Hexosamines stimulate apoptosis by altering SIRT1 action and levels in rodent pancreatic β-cells

in Journal of Endocrinology

The activity and levels of SIRT1, which promotes cell survival in several models, are linked to glucose concentrations and cellular energy metabolism. The present study aimed to determine whether impaired Sirt1 activity is involved in the induction of apoptosis by the nutrient-sensing hexosamine biosynthesis pathway (HBP). Pancreatic Nit-1, Rin-m5F, and Min6 β-cells were acutely treated at different doses and times with glucosamine, which enters and stimulates the HBP. Sirt1 levels were genetically modulated by retroviral infection. Expression levels, cellular localization, and activity of apoptosis-related markers were determined by qPCR, immunoblotting, and co-immunoprecipitation. Glucosamine treatment dose- and time dependently induced cell apoptosis in all cell lines studied. HBP stimulation time dependently modified SIRT1 protein levels, notably in the cytoplasm. This was concomitant with increased E2F1 binding to the c-myc promoter. In both NIT-1 and min6 β-cells, genetic knockdown of Sirt1 expression resulted in higher susceptibility to HBP-stimulated apoptosis, whereas overexpression of Sirt1 had the opposite impact. These findings indicate that reduction of SIRT1 levels by hexosamines contributes to β-cell apoptosis. Methods to increase SIRT1 levels or activity could thus prevent the decrease in β-cell mass, notably that observed in type 2 diabetes.

Abstract

The activity and levels of SIRT1, which promotes cell survival in several models, are linked to glucose concentrations and cellular energy metabolism. The present study aimed to determine whether impaired Sirt1 activity is involved in the induction of apoptosis by the nutrient-sensing hexosamine biosynthesis pathway (HBP). Pancreatic Nit-1, Rin-m5F, and Min6 β-cells were acutely treated at different doses and times with glucosamine, which enters and stimulates the HBP. Sirt1 levels were genetically modulated by retroviral infection. Expression levels, cellular localization, and activity of apoptosis-related markers were determined by qPCR, immunoblotting, and co-immunoprecipitation. Glucosamine treatment dose- and time dependently induced cell apoptosis in all cell lines studied. HBP stimulation time dependently modified SIRT1 protein levels, notably in the cytoplasm. This was concomitant with increased E2F1 binding to the c-myc promoter. In both NIT-1 and min6 β-cells, genetic knockdown of Sirt1 expression resulted in higher susceptibility to HBP-stimulated apoptosis, whereas overexpression of Sirt1 had the opposite impact. These findings indicate that reduction of SIRT1 levels by hexosamines contributes to β-cell apoptosis. Methods to increase SIRT1 levels or activity could thus prevent the decrease in β-cell mass, notably that observed in type 2 diabetes.

Keywords:

Introduction

Hyperglycemia, occurring after a meal or during diabetes or aging, is detrimental to several systems, especially in sensitive tissues such as the brain and the pancreas, in which it has been shown that prolonged hyperglycemia leads to cell death through apoptotic signals. Pancreatic β-cells suffer from long-term high glucose levels via the induction of oxidative stress, stimulation of pro-apoptotic events, and ATP depletion from reduced glucose metabolism (Kaneto et al. 2001, Anello et al. 2004, Zachara & Hart 2004). Although apoptosis has been established as the primary factor for reduced β-cell mass in type 2 diabetes (Hanley et al. 2010), the molecular mechanisms linking hyperglycemia and the induction of apoptotic gene expression have not been fully determined.

Glucose metabolism through the hexosamine biosynthesis pathway (HBP) has been hypothesized to mediate many of the adverse effects of hyperglycemia and to be involved in the pathogenesis of type 2 diabetes and glucose toxicity (Brownlee et al. 1988, Rossetti et al. 1995, Hebert et al. 1996). Rate-limiting enzymes in the HBP are glutamine:fructose-6-phosphate amidotransferase (GFAT), which catalyzes the formation of glucosamine-6-phosphate from fructose-6-phosphate and glutamine, and O-glucosamine acetyl transferase, which adds a UDP-acetylglucosamine group to a target protein and is overexpressed in pancreas. Adipocytes (Considine et al. 2000), fibroblasts, and rodents (Brownlee et al. 1988, Shankar et al. 1998, Veerababu et al. 2000, Einstein et al. 2008) exposed to glucosamine, which directly enters and stimulates the HBP, develop insulin resistance. In addition, HBP activation induces defects in insulin production and secretion by attenuating the activity of glucokinase in β-cells (Balkan & Dunning 1994, Shankar et al. 1998, Yoshikawa et al. 2002). Similar to animals exposed to glucosamine, transgenic animals overexpressing GFAT either in skeletal muscle, fat, or in liver also develop insulin resistance (Hebert et al. 1996, Veerababu et al. 2000). Less is known about the effects of HBP in humans. Although GFAT levels have been shown to correlate inversely with glucose disposal rate (Daniels et al. 1996) and fasting glycemia (Monauni et al. 2000) in humans, this issue remains controversial, as others have found that HBP has no impact on insulin sensitivity (Pouwels et al. 2001). In contrast, stimulation of HBP by glucosamine was demonstrated to cause apoptosis in both rodent (Konrad et al. 2001, Okuyama & Yachi 2001, Anello et al. 2004) and human (D'Alessandris et al. 2004) pancreatic β-cells, despite the up-regulation of cell survival factors such as MAFA (Vanderford et al. 2007).

