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Activation of the Hexosamine Pathway Leads to Phosphorylation of Insulin Receptor Substrate-1 on Ser³⁰⁷ and Ser⁶¹² and Impairs the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Insulin Biosynthetic Pathway in RIN Pancreatic ß-Cells

Department of Experimental and Clinical Medicine (F.A., E.L., F.P., G.S.), University of Catanzaro-Magna Græcia, 88100 Catanzaro, Italy; Laboratory of Molecular Medicine (C.D., M.F., R.L.), Department of Internal Medicine, University of Rome-Tor Vergata, 00133 Rome, Italy; and Department of Endocrinology and Metabolism (S.D.G., S.D.P., P.M.), Metabolic Unit, University of Pisa, 56100 Pisa, Italy

Address all correspondence and requests for reprints to: Giorgio Sesti, M.D., Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro-Magna Græcia, Via Tommaso Campanella, 115, 88100 Catanzaro, Italy. E-mail: sesti{at}unicz.it.

	Abstract

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Many adverse effects of glucose were attributed to its increased routing through the hexosamine pathway (HBP). There is evidence for an autocrine role of the insulin signaling in ß-cell function. We tested the hypothesis that activation of the HBP induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling. Exposure of human pancreatic islets and RIN ß-cells to glucosamine resulted in reduction in glucose- and insulin-stimulated insulin biosynthesis, which in RIN ß-cells was associated with impairment in insulin-stimulated insulin receptor substrate-1 (IRS-1) phosphorylation at Tyr⁶⁰⁸ and Tyr⁶²⁸, which are essential for engaging phosphatidylinositol 3-kinase (PI 3-kinase). These changes were accompanied by impaired activation of PI 3-kinase, and activation of Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway. RIN ß-cells exposed to high glucose exhibited increased c-Jun N-terminal kinase (JNK) and ERK1/2 activity, which was associated with increased IRS-1 phosphorylation at serine (Ser)³⁰⁷ and Ser⁶¹², respectively, that inhibits coupling of IRS-1 to the insulin receptor and is upstream of the inhibition of IRS-1 tyrosine phosphorylation. Azaserine reverted the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹². Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹². Inhibition of JNK and MAPK kinase-1 activity reverted the negative effects of glucosamine on insulin-mediated protein synthesis. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser³⁰⁷ and Ser⁶¹² of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, and activation of the Akt/mammalian target of rapamycin/phosphorylated heat- and acid-stable protein-1/p70S6 kinase pathway.

	Introduction

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THE PATHOGENESIS OF type 2 diabetes is characterized by a combination of peripheral insulin resistance and a progressive decline in pancreatic ß-cell function. When insulin secretion by ß-cells fails to compensate for insulin resistance, glucose intolerance and, eventually, overt hyperglycemia occurs (1). Chronic elevation of glucose concentrations deteriorates ß-cell function through a process referred to as glucose toxicity (2, 3). Exposure to a hyperglycemic environment of human or murine islets and ß-cell lines leads to alterations in insulin gene transcription, insulin biosynthesis, glucose-induced insulin secretion, and ß-cell survival (4, 5, 6, 7, 8, 9).

Several of the adverse effects of glucose have been attributed to increased routing of carbohydrate through the hexosamine pathway (HBP) (10, 11, 12). In the HBP, glutamine/fructose-6-phosphate amidotransferase (GFAT) catalyzes the conversion of fructose-6-phosphate to glucosamine-6-phosphate with glutamine acting as an aminodonor. The final product of this pathway is the substrate uridine 5'-diphosphate-N-acetylglucosamine (GlcNAc), which is the donor sugar used by the enzyme O-linked N-acetylglucosamine transferase for O-linked glycosylation of proteins. In this reaction, a single GlcNAc moiety is enzymatically attached to the hydroxyl group of either Ser or Thr residue (13).

