Glycobiology Advance Access originally published online on November 9, 2006
Glycobiology 2007 17(2):127-140; doi:10.1093/glycob/cwl067
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Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto–Kakizaki rats
2 Department of Anatomy
3 Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
4 Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205
5 Medicinal Research Laboratories, Taisho Pharmaceutical Co., LTD, Saitama 331-9530, Japan
7 Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 North 15th Street, M.S. 497, PA 19102
1 To whom correspondence should be addressed; Tel: +81-422-47-5511; Fax: +81-422-44-0866; e-mail: yakimoto{at}kyorin-u.ac.jp
Received on April 26, 2006; revised on November 2, 2006; accepted on November 3, 2006
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Abstract |
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Many nuclear and cytoplasmic proteins are O-glycosylated on serine or threonine residues with the monosaccharide ß-N-acetylglucosamine, which is then termed O-linked N-acetylglucosamine (O-GlcNAc). It has been shown that abnormal O-GlcNAc modification (O-GlcNAcylation) of proteins is one of the causes of insulin resistance and diabetic complications. In this study, in order to examine the relationship between O-GlcNAcylation of proteins and glucose-stimulated insulin secretion in noninsulin-dependent type (type 2) diabetes, we investigated the level of O-GlcNAcylation of proteins, especially that of PDX-1, and the expression of O-GlcNAc transferase in Goto–Kakizaki (GK) rats, which are an animal model of type-2 diabetes. By immunoblot and immunohistochemical analyses, the expression of O-GlcNAc transferase protein and O-GlcNAc-modified proteins in whole pancreas and islets of Langerhans of 15-week-old diabetic GK rats and nondiabetic Wistar rats was examined. The expression of O-GlcNAc transferase at the protein level and O-GlcNAc transferase activity were increased significantly in the diabetic pancreas and islets. The diabetic pancreas and islets also showed an increase in total cellular O-GlcNAc-modified proteins. O-GlcNAcylation of PDX-1 was also increased. In the diabetic GK rats, significant increases in the immunoreactivities of both O-GlcNAc and O-GlcNAc transferase were observed. PUGNAc, an inhibitor of O-GlcNAcase, induced an elevation of O-GlcNAc level and a decrease of glucose-stimulated insulin secretion in isolated islets. These results indicate that elevation of the O-GlcNAcylation of proteins leads to deterioration of insulin secretion in the pancreas of diabetic GK rats, further providing evidence for the role of O-GlcNAc in the insulin secretion.
Key words: diabetic Goto–Kakizaki rat / hexosamine biosynthetic pathway / pancreas / PDX-1 / O-GlcNAc / diabetes
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Introduction |
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ß-O-linked N-acetylglucosamine (O-GlcNAc) is one of the post-translational modifications of nuclear and cytosolic proteins. O-GlcNAc transferase is a nucleocytoplasmic enzyme that catalyzes the addition of GlcNAc moieties to Ser/Thr residues of proteins (Haltiwanger et al. 1992
). The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability (Shafi et al. 2000). It has been shown that transgenic over-expression of O-GlcNAc transferase in muscle and fat produces insulin resistance and hyperleptinemia (MaClain et al. 2002).
O-GlcNAcylation often occurs at the same sites as phosphorylation or at sites adjacent to those of phosphorylation. A reciprocal relation between phosphorylation and O-GlcNAcylation has been observed for some proteins. O-GlcNAc transferase is abundant in the cell nucleus and regulates gene expression there (Comer and Hart 1999; Yang et al. 2002). In view of the interplay between O-GlcNAc and phosphate in signal transduction, we considered that a high level of O-GlcNAcylation would antagonize phosphorylation-dependent signal transduction and thus cause diabetic complications.
The end products of the hexosamine biosynthetic pathway, UDP-ß-N-acetylglucosamine (UDP-GlcNAc) and UDP-ß-N-acetylgalactosamine (UDP-GalNAc), are substrates for the glycosylation of proteins and lipids. In addition to being used in the process of classical complex glycosylation, UDP-GlcNAc is used as the donor sugar for a carbohydrate modification of proteins, in which single GlcNAc moieties are dynamically attached to serine and threonine residues of cytosolic and nuclear proteins (after which they are termed O-GlcNAc) (Torres and Hart 1984; Haltiwanger et al. 1990; Hart 1997; Comer and Hart 2000; Wells et al. 2001; Slawson et al. 2006). Activation of the hexosamine pathway by hyperglycemia may result in changes in gene expression and protein function, which may lead to insulin resistance and diabetic complications (Marshall et al. 1991; McClain and Crook 1996; Brownlee 2001; McClain 2002; Vosseller et al. 2002).
In previous studies, we showed that (1) O-GlcNAc transferase was highly expressed in the pancreas, especially in the islets (Akimoto et al. 1999); (2) expression of O-GlcNAc transferase and O-GlcNAcylation were elevated in the pancreas of rats with streptozotocin (STZ)-induced diabetes (type-1 diabetes) (Akimoto et al. 2000); and (3) PDX-1, the pancreatic/duodenal homeobox-1 protein, which is a homeodomain transcription factor that plays an important role in insulin and somatostatin transcription and in pancreatic development (Jonsson et al. 1994; Miller et al. 1994), was modified by O-GlcNAc; and its hypermodification correlated with increases in DNA-binding activity of PDX-1 and insulin secretion by MIN6 ß cells (Gao et al. 2003).