SIRT1 is a deacetylase enzyme mostly located in the nucleus but also found in the cytoplasm under certain conditions (Tanno et al. 2007). Its NAD-dependent activity suggests that this protein may sense the metabolic state of cells and modulate gene expression accordingly. SIRT1 was shown to impact on cell survival through physical binding with p53, HSF1, Hif-2α, FOXO, and Ku70 (Dioum et al. 2009, Westerheide et al. 2009, Gagarina et al. 2010) and reviewed in Yamamoto et al. (2007). Importantly, SIRT1 and its yeast homolog SIR2 appear to be very sensitive to changes in cellular glucose content such as those occurring upon caloric restriction (Lin et al. 2000, Cohen et al. 2004, Nisoli et al. 2005, Civitarese et al. 2007, Chen et al. 2008, Kanfi et al. 2008, Nedachi et al. 2008). In cultured pancreatic β-cells, overexpression of Sirt1 was shown to protect against cytokine toxicity (Lee et al. 2009). Moreover, glucosamine treatment in the hepatic HepG2 cell line acutely reduces SIRT1 protein levels (Sun et al. 2007). In this context, we postulated the hypothesis that HBP stimulation could induce β-cell death through a reduction in SIRT1 level and/or activity.

Materials and Methods

Cell culture and treatment

Mouse Nit-1 and rat Rin-m5F cell lines were bought from ATCC (Rockville, MD, USA). Mouse Min6 cells were a kind gift from Dr André Marette (Université Laval, Canada). Nit-1 and Rin-m5F cells were cultured in Ham's F12K medium (Sigma–Aldrich) containing 5 mM glucose added with 1.5 g/l sodium bicarbonate and 10% heat-inactivated dialyzed fetal bovine serum (FBS). Min6 cells were cultured in DMEM high glucose supplemented with 15% FBS, 5 μl/l β-mercaptoethanol, and 10 mM HEPES. Cells were cultured at 37 °C in a humidified incubator at 5% CO2. Experiments were typically performed at 80% confluence. Following the treatments, cells were harvested, centrifuged in collection tubes for mRNA and protein extractions, and were frozen at −20 °C until subsequent analysis. Viral production and infection were performed exactly as previously described (Picard et al. 2004). Infected cells were kept in a medium containing puromycin throughout the study.

Measurement of cell death

Cell death and apoptosis were quantified using three complementary approaches. Cells were harvested by treatment with a cell dissociation buffer (an enzyme free Hank's based solution; Gibco). Dead cells were counted based on dye exclusion after trypan blue staining in a hemacytometer. Annexin V assays were used to measure cell death through reorganization of phosphatidylserine in cell membrane. Briefly, cells were resuspended in annexin binding buffer (10 mM HEPES, pH=7.4, 140 mM NaCl, and 5 mM CaCl2) containing annexin V-FITC (Roche Diagnostic) and Hoechst 33258 (Sigma–Aldrich) for 10 min in the dark. Cells were washed three times with ice-cold PBS, plated on slides, and allowed to dry. Cells were then coverslipped with MOWIOL 4-88 mounting medium (Calbiochem, Gibbstown, NJ, USA). TUNEL assays were conducted following the manufacturer's instructions (Roche Diagnostic). For TUNEL assays, floating cells were not taken into account, which at least underestimated apoptosis ratios. Slides were analyzed by microscopy (IX-81 by Olympus, Markham, ON, Canada). Pictures were taken using an Evolution QEi camera and image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA).

RNA analysis and real-time quantitative RT-PCR

Total RNA from cultured cells was extracted according to the standard protocol provided with RNAspin mini isolation kit (GE Healthcare, Baie d'Urfe, QC, Canada) and quantified by Experion automated electrophoresis system (Bio-Rad). A reverse transcription step was carried on 1 μg purified RNA by using oligo dT and SuperScript II reverse transcriptase (Invitrogen) at 42 °C for 1 h. Relative quantification of cDNA amplification was performed on reactions containing 40 ng cDNA, 0.25 μM sense and antisense primers, 2 μM MgCl2, and SYBR Green JumpStart Taq ReadyMix without MgCl2 (Sigma–Aldrich) in a final volume of 20 μl for each reaction. Amplification and detection of specific products were obtained with the Rotor-Gene RG-3000 (Corbett Research, Concorde, NSW, Australia) (primer sequences and qPCR protocols available on request). Relative level of gene expression was determined via a standard curve composed of a cDNA mix from all groups. PCR data were normalized over HPRT expression (hypoxanthine–guanine phosphoribosyltransferase) used as a housekeeping gene.

Protein extraction and western blot analysis

Cells were scraped in lysis buffer A (10 mM HEPES, pH=7.9, 10 mM KCl, 100 μM EDTA, 100 μM EGTA, 1 M dithiothreitol (DTT) (1:1000), and Protease Inhibitor Cocktail (Sigma–Aldrich) (1:1000)) and combined with floating cells fraction. NP40 was added to each sample before vigorous mixing and nucleus recovery through centrifugation. Then, nuclei were resuspended in lysis buffer C (20 mM HEPES, pH=7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 M DTT (1:1000), and Protease Inhibitor Cocktail (1:1000)) and mixed. Proteins were recovered in the supernatant and dosed on a u.v. plate reader by DC Protein Assay (Bio-Rad). A total of 20 μg of each sample were resolved by SDS-PAGE and transferred to PVDF membranes. After a 2 h transfer, membranes were blocked in a 5% milk (in BSA-1X) for 35 min. Then, membranes were incubated with different primary antibodies at 4 °C overnight against Bax (1:500) (Santa Cruz sc-526, Santa Cruz, CA, USA), c-Jun (1:700) (Santa Cruz sc-45), E2F1 (1:700) (Santa Cruz sc-193), p53 (1:700) (Santa Cruz sc-6243), p65-NFKB (1:700) (Santa Cruz sc-372), Sirt1 (1:2000) (Upstate 07-131), ubiquitin (1:700) (Santa Cruz sc-9133), and glycosylated proteins (Affinity BioReagents MA1-072). The measure of actin (1:25 000) (Upstate MAB1501) was used as a loading control. The next day, membranes were rinsed four times in wash buffer (TBS 1× with Tween 20 and NP40) before incubation for 1 h at 4 °C with respective HRP-conjugated secondary antibodies (1:5000) (Amersham). Membranes were washed four times in wash buffer, and tagged proteins were visualized by ECL system (GE Healthcare).

