose and glucosamine (GlcN) on the activity of several kinases that phosphorylate and inactivate GS.

Methods

Glycogen synthase kinase (GSK) 3, CAMP-dependent protein kinase (PKA), protein kinase C (PKC), casein kinase (CK) 1, and phosphorylase kinase (PhK) activities were assayed in cellular extracts from control rat-1 fibroblasts and those that overexpress human cDNA for glutamine:fructose 6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the HBP.

Results

Culturing rat-1 fibroblasts in HG (20 mmol/L) or GlcN (3-5 mmol/L) for 16-20 hours increases GSK-3 activity by 23.9 and 50%, respectively, when compared to activity at low glucose (LG, 1 mmol/L). The effects of HG on GSK-3 activity are greater in cells overexpressing GFA (38.8% increase). Insulin (1.7 nmol/L) treatment leads to a 20-25% decrease in GSK-3 activity that is not affected by HG, GlcN, or GFA overexpression. Culturing control cells in HG increases PKA and CK-1 activities by 56 and 95%, respectively, and HG diminishes insulin action on CK-1 activity. GlcN inhibits insulin action on both PKA and CK-1 activities. HG, GlcN, and GFA overexpression blunted insulin's ability to downregulate PhK activity in LG conditions. PKC activity is not significantly altered in either cell line in the above conditions.

Conclusions

These results suggest that HG alters both basal and insulin-regulated activity of several kinases that phosphorylate GS, and some of the effects of glucose may be mediated by its metabolism via the HBP.

Keywords

glycogen synthase hexosamines glycogen synthase kinase-3 phosphorylase kinase glutamine fructose-6-P amidotransferase

Introduction

In mammals, insulin is the principal hormone controlling blood glucose levels by stimulating glucose influx and metabolism in muscles and adipocytes, and by inhibiting gluconeogenesis in the liver. Defects in insulin signal transduction, leading to insulin resistance of the target cells, are characteristics of both type 1 and type 2 diabetes mellitus. The resultant hyperglycemia worsens insulin resistance in muscle and adipocytes and impairs pancreatic secretion of insulin.1,2 The mechanisms mediating the development of insulin resistance and other adverse effects of glucose are not yet fully understood. Several studies suggest that the hexosamine biosynthesis pathway that converts fructose 6-phosphate to glucosamine (GlcN) 6-phosphate via the first and rate-determining enzyme, glutamine:fructose 6-phosphate amidotransferase (GFA), is involved in mediating many of the effects of glucose.3–6 We have previously demonstrated that overexpression of the cDNA for yeast or human GFA in rat-1 fibroblasts results in insulin resistance.5,6 This resistance is manifested as a reduction in the ability of insulin to stimulate glycogen synthase (GS) activity. Furthermore, chronic exposure of rat-1 fibroblasts to high glucose (HG) decreases both basal and insulin-stimulated GS activity, and the influence of glucose is greater in cells overexpressing GFA. Similar effects of glucose, GlcN, and GFA overexpression are observed on the type 1 protein phosphatase (PP1) responsible for dephosphorylating (and activating) GS.7

GS plays an important role in glycogen metabolism and its activity is controlled by phosphorylation and/or de-phosphorylation in response to insulin or glucagon.8,9 GS is a 85-kDa protein and is known to be phosphorylated at least at nine Ser residues in vivo, and mutational analyses of the phosphorylation sites indicate that phosphorylation at sites 2a, 2b, 3a, and 3b particularly correlates with the inactivation of GS.10–12 Several protein kinases that may phosphorylate GS in vivo and inhibit its activity are illustrated Figure 1. These kinases are Ser/Thr kinases that are also involved in various other cellular processes.13 For example, glycogen synthase kinase 3 (GSK-3) has been implicated in the regulation of gene transcription and developmental programs in Drosophila.14 PKA is involved in the phosphorylation and activation of phosphorylase kinase (PhK), which in turn phosphorylates and converts an inactive phosphorylase b to an active phosphorylase a.14 In addition to the activation of phosphorylase a, PhK also phosphorylates GS at site 2a and inactivates the synthase activity.8,13 Protein kinase C (PKC) and CK-1 are also involved in the phosphorylation of a large number of other proteins that are involved in signal transduction, transcription, and translation.15–18

In the present study, we investigate further the mechanism(s) by which HG, GlcN, and GFA overexpression lead to insulin resistance and enhancement of the effects of glucose on GS. Specifically, we examined the effect of glucose and GlcN on the activity of kinases that phosphorylate (and inactivate) GS: GSK-3, PKA, PhK, CK-1, and PKC. The results suggest that the effects of altered hexosamine flux on GS are mediated via HBP regulation of GSK-3 PhK.