The spontaneously diabetic GK rat is a nonobese model of noninsulin-dependent diabetes mellitus that was developed by the selective breeding of glucose-intolerant Wistar rats (Galli et al. 1996; Goto et al. 1988; Zong-Chao et al. 1998). In the GK rat, we observed that the levels of O-GlcNAcylated proteins were elevated in various tissues known to be affected by diabetic complications, for example, sciatic nerve, kidney, liver, and cornea (Akimoto et al. 2003, 2005). In the present study, to further examine whether levels of O-GlcNAc and O-GlcNAc transferase are elevated in the pancreas of type-2 diabetes, as they are in STZ-induced type-1 diabetes, we investigated the level of O-GlcNAcylated proteins and the expression of O-GlcNAc transferase in the pancreas of GK rats. Also, we looked for the changes in the localization of O-GlcNAcylated proteins in the islet ß and 
cells by immuno-electron microscopy. Furthermore, to study the role of O-GlcNAc in glucose-stimulated insulin secretion, we examined PUGNAc effects on glucose-stimulated insulin secretion.
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Results |
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Systemic parameters
The body weight was significantly (P < 0.01) lower in the GK rats than in the Wistar ones: the values (mean ± SEM) in Wistar and GK rats were 522.7 ± 3.1 and 366.3 ± 7.8 g, respectively (Figure 1A). Serum glucose levels, which were measured after an overnight fast, were significantly (P < 0.01) higher in GK rats than in Wistar rats, being 158.0 ± 12.0 and 375.9 ± 11.6 mg/dL in Wistar and GK rats, respectively (Figure 1B). Serum insulin levels were significantly (P < 0.01) higher in GK rats than in Wistar rats, being 1.622 ± 0.067 and 5.475 ± 0.489 ng/mL in Wistar and GK rats, respectively (Figure 1C).
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) and diabetic GK (
) rats (both 15 weeks old). Data represent the mean ± SEM (n = 6); *P < 0.01.Immunoblot analysis of O-GlcNAc transferase in the normal and diabetic pancreas by use of anti-O-GlcNAc transferase antibody (AL-28)
O-GlcNAc transferase in the rat pancreas consists of one 110-kDa subunit and one 78-kDa subunit (Haltiwanger et al. 1992
). To examine the protein level of O-GlcNAc transferase in the pancreas, we immunoblotted proteins with anti-O-GlcNAc transferase antibody (AL-28). The AL-28 antibody recognized both the 110-kDa subunit, which contains the active site of the enzyme, and the 78-kDa subunit (Figure 2A). The density of protein bands obtained after electrophoresis of homogenates of normal Wistar and diabetic GK rat pancreas was quantified by scanning densitometry (Figure 2B). The amounts of both 110-kDa subunit (p110) and 78-kDa subunit (p78) were significantly (P < 0.05) increased in the diabetic pancreas, because the same amount of total protein was loaded into each lane of the gel (Figure 2). The ß-actin protein level did not change significantly in the GK rats (Figure 2).
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Assay of O-GlcNAc transferase activity in pancreatic extracts from Wistar and GK rats
To examine the effect of diabetes on O-GlcNAc transferase, the activity of the enzyme in the pancreas was assayed. The 30% ammonium sulfate cytosolic pellets were assayed for enzymatic activity. The O-GlcNAc transferase activity of the diabetic GK rat pancreas (306.3 ± 19.4 dpm/µg protein) was significantly higher (P < 0.01) than that of the normal pancreas (164.1 ± 28.1 dpm/µg protein) (Figure 3).
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Immunohistochemical localization of O-GlcNAc and O-GlcNAc transferase in diabetic and nondiabetic pancreas
The distribution of O-GlcNAc, O-GlcNAc transferase, and insulin was examined by laser confocal scanning microscopy (Figure 4A). The immunofluorescence intensity of islets of Langerhans and exocrine cells was quantified in the control and diabetic GK pancreas (Figure 4B–D).
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The multiple staining method showed that both islet cells and exocrine cells of the normal pancreas were enriched in O-GlcNAc and O-GlcNAc transferase (Figure 4A). In the normal islet cells, intense immunoreactivities of O-GlcNAc and O-GlcNAc transferase were observed in the nucleus, whereas the cytoplasm was diffusely and only weakly immunoreactive (Figure 4Aa and c). In the normal exocrine cells, intense immunoreactivities of O-GlcNAc and O-GlcNAc transferase were observed in the nuclei, in the zymogen granule region, and along the contour of each cell (Figure 4A a and c). In both the islets and exocrine cells of the diabetic pancreas, the immunoreactivities of O-GlcNAc and O-GlcNAc transferase were increased (Figure 4A–C). But the insulin immunoreactivity showed little change (Figure 4D), whereas the serum insulin levels were higher in the diabetic GK rats (Figure 1C). The reason for the discrepancy between serum insulin level and insulin-immunohistochemical results is unclear and to be clarified. In the control experiment in which the primary antibody was omitted or replaced with normal mouse IgM or rabbit IgG, no positive staining was observed (data not shown).