Recently it has been demonstrated that insulin released by ß-cells in response to glucose stimulus enhanced its own biosynthesis, and exogenous insulin mimicked the stimulatory effects of glucose on insulin biosynthesis (14, 15, 16, 17, 18). The main signaling pathway used by the insulin receptor for promoting insulin biosynthesis originates with tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) leading to sequential activation of phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream effector, the Ser/Thr kinase Akt. Akt plays a central role in promoting protein synthesis by modulating downstream elements involved in the control of the translation process including the kinase mammalian target of rapamycin (mTOR) and its downstream targets p70S6 kinase and phosphorylated heat- and acid-stable protein-1 (PHAS-1), also termed eucaryotic initiation factor 4E binding protein 1 (18, 19, 20, 21). We have recently shown that exposure to a hyperglycemic environment of human pancreatic islets or ß-cell lines leads to alterations in insulin gene transcription and insulin biosynthesis (9). These abnormalities have been associated with decreased insulin receptor expression and increased relative abundance of the Ex11⁺ isoform of the insulin receptor in both human pancreatic islets and RIN ß-cells (9). These changes were paralleled by defects in insulin signaling involving IRS-associated PI 3-kinase/Akt/PHAS-1 pathway in RIN ß-cells. Interestingly, reexpression in RIN ß-cells chronically exposed to high glucose of Ex11–, but not Ex11+, isoform restored insulin mRNA expression (9). These data suggest that changes in early steps of the insulin receptor signaling may play a role in determining ß-cell dysfunction caused by chronic hyperglycemia.

Serine (Ser) phosphorylation of IRS-1 has been implicated as a negative regulator of insulin signaling and may be a contributing factor in the development of insulin resistance (22, 23, 24, 25, 26). Upon phosphorylation on Ser residues, IRS-1 has been shown to have a reduced ability to interact with the insulin receptor, to be phosphorylated on tyrosine residues, and to engage the src homology 2 (SH2) domains of the p85 regulatory subunit of PI 3-kinase (22, 23, 26). Several Ser residues in IRS-1 have been identified as negative regulatory sites including Ser³⁰⁷, which is activated by c-Jun N-terminal kinase (JNK) and Ser⁶¹², which is activated by MAPK (22, 23, 24, 25). There is evidence that high glucose and glucosamine, which is a product of the HBP downstream to GFAT, induces activation of both JNK and MAPK (27, 28, 29). However, it is unknown whether activation of the HBP would affect phosphorylation of IRS-1 at Ser³⁰⁷ and Ser⁶¹² residues, thus impairing activation of the insulin biosynthetic pathway involving PI3-kinase/Akt/mTOR. The present study was designed to examine the effect of activation of the HBP on insulin biosynthesis in human pancreatic islets and the ß-cell line RIN 1046–38, a well-characterized cell model, which exhibits the key regulatory elements of pancreatic ß-cells (9, 30, 31, 32, 33). We found that exposure of ß-cells to glucosamine resulted in hyperphosphorylation of IRS-1 at Ser³⁰⁷ and Ser⁶¹² and inhibition of tyrosine phosphorylation at positions 608 and 628 (Tyr⁶⁰⁸ and Tyr⁶²⁸), which play a major role in binding the SH2 domains of p85 subunit of PI 3-kinase (34). These changes in IRS-1 phosphorylation resulted in reduction in insulin biosynthesis due to impaired activation of the PI3-kinase/Akt/mTOR protein translation initiation pathway.

	Materials and Methods

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Proinsulin biosynthesis in human islet and RIN ß-cells
The pancreata were procured through the local organ procurement agency, within the framework of a project aiming to clinical islet allotransplantation into diabetic patients, approved by the Ethics Committee of the University of Pisa. Human pancreatic islets were obtained by collagenase digestion and density-gradient purification from pancreata of heart beating multiorgan donors as previously described (8, 9, 31). After isolation, the islets were plated on collagen I-coated dishes, allowing the cells to attach to the dishes. Islets were cultured in M199 medium containing 10% fetal bovine serum. Two days after plating, when most islets were attached, the islets were incubated for 24 h in the presence or absence of glucosamine (7.5 mM) in M199 medium containing 0.25% BSA. In experiments with protein kinase inhibitors, these were added to cells 30 min before glucosamine addition. Proinsulin biosynthesis was determined by preincubating islets for 2 h in modified Krebs-Ringer bicarbonate buffer containing 20 mM HEPES and 0.25% BSA, supplemented with 2.8 mM glucose, minimal essential medium amino acid solution, minimal essential medium nonessential amino acid solution, and L-glutamine. Then the islets were stimulated with glucose (16 mM) or insulin (100 nM) for 60 min followed by incubation for 60 min in buffer containing 50 µCi [³⁵S] methionine (Amersham Pharmacia Biotech, Milan, Italy). Thereafter cells were washed and lysed, and insulin was immunoprecipitated with antiinsulin antibody. Total labeled proteins were precipitated in 10% trichloroacetic acid and pelleted by centrifugation, and radioactivity was determined by scintillation counting. RIN1046–38 rat pancreatic ß-cells were kindly provided by Dr. W. L. Chick (Transplantation Biology, BioHybrid Technologies, Inc., Shrewsbury, MA). Proinsulin biosynthesis was determined in RIN ß-cells treated for 15 min with maximally stimulating concentrations of glucose (3 mM) or insulin (100 nM) according to the procedure above described. In preliminary experiments, the effect of glucose and glucosamine on O-linked GlcNAc modification of proteins in RIN ß-cells had been determined in total cell lysates by immunoblotting with the anti-O-linked GlcNAc monoclonal antibody RL2 (Affinity Bioreagents, Golden, CO). In agreement with our previous results in a different cell model (35), exposure of RIN ß-cells to different glucose or glucosamine concentrations for 24 h resulted in a dose-dependent increase in O-GlcNAcylation levels with maximal effect occurring at 25–33 mM glucose that was equivalent to that exerted by 7.5 mM glucosamine (data not shown).