Materials and Methods

Materials

γ-³²P[ATP] (4500 Ci/mmol) was purchased from ICN Pharmaceuticals, Inc. (Costa Mesa, Calif) CK-1, CK-2, PKC and PKA were obtained from New England Biolabs. Phosphorylase kinase and phosphorylase b were purchased from Sigma Chemical Co. (St. Louis, Mo). P-81 phosphocellulose papers were from Whatman (Princeton, NJ). All other reagents and chemicals were of reagent or analytical grade.

Cell Culture

Rat-1 fibroblasts that stably overexpress the human cDNA for GFA were used for these studies. Wild type rat-1 fibroblasts were control cells. These GFA overexpressing cells have been characterized previously5,6 and have 1.5- to 2-fold increases in GFA activity when compared to control. Increased GFA activity was verified in cells used for these studies. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM), 10% fetal calf serum, and 0.5 mg/mL gentamicin, and routinely passaged at confluence every 4 days using 10-cm culture dishes. Eighty-five to 95% confluent monolayers were incubated in DMEM supplemented with 0.1% fetal calf serum and the desired concentrations of glucose and GlcN.7 To examine the effect of glucose or GlcN on insulin action, insulin (0.17 or 1.7 nmol/L) was added for 10 minutes before harvesting the cells. The dishes were rinsed twice with extraction buffer (50 mmol/L β-glycerophosphate, pH 7.3, 1.5 mmol/L EGTA, 1 mmol/L dithiothreitol, 0.2 mmol/L Na orthovanadate, 1 mmol/L benzamidine, 10 µg/mL aprotinin, 20 µg/mL leupeptin, 1 mmol/L NaF, 0.5 µg/mL microcystine, and 2 µg/mL pepstatin A) then harvested in 1 mL of the same buffer using a rubber policeman. The cells were centrifuged at 16,000g for 5 seconds, resuspended in 200 µL of extraction buffer, and frozen in liquid nitrogen. Cells were subsequently thawed, sonicated for 20 seconds, and centrifuged as above for 10 minutes. For GSK-3 activity, supernatants were centrifuged at 100,000g, 4°C for 20 minutes. Protein concentration in cell extracts was determined by the method of Bradford,19 using BSA as the standard.

Determination of GSK-3 Activity

GSK-3 substrate, (GS-1) YRRAAVPPSPSLSRHSSPHQSEDEEE20,21 was purchased from Quality Controlled Biochemical (Hopkinton, Mass). Prior phosphorylation of GS-1 by CK-2 is required for GSK-3 to phosphorylate this peptide. The priming site phosphorylated by CK-2 is underlined and the residues targeted by GSK-3 are shown in bold. Phosphorylation of GS-1 with recombinant CK-2 (New England Biolabs) was performed in 100 µL containing 20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L KCl, 1 mmol/L Dithiothreitol (DTT), 10 mmol/L Mg-acetate, 1 mmol/L peptide, and 0.5 mmol/L cold ATP.20 After 2 hours incubation at 30 °C, 2 µg/mL heparin was added to inactivate CK-2.

CK-2-phosphorylated GS-1 peptide (phospho-GS-1) was used to determine GSK-3 activity in cell extracts using 5 µg protein. The reaction mixture (30 µL) contained 20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L KCl, 1 mmol/L DTT, 10 mmol/L Mg-acetate, 60 µmol/L phospho-GS-1, 0.1 mmol/L [γ- $^{32}$ P]ATP (1000-2000 cpm/pmol), and 5 µg/mL heparin.21 The mixture was incubated at 30 °C for 15 minutes. $^{32}$ P incorporated into the peptides was determined by spotting 25 µL of the reaction mixtures on P-81 phosphocellulose papers and washing the papers 4 times (10 minutes per wash) in 0.5% (wt/vol) phosphoric acid and counting in the liquid-based Scintillation counter. For each extract the reaction was run in duplicate and values averaged to obtain activity for that plate of cells. $^{32}$ P incorporated into extracts in the absence of peptide substrates was used as background and was subtracted from each experimental value. The incorporation of radioactivity into peptides in the absence of extract was negligible for GSK-3 and all other assays listed below.