Immuno-electron microscopic localization of O-GlcNAc in the islets of diabetic and nondiabetic pancreas
In the islets of Langerhans of the diabetic pancreas, a decreased number of ß cells were observed (data not shown), as previously reported by Movassat et al. (1997). However, no obvious difference in the granule morphology of the and ß cells was seen between GK rats and Wistar rat, as previously reported by Leckström et al. (1996) (Figure 5).
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The precise localization of O-GlcNAc in the islets was examined by immuno-electron microscopy, using colloidal gold-labeled antibodies (Figure 5). The O-GlcNAc immunoreactivity was observed mainly in the nucleus and also in the cytoplasm around the secretory granules in both
cells (Figure 5A and B) and ß cells (Figure 5C and D). Compared with Wistar rats, GK rats showed a significant increase in the density of colloidal gold in both cell types (Figure 5E).
Immunoblot analysis of O-GlcNAcylated proteins in the pancreas
To examine the O-GlcNAcylation level in the pancreas, we immunoblotted proteins with monoclonal anti-O-GlcNAc antibody (CTD110.6). Proteins extracted from the diabetic pancreas of GK rats had a higher O-GlcNAc content than those from the normal pancreas of Wistar rats (Figure 6). At least seven major proteins [molecular weights (Mr) of 61 000, 52 000, 44 000, 40 000, 32 000, 28 000, and 25 000] from the diabetic pancreas showed a significant (P < 0.05) increase in the amount of O-GlcNAcylation [The increases were (in folds) 1.34, 1.46, 1.61, 1.50, 1.43, 1.39, and 1.84, respectively, with an average increase of 1.5-fold.] Although the intensity of two-protein bands indicated as 1 and 2 in Figure 6B had a tendency to increase, no statistically significant difference was found between normal and diabetic pancreas.
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Immunoblot analysis of O-GlcNAc transferase and O-GlcNAcylated proteins in the islets of normal and diabetic pancreas
To further examine whether the differences in the O-GlcNAcylation level and O-GlcNAc transferase expression in the pancreas of Wistar and GK rats were a reflection of the differences in islets or exocrine cells, we isolated pancreatic islets from both strains and examined the cells.
The protein level of O-GlcNAc transferase was examined by immunoblotting with anti-O-GlcNAc transferase antibody (AL-28). The AL-28 antibody recognized one major band having a molecular weight of 110 kDa (Figure 7A). The density of the band was significantly (P < 0.01) increased in the diabetic islets (Figure 7C), whereas the ß-actin level did not change (Figure 7B).
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The O-GlcNAcylation levels in the islets were analyzed by immunoblotting with anti-O-GlcNAc antibody (CTD110.6). Proteins extracted from the islets of diabetic rats had a higher O-GlcNAc content than islets from normal rats (Figure 7E and F), whereas total protein content was the same (Figure 7D). At least seven major proteins (Mr of 270 000, 253 000, 173 000, 135 000, 84 000, 55 000, and 39 000) from the diabetic islets showed a significant increase in the amount of O-GlcNAc, whereas two proteins (Mr of 120 000, 52 000] showed a significant decrease [The increases were (in folds) 2.01, 1.24, 1.31, 1.13, 1.91, 1.08, and 1.12, respectively; and the decreases, 0.84 and 0.74, respectively, with an average increase of 1.3-fold.] Immunoblots of O-GlcNAcylated-protein patterns were different between whole pancreas and isolated islets (Figures 6A and 7E).
As the islets constitute only 1–3% of the total pancreatic tissue, these data show that the higher O-GlcNAc level and O-GlcNAc transferase expression in the GK pancreas as detected by immunoblot analysis were due to a higher expression in both islets and exocrine cells.
Immunohistochemical and immunoblot analysis of PDX-1 and its O-GlcNAcylation
Our previous study showed that the transcription factor PDX-1 is modified by O-GlcNAc and that its modification is correlated with its DNA-binding activity and insulin secretion in MIN 6 ß cells (Gao et al. 2003). On the basis of the results of the immunohistochemical study showing that the O-GlcNAcylation level was elevated, especially in the nuclei of ß cells, in the diabetic GK rats, we studied immunohistochemically the localization of PDX-1 and biochemically the O-GlcNAcylation of PDX-1 and the PDX-1 protein level in the pancreas of Wistar and GK rats. Immunohistochemical examination of the normal pancreas revealed PDX-1 reactivity to be localized mainly in the nucleus of the ß cells, and its localization and immunoreactivity did not change in the diabetic pancreas (Figure 8).
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The protein level of PDX-1 was examined in the isolated islets from both strains by immunoblotting. The PDX-1 level was significantly decreased (63.1% of control) in the diabetic islets, whereas the ß-actin level did not change (Figure 9A and B). This decrease could be ascribed to the decrease in the number of ß cells in the GK rat islets.