Immunofluorescence

Cells were plated on coverslips and treated with 5 mM glucosamine for 1, 5, and 24 h. Cells were fixed in methanol at 4 °C for 10 min. After three washes with ice-cold PBS, non-specific binding sites were saturated with 1% BSA–PBS for 10 min at room temperature. Then, cells were incubated with Sirt1 antibody (1:25) in 1% BSA–PBS. Secondary antibodies used were FITC-conjugated affinipure goat anti-rabbit IgG (1:1000) (Jackson ImmunoResearch, West Grove, PA, USA) with nuclear staining with Hoechst 33258 (1:1000) in 1% BSA–PBS. Coverslips were mounted on slides using Vectashield mounting medium (VectorLabs, Burlington, ON, Canada) and analyzed by microscopy (IX-81 by Olympus). Pictures were acquired using an Evolution QEi camera and image Pro Plus 6.0 software (Media Cybernetics).

Chromatin immunoprecipitation

Nit-1 cells were cross-linked with 1% paraformaldehyde at room temperature after 6, 12, 18, or 24 h of 5 mM glucosamine treatment. Cells were washed three times with ice-cold PBS and scraped in ChIP lysis buffer (50 mM HEPES, pH=7.5, 140 mM NaCl, 1% (v/v) Triton X-100, and Protease Inhibitor Cocktail 1:1000). Cells were sonicated to shear DNA by repeating seven cycles of sonication: 9 s at 30% of the maximum amplitude (Sonifier Cell Disrupter 185, Branson, Danbury, CT, USA) with 1 min pauses of 10 s on dry ice followed by 50 s on ice. After taking fractions of sheared DNA for inputs, samples were pre-cleared with 50 μl of Protein A Sepharose Fast Flow (GE Healthcare) beads and 20 μg salmon sperm ssDNA (Invitrogen) for 1 h at 4 °C. Supernatants were then split to incubate with 50 μl beads, 20 μg ssDNA, and 4 μg rabbit IgG (Santa-Cruz, sc-2027) as a negative control, and with 4 μg E2F1 or 2 μg Sirt1 antibodies for ChIP assay and were rocked overnight at 4 °C. On the next day, samples were washed twice with ChIP lysis buffer, one time with ChIP lysis buffer high salt (50 mM HEPES, pH=7.5, 0.5 M NaCl, 1% (v/v) Triton X-100, and Protease Inhibitor Cocktail 1:1000), twice with wash buffer (10 mM Tris, pH=8.0, 250 mM LiCl, 0.5% (v/v) NP40, and 1 mM EDTA), and twice with TE (10 mM Tris, pH=8.0, and 10 mM EDTA). Beads were eluted (10 mM Tris, pH=8.0, 1% (v/v) SDS, and 10 mM EDTA) with two rounds of heating at 65 °C separated by vortexing. Combined supernatants and inputs were heated at 65 °C overnight. The next day, DNA was purified using PCR Purification Kit (Qiagen) followed by PCR on the c-myc promoter with specific primers (forward, 5′-GCGGGGGCTCCTAGATAACTC-3′; reverse, 5′-CGGCTCCGGGGTGTAAAC-3′) or on the Hsp70 promoter (forward, 5′-GAACCCACAACTCCGATTACTCA-3′; reverse, 5′-TCTTGTCTTCGCTTGTCTCTGGAT-3′). Amplification results were obtained by migration on 1% EtBr agarose gel.

Statistical analysis

Data are presented as means±s.e.m. The main and interactive effects were analyzed by ANOVA. When justified by ANOVA, differences between individual group means were analyzed by Fisher's PLSD test. Differences were considered statistically significant at P<0.05.

Results

HBP stimulation induces apoptosis in β-cells

HBP stimulation by glucosamine treatment induced a large increase in the number of floating cells resulting from disorganization of β-cell colonies (Fig. 1A and B). Cells treated with glucosamine lost almost every ultrastructural feature such as beta granules and well-developed rough endoplasmic reticulum. Cell death was also apparent following nucleus staining (Fig. 1C and D). These findings were confirmed by annexin V staining (Fig. 1E–H). The impact of glucosamine treatment was not due to osmotic or pH modification, as control solutions with mannitol or pH comparable to that of glucosamine did not result in any change in cell death (data not shown). Statistical analysis of cell death ratio indicated that 5 mM glucosamine stimulated cell death within 18–24 h of treatment (Fig. 1I). The significant impact of glucosamine was also observed in rat Rin-m5F β-cells (Fig. 1J), indicating that the effect of HBP stimulation on cell death is not species-specific. Further tests using highly sensitive TUNEL assays indicated that a 48 h treatment with a lower dose of 1 mM glucosamine also increased cell death (Fig. 1K). These findings demonstrate that HBP stimulation even by a low dose of glucosamine induces β-cell apoptosis.

Figure 1
Figure 1

Glucosamine treatment dose- and time dependently triggers β-cell death. Cells were treated with or without glucosamine at the indicated time and doses. Bright-field pictures (A and B), nucleus staining (Hoechst) (C and D), and immunofluorescent annexin V staining (E and F) of Nit-1 cells taken 24 h after glucosamine treatment (24 h). Loss of β-cells was then scored by cell count of trypan blue (G) or annexin staining (H) after correction for total cell number. (I) Time course of positive annexin staining in Nit-1 cells after 5 mM glucosamine treatment. (J) Dose–response of cell death in Rin-m5F cells treated with glucosamine for 24 h. (K) Dose–response of cell death in Nit-1 cells treated with glucosamine for 48 h. TUNEL assays (representative left panels) were done at the end of the incubation period. Data shown were obtained from at least three independent experiments done in duplicate. Bars are mean±s.e.m. * indicates a significant effect (P<0.05) compared to the control group (white bars). Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0243.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

