HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/9/959 03-0725fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by D’ALESSANDRIS, C. Articles by SESTI, G. Search for Related Content PubMed PubMed Citation Articles by D’ALESSANDRIS, C. Articles by SESTI, G.

Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic ß-cells¹

CRISTINA D’ALESSANDRIS^*,, FRANCESCO ANDREOZZI, MASSIMO FEDERICI^*, MARINA CARDELLINI^*, ANTONIO BRUNETTI, MARCO RANALLI, SILVIA DEL GUERRA, DAVIDE LAURO^*, STEFANO DEL PRATO, PIERO MARCHETTI, RENATO LAURO^* and GIORGIO SESTI^,2

^* Laboratory of Molecular Medicine, Department of Internal Medicine, University of Rome-Tor Vergata, Rome, Italy;
Department of Experimental and Clinical Medicine, University of Catanzaro-Magna Græcia, Catanzaro, Italy;
IDI-IRCCS, Biochemistry Laboratory, c/o Department of Experimental Medicine, University of Rome-"Tor Vergata," Rome, Italy, and
Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, Pisa, Italy

²Correspondence: Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro-Magna Græcia, Via Tommaso Campanella, 115; 88100 Catanzaro-Italy. E-mail: sesti{at}unicz.it

SPECIFIC AIMS

Many of the adverse effects of glucose have been attributed to increased routing of glucose through the hexosamine biosynthesis pathway (HBP), which results in increased O-glycosylation of proteins at Ser/Thr residues. Because the insulin signaling is thought to play an important role in maintaining ß-cell function and survival, we inquired whether increased routing of glucose through the HBP affects ß-cell survival by enhancing O-glycosylation of anti-apoptotic insulin signaling proteins.

PRINCIPAL FINDINGS

1. Effects of high glucose and glucosamine on human pancreatic ß-cell survival
To investigate whether increased routing of glucose through the HBP affects islet cells survival, we analyzed pancreatic islets isolated from human donors. Flow cytometry analysis revealed that dissociated human islet cell exposed to high glucose show higher apoptotic events upon serum deprivation compared with islets cultured at normal glucose concentration or high mannitol concentration used as appropriate hyperosmolar control. Inhibition of HBP by the GFAT inhibitor azaserine reversed the proapoptotic effects of high glucose. Consistent with these results, exposure of human islets to glucosamine, a direct precursor of the downstream product of the HBP, resulted in increased apoptosis upon serum deprivation compared with control islets. BADGP has been shown to limit O-glycosylation by preventing addition of N-acetylglucosamine to proteins and has been used to attenuate the activity of HBP. Treatment of human islet with BADGP reverted the proapoptotic effects of high glucose and glucosamine. Immunofluorescence analysis of dissociated human islet cells revealed increased ß-cells apoptosis upon serum deprivation in islets exposed to high glucose or glucosamine compared with control islets (Fig. 1 a, b).

View larger version (35K): [in this window] [in a new window]   Figure 1. Assessment of apoptosis in human islets cultured in high glucose vs. normal glucose. a) Dissociated human islet were cultured in the presence of 5.5 mM glucose, 16.7 mM glucose, 5.5 mM glucose plus 11.2 mM mannitol, 16.7 mM glucose plus 5.8 µM azaserine, 16.7 mM glucose plus BADGP, 7.5 mM glucosamine (GlcNAc), or 7.5 mM GlcNAc plus 0.5 mM BADGP in the presence or absence of 10% FBS. At the end of treatment, cells were fixed and analyzed for apoptosis after propidium iodide staining by flow cytometry. b) TUNEL assay and immunostaining for insulin were performed in dissociated human islets cultured in the presence of 5.5 mM glucose (LG), 16.7 mM glucose (HG), or 7.5 mM GlcNAc. Fluorescence microscopic fields were photographed individually and merged to detect ß-cells death under normal and high glucose conditions. Data represent means ± SD from 3 independent preparations.

2. Effects of high glucose and glucosamine on RIN ß- cells survival
To further investigate molecular mechanisms of the increased susceptibility to apoptosis caused by activation of the HBP, we used the RINr1046-38 ß-cell line. Apoptosis induced by serum deprivation was increased by 2.5-fold in RIN ß-cells incubated with 33 mM glucose compared with control cells incubated with 5.5 mM glucose + 27.5 mM mannitol, whereas inhibition of HBP by azaserine reversed the effect of glucose by 40%. Accordingly, exposure of cells to glucosamine resulted in a dose-dependent increase in apoptosis induced by serum deprivation, which is correlated with a marked increase in caspase-3 activity compared with control ß-cells.

3. Effect of glucosamine on O-glycosylation of the insulin receptor, IRS-1, and IRS-2 in RIN ß-cells
Analysis by YinOYang prediction server allowed the identification of O-GlcNAcylation sites in several molecules involved in insulin signaling, including the insulin receptor, IRS-1, and IRS-2. Immunoblotting analysis of these signaling proteins with anti-O-linked GlcNAc antibody RL2 showed a significant increase in O-glycosylation in RIN ß-cells exposed to glucosamine compared with control ß-cells. Treatment of RIN ß-cells with BADGP reverted the glucosamine-induced increase in O-glycosylation of the insulin receptor, IRS-1, and IRS-2.