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After the immunoprecipitation of PDX-1, isolated proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophorosis (SDS–PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and probed with O-GlcNAc antibody (CTD110.6, Figure 9). Then the membrane was stripped and reprobed with PDX-1 antibody. The PDX-1 protein level was decreased in GK rats compared with that in the Wistar rats (Figure 9C). The ratio between the intensity of CTD110.6 bands and that of the corresponding PDX-1 bands was significantly higher (P < 0.05) for GK rats than for Wistar rats (Figure 9D). These results indicate that the PDX-1 protein level had dropped, but that the O-GlcNAcylation level of the remaining PDX-1 had increased, in the GK rats.
Induction of O-GlcNAcylation decreases glucose-induced insulin secretion
To examine the involvement of O-GlcNAc in glucose-stimulated insulin secretion, we enhanced O-GlcNAcylation by using PUGNAc, an O-GlcNAcase inhibitor. Previous study by Kaneto et al. (2001), who incubated islets for 24 h with 20 or 50 µM PUGNAc, reported that induction of O-GlcNAcylation does not suppress ß cell function. As the half-life of PUGNAc is 12 h and 100 µM PUGNAc is not toxic to ß cells (Gao et al. 2000), in this study, we incubated islets for 12 h with 100 µM PUGNAc, then incubated for 6 h with fresh medium containing 100 µM PUGNAc, and further incubated with 100 µM PUGNAc during the glucose-stimulation experiments. PUGNAc treatment increased O-GlcNAc levels significantly (Figure 10B), whereas PUGNAc caused little change in total proteins and ß-actin (Figure 10A and C). As shown in Figure 10D, glucose-stimulated insulin secretion was decreased by PUGNAc treatment. These results suggest that O-GlcNAc is likely to be involved in hexosamine biosynthetic pathway-mediated dysfunction of ß cells.
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O-GlcNAc transferase RNA interference reduces intracellular O-GlcNAc and glucose-stimulated insulin secretion in MIN6 cells
To further clarify the role of O-GlcNAc in glucose-stimulated insulin secretion, MIN6 cells were transfected with O-GlcNAc transferase small interfering RNA (siRNA) in an effort to downregulate intracellular O-GlcNAc levels. O-GlcNAc transferase siRNA decreased the expression of O-GlcNAc transferase, whereas it scarcely affected the expression of ß-actin (Figure 11A and B). Intracellular O-GlcNAc levels were significantly reduced by RNA interference (RNAi), with a concomitant reduction in glucose-stimulated insulin secretion (Figure 11C and D).
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Discussion |
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The present study showed that expression of O-GlcNAc transferase and O-GlcNAcylation of proteins were increased in the pancreas and islets of GK rats with spontaneous noninsulin-dependent diabetes, a model of type-2 diabetes. O-GlcNAc transferase is highly expressed in the pancreas and brain, relative to its expression in other tissues (Kreppel et al. 1997
; Lubas et al. 1997; Nolte and Müller 2002). Northern blot analysis of the gene expression of O-GlcNAc transferase in various human tissues revealed it to be very strong in the pancreas, approximately 12 times higher than that in other tissues (Lubas et al. 1997). This transferase is found not only in the endocrine cells, that is, the islets of Langerhans, where its expression is especially high (Hanover et al. 1999), but also in the exocrine cells of the pancreas (Akimoto et al. 1999). In pancreatic ß cells, the O-GlcNAcylation level is very high, suggesting that O-GlcNAc transferase may play a role in insulin secretion or regulation of the glucose-sensing mechanism in the pancreas (Hanover et al. 1999). A previous study of ours showed that the expression of O-GlcNAc transferase and O-GlcNAcylation of proteins are enhanced in the pancreas of rats with STZ-induced diabetes, a model of type-1 diabetes (Akimoto et al. 2000). Increased flow through the hexosamine biosynthetic pathway in response to hyperglycemia may be a major factor in the increased O-GlcNAcylation.
Our immunohistochemical study revealed that both O-GlcNAc and O-GlcNAc transferase were localized not only in endocrine cells of the Langerhans islets but also in exocrine cells (Figure 4), as previously reported (Akimoto et al. 1999; Vosseller et al. 2002). Furthermore, by immuno-electron microscopy, O-GlcNAc was detectable not only in the nucleus but also in the cytoplasm around the secretory granules of islet cells (Figure 5). These immunoreactivities increased in the diabetic islets (Figures 4 and 5). Since pancreatic islets consist of only 1–3% of total pancreatic tissue and, in adult GK rats, ß cell mass is reduced 50% (Movassat et al. 1997), the differences in O-GlcNAcylation between whole pancreas tissue of the Wistar and GK rats (Figure 6) could be due mainly to the differences in the exocrine cell proteins, which represented most of the proteins in the extracts. The higher O-GlcNAc level in the GK rat pancreas as detected by immunoblot analysis (Figures 6 and 7) may be due to a higher expression in not only the islets but also the exocrine cells.