Determination of CK-1 Activity

CK-1 substrate, DDDDVASLPGLRRR (D4), was synthesized at the Protein Chemistry Core Facility at the University of Florida, Gainesville. The CK-1 site is shown in bold. CK-1 activity was determined in a 30-µL assay with 20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L KCl, 1 mmol/L DTT, 2.5 mmol/L Mg-acetate, 1 mmol/L peptide, and cell extracts containing 7.5 µg protein and 0.25 mmol/L [γ- $^{32}$ P]ATP (800-1000 cpm/pmol).22,23 After incubating for 15 minutes at 30 °C, the amount of radioactivity incorporated into the peptides was determined by binding to P-81 papers.

Determination of PKA Activity

PKA peptide substrate (Kemptide), LRRASLG, was obtained from Pierce Chemical Co. PKA activity was determined in a 30-µL assay with 20 mmol/L Tris-HCl, pH 7.5, 100 mmol/L KCl, 1 mmol/L DTT, 15 mmol/L Mg-acetate, 250 µmol/L peptide, 0.25 mmol/L [γ- $^{32}$ P]ATP (800-1000 cpm/pmol), and 5 µg protein of cell extracts.24 After incubating for 15 minutes at 30 °C, the amount of radioactivity incorporated into the peptides is determined by binding to P-81 papers.

Determination of PKC Activity

PKC peptide (Pseudosubstrate), RFARKGSLRQKNV was purchased from Pierce. PKC activity was determined in a 30-µL assay with 20 mmol/L Tris-HCl, pH 7.5, 10 mmol/L Mg-acetate, 0.9 mmol/L CaCl₂, 0.4 mmol/L EGTA, 30 mmol/L β-mercaptoethanol, 25 µg/mL micellar phosphatidylserine, 100 µmol/L peptide, 5 µg extract, and 0.25 mmol/L [γ-³²P]ATP (800-1000 cpm/pmol).25 After incubating for 15 minutes at 30 °C, the amount of radioactivity incorporated into the peptides was determined by binding to P-81 papers.

Determination of PhK Activity

The activity of phosphorylase kinase was determined by following the incorporation of ³²P from [γ-³²P]ATP into phosphorylase b.26 Final concentration in a 20-µL assay mixture was as follows: 50 mmol/L Tris, 50 mmol/L β-glycerophosphate, pH 8.6, 0.2 mmol/L CaCl₂, 0.1 mmol/L EGTA, 13 mmol/L β-mercaptoethanol, 10 mmol/L Mg-acetate, 5 µg phosphorylase b, 5 µg protein of cell extracts and 100 µmol/L [γ-³²P]ATP (1000-2000 cpm/pmol). After incubation for 30 minutes at 30 °C, the reaction was stopped by the addition of 5 µL of a 5×SDS-sample buffer, boiled for 5 minutes and then applied on 10% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue R-250, destained, dried, and exposed at −70 °C to X-ray films. The radioactivity incorporated into phosphorylase b (97-kDa band) was determined by cutting the bands and counting in a liquid-based Scintillation counter.

Statistical Analysis

Results are expressed as mean±SE of the indicated number of experiments. For statistical analysis, both the unpaired Student's t test and ANOVA were used. Analysis was performed with STATVIEW from Abacus. P<0.05 or <0.0033 were considered significant for t test and ANOVA, respectively.