HBP stimulation modulates the levels and cellular location of pro-apoptotic factors

Previous studies suggested that glucosamine treatment of rat β-cells results in reduced insulin synthesis (Andreozzi et al. 2004), and in the inactivation of the IRS1/PI3K pathway, leading to increased c-Jun/JNK and a down-regulation of FOXO1 activity (Andreozzi et al. 2004, D'Alessandris et al. 2004). In the present study, further analysis of apoptotic pathways by real-time quantitative PCR revealed that glucosamine treatment dose dependently stimulated the mRNA expression of Myc, Bax, Jun, and the p65 unit of NFκB (Fig. 2). In contrast, fatty acid synthase (FAS) expression levels were significantly down-regulated by glucosamine treatment. However, no change in glucokinase mRNA levels was found. Insulin, Apaf-1, and Sirt1 mRNA levels also remained unchanged after incubation with glucosamine (Fig. 2).

Figure 2
Figure 2

Glucosamine triggers an apoptotic transcriptional program in Nit-1 cells after 24 h of treatment. Quantitative real-time PCR results were corrected for mRNA levels of GAPDH as a housekeeping gene. Data shown were obtained from three independent experiments done in duplicate. Bars represent fold change compared to the control condition (dotted line). Asterisks indicate a statistical difference from the control group (*P<0.05; **P≤0.01; ***P≤0.0001). Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0243.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

Determination of protein cellular location and expression levels confirmed the increase in BAX levels upon glucosamine treatment, which was mainly observed in the nucleus (Fig. 3). Overall, ubiquitination of proteins did not change upon treatment (Fig. 3). Cytoplasmic protein levels of p65-NFkB and p53 were reduced (Fig. 3). Interestingly, E2F1 levels were slightly diminished in the nucleus 24 h after glucosamine treatment (Fig. 3).

Figure 3
Figure 3

Effects of glucosamine treatment on the protein cellular location and levels in Nit-1 cells harvested after 24 h of glucosamine treatment (A). Actin levels are shown as loading controls. Each lane from both groups represents an independent experiment. (B) Quantification of the protein levels shown in (A) relative to those of actin. *P<0.05 compared to the vehicle treatment.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

HBP affects SIRT1 level and activity

Consistent with previous findings in hepatic HepG2 cells (Sun et al. 2007), western blotting revealed a robust decline in the cytoplasmic fraction of SIRT1 24 h after incubation with glucosamine (Fig. 3). Given the possibility that SIRT1 serves as a node interacting with and inhibiting pro-apoptotic factors such as E2F1 and p53 while stimulating anti-apoptotic factors such as HSF1, a time-course study was performed to document the decrease in SIRT1 protein level upon glucosamine treatment. The reduction (58%, P<0.0001) was noticeable only 24 h after the beginning of the incubation period (Fig. 4A and B). At that time point, most of SIRT1 protein was observed in the nucleus (Fig. 4C). This diminution in SIRT1 protein expression while mRNA levels remained constant suggests that SIRT1 protein degradation might be increased by HBP stimulation.

Figure 4
Figure 4

Stimulation of HBP by glucosamine alters SIRT1 protein levels and location. (A) Western blot of SIRT1 protein at 0–24 h after glucosamine exposure in Nit-1 cells. (B) Quantitative analysis of the changes in SIRT1 protein levels upon glucosamine treatment (n=3). Signals for SIRT1 were divided by those for actin to account for loading. * indicates P<0.0001. (C) Time course showing localization of SIRT1 (green) using immunofluorescence in Nit-1 cells treated with 5 mM glucosamine. Cell nuclei are shown in blue (Hoechst). Data shown are representative of three independent experiments done in duplicate. Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0243.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

To test the effect of this phenomenon on Sirt1 transcriptional activity, ChIP assays were first performed on the promoter of the heat shock protein 70 (Hsp70), which has a protective, anti-apoptotic effect against stress (Daugaard et al. 2007). SIRT1 binding to the Hsp70 promoter was induced within 1 h by glucosamine treatment and remained high 5 h after HBP stimulation (Fig. 5A). Then, as SIRT1 protein levels are decreasing as a function of glucosamine treatment (Fig. 4), possibly because of progressing changes in protein conformation, we postulated that its inhibitory activity on E2F1 would also diminish, thus allowing E2F1 to stimulate pro-apoptotic gene transcription. Consistent with this concept, binding of E2F1 on the Myc promoter, as evidenced by ChIP, was increased from 6 to 18 h of HBP stimulation, but was greatly reduced at 24 h (Fig. 5B).

Figure 5
Figure 5

Time-dependent transactivation of Hsp70 and Myc promoters by HBP stimulation. (A) ChIP assay on Sirt1 and IgG (control)-immunoprecipitated DNA from Nit-1 cells treated with 5 mM glucosamine for up to 5 h. A sequence containing the Hsp70 promoter was amplified by specific primers. (B) ChIP assay on E2F1 and IgG (control)-immunoprecipitated DNA from Nit-1 cells treated with 5 mM glucosamine for 6–24 h. A sequence containing E2F1 response elements from the Myc promoter was amplified by specific primers. For both A and B, quantification of the binding signals is shown on the right panels. Data shown are representative of three independent experiments. *P<0.05 compared to 0 h of treatment.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

Genetic manipulation of SIRT1 levels modulates apoptotic susceptibility to HBP

To confirm that reduced SIRT1 contributes to HBP-induced apoptosis, virus-mediated, specific Sirt1 knockdown (T1 KD) was performed in Nit-1 cells, which resulted in a 60% decrease in its protein level (Fig. 6A). In normal conditions, death ratios were similar between control and T1 KD cells (Fig. 6B). In contrast, compared to control cells, T1 KD cells were significantly more susceptible to apoptosis when incubated with glucosamine (Fig. 6B). These findings indicate that reduction of SIRT1 exacerbates HBP-induced cell death in Nit-1 β-cells.