In the pancreas of GK rats, the number of ß cells decreased, as already reported by Movassat et al. (1997). Increases in the levels of both O-GlcNAc transferase protein and O-GlcNAcylation in the ß cells may be involved in the maintenance of insulin production by the remaining ß cells in the diabetic pancreas. Although in the present study it was not established whether increased O-GlcNAcylation of proteins accounts for ß cell insensitivity to glucose, we may propose that, in the diabetic pancreas, changes in the expression level of O-GlcNAc transferase may cause aberrant O-GlcNAcylation of proteins involved in the maintenance of the glucose concentration and in the release of granules.
The ß cells of GK rats are known to be insensitive to glucose (Portha et al. 2001). Also, O-GlcNAcylation plays a role in insulin secretion by ß cells (Liu et al. 2000, 2002; Zraika et al. 2002). A previous study showed that O-GlcNAcylation of PDX-1 protein is enhanced in MIN6 ß cells when the cells are cultured in high-glucose medium (Gao et al. 2003). The present results showed that the PDX-1 level was significantly reduced in the diabetic GK pancreas but that the remaining PDX-1 was more extensively O-GlcNAc-modified (Figure 9). The lower level of PDX-1 may be due to the lower number of ß cells in the GK rat. The results of our present animal study are very consistent with those of in vitro study just mentioned. The O-GlcNAcylation of PDX-1 protein is positively correlated with an increase in PDX-1 DNA-binding activity (Gao et al. 2003). Such findings suggest that in the GK rat pancreas, the increase in the O-GlcNAcylation of PDX-1 might enhance its activity and play a role in insulin secretion by ß cells. The elevation of PDX-1 DNA-binding activity caused by the increase in O-GlcNAcylation of PDX-1 may be a potential mechanism to maintain insulin production by the remaining ß cells. PDX-1 is also regulated by phosphorylation (Rafiq et al. 2000). The relation between O-GlcNAcylation and phosphorylation of PDX-1 remains to be elucidated.
It has been suggested that increased O-GlcNAcylation could eventually cause destruction of ß cells (Liu et al. 2000). STZ is an alkylating diabetic agent and is thought to induce ß cell death by causing DNA damage, which results in poly-(adenosine diphosphate-ribose) synthase activation followed by nicotinamide adenine dinucleotide depletion (Yamamoto et al. 1981; Elsner et al. 2000). Streptozocin is also a glucosamine analog and a weak inhibitor of O-GlcNAcase (Hanover et al. 1999; Roos et al. 1998). For example, streptozocin raises the O-GlcNAc level in ß cells (Konrad, Janowski, et al. 2000; Konrad et al. 2001; Liu et al. 2000). However, a study using PUGNAc, which is a stronger inhibitor of O-GlcNAcase (Haltiwanger et al. 1998), showed that elevation of O-GlcNAcylation alone does not induce ß cell death (Gao et al. 2000; Okuyama and Yachi 2001). It was earlier shown that activation of the hexosamine biosynthetic pathway leads to ß cell death through the induction of oxidative stress (Kaneto et al. 2001) and that oxidative stress is a mediator of glucose toxicity in ß cells (Wu et al. 2004). Oxidative stress itself increases O-GlcNAcylation of proteins (Zachara et al. 2004). On the other hand, alloxan, which is an uracil analog and induces ß cell death, was shown to be an inhibitor of O-GlcNAc transferase (Konrad et al. 2002). Our study showed that more O-GlcNAc-modified proteins were present in the diabetic GK rat than in control Wistar rat. A decrease in the number of ß cells was also observed in the GK rat. Although it is not certain that elevation of O-GlcNAc is a cause of the decrease in the number of ß cells, there is a possibility that the combination of oxidative stress (Kaneto et al. 2001; Wu et al. 2004), DNA damage (Yamamoto et al. 1981; Elsner et al. 2000), and aberrant O-GlcNAcylation (Liu et al. 2000) may work synergistically to lower their number.
A role for O-GlcNAc in modulating insulin secretion by ß cells is supported by mice overexpressing GFAT or O-GlcNAc transferase (Hebert et al. 1996; McClain et al. 2002). These mice exhibit an increase in O-GlcNAc-modified proteins and secrete more insulin than wild-type mice. So we expected that PUGNAc induced the elevation of O-GlcNAcylation, which would concomitantly increase glucose-stimulated insulin secretion. However, as shown in Figure 10, PUGNAc increased O-GlcNAcylation but actually decreased glucose-stimulated insulin secretion. This result is consistent with those of Kaneto et al. (2001). These workers showed that overexpression of GFAT or treatment with glucosamine induces the elevation of O-GlcNAcylation but decreased glucose-stimulated insulin secretion. Our results, obtained by using RNAi to reduce O-GlcNAc transferase levels, also suggest that O-GlcNAcylation of proteins has an important role in the glucose-stimulated insulin secretion (Figure 11). Although the data in Figure 11 are apparently contradictory to those in Figure 10, we hypothesize that the dynamic turnover of O-GlcNAc in some proteins may be necessary for glucose-stimulated insulin secretion. These effects could occur at several steps in the insulin biosynthetic pathway, including transcription, translation, and the secretory processes. The clarification of the role of O-GlcNAcylated proteins in the insulin release may provide the clues as to the cause of insulin secretion dysfunction in type-2 diabetes.