Results

Effect of HG and GlcN on GSK-3 Activity

We have previously demonstrated that HG and GlcN decrease both basal and insulin-stimulated GS activity, and the influence of glucose is greater in cells overexpressing GFA.5,6 We measured GSK-3 activity in cellular extracts to see if the effects of glucose and GlcN on GS activity were similar to the effects on GSK-3. Cells were cultured in LG (1 mmol/L), HG (20 mmol/L), or GlcN (1 mmol/L glucose with 3-5 mmol/L GlcN) for 16-20 hours before harvest. These conditions are identical to those in which we observed the effects of HG and GlcN on basal and insulin-stimulated GS and PP1 activities.6,7 In control cells, HG results in a 23.9±8.3% (P<0.025) increase in GSK-3 activity when compared to activity at 1 mmol/L glucose (Figure 2). Likewise, GlcN (5 mmol/L) results in a 50.0±9.0% (P<0.05) increase in GSK-3 activity (Figure 2). These effects of glucose and GlcN on basal GSK-3 activity are not due to osmolar effects, as cells cultured in 1 mmol/L d-glucose with or without 20 mmol/L l-glucose had similar GSK-3 activity. In GFA cells HG treatment results in a 38.8±5.7% (P<0.05) increase in GSK-3 activity. There was a trend for GFA cells to be more responsive to HG with regards to GSK-3 activity (39 vs 24, GFA vs control).

Insulin treatment (0-1.7 nmol/L) for 10 minutes results in a 20-25% decrease in GSK-3 activity. This downregulation is near maximal at the lowest insulin dose used (0.17 nmol/L). As shown in Figure 3, neither glucose, GlcN, nor GFA overexpression significantly affects insulin action on GSK-3.

Influence of Glucose and GlcN on PKA, PKC, and CK-1 Activities

Although HG and GlcN increase GSK-3 activity, glucose and GlcN had no effect on insulin's ability to inactivate its activity. Hence, the decrease in the insulinstimulated GS activity, observed with HG and GlcN in these cells,5–7 may involve other kinases that phosphorylate GS. Therefore, PKA, PKC, and CK-1 activity were determined after exposure of cells to HG or GlcN. The results for control cells are summarized in Table 1. All kinase activities are normalized to specific activity at 1 mmol/L glucose without insulin (basal) that is designated as 100% activity. The activity of PKA in cells treated with 20 mmol/L glucose is increased when compared to 1 mmol/L, but GlcN does not have a significant influence on basal PKA activity. There was no evidence of insulin resistance with PKA activity. As was observed with PKA, GlcN does not mimic the HG-induced increase in basal activity of CK-1. Unlike the effects of insulin on CK-1 activity in cells cultured in 1 mmol/L glucose, insulin did not increase CK-1 activity further in HG or GlcN conditions. PKC activity was not significantly altered in these conditions.

Table 1.

PKA, CK-1, and PKC activity in rat-1 fibroblasts.

Culture Condition	PKA, % Activity	CK-1, % Activity	PKC, % Activity
Basal (no insulin)
1 mmol/L Glc	100	100	100
20 mmol/L Glc	157±14*	195±10*	109±7.7
1 mmol/L Glc/3 mM GlcN	85±9.0	86±11	106±5.6
Insulin (1.7 nmol/L)
1 mmol/L Glc	87±5.0	157±10*	92±9.6
20 mmol/L Glc	135±4.0	191±10	115±4.2
1 mmol/L Glc/3 mmol/L GlcN	88±7.0	83±17	99±6.7

P<0.0033, by ANOVA, compared to appropriate control.

Cells were serum-starved in media supplemented with the indicated Glc or GlcN concentrations for 16–20 hours before harvest. Insulin treatment was for 10 minutes. PKA, CK-1, and PKC activities were assayed as described in Methods. All data are normalized to activity determined at 1 mmol/L Glc (basal, without insulin) for each kinase and expressed as a percentage of that activity. Specific activity at 1 mmol/L Glc (basal): PKA=0.72±0.11 pmol/mg/min; CK-1=20,9±2.4 pmol/mg protein/min; and PKC=1.02±0.08 nmol/mg protein/min. All values are for n=5 for each kinase.

The results for PKA, CK-1, and PKC activity in GFA overexpressing cells are presented in Table 2. Similar to control cells, PKA activity increases with HG. However, the degree of upregulation with HG in GFA is approximately half that seen in controls (25 vs 57%, GFA vs control, P<0.07). This blunting of glucose's effect may be secondary to excess hexosamines, as GlcN did not significantly change basal PKA activity in controls (Table 1). The trend for both CK-1 and PKC activities are not different from controls. Basal activities, measured at 1 mmol/L glucose, for these three kinases are not significantly different between GFA and controls (see footnotes for Tables 1 and 2), however, there is a trend toward higher basal PKC activity in GFA cells.