Figure 6
Figure 6

Reduction in Sirt1 levels exacerbates HBP-induced apoptosis in Nit-1 β-cells. (A) SIRT1 protein levels in Nit-1 cells infected with viruses produced through an empty (pSuper) plasmid, or with a sequence specifically targeting Sirt1 (T1 KD) (Picard et al. 2004). (B) Cell death in infected cells incubated or not with 5 mM glucosamine for 12 h. Results are mean±s.e.m. of three independent experiments done in triplicate. * indicates significant effect of glucosamine; † indicates significant difference with empty virus.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

To further validate this observation, levels of the Sirt1 gene were increased (pBabe-Sirt1) or reduced (pSuper-Sirt1-RNAi) in mouse Min6 cells by retroviral infection. On the one hand, overexpression of Sirt1 resulted in a protection against glucosamine-induced cell death as quantified by trypan blue (Fig. 7A) and annexin (Fig. 7B) staining. On the other hand, cells with down-regulation of Sirt1 levels showed increased basal and glucosamine-stimulated apoptosis (Fig. 7A and B). These results confirmed that modulation of SIRT1 levels impacts on HBP-induced apoptosis.

Figure 7
Figure 7

Overexpression of Sirt1 protects against HBP-induced apoptosis in Min6 β-cells. Overexpression (pBabe-Sirt1) or down-regulation (pSuper-Sirt1-RNAi) of Sirt1 was achieved by retroviral infection. After at least 10 days of antibiotic selection, Min6 cells were incubated with glucosamine. Trypan blue- (A) and annexin- (B) positive cells were quantified 48 h after. * indicates a significant effect of glucosamine (P<0.05). † indicates a significant effect of the virus compared to the control pSuper group (P<0.05). Results are mean±s.e.m. of two independent experiments done in triplicate. pS-Sirt1-scr represents the same plasmid as pS-Sirt1-RNAi except that the RNAi sequence was scrambled as a negative control.

Citation: Journal of Endocrinology 208, 1; 10.1677/JOE-10-0243

Discussion

It has previously been shown that stimulation of the HBP results in apoptosis in several models of pancreatic β-cells. The findings of the present study indicate that this pathway can modify SIRT1 protein levels and location, resulting in reduced activity and a concomitant transactivation of pro-apoptotic target genes such as Myc.

The present study confirms in three different cell lines that HBP stimulation by glucosamine triggers β-cell apoptosis and subsequent cell death at a dose even lower than that used in previous reports (Konrad et al. 2001, Okuyama & Yachi 2001, Anello et al. 2004, D'Alessandris et al. 2004). On the one hand, consistent with strong annexin staining, Myc and NFκB transactivation, morphologic differences observed after glucosamine exposure as well as modified expression patterns of genes such as Fas showed that cell metabolism was robustly altered, likely in a time-dependent manner. On the other hand, the mRNA expression of some, but not other, pro-apoptotic factors was not significantly changed upon 5 mM glucosamine treatment, suggesting activation of specific pathways implicated in apoptosis. These could be linked to altered mitochondrial function (Balestrieri et al. 2008) and production of reactive oxygen species, as SIRT1 controls the expression of UCP2 in pancreatic β-cells (Bordone et al. 2005, Moynihan et al. 2005) and consequently insulin secretion.

Our findings demonstrate that modulation of SIRT1 levels robustly interferes with HBP-induced β-cell apoptosis. The glucosamine-stimulated reduction of SIRT1 protein would likely affect many pathways involved in cell cycle and apoptosis. Our data suggest that SIRT1 can initially protect against HBP-induced metabolic stress by activating the Hsp70 promoter (Fig. 5A), but that, in the longer term, loss of SIRT1 function enables pro-apoptotic factors. Previous data indicate that SIRT1 can inhibit E2F1 activity through direct physical interaction (Wang et al. 2006). From our present findings, it could be postulated that reduced SIRT1 levels could diminish its docking to E2F1, which could then stimulate the binding of E2F1 to the MYC promoter (Fig. 5B), triggering transactivation of the gene and induction of apoptosis. Reduced E2F1 DNA binding at 24 h might be due to a slightly reduced protein level (Fig. 3) or to the fact that other pro-apoptotic mechanisms take place at this time point. However, to date, attempts to pinpoint one exclusive specific mechanism (e.g. via altered SIRT1 docking to E2F1, FOXO, or HSF1) have not been conclusive, leading to the speculation that SIRT1 may act through multiple networks.

Several studies have reported increased SIRT1 protein levels (Balestrieri et al. 2008, Kanfi et al. 2008, Nedachi et al. 2008) and nuclear accumulation (Nedachi et al. 2008) under conditions of low glucose concentrations. In line with this, a report showed that glucosamine reduced SIRT1 protein expression in HepG2 cells (Sun et al. 2007). These observations suggest that stimulation of HBP by glucosamine affects SIRT1 stability, a concept supported by our findings that glucosamine reduces SIRT1 protein but not mRNA levels (Figs 24). Phosphorylation of SIRT1 has been hypothesized to play a significant role in its deacetylase activity (Beausoleil et al. 2004), and glycosylation was shown to affect the same protein motifs as phosphorylation (Zachara & Hart 2004). Transcription factors and other protein components of the nucleus can become glycosylated, which triggers rapid changes in affinity, specificity, and stability of components in protein complexes. For example, this applies in β-cells to PDX-1 (Gao et al. 2003, Akimoto et al. 2007) and H6451sp60, which detaches from BAX upon HBP stimulation and triggers BAX translocation to the mitochondria (Kim et al. 2006). The balance between phosphorylation and glycosylation, in association with the reported sumoylation modification of SIRT1 (Yang et al. 2007), could thus represent a fine-tuning regulatory mechanism of SIRT1 activity. Moreover, prolonged glycosylation was previously shown to modulate protein ubiquitination (Guinez et al. 2008), a mechanism that could have contributed, in the present study, to the decrease in SIRT1 observed after 24 h of glucosamine treatment. Given the proposed links between SIRT1 and longevity (Bordone et al. 2007, Guarente 2008) and the increase in HBP activity shown in aged rats (Einstein et al. 2008), more studies are required to better analyze how HBP stimulation controls SIRT1 protein status.