4. Effect of glucosamine on insulin receptor, IRS-1, and IRS-2 phosphorylation, and PI 3-kinase activity
Insulin-stimulated tyrosine phosphorylation of the insulin receptor was decreased by 65% in RIN ß-cells exposed to glucosamine compared with control ß-cells. Similarly, the extent of insulin-stimulated tyrosine phosphorylation of IRS-1 or IRS-2 was decreased by 60 and 55%, respectively, in RIN ß-cells exposed to glucosamine compared with control ß-cells. A potential O-glycosylation site in the rat insulin receptor sequence is located at site 978 (orthologous to Tyr-976 in human insulin receptor) in close proximity to the NPXY⁹⁷⁴ (orthologous to Tyr-972 in human insulin receptor) site in the juxtamembrane domain of the ß subunit. Insulin-stimulated Y⁹⁷⁴ phosphorylation of the insulin receptor was decreased by 47% in RIN ß-cells exposed to glucosamine compared with control RIN ß-cells. Treatment of RIN ß-cells with BADGP completely reverted the glucosamine-induced reduction in tyrosine phosphorylation of the insulin receptor, pY⁹⁷⁴, IRS-1, and IRS-2. PI 3-kinase activity associated with either IRS-1 or IRS-2 in response to insulin was decreased by 36 and 39%, respectively, in RIN ß-cells exposed to glucosamine compared with control ß-cells.

5. Effect of glucosamine on Akt phosphorylation/activation and GSK-3 phosphorylation in RIN ß-cells
Insulin-induced Ser473 Akt phosphorylation and in vitro Akt activity were reduced in RIN ß-cells exposed to glucosamine compared with control ß-cells. One mechanism by which Akt promotes cell survival is through the phosphorylation and inactivation of downstream proapoptotic proteins such as GSK-3. Insulin-induced GSK-3/ß phosphorylation was reduced in RIN ß-cells exposed to glucosamine compared with control ß-cells at all times tested. Treatment with BADGP reverted the glucosamine-induced reduction in Akt and GSK-3/ß phosphorylation in response to insulin.

6. Effect of glucosamine on FOXO transcription factors phosphorylation in RIN ß-cells
There is evidence that Akt regulates the activity of FOXO transcription factors comprising FOXO1a, FOXO3a, and FOXO4 (formerly named FKHR, FKHRL1, and AFX). Under conditions of growth deprivation, Akt is inactive; FOXO transcription factors are dephosphorylated and localized in the nucleus, where they activate transcription of death genes. In response to survival factors such as insulin, Akt is activated and FOXO transcription factors are phosphorylated. This leads to their retention in the cytoplasm, where FOXO transcription factors are sequestered by interacting with 14-3-3 docking proteins. To investigate whether glucosamine alters the phosphorylation of FOXO transcription factors as a consequence of impaired activation of the PI 3-kinase/Akt pathway, FOXO1a phosphorylation at Thr24 was measured in glucosamine-treated RIN ß-cells. Immunoblotting analyses revealed that FOXO1a phosphorylation at Thr24 was significantly decreased in ß-cells exposed to glucosamine upon stimulation with insulin compared with control RIN ß-cells. Treatment with BADGP reverted the inhibitory effects of glucosamine.

7. Effect of glucosamine on Bim, Bad, Bcl-X_L, and Bcl-2 expression in RIN ß-cells
A death gene whose expression is negatively regulated by FOXO transcription factors is Bim, a member of the Bcl-2 family of proteins known to play a central role in the regulation of cell death. Like other proapoptotic BH3-only members of the Bcl-2 family, Bim exerts its proapoptotic effect by sequestering anti-apoptotic proteins such as Bcl-2 and Bcl-X_L in nonfunctional heterodimers. Thus, unbalance in the expression of the proapoptotic Bim vs. the anti-apoptotic Bcl-2 and Bcl-X_L proteins may promote apoptosis. The inhibitory effect of insulin on Bim expression was decreased by 40% in RIN ß-cells exposed to glucosamine compared with control ß-cells. By contrast, expression of Bad, Bcl-X_L, or Bcl-2 did not differ between RIN ß-cells exposed to glucosamine and control RIN ß-cells.