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Materials and methods |
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Animals and tissues
All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of Kyorin University School of Medicine. Pancreatic tissues from 15-week-old male (n = 6) GK rats and Wistar rats (as controls), both obtained from CLEA (Tokyo, Japan), were used in the present study.
Antibodies
Rabbit polyclonal anti-O-GlcNAc transferase antibodies (AL-25 and -28, purified IgG) and mouse monoclonal anti-O-GlcNAc antibodies (RL2 and CTD110.6) were used. The generation of AL-25, -28, and CTD110.6 was previously described (Kreppel et al. 1997; Comer et al. 2001; Iyer et al. 2003). RL2 was obtained from Affinity BioReagents (Golden, CO). RL2 and CTD110.6 specifically recognize O-GlcNAc in the ß-O-glycosidic linkage to either serine or threonine (Snow et al. 1987; Comer et al. 2001). Mouse monoclonal anti-ß-actin antibody and mouse monoclonal anti-insulin antibody were purchased from Sigma (St Louis, MO). Guinea pig polyclonal anti-insulin antibody was from Linco Research (St. Charles, MO). Rabbit polyclonal anti-PDX-1 antibody was obtained from Transgenic (Kumamoto, Japan). Horseradish peroxidase (HRP), Cy2- and Cy3-conjugated donkey antirabbit IgG antibodies, HRP- and Cy3-conjugated goat antimouse IgM antibodies, Cy2-conjugated goat antimouse IgG antibody, Cy5- conjugated goat antiguinea pig IgG antibody, normal rabbit IgG, and normal mouse IgM were from Jackson Immunoresearch (West Grove, PA).
Immunoblot analysis of O-GlcNAc transferase and O-GlcNAc-modified proteins
Crude protein extracts were prepared from pancreas dissected from either normal Wistar rats or diabetic GK rats (male, 15 weeks old). The tissue was homogenized in a homogenization buffer [20 mM Tris–HCl (pH 7.4), containing 5 mM EDTA, 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1 mM DTT, 2 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktails 1 and 2 (1:1000 dilution), and 0.1 mM O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenyl-carbamate (PUGNAc, Carbogen, Aarau, Switzerland), which is an inhibitor of the endogenous O-GlcNAc-N-acetylglucosaminidase (O-GlcNAcase), the enzyme responsible for the removal of O-GlcNAc from proteins (Gao et al. 2001)]. Equal amounts of proteins from each sample were separated by 7.5% SDS–PAGE and transferred to PVDF membranes. Purified rabbit polyclonal IgG (AL-28, 1:5000) or mouse monoclonal antibody (CTD110.6, 1:3000) against O-GlcNAc was used as a primary antibody, and antirabbit or antimouse IgG or IgM coupled to HRP, as the secondary antibody (1:20 000 dilution). The HRP activity was detected by using enhanced chemiluminescence (ECL), as described by the manufacturer (Amersham Biosciences, Buckinghamshire, UK), following exposure to CL-X Posure Film (Pierce, Rockford, IL) for 1–15 min. The intensity of protein bands of normal and diabetic rat tissues was quantified by scanning densitometry. Blots were stripped and then reprobed with anti-ß-actin antibody to normalize the data.
O-GlcNAc transferase activity assay
Pancreas tissues of Wistar rats and GK rats were homogenized in buffer [10 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM EDTA, and 1 mM PMSF] by use of a Dounce homogenizer and then sonicated. The insoluble material was pelleted by centrifugation at 27 500g for 30 min and discarded. The supernatant was made 30% saturated with ammonium sulfate. The precipitate was collected by centrifugation at 12 000g for 20 min. The supernatants were discarded, and the pellets were resuspended in buffer (20 mM Tris–HCl, pH 7.8, containing 20% glycerol). The insoluble material was removed by centrifugation at 12 000g for 15 min, and the supernatant was used as the source of the enzyme.
O-GlcNAc transferase assays were done as described previously (Akimoto et al. 2000). The reaction mixture for the standard assay contained 50 mM sodium cacodylate (pH 6.0), 150 µg of casein kinase II acceptor peptide, 2.5 mM 5'-adenosine monophosphate, and 18.5 kBq of UDP-[6-3H]GlcNAc. The reaction was started by the addition of enzyme and continued for 30 min at 20 °C. After the reaction had been stopped by the addition of 50 mM formic acid, the mixture was loaded onto a 0.5-mL SP-Sephadex (SP-C25-120, Sigma) column equilibrated with the same buffer. The column was then washed with 50 mM formic acid, and the peptides were eluted with 0.5 M NaCl. Incorporation of [3H]GlcNAc into the peptide was quantified by liquid scintillation spectrophotometry.