Phospho-specific phosphorylation of IRS-1
RIN 1046–38 ß-cells were cultured for 24 h in serum-deprived M-199 medium containing 0.1% BSA in the presence or absence of 7.5 mM glucosamine and then incubated in the presence or absence of 100 nM insulin for 5 min. The cells were then washed with ice-cold PBS and lysed for 1 h at 4 C in lysis buffer [94 mM NaCl, 14 mM Tris (pH 7.6), 0.7 mM MgCl₂, 0.7 mM CaCl₂, 1.5% Nonidet P-40, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride, 2 mM Na₃VO₄, 2 mM EDTA, 10 mM NaPP, 10 mM NaF, 8 µg/ml leupeptin]. Insoluble material was removed by centrifugation in microfuge for 15 min at 4 C, and the supernatants were incubated for 16 h at 4 C with anti-IRS-1 (Upstate Biotechnolocy Inc., Lake Placid, NY). Immune complexes were collected by incubation with protein A-Sepharose for 2 h at 4 C and resuspended in 2x Laemmli buffer. Equal amounts of immunoprecipitated proteins were subjected to SDS-PAGE under reducing conditions. Proteins resolved by SDS-PAGE were electrophoretically transferred to nitrocellulose membrane. The membranes were blotted with antiphosphotyrosine antibody RC20 (Transduction Laboratories, Lexington, KY), anti-Ser³⁰⁷ IRS-1, anti-Ser⁶¹² IRS-1 (Cell Signaling, Beverly, MA), anti-Tyr⁶⁰⁸ IRS-1 (Biosource, Camarillo, CA), or anti-Tyr⁶²⁸ IRS-1 (Santa Cruz Biotechnology, Santa Cruz, CA). After extensive washings, the blots were incubated with peroxidase-conjugated secondary antibodies. To normalize the blots for protein levels, after being immunoblotted with antiphosphospecific antibodies, the blots were stripped and reprobed with anti-IRS-1 antibody. Proteins were detected by using enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech) and band densities were quantified by densitometry.

IRS-1-associated PI 3-kinase activity
RIN 1046–38 ß-cells cultured as described above were incubated in the presence or absence of 100 nM insulin for 5 min and then lysed. Cell lysates were subjected to immunoprecipitation with anti-IRS-1 antibody and PI 3-kinase activity was assayed in the immunoprecipitates according to previously described method (9, 31, 35) with phosphatidylinositol used as substrate. The PI 3-phosphate products were visualized by autoradiography, identified by its comigration with a PI 4-phosphate standard (Calbiochem, La Jolla, CA), and quantified by scanning densitometry.

Phosphorylation of Akt, tuberin, mTOR, PHAS-1, and p70 S6 kinase
After insulin stimulation, RIN 1046–38 ß-cells were lysed, and equal amounts of protein from cell lysates were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were blotted with anti-Ser⁴⁷³ Akt, anti-Thr³⁸⁹ p70S6 kinase, anti-Ser²²⁴⁸ mTOR, or anti-Thr¹⁴⁶² tuberin antibody (Cell Signaling). PHAS-1 phosphorylation was detected using a polyclonal anti-PHAS-1 antibody (Santa Cruz Biotechnology) and determining the electrophoretic mobility pattern. Blots were visualized using peroxidase-conjugated secondary antibodies followed ECL detection, and band densities were quantified by densitometry.