While confirming that stimulation of the HBP triggers β-cell apoptosis, our present findings reveal that this effect is associated with robust changes in SIRT1 protein levels, which leads to impaired activity, allowing transactivation of pro-apoptotic genes. The relevance of this novel mechanism remains, however, to be demonstrated in vivo or using isolated islets. In addition, compared to that of other established HBP-induced pro-apoptotic pathways, the relative contribution of this molecular mechanism to overall cell death is still to be clarified. Finally, the findings described herein suggest that post-translational modifications of SIRT1 might play an important role in the responses of β-cell to metabolic stresses.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

M L-L was a recipient of FER, FRSQ, and IRSC studentships. G D is a recipient of FER and CRIUPQ studentships. F P holds a FRSQ Junior 2 Investigator Award. This work was supported by operating grants from the CIHR (MOP-66967 and CCI-85677) and Diabète Québec.

Author contribution statement

M L-L designed the experiments, performed experiments shown in Figs 1, 3, 4A and B, 5, 6, and 7, analyzed the data, and wrote the manuscript. G D performed the experiments shown in Figs 1, 4C, and 7, and analyzed the data. F P initiated the study, designed the experiments, analyzed the data, and wrote the manuscript.

Acknowledgements

We would like to thank Yves Deshaies for critical reading of the manuscript.

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  • BalestrieriMLRienzoMFeliceFRossielloRGrimaldiVMiloneLCasamassimiAServilloLFarzatiBGiovaneA2008High glucose downregulates endothelial progenitor cell number via SIRT1. Biochimica et Biophysica Acta1784936945doi:10.1016/j.bbapap.2008.03.004.

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    • Export Citation
  • BalkanBDunningBE1994Glucosamine inhibits glucokinase in vitro and produces a glucose-specific impairment of in vivo insulin secretion in rats. Diabetes4311731179doi:10.2337/diabetes.43.10.1173.

    • Search Google Scholar
    • Export Citation
  • BeausoleilSAJedrychowskiMSchwartzDEliasJEVillenJLiJCohnMACantleyLCGygiSP2004Large-scale characterization of HeLa cell nuclear phosphoproteins. PNAS1011213012135doi:10.1073/pnas.0404720101.

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  • BordoneLMottaMCPicardFRobinsonAJhalaUSApfeldJMcDonaghTLemieuxMMcBurneyMSzilvasiA2005Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS Biology4e31doi:10.1371/journal.pbio.0040031.

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  • BordoneLCohenDRobinsonAMottaMCvan VeenECzopikASteeleADCroweHMarmorSLuoJ2007SIRT1 transgenic mice show phenotypes resembling calorie restriction. Aging Cell6759767doi:10.1111/j.1474-9726.2007.00335.x.

    • Search Google Scholar
    • Export Citation
  • BrownleeMCeramiAVlassaraH1988Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. New England Journal of Medicine31813151321doi:10.1056/NEJM198805193182007.

    • Search Google Scholar
    • Export Citation
  • ChenDBrunoJEaslonELinSJChengHLAltFWGuarenteL2008Tissue-specific regulation of SIRT1 by calorie restriction. Genes and Development2217531757doi:10.1101/gad.1650608.

    • Search Google Scholar
    • Export Citation
  • CivitareseAECarlingSHeilbronnLKHulverMHUkropcovaBDeutschWASmithSRRavussinE2007Calorie restriction increases muscle mitochondrial biogenesis in healthy humans. PLoS Medicine4e76doi:10.1371/journal.pmed.0040076.

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    • Export Citation
  • CohenHYMillerCBittermanKJWallNRHekkingBKesslerBHowitzKTGorospeMde CaboRSinclairDA2004Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science305390392doi:10.1126/science.1099196.

    • Search Google Scholar
    • Export Citation
  • ConsidineRVCookseyRCWilliamsLBFawcettRLZhangPAmbrosiusWTWhitfieldRMJonesRInmanMHuseJ2000Hexosamines regulate leptin production in human subcutaneous adipocytes. Journal of Clinical Endocrinology and Metabolism8535513556doi:10.1210/jc.85.10.3551.

    • Search Google Scholar
    • Export Citation
  • D'AlessandrisCAndreozziFFedericiMCardelliniMBrunettiARanalliMDel GuerraSLauroDDel PratoSMarchettiP2004Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells. FASEB Journal18959961doi:10.1096/fj.03-0725fje.

    • Search Google Scholar
    • Export Citation
  • DanielsMCCiaraldiTPNikoulinaSHenryRRMcClainDA1996Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells: relationship to glucose disposal rate in control and non-insulin-dependent diabetes mellitus subjects and regulation by glucose and insulin. Journal of Clinical Investigation9712351241doi:10.1172/JCI118538.

    • Search Google Scholar
    • Export Citation
  • DaugaardMRohdeMJaattelaM2007The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Letters58137023710doi:10.1016/j.febslet.2007.05.039.

    • Search Google Scholar
    • Export Citation
  • DioumEMChenRAlexanderMSZhangQHoggRTGerardRDGarciaJA2009Regulation of hypoxia-inducible factor 2alpha signaling by the stress–responsive deacetylase sirtuin 1. Science32412891293doi:10.1126/science.1169956.

    • Search Google Scholar
    • Export Citation
  • EinsteinFHFishmanSBaumanJThompsonRFHuffmanDMAtzmonGBarzilaiNMuzumdarRH2008Enhanced activation of a "nutrient-sensing" pathway with age contributes to insulin resistance. FASEB Journal2234503457doi:10.1096/fj.08-109041.