Effect of Glucose and GlcN on PhK Activity

Although HG has a significant effect on the activities of PKA and CK-1, the effect of GlcN on these kinases are minimal and does not correlate with the inhibition of GS by GlcN.5–7 We, therefore, examined the activity of PhK in cell extracts after rat-1 fibroblasts were exposed to 1 mmol/L glucose, 20 mmol/L glucose, or 1 mmol/L glucose plus 3 mmol/L GlcN with or without insulin (1.7 nmol/L) treatment. Neither HG nor GlcN significantly stimulate basal PhK activity in control cells (Figure 4, lanes 1-3). At LG, the addition of insulin for 10 minutes decreases PhK activity by 29±2.6% (Figure 4, lane 4). However, insulin's ability to decrease PhK was inhibited in cells cultured in HG and GlcN (Figure 4, lanes 5 and 6). Similar results were seen in GFA cells. However, in GFA cells the decrease in PhK activity with insulin in cells cultured in LG (18.1±3.1%) was less than that seen in control cells and did not reach statistical significance. This indicates that GFA cells are insulin-resistant with respect to PhK, even at LG. These results suggest that HG, GlcN, and GFA overexpression inhibit insulin's effect on PhK activity and may explain, in part, the decrease in insulin-sensitivity of GS activity observed with HG, GlcN, and GFA overexpression.5–7

Table 2.

PKA, CK-1, and PKC activity in rat-1 fibroblasts overexpressing GFA.

Culture Condition	PKA, % Activity	CK-1, % Activity	PKC, % Activity
Basal (no insulin)
1 mmol/L Glc	100	100	100
20 mmol/L Glc	125±10*	205±10*	93±2.1
Insulin (1.7 nmol/L)
1 mmol/L Glc	90.3±2.0	175±26*	97±3.1
20 mmol/L Glc	109±5.6	188±44	95.5±5.3

P<0.05 compared to appropriate control by t test. *P<0.04 by ANOVA.

Cells were serum-starved in media supplemented with the indicated Glc or GlcN concentrations for 16–20 hours before harvest. Insulin treatment was for 10 minutes. PKA, CK-1, and PKC activities were assayed as described in Methods. All data are normalized to activity determined at 1 mmol/L Glc (basal, without insulin) for each kinase and expressed as a percentage of that activity. Specific activity at 1 mmol/L Glc (basal): PKA=0.78±0.16 pmol/mg/min; CK-1=22.3±3.0 pmol/mg protein/min; and PKC=1.91±0.43 nmol/mg protein/min. All values are for n=4–5 for each kinase.

Discussion

Glucose is an important regulator of cell growth and metabolism. However, hyperglycemia interferes widely with cellular metabolism and the mechanisms for insulininduced glucose disposal. There is substantial evidence that the products of the hexosamine biosynthesis pathway may exert some regulatory effect(s) on glucose uptake,4 glycogen synthesis,6,27 and synthesis of growth factors.3,7 Studies in whole animals support the importance of this pathway in mediating glucose disposal. For example, infusion of animals with GlcN results in a decrease in glucose disposal in rats,28 which is secondary to an impaired translocation of insulin-stimulated glucose transporters.29 Similarly, the overexpression of GFA in skeletal muscle and fat in transgenic mice results in decreased glucose disposal when measured during hyperinsulinemic euglycemic clamps.30 This defect is related to decreased GLUT4 translocation and is reversed in the transgenic animal with troglitazone.31

GS, which catalyzes the elongation of the glycogen chain, is the rate-limiting enzyme for glycogen synthesis and is stimulated by insulin in the liver and muscle. We have previously shown that culturing cells in HG leads to a decrease in the insulin-sensitivity of GS in rat-1 fibroblasts, and that overexpression of GFA made the cells more sensitive to the effects of glucose.5–7 In addition, HG leads to a decrease in basal GS activity in these cells and GFA overexpression shifts the IC50 for glucose to the left. These alterations in GS activity were due to posttranslational modifications of GS. Consistent with this hypothesis, we have shown that chronic exposure of rat-1 fibroblasts to HG and GlcN leads to a decrease in basal and insulin-stimulated PP1 activity.7

The data presented in this study further demonstrate that glucose and excess hexosamines have profound effects on kinases that target GS. For example, the increase in GSK-3 activity in cells cultured in HG or GlcN correlates with the decrease in basal GS activity by HG.6,7 The effects of hexosamines on GSK-3 activity appear to be specific to basal GS activity, as neither glucose, GlcN, nor GFA overexpression affect inactivation of GSK-3 by insulin. Therefore the effects of HG and excess hexosamines on insulin-stimulated GS activity may involve additional kinase(s).