Phosphorylation of c-Jun, ERK1/2, and Ser^307/312 phosphorylation of IRS-1
RIN 1046–38 ß-cells were cultured for 24 h in serum-free medium with the indicated concentrations of glucose and glucosamine in the presence or absence of azaserine (Sigma-Aldrich, St. Louis, MO), the cell-permeable peptide JNK inhibitor I (Calbiochem), and PD98059 (Calbiochem). Cells were then lysed, and equal amounts of proteins were subjected to SDS-PAGE and immunoblotted with phospho-specific anti-Ser⁶³cJun, anti-Thr²⁰²/Tyr²⁰⁴ ERK1/2, anti-Ser³⁰⁷ IRS-1, or Ser⁶¹² IRS-1 antibody (Cell Signaling). To normalize the blots for protein levels, after being immunoblotted with antiphospho-specific antibody, the blots were stripped and reprobed with appropriate primary antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry.

Statistical analysis
All results are given as means ± SEM. Differences between values were evaluated by one-way ANOVA or unpaired Student’s t test as appropriate. Values of P < 0.05 were considered statistically different (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

	Results

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Impaired insulin biosynthesis stimulated by glucose or insulin in human pancreatic islets and RIN ß-cells exposed to glucosamine
To examine the possible involvement of the HBP in toxic effect of glucose, human pancreatic islets and RIN ß-cells were incubated in the presence or absence of glucosamine and then stimulated with glucose or exogenous insulin. In human pancreatic islets, specific insulin biosynthesis, i.e. after normalization to total protein biosynthesis, increased by 2-fold (P < 0.01) upon stimulation with glucose and by 2.3-fold (P < 0.01) upon stimulation with insulin (Fig. 1). Insulin biosynthesis in human pancreatic islets exposed to glucosamine was reduced by 39% upon glucose stimulation (P < 0.01) and by 24% upon insulin stimulation (P < 0.01) as compared with untreated control human islets. Only a limited number of human pancreatic islets can be obtained from donors; thus, to further investigate the molecular mechanisms by which glucosamine would affect glucose- and insulin-stimulated insulin biosynthesis, we used the pancreatic RIN ß-cell line that exhibits the key regulatory elebments of the pancreatic ß-cell. In control RIN ß-cells, specific insulin biosynthesis increased by 1.9-fold (P < 0.001) upon stimulation with glucose and by 2.0-fold (P < 0.001) upon stimulation with insulin (Fig. 2). Insulin biosynthesis in RIN ß-cells exposed to glucosamine was reduced by 40% upon glucose stimulation (P < 0.001) and by 45% upon insulin stimulation (P < 0.01) as compared with untreated control ß-cells. To examine whether glucosamine affects cell survival, apoptosis was evaluated by flow cytometry by measuring the number of hypodiploid events after propidium iodide staining of fixed cells. Exposure of RIN ß-cells to glucosamine in the presence of 0.2% BSA for 24 h resulted in apoptosis of less than 10% of total cells. These data were confirmed by assessment of caspase-3 activity, which serves as a biochemical marker for the execution phase of apoptosis (data not shown). To analyze the intracellular signaling that in human pancreatic islets and RIN ß-cells contributes to regulate insulin biosynthesis in response to both stimuli, we combined the stimulation with glucose or insulin with the cotreatment with inhibitors of the insulin receptor tyrosine kinase [hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester (HNMPA)], PI 3-kinase (wortmannin and LY294002), and mTOR (rapamycin). As shown in Figs. 1 and 2, HNMPA, wortmannin, LY294002, and rapamycin inhibited both glucose- and insulin-stimulated insulin biosynthesis in both human pancreatic islets and RIN ß-cells. These data are consistent with previous studies showing a role for the insulin receptor, PI3-kinase, and mTOR in the signaling pathway involved in insulin biosynthesis by pancreatic ß-cells in response to secreted insulin or exogenous insulin (15, 16).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Effect of glucosamine on glucose- and insulin-stimulated insulin biosynthesis in human pancreatic islets. Human islets were incubated for 24 h in serum-free medium in the indicated combinations of glucosamine with or without the kinase inhibitors PD98059 and JNK inhibitor I. The cells were then stimulated with glucose and insulin, as described in Materials and Methods, in the presence or absence of the indicated inhibitors. Specific insulin biosynthesis was calculated as the ratio of insulin to total protein biosynthesis, and values are given in percentages relative to those obtained under basal unstimulated conditions, which were set to 100%. Each bar represents the mean ± SEM of three independent experiments. Differences between values were evaluated by one-way ANOVA.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Effect of glucosamine on glucose- and insulin-stimulated insulin biosynthesis in RIN ß-cells. RIN ß-cells were incubated for 24 h in serum-free medium in the indicated combinations of glucosamine with or without the kinase inhibitors PD98059 and JNK inhibitor I. The cells were then stimulated with glucose and insulin, as described in Materials and Methods, in the presence or absence of the indicated inhibitors. Specific insulin biosynthesis was calculated as the ratio of insulin to total protein biosynthesis, and values are given in percentages relative to those obtained under basal unstimulated conditions, which were set to 100%. Each bar represents the mean ± SEM of six independent experiments carried out in triplicate. Differences between values were evaluated by one-way ANOVA.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Effect of glucosamine on insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1-associated PI 3-kinase activity RIN ß-cells. RIN ß-cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated for 5 min in the presence or absence of 100 nM insulin. The cells were then lysed, and equal amounts of total proteins were immunoprecipitated with anti-IRS-1 antibody, subjected to SDS-PAGE, and immunoblotted with anti-pY (phosphotyrosine) antibody (A, top panel). Kinase assays were performed on anti-IRS-1 immunoprecipitates (B) using phosphatidylinositol as an in vitro substrate. Each bar represents the mean ± SEM of at least three independent experiments. To normalize the blots for protein levels, after being immunoblotted with antiphosphotyrosine antibody, the blots were stripped and reprobed with anti-IRS-1 antibody (A, lower panel). Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of at least three independent experiments, and autoradiographs of a representative experiment are shown. *, P < 0.05; **, P < 0.01.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Effect of glucosamine on insulin-stimulated Tyr608 and Tyr628 phosphorylation of IRS-1 in RIN ß-cells. RIN ß-cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated for 5 min in the presence or absence of 100 nM insulin. The cells were then lysed, and equal amounts of total proteins were immunoprecipitated using anti-IRS-1 antibody, separated by SDS-PAGE, and immunoblotted with anti-Tyr608 (A, top panel) or anti Tyr628 (A, middle panel) phospho-specific IRS-1 antibody. To normalize the blots for protein levels, after being immunoblotted with antiphosphotyrosine antibody, the blots were stripped and reprobed with anti-IRS-1 antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of three independent experiments, and autoradiographs of a representative experiment are shown (*, P < 0.05).