CONCLUSIONS AND SIGNIFICANCE

We found that exposure of human islets to high glucose resulted in increased apoptosis of ß-cells upon serum deprivation that was significantly reversed by azaserine and BADGP, suggesting that elevated glucose concentration impairs ß-cell survival at least in part via the excessive flux of glucose through HBP. Exposure of human islets to glucosamine resulted in an increased apoptosis upon serum deprivation compared with control islets, which was reverted by BADGP. Results in human pancreatic islets were reproduced in RIN ß-cells. Exposure of RIN ß-cells to glucosamine resulted in a significant inhibition in the early steps of insulin signaling, including tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2 as well as activation of PI 3-kinase associated with IRS-1 and IRS-2. These changes also resulted in a significant impairment in the sequential activation of Akt, which is known to play a central role in the regulation of survival of many cell models. We observed a marked increase in O-glycosylation of the insulin receptor, IRS-1, and IRS-2 upon exposure of RIN ß-cells to glucosamine. Inhibition of O-glycosylation by treatment with BADGP reverted the negative effect of glucosamine on insulin-induced tyrosine phosphorylation of the insulin receptor, IRS-1, and IRS-2. A potential O-glycosylation site in the rat insulin receptor sequence is located at serine 978 in close proximity to the NPXY⁹⁷⁴ site in the juxtamembrane domain of the ß subunit that is involved in interaction with the PTB domain of the IRS proteins. We noted that glucosamine induced a marked impairment in insulin-stimulated phosphorylation at tyrosine 974 that was reverted by treatment of RIN ß-cells with BADGP.

FOXO1a, a member of the forkhead family of transcription factors, is a critical regulator of apoptosis by activating transcriptions of death genes. Under conditions of deprivation of insulin or other stimulators of the PI 3-kinase/Akt pathway, FOXO1a is unphosphorylated at the Akt sites, and accumulates in the nucleus, where it can activate genes regulating apoptosis including Bim. We found that in RIN ß-cells exposed to glucosamine, basal and insulin-stimulated FOXO1a phosphorylation on Thr24 was reduced compared with control RIN ß-cells, resulting in impaired capability of insulin to down-regulate expression of Bim but not that of anti-apoptotic Bcl-X_L and Bcl-2 (Fig. 2 ). Our findings of unbalanced expression of the proapoptotic member Bim vs. the anti-apoptotic members Bcl-X_L and Bcl-2 of the Bcl family indicate that activation of the HBP might ultimately favor ß-cell apoptosis by modulating specific expression of Bim via the PI 3-kinase/Akt/Foxo pathway. Recent evidence indicates that activation of GSK-3 contributes to apoptotic signaling by a mechanism that remains to be elucidated. It is conceivable that impairment in the PI 3-kinase/Akt pathway may result in increased GSK-3 activity. Accordingly, we observed a reduction in both Ser 21 and Ser 9 GSK-3/ß phosphorylation in RIN ß-cells exposed to glucosamine.

View larger version (77K): [in this window] [in a new window]   Figure 2. Schematic diagram of the hypothesized effects of high glucose and glucosamine on insulin anti-apoptotic pathway in pancreatic ß-cell. Exposure of pancreatic ß-cells to high glucose or glucosamine may increase susceptibility to apoptosis of human and rat ß-cell through the activation of the HBP. Increased routing of glucose through this metabolic pathway causes impaired activation of the IR/IRSs/PI3-kinase/Akt survival pathway by induction of O-glycosylation of signaling molecules.

In conclusion, the results demonstrate that exposure of pancreatic ß-cells to glucosamine increases susceptibility to apoptosis induced by growth factor deprivation by impairing the IR/IRS/PI3-kinase/Akt insulin survival pathway. These data indicate an important role for insulin signaling cascade in ß-cell survival and suggest that defects in the early steps of insulin signaling may play a role in ß-cell death caused by hyperglycemia.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0725fje;

This article has been cited by other articles:

				L. Shu, N. S. Sauter, F. T. Schulthess, A. V. Matveyenko, J. Oberholzer, and K. Maedler Transcription Factor 7-Like 2 Regulates {beta}-Cell Survival and Function in Human Pancreatic Islets Diabetes, March 1, 2008; 57(3): 645 - 653. [Abstract] [Full Text] [PDF]

				B.-C. Jang, S.-H. Sung, J.-G. Park, J.-W. Park, J. H. Bae, D. H. Shin, G.-Y. Park, S.-B. Han, and S.-I. Suh Glucosamine Hydrochloride Specifically Inhibits COX-2 by Preventing COX-2 N-Glycosylation and by Increasing COX-2 Protein Turnover in a Proteasome-dependent Manner J. Biol. Chem., September 21, 2007; 282(38): 27622 - 27632. [Abstract] [Full Text] [PDF]

				N. Fulop, R. B. Marchase, and J. C. Chatham Role of protein O-linked N-acetyl-glucosamine in mediating cell function and survival in the cardiovascular system Cardiovasc Res, January 15, 2007; 73(2): 288 - 297. [Abstract] [Full Text] [PDF]

				R. C. Cooksey, S. Pusuluri, M. Hazel, and D. A. McClain Hexosamines regulate sensitivity of glucose-stimulated insulin secretion in {beta}-cells Am J Physiol Endocrinol Metab, February 1, 2006; 290(2): E334 - E340. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/9/959 03-0725fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by D’ALESSANDRIS, C. Articles by SESTI, G. Search for Related Content PubMed PubMed Citation Articles by D’ALESSANDRIS, C. Articles by SESTI, G.