Other kinases involved in regulation of GS activity are PKA, CK-1, PKC, and PhK.32,33 HG stimulates PKA and CK-1 activity. The increases in activity of the kinases with HG would presumably lead to increased phosphorylation of sites 2a and 2b on GS (see Figure 1) and decreased GS activity. The increase in activity of CK-1 and PKA with HG indicates a role for these kinases in the glucoseinduced downregulation of basal GS activity. Interestingly, the changes in PKA and CK-1 activities with HG are not mimicked by GlcN and may represent a hexosamine independent pathway for glucose regulation of GS activity. This latter finding is unique and indicates the complexity of cellular regulation of glycogen metabolism.

In contrast to GSK-3, PKA, and CK-1, the effects of glucose and hexosamines on PhK appear to be targeted to insulin sensitivity. PhK, another kinase with 2a as its target site, demonstrates insulin resistance in cells cultured in HG and GlcN. Specifically, inactivation of PhK by insulin was observed in LG, but this insulin-induced inactivation of activity is inhibited by HG or GlcN (Figure 4). Therefore, in addition to regulation of PP1,7 regulation of PhK may be a potential mechanism by which the HBP regulates insulin sensitivity of GS.

We did not observe significant changes in PKC activity in any of the conditions studied. It appears that the effects of glucose and GlcN on GS are not mediated via effects on PKC. However, there are several isoforms of PKC that have unique functions. Our assay did not determine isoform specific PKC activity, but rather total activity. Therefore, we can not conclusively rule out a role for PKC in mediating the effects of hexosamines on basal and insulinstimulated GS activity. In fact overexpression of GFA resulted in increased basal PKC activity (see below).

A limitation of these studies is the failure to reproduce all of the effects of GlcN in GFA overexpressing cells. Some effects of GFA overexpression were observed, however. Overexpression of GFA in rat-1 fibroblasts results in increases in basal PKC activity at 1 mmol/L glucose (control 1.02±0.08, GFA 1.91±0.43 nmol/mg protein/min). The effects of HG on basal GSK-3 activity were enhanced in GFA cells (28.9 vs 38.8% increase over basal for control and GFA, respectively). Basal PKA activity was stimulated less by HG in GFA cells when compared to controls. Most important was the blunting of insulin's ability to downregulate PhK activity in LG conditions. These results with GSK-3 and PhK parallel the effects of GlcN in controls and indicate possible sites for the effects of the HBP on GS.

Although not completely understood, there are several possibilities to explain the lack of effects of GFA overexpression. First, only modest increases in hexosamine flux would be expected with the 1.5- to 2-fold increases in GFA activity observed in these cells. In fact, the level of UDP-GlcNAc, a downstream product of the HBP, seen in GFA cells cultured at LG is equal to that in controls cultured at HG (2-fold higher than LG).34 Second, the effects of GlcN are most prominent on insulin sensitivity, which is difficult to detect given the relatively small degree of insulin stimulation seen in these studies. Third, the effects of GFA overexpression on GS activity is representative of the sum of several (and perhaps more modest) effects on the many regulators of GS. Therefore, modest hexosamine excess would result in small and perhaps experimentally undetectable effects on those regulators when examined individually.

In conclusion, the results of the present study, when combined with our previous work,5–7 provide insight into the mechanisms by which hexosamines regulate glycogen synthesis and its rate-limiting enzyme GS. We have demonstrated that HG and GlcN affect the activity of important protein kinases involved in the phosphorylation of GS. Downregulation of basal GS activity by HG is associated with increases in basal GSK-3, PKA, and CK-1 activities (as shown here), and a parallel decrease in PP1 activity.7 In the case of GSK-3 and PP1, GlcN mimics glucose's effects, but the effects of glucose on PKA and CK-1 appear to be independent of its metabolism via the HBP. The downregulation of insulin sensitivity of GS by HG may occur by altering PhK and PP1 activity; here again, these effects are mediated by the metabolism of glucose to hexosamines.

Footnotes

Acknowledgments

E.D.C. was supported by a grant from the Robert Wood Johnson Foundation.

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