View larger version (32K): [in this window] [in a new window]   FIG. 5. Effect of glucosamine on insulin-stimulated tyrosine phosphorylation of IRS-2 and phosphotyrosine (pY)-associated PI 3-kinase activity. RIN ß-cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated for 5 min in the presence or absence of 100 nM insulin. The cells were then lysed, and equal amounts of total proteins were immunoprecipitated with anti-IRS-2 antibody, subjected to SDS-PAGE, and immunoblotted with antiphosphotyrosine antibody (A, top panel). To normalize the blots for protein levels, after being immunoblotted with anti-pY antibody, the blots were stripped and reprobed with anti-IRS-2 antibody (A, lower panel). Proteins were detected by using ECL, and band densities were quantified by densitometry. Kinase assays were performed on anti-pY immunoprecipitates (B) using phosphatidylinositol as an in vitro substrate. Each bar represents the mean ± SEM of at least three independent experiments, and autoradiographs of a representative experiment are shown (**, P < 0.01).

View larger version (12K): [in this window] [in a new window]   FIG. 6. Effect of glucosamine on Akt phosphorylation in RIN ß-cells. RIN ß-cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated for 15 min in the presence or absence of 100 nM insulin. The cells were lysed, and equal amounts of proteins were subjected to SDS-PAGE and immunoblotted with anti-Ser473 phospho-specific Akt antibody. To normalize the blots for protein levels, after being immunoblotted with antiphospho-specific antibody, the blots were stripped and reprobed with anti-Akt antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of three independent experiments, and autoradiographs of a representative experiment are shown (**, P < 0.01).

View larger version (25K): [in this window] [in a new window]   FIG. 7. Effect of glucosamine on phosphorylation of mTOR and tuberin in RIN ß-cells. RIN ß-cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated 15 min in the presence or absence of 100 nM insulin. The cells were lysed, and equal amounts of proteins were subjected to SDS-PAGE and immunoblotted with phosphor-specific anti-Ser2248 mTOR (A) or anti-Thr1462 tuberin (B) antibody. To normalize the blots for protein levels, after being immunoblotted with antiphosphospecific antibodies, the blots were stripped and reprobed with anti-mTOR or antituberin antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of three independent experiments, and autoradiographs of a representative experiment are shown (*, P < 0.05; **, P < 0.01).

View larger version (21K): [in this window] [in a new window]   FIG. 8. Effect of glucosamine on phosphorylation of p70S6 kinase and PHAS-1 in RIN ß-cells. RIN ß-cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated for 30 min in the presence or absence of 100 nM insulin. The cells were lysed, and equal amounts of proteins were subjected to SDS-PAGE and immunoblotted with phospho-specific anti-Thr389 p70S6 kinase (A) or anti-PHAS-1 (B) antibody. To normalize the blots for protein levels, after being immunoblotted with antiphospho-specific antibody, the blots were stripped and reprobed with anti-p70S6 kinase antibody (A). Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of three independent experiments, and autoradiographs of a representative experiment are shown (*, P < 0.05; **P < 0.01).