    • Search Google Scholar
    • Export Citation
  • GagarinaVGabayODvir-GinzbergMLeeEJBradyJKQuonMJHallDJ2010SirT1 enhances survival of human osteoarthritic chondrocytes by repressing PTP1B and activating the IGF receptor pathway. Arthritis and Rheumatism6213831392doi:10.1002/art.27369.

    • Search Google Scholar
    • Export Citation
  • GaoYMiyazakiJHartGW2003The transcription factor PDX-1 is post-translationally modified by O-linked N-acetylglucosamine and this modification is correlated with its DNA binding activity and insulin secretion in min6 beta-cells. Archives of Biochemistry and Biophysics415155163doi:10.1016/S0003-9861(03)00234-0.

    • Search Google Scholar
    • Export Citation
  • GuarenteL2008Mitochondria – a nexus for aging, calorie restriction, and sirtuins?Cell132171176doi:10.1016/j.cell.2008.01.007.

  • GuinezCMirAMDehennautVCacanRHarduin-LepersAMichalskiJCLefebvreT2008Protein ubiquitination is modulated by O-GlcNAc glycosylation. FASEB Journal2229012911doi:10.1096/fj.07-102509.

    • Search Google Scholar
    • Export Citation
  • HanleySCAustinEAssouline-ThomasBKapelutoJBlaichmanJMoosaviMPetropavlovskaiaMRosenbergL2010{beta}-Cell mass dynamics and islet cell plasticity in human type 2 diabetes. Endocrinology15114621472doi:10.1210/en.2009-1277.

    • Search Google Scholar
    • Export Citation
  • HebertLFJrDanielsMCZhouJCrookEDTurnerRLSimmonsSTNeidighJLZhuJSBaronADMcClainDA1996Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance. Journal of Clinical Investigation98930936doi:10.1172/JCI118876.

    • Search Google Scholar
    • Export Citation
  • KanetoHXuGSongKHSuzumaKBonner-WeirSSharmaAWeirGC2001Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress. Journal of Biological Chemistry2763109931104doi:10.1074/jbc.M104115200.

    • Search Google Scholar
    • Export Citation
  • KanfiYPeshtiVGozlanYMRathausMGilRCohenHY2008Regulation of SIRT1 protein levels by nutrient availability. FEBS Letters58224172423doi:10.1016/j.febslet.2008.06.005.

    • Search Google Scholar
    • Export Citation
  • KimHSKimEMLeeJYangWHParkTYKimYMChoJW2006Heat shock protein 60 modified with O-linked N-acetylglucosamine is involved in pancreatic beta-cell death under hyperglycemic conditions. FEBS Letters58023112316doi:10.1016/j.febslet.2006.03.043.

    • Search Google Scholar
    • Export Citation
  • KonradRJMikolaenkoITolarJFLiuKKudlowJE2001The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic beta-cell O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase. Biochemical Journal3563141doi:10.1042/0264-6021:3560031.

    • Search Google Scholar
    • Export Citation
  • LeeJHSongMYSongEKKimEKMoonWSHanMKParkJWKwonKBParkBH2009Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway. Diabetes58344351doi:10.2337/db07-1795.

    • Search Google Scholar
    • Export Citation
  • LinSJDefossezPAGuarenteL2000Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science28921262128doi:10.1126/science.289.5487.2126.

    • Search Google Scholar
    • Export Citation
  • MonauniTZentiMGCrettiADanielsMCTargherGCarusoBCaputoMMcClainDDel PratoSGiaccariA2000Effects of glucosamine infusion on insulin secretion and insulin action in humans. Diabetes49926935doi:10.2337/diabetes.49.6.926.

    • Search Google Scholar
    • Export Citation
  • MoynihanKAGrimmAAPluegerMMBernal-MizrachiEFordECras-MeneurCPermuttMAImaiS2005Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice. Cell Metabolism2105117doi:10.1016/j.cmet.2005.07.001.

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    Glucosamine treatment dose- and time dependently triggers β-cell death. Cells were treated with or without glucosamine at the indicated time and doses. Bright-field pictures (A and B), nucleus staining (Hoechst) (C and D), and immunofluorescent annexin V staining (E and F) of Nit-1 cells taken 24 h after glucosamine treatment (24 h). Loss of β-cells was then scored by cell count of trypan blue (G) or annexin staining (H) after correction for total cell number. (I) Time course of positive annexin staining in Nit-1 cells after 5 mM glucosamine treatment. (J) Dose–response of cell death in Rin-m5F cells treated with glucosamine for 24 h. (K) Dose–response of cell death in Nit-1 cells treated with glucosamine for 48 h. TUNEL assays (representative left panels) were done at the end of the incubation period. Data shown were obtained from at least three independent experiments done in duplicate. Bars are mean±s.e.m. * indicates a significant effect (P<0.05) compared to the control group (white bars). Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0243.

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    Glucosamine triggers an apoptotic transcriptional program in Nit-1 cells after 24 h of treatment. Quantitative real-time PCR results were corrected for mRNA levels of GAPDH as a housekeeping gene. Data shown were obtained from three independent experiments done in duplicate. Bars represent fold change compared to the control condition (dotted line). Asterisks indicate a statistical difference from the control group (*P<0.05; **P≤0.01; ***P≤0.0001). Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0243.

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    Effects of glucosamine treatment on the protein cellular location and levels in Nit-1 cells harvested after 24 h of glucosamine treatment (A). Actin levels are shown as loading controls. Each lane from both groups represents an independent experiment. (B) Quantification of the protein levels shown in (A) relative to those of actin. *P<0.05 compared to the vehicle treatment.