Isolation of islets from Wistar and GK rat pancreas
Pancreatic islets were isolated from male Wistar or GK rats by collagenase digestion, as described previously with some modification (Nagamatsu et al. 1999). The common bile duct of the pancreas was cannulated and the pancreas was inflated with 10 mL collagenase P (1 mg/mL)-Hank's balanced salt solution (HBSS). The distended pancreas was excised, chopped, and digested with collagenase P-HBSS at 37 °C for 30 min using a shaker. Digested tissue was rinsed three times with HBSS. Islets were purified on a Ficoll discontinuous gradient centrifugation. Islets were drawn with mouth pipette to try to select the same size of islets from both GK and Wistar rats. To mitigate a loss of O-GlcNAc from proteins, all isolation buffers were supplemented to contain 11 mM glucose and 1 mM glutamine according to Konrad, Liu, et al. (2000).
Incubation of islets for insulin secretion
Freshly isolated islets from Wistar or GK rats were incubated in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and 2.2 mM glucose with or without 100 µM PUGNAc for 12 h, and then incubated for 6 h with the same fresh medium. For each experiment, 30 islets were placed into each tube. The islets were washed twice with RPMI 1640 containing 0 mM glucose and 10% FBS with or without 100 µM PUGNAc, incubated in this medium for 1 h, and then aspirated. RPMI 1640 containing 22 mM glucose and 10% FBS with or without 100 µM PUGNAc was added to each tube and the islets were incubated with this medium for 1 h under an atmosphere of 95% O2 and 5% CO2 at 37 °C. The media were collected and aliquots of the medium were analyzed for immunoreactive insulin (IRI) by enzyme-linked immunosorbent assay.
Immunohistochemical localization of O-GlcNAc, O-GlcNAc transferase, PDX-1, glucagon, and insulin
Tissues from diabetic and nondiabetic rats were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at 4 °C. The specimens were then immersed and frozen in optimal cutting temperature compound (Miles Inc., Elkhart, IN) and cut into 4-µm-thick sections. After having been rinsed with PBS, the sections were incubated with 5% normal bovine serum albumin (BSA) in PBS for 20 min at room temperature. They were then incubated with rabbit anti-O-GlcNAc transferase polyclonal antibody (AL-25 or -28, 1:200) in 0.1% BSA in PBS for 1 h at room temperature. After another wash in PBS, the sections were incubated with Cy2-conjugated goat antirabbit IgG (1:200) for 1 h at room temperature and washed again with PBS. Next they were incubated with mouse anti-O-GlcNAc monoclonal antibody (RL2 or CTD110.6, 1:200) in 0.1% BSA in PBS for 1 h. Another wash in PBS, the sections were incubated with Cy3-conjugated goat antimouse IgG or IgM (1:200) for 1 h, and washed again with PBS. Next they were incubated with guinea pig anti-insulin antibody (1:1000) for 1 h. Another wash with PBS, the sections were incubated with Cy5-conjugated goat antiguinea pig IgG (1:200). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, 1 µg/mL, Boehringer Mannheim, Germany). Finally, the sections were observed under a laser confocal scanning microscope (MRC-1024, BioRad, Hercules, CA). For a control experiment, other specimens were incubated with normal rabbit or mouse IgG or with 0.1% BSA-PBS alone instead of the primary antibodies. No positive staining was observed in the control experiment (data not shown). Almost the same results were obtained by using either AL-25 or -28, and either RL2 or CTD110.6. The immunohistochemical data presented in this paper were obtained by using AL-25 and CTD110.6 antibodies.
The distribution of anti-PDX-1 and anti-insulin reactivity in the islets of Langerhans from normal Wistar rat or diabetic GK rat pancreas was examined by the immunofluorescence method. Pancreas was fixed in 4% paraformaldehyde for 1 h at 4 °C. Frozen sections (10 µm in thickness) were reacted with rabbit polyclonal anti-PDX-1 antibody and then with Cy3-donkey antirabbit IgG. The sections were subsequently incubated with mouse monoclonal anti-insulin antibody and then with Cy2-donkey antimouse IgG. Nuclei were stained with DAPI.
Quantification of immunostaining intensity was done on a Macintosh computer using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet). The results (mean ± SEM) represent duplicate measurements made in three separate experiments. The terms "increase" and "decrease" were used only when the results were statistically significant (the Student t test, P < 0.05).
Immuno-electron microscopy
Tissues were fixed in 4% paraformaldehyde in PBS for 1 h at 4 °C. After dehydration with ethanol, they were embedded in LR white (London Resin Co., Basingstoke, UK) and sectioned. Then double-immunogold labeling was performed. One grid face was incubated with either anti-insulin or antiglucagon and the other with anti-O-GlcNAc. Then the grids were incubated with the appropriate secondary antibody labeled with gold particles of different sizes (18, 12, and 6 nm; Jackson Immunoresearch). The sections were finally stained with uranyl acetate and observed with a JEM-1010C (JEOL, Tokyo, Japan).