Increased IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² in RIN ß-cells exposed to glucosamine
Increased flux of glucose through the hexosamine pathway has been reported to activate both JNK and MAPK (28). Stimulation of JNK has been shown to inhibit insulin signaling by promoting IRS-1 phosphorylation at Ser³⁰⁷, whereas stimulation of MAPK has been shown to inhibit insulin signaling by promoting IRS-1 phosphorylation at Ser⁶¹². We therefore investigated whether activation of the HBP induces IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² by activating JNK and MAPK, respectively. As shown in Fig. 9, A–C, exposure of RIN ß-cells to high glucose resulted in an increased phosphorylation of both JNK and the transcription factor c-Jun, a physiological substrate of JNK as well as the ERK-1 and -2 members of MAPK family. In addition, exposure of RIN ß-cells to high glucose increased phosphorylation of both IRS-1 Ser³⁰⁷ and IRS-1 Ser⁶¹² (Fig. 10, A and B), causing a decrease in the electrophoretic mobility shift of IRS-1 (Fig. 10C). Inhibition of hexosamine biosynthesis by the GFAT inhibitor azaserine reversed the stimulatory effects of glucose on JNK, c-Jun, and ERK1/2 activation (Fig. 9, A–C) as well as IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² (Fig. 10, A and B). Accordingly, exposure of RIN ß-cells to glucosamine resulted in an increased phosphorylation of JNK, c-Jun, and ERK1/2 as compared with control RIN ß-cells (Fig. 9, A–C). Exposure of RIN ß-cells to glucosamine increased phosphorylation of both IRS-1 Ser³⁰⁷ and IRS-1 Ser⁶¹², which was reversed by treatment with either a cell-permeable JNK inhibitor or PD98059, a reversible MAPK kinase (MEK)1 inhibitor, the enzyme that directly activates ERK1/2, respectively (Fig. 10, A and B). Taken together, these results suggest that increased flux of glucose through the HBP induces IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² by activation of JNK and ERK1/2, respectively, which can be effectively reversed by their respective inhibitors.

View larger version (40K): [in this window] [in a new window]   FIG. 9. Effect of high glucose or glucosamine on activation of JNK, c-Jun, and ERK1/2 in RIN ß-cells. RIN ß-cells were incubated for 24 h in serum-free medium in the indicated combinations of glucose with or without azaserine and glucosamine with or without cell-permeable peptide JNK inhibitor or PD98059. Cells were then lysed, and equal amounts of total proteins were subjected to SDS-PAGE and immunoblotted with phospho-specific anti-JNK, anti-Ser63cJun (used as an indirect measure of JNK activity) (B), or anti-Thr202/Tyr204 ERK1/2 (C). To normalize the blots for protein levels, after being immunoblotted with antiphospho-specific antibody, the blots were stripped and reprobed with appropriate primary antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of three independent experiments, and autoradiographs of a representative experiment are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

View larger version (38K): [in this window] [in a new window]   FIG. 10. Effect of high glucose or glucosamine on phosphorylation of IRS-1 at Ser307 and Ser612 and on IRS-1 mobility shift in RIN ß-cells. RIN ß-cells were incubated for 24 h in serum-free medium in the indicated combinations of glucose with or without azaserine and glucosamine with or without cell-permeable peptide JNK inhibitor or PD98059. Cells were then lysed, and lysates were immunoprecipitated using anti-IRS-1 antibody, separated by SDS-PAGE, and immunoblotted with phospho-specific anti-Ser307 IRS-1 (A), anti-Ser612 IRS-1 (B), and anti-IRS-1 (C) antibodies. To normalize the blots for protein levels after being immunoblotted with antiphospho-specific antibody, the blots were stripped and reprobed with anti-IRS-1 antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean ± SEM of three independent experiments, and autoradiographs of a representative experiment are shown (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glucose is the main physiological nutrient regulator of ß-cell function including insulin secretion and biosynthesis. However, chronic hyperglycemia is accompanied by a decline in glucose-stimulated insulin secretion and biosynthesis, a phenomenon referred to as glucose toxicity (2, 3, 4, 5, 6, 7, 8, 9). An increased flux of glucose through the HBP has been hypothesized to mediate many of the adverse effects of chronic hyperglycemia (10, 11, 12). Recent investigations (14, 15, 16, 17, 18) have provided functional and genetic evidence for an autocrine role of the insulin signaling cascade in ß-cell growth and function. Because activation of the HBP has been reported to alter insulin signaling in target tissues such as skeletal muscle, adipocytes, and endothelium (35, 42, 43), we tested the hypothesis that activation of this metabolic pathway induces defects in insulin biosynthesis in both human pancreatic islets and the RIN pancreatic ß-cell line by affecting the insulin-mediated protein translation signaling. Our initial approach was to determine whether insulin secreted by ß-cells contributes to its biosynthesis via an insulin receptor-mediated mechanism and whether this effect could be mimicked by exogenous insulin. Consistent with previous studies (16, 18), we found that glucose and exogenous insulin equally stimulate insulin biosynthesis in both human pancreatic islets and RIN ß-cells, and inhibitors of the insulin receptor tyrosine kinase, PI 3-kinase, and mTOR abolished both glucose- and insulin-stimulated insulin biosynthesis. To exclude the possibility that the observed stimulatory effects of glucose and insulin were a result of recovery from a lack of glucose and amino acids such as leucine, which has been shown to be essential for glucose and insulin signaling through mTOR (19), in vitro incubations were routinely performed in medium containing both glucose and amino acids, and labeled methionine was used as tracer instead of the most commonly used leucine. Exposure of human pancreatic islets and RIN ß-cells to glucosamine resulted in a marked reduction in glucose- and insulin-stimulated insulin biosynthesis as compared with control ß-cells. In the presence of glucosamine, we observed in RIN ß-cells a significant impairment in insulin-stimulated total tyrosine phosphorylation of IRS-1 and IRS-2 as well as a reduction in specific phosphorylation of tyrosine residues of IRS-1 at positions 608 and 628, which have an important role for engaging the SH2 domains of the p85 regulatory subunit of PI 3-kinase (34). These changes were accompanied by a significant impairment activation of IRS-1-associated PI 3-kinase, and the sequential activation of Akt.

The initial signaling events that are thought to be responsible for initiating mRNA translation involve activation of p70S6 kinase and PHAS-1 through a mTOR-mediated pathway (19, 20, 21). Akt promotes protein synthesis by modulating mTOR activity by a direct and indirect way. Akt phosphorylates mTOR at Ser²⁴⁴⁸, and this event has been correlated with mTOR activity, although there is also evidence that substitution of Ser²⁴⁴⁸ by alanine does not affect the ability of mTOR to activate p70S6 kinase (44). More recently it has been proposed an alternative way by which Akt regulates mTOR. Indeed, it has been demonstrated that Akt stimulates tuberin at Thr¹⁴⁶² upon insulin stimulation, thus relieving the inhibitory function of the tuberin-hamartin complex on the activation of mTOR and its downstream targets p70S6 kinase and PHAS-1 (38, 39). Interestingly, we found that exposure of RIN ß-cells to glucosamine resulted in a significant impairment in insulin-stimulated phosphorylation of both mTOR at Ser²⁴⁴⁸ and tuberin at Thr¹⁴⁶². These changes were accompanied by a significant impairment of the sequential activation of p70S6 kinase and PHAS-1. These results are consistent with the idea that activation of the HBP might interferes with insulin protein biosynthesis by affecting at multiple steps the Akt/mTOR/PHAS-1/p70S6 kinase protein translation initiation pathway.

A growing body of evidence has implicated Ser phosphorylation of IRS-1 as a mechanism of insulin resistance (22, 23, 24, 25, 26). Several studies have shown that enhanced Ser phosphorylation of IRS-1 stimulated by a variety of factors interferes with both the ability of this substrate to be tyrosine phosphorylated upon insulin stimulation and reduces its ability to engage the p85 regulatory subunit of PI 3-kinase. More recent investigations (22, 23, 24, 25) have characterized several specific Ser phosphorylation sites in IRS-1 as responsible for these inhibitory effects and have identified the activating kinases including JNK and ERK1/2. Activation of JNK has been shown to result in stimulation of Ser³⁰⁷ of IRS-1, whereas activation of ERK1/2 has been shown to result in an increased phosphorylation of Ser⁶¹². Consistent with previous studies, we found that RIN ß-cells exposed to high glucose exhibited increased JNK and ERK1/2 activity, which was associated with a concomitant increase in IRS-1 phosphorylation on Ser³⁰⁷ and Ser⁶¹², respectively. Interestingly, azaserine, a GFAT inhibitor that blocks glucose flux through the HBP, inhibited the stimulatory effects of glucose on JNK and ERK1/2 activity and reverted the enhanced Ser³⁰⁷ and Ser⁶¹² phosphorylation of IRS-1 stimulated by high glucose. Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity as well as IRS-1 phosphorylation on Ser³⁰⁷ and Ser⁶¹². These results indicate that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser³⁰⁷ and Ser⁶¹² of IRS-1 mediated by JNK and ERK1/2, respectively, and thus for inhibition of insulin protein translation signaling involving the PI 3-kinase/Akt/mTOR pathway. We further demonstrated the cause-effect relationship between these two events by using inhibitors of JNK and MEK1. Indeed, we found that inhibition of JNK and MEK1 activity prevented the negative effects of glucosamine on insulin protein translation signaling by reverting its adverse effect of insulin biosynthesis in both human pancreatic islets and the RIN pancreatic ß-cell line.