Immunoprecipitation and immunoblotting
Pancreatic tissues dissected from Wistar and GK rats were washed with ice-cold PBS, homogenized in lysis buffer (50 mM Tris–HCl/50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.5, containing 1% Nonidet P-40; 150 mM NaCl; 1 mM EGTA, and 0.25% sodium deoxycholate) supplemented with O-GlcNAcase inhibitor (100 µg/mL PUGNAc) and protease inhibitors (1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 M 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride, and 0.5 mg/mL pepstatin). The insoluble materials were removed by centrifugation at 15 000g for 40 min, and the supernatant (same amount of proteins) was incubated overnight at 4 °C with 3 µg/mL anti-PDX-1 polyclonal antibody. The immunocomplexes were incubated with protein G-Sepharose (Pharmacia Biotech, Uppsala, Sweden) at 4 °C for 2 h, and the immunocomplexes immobilized on the protein G-Sepharose were sedimented by centrifugation at 10 000g for 1 min, washed four times with the same lysis buffer, and resuspended in 50 µL of 2 x reducing-SDS sample buffer. The immunoprecipitated proteins were subjected to 7.5% SDS–PAGE under reducing conditions and electrophoretically transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were blocked for 4 h at room temperature with 5% nonfat dried milk, 0.1% Tween 20 in Tris-buffered saline (10 mM Tris, pH 7.4, 140 mM NaCl), and then incubated overnight at 4 °C with anti O-GlcNAc monoclonal antibody (CTD110.6) diluted with blocking buffer (1:3000). After several washes with washing buffer [Tris-buffered saline containing 0.1% (v/v) Tween 20], immunoreactive proteins were identified by a 2-h incubation at room temperature with HRP-conjugated goat antimouse IgM diluted with blocking buffer (1:5000). Following several washes with the washing buffer, the membranes were visualized by using ECL and exposure to CL-X Posure Film. Then to examine the expression level of the PDX-1 protein, we stripped the O-GlcNAc monoclonal antibody by using western blot stripping buffer (Chemicon International, Temecula, CA) and reblotted the membranes with the anti-PDX-1 antibody that had been used for the immunoprecipitation.
RNA interference
The following three 25-mer oligonucleotide pairs were used as siRNA against mouse O-GlcNAc transferase: OGT1 (5'-AAAGUUUGUACCAUCAUCCGGGCUC-3' and 5'-GAGCCCGGAUGAUGGUACAAACUUU-3'), OGT2 (5'-UAUUGUUUGGUGUUGAACAGAGGGC-3' and 5'-GCCCUCUGUUCAACACCAAACAAUA-3'), and OGT3 (5'-AUGAAAUCAGGCUUCAGCCGCAAGG-3' and 5'-CCUUGCGGCUGAAGCCUGAUUUCAU-3') (Stealth Select siRNA synthesized by Invitrogen, Carlsbad, CA). An equal mixture of OGT1, OGT2, and OGT3 was used. A green fluorescent protein (GFP) siRNA (5'-GGCUACGUCCAGGAGCGCACC-3' and 5'-UGCGCUCCUGGACGUAGCCUU-3') was used as a nonspecific control. MIN6 cells (a gift from Dr J-I Miyazaki, Osaka University, Osaka, Japan) at passage 15–30 were cultured as described previously (Nagamatsu et al. 1999). The O-GlcNAc transferase (OGT) siRNA and GFP siRNA were transfected into MIN6 cells by using Lipofectamine RNAi MAX (Invitrogen) according to the manufacturer's protocol.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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The authors thank Mr Minoru Fukuda, Ms Sachie Matubara, Ms Miki Kanai, and Ms Tomoko Miura (Laboratory for Electron Microscopy and Department of Anatomy, Kyorin University School of Medicine) for technical assistance and Dr Chad Slawson (Department of Biological Chemistry, Johns Hopkins University School of Medicine) and Dr Akihiko Kudo and Dr Masami Kanai-Azuma (Department of Anatomy, Kyorin University School of Medicine) for invaluable discussions.
This study was supported in part by grants from Pancreatic Research Foundation of Japan, by Japan Diabetes Foundation, from by the Kazato Research Foundation to Y.A., and by NIH grant DK61671 to G.W.H.
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6 Present address: Department of Biochemistry and Molecular Biology, Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, GA 30602
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Abbreviations |
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BSA, bovine serum albumin; DAPI, 4',6-diamidino-2-phenylindolle dihydrochloride; ECL, enhanced chemiluminescence; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; FBS, fetal bovine serum; GFP, green fluorescent protein; GK rat, Goto–Kakizaki rat; HBSS, Hank's balanced salt solution; HRP, horseradish peroxidase; IRI, immunoreactive insulin; O-GlcNAc, O-linked N-acetylglucosamine; O-GlcNAcase, O-linked-N-acetylglucosaminidase; OGT, O-GlcNAc transferase; PBS, phosphate-buffered saline; PDX-1, pancreatic/duodenal homeobox-1 transcription factor; PMSF, phenylmethylsulfonyl fluoride; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylikene)-amino-N-phenyl-carbamate; PVDF, polyvinylidene difluoride; RNAi, RNA interference; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophorosis; STZ, streptozotocin; UDP-GalNAc, UDP-ß-N-acetylgalactosamine; UDP-GlcNAc, UDP-ß-N-acetylglucosamine.
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