In summary, the present study has confirmed previous findings demonstrating that insulin promotes its own biosynthesis by an autocrine pathway involving the insulin receptor kinase/IRS-1/PI3-kinase/Akt/mTOR protein translation pathway. We show that activation of the HBP exerts an inhibitory effect on insulin biosynthesis and, for the first time, have correlated these changes with activation of JNK and ERK1/2. Our data suggest that the uncoupling of IRS-1 and PI 3-kinase in glucosamine-treated RIN ß-cells may be linked to an increased phosphorylation at Ser³⁰⁷ and Ser⁶¹² of IRS-1 mediated by JNK and ERK1/2, respectively. These changes were associated with a concomitant reduction in phosphorylation of Tyr⁶⁰⁸ and Tyr⁶²⁸ in two YXXM motifs essential for engaging p85 regulatory subunit of PI 3-kinase, resulting in impairment in binding of IRS-1 to p85 subunit, activation of IRS-1-associated PI 3-kinase, and sequential activation of the Akt/mTOR/PHAS-1/p70S6 kinase protein translation initiation pathway (Fig. 11). Obviously, we cannot exclude the possibility that other Ser/Thr kinase may phosphorylate IRS-1 under the conditions used in the present study, leading to impairment in the activation of downstream protein translation pathway. Notwithstanding, the present results suggest that glucose-induced activation of JNK and ERK1/2 might be an important negative regulator for insulin biosynthetic pathway, and thus inhibiting these two kinases might be a plausible pharmacological target to treat pancreatic ß-cells dysfunction associated with type 2 diabetes.

View larger version (37K): [in this window] [in a new window]   FIG. 11. Schematic diagram of the hypothesized effects of high glucose and glucosamine on insulin-mediated protein synthesis in pancreatic ß-cells. Exposure of pancreatic ß-cells to high glucose or glucosamine may induces defects in insulin biosynthesis by affecting the insulin-mediated protein translation signaling through the activation of the HBP. Increased routing of glucose through HBP was associated with impairment in insulin-stimulated IRS-1 phosphorylation at Tyr608 and Tyr628, which are essential for engaging PI 3-kinase. Glucosamine mimicked the stimulatory effects of high glucose on JNK and ERK1/2 activity and IRS-1 phosphorylation at Ser307 and Ser612. These results suggest that activation of the HBP accounts, in part, for glucose-induced phosphorylation at Ser307 and Ser612 of IRS-1 mediated by JNK and ERK1/2, respectively. These changes result in impaired coupling of IRS-1 and PI 3-kinase, impaired activation of PI 3-kinase, and impaired activation of the Akt/mTOR/PHAS-1/p70S6 kinase protein translation initiation pathway.

	Footnotes

 
F.A. and C.D.A. contributed equally to this manuscript.

This work was supported in part by Grant E.1309 from Telethon-Italy (to G.S.), Grant QLG1-CT-1999-00674 from the European Community "EuroDiabetesGene" (to G.S.), and Grant PRIN-COFIN from Ministero dell’Istruzione, dell’Università e della Ricerca (to G.S. and R.L.).

Abbreviations: ECL, Enhanced chemiluminescence; GFAT, glutamine/fructose-6-phosphate amidotransferase; GlcNAc, N-acetylglucosamine; HBP, hexosamine pathway; IRS-1, insulin receptor substrate-1; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; mTOR, mammalian target of rapamycin; PHAS-1, phosphorylated heat- and acid-stable protein-1; PI 3-kinase, phosphatidylinositol 3-kinase; RIN, rat insulinoma; SH2, src homology 2.

Received July 24, 2003.

Accepted for publication February 26, 2004.

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