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    Stimulation of HBP by glucosamine alters SIRT1 protein levels and location. (A) Western blot of SIRT1 protein at 0–24 h after glucosamine exposure in Nit-1 cells. (B) Quantitative analysis of the changes in SIRT1 protein levels upon glucosamine treatment (n=3). Signals for SIRT1 were divided by those for actin to account for loading. * indicates P<0.0001. (C) Time course showing localization of SIRT1 (green) using immunofluorescence in Nit-1 cells treated with 5 mM glucosamine. Cell nuclei are shown in blue (Hoechst). Data shown are representative of three independent experiments done in duplicate. Full colour version of this figure available via http://dx.doi.org/10.1677/JOE-10-0243.

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    Time-dependent transactivation of Hsp70 and Myc promoters by HBP stimulation. (A) ChIP assay on Sirt1 and IgG (control)-immunoprecipitated DNA from Nit-1 cells treated with 5 mM glucosamine for up to 5 h. A sequence containing the Hsp70 promoter was amplified by specific primers. (B) ChIP assay on E2F1 and IgG (control)-immunoprecipitated DNA from Nit-1 cells treated with 5 mM glucosamine for 6–24 h. A sequence containing E2F1 response elements from the Myc promoter was amplified by specific primers. For both A and B, quantification of the binding signals is shown on the right panels. Data shown are representative of three independent experiments. *P<0.05 compared to 0 h of treatment.

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    Reduction in Sirt1 levels exacerbates HBP-induced apoptosis in Nit-1 β-cells. (A) SIRT1 protein levels in Nit-1 cells infected with viruses produced through an empty (pSuper) plasmid, or with a sequence specifically targeting Sirt1 (T1 KD) (Picard et al. 2004). (B) Cell death in infected cells incubated or not with 5 mM glucosamine for 12 h. Results are mean±s.e.m. of three independent experiments done in triplicate. * indicates significant effect of glucosamine; † indicates significant difference with empty virus.

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    Overexpression of Sirt1 protects against HBP-induced apoptosis in Min6 β-cells. Overexpression (pBabe-Sirt1) or down-regulation (pSuper-Sirt1-RNAi) of Sirt1 was achieved by retroviral infection. After at least 10 days of antibiotic selection, Min6 cells were incubated with glucosamine. Trypan blue- (A) and annexin- (B) positive cells were quantified 48 h after. * indicates a significant effect of glucosamine (P<0.05). † indicates a significant effect of the virus compared to the control pSuper group (P<0.05). Results are mean±s.e.m. of two independent experiments done in triplicate. pS-Sirt1-scr represents the same plasmid as pS-Sirt1-RNAi except that the RNAi sequence was scrambled as a negative control.

  • AkimotoYHartGWWellsLVossellerKYamamotoKMunetomoEOhara-ImaizumiMNishiwakiCNagamatsuSHiranoH2007Elevation 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. Glycobiology17127140doi:10.1093/glycob/cwl067.

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  • AndreozziFD'AlessandrisCFedericiMLarattaEDel GuerraSDel PratoSMarchettiPLauroRPerticoneFSestiG2004Activation 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 beta-cells. Endocrinology14528452857doi:10.1210/en.2003-0939.

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  • AnelloMSpampinatoDPiroSPurrelloFRabuazzoAM2004Glucosamine-induced alterations of mitochondrial function in pancreatic beta-cells: possible role of protein glycosylation. American Journal of Physiology. Endocrinology and Metabolism287E602E608doi:10.1152/ajpendo.00320.2003.

    • Search Google Scholar
    • Export Citation
  • BalestrieriMLRienzoMFeliceFRossielloRGrimaldiVMiloneLCasamassimiAServilloLFarzatiBGiovaneA2008High glucose downregulates endothelial progenitor cell number via SIRT1. Biochimica et Biophysica Acta1784936945doi:10.1016/j.bbapap.2008.03.004.

    • Search Google Scholar
    • Export Citation
  • BalkanBDunningBE1994Glucosamine inhibits glucokinase in vitro and produces a glucose-specific impairment of in vivo insulin secretion in rats. Diabetes4311731179doi:10.2337/diabetes.43.10.1173.

    • Search Google Scholar
    • Export Citation
  • BeausoleilSAJedrychowskiMSchwartzDEliasJEVillenJLiJCohnMACantleyLCGygiSP2004Large-scale characterization of HeLa cell nuclear phosphoproteins. PNAS1011213012135doi:10.1073/pnas.0404720101.

    • Search Google Scholar
    • Export Citation
  • BordoneLMottaMCPicardFRobinsonAJhalaUSApfeldJMcDonaghTLemieuxMMcBurneyMSzilvasiA2005Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS Biology4e31doi:10.1371/journal.pbio.0040031.

    • Search Google Scholar
    • Export Citation
  • BordoneLCohenDRobinsonAMottaMCvan VeenECzopikASteeleADCroweHMarmorSLuoJ2007SIRT1 transgenic mice show phenotypes resembling calorie restriction. Aging Cell6759767doi:10.1111/j.1474-9726.2007.00335.x.

    • Search Google Scholar
    • Export Citation
  • BrownleeMCeramiAVlassaraH1988Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. New England Journal of Medicine31813151321doi:10.1056/NEJM198805193182007.

    • Search Google Scholar
    • Export Citation
  • ChenDBrunoJEaslonELinSJChengHLAltFWGuarenteL2008Tissue-specific regulation of SIRT1 by calorie restriction. Genes and Development2217531757doi:10.1101/gad.1650608.

    • Search Google Scholar
    • Export Citation
  • CivitareseAECarlingSHeilbronnLKHulverMHUkropcovaBDeutschWASmithSRRavussinE2007Calorie restriction increases muscle mitochondrial biogenesis in healthy humans. PLoS Medicine4e76doi:10.1371/journal.pmed.0040076.

    • Search Google Scholar
    • Export Citation
  • CohenHYMillerCBittermanKJWallNRHekkingBKesslerBHowitzKTGorospeMde CaboRSinclairDA2004Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science305390392doi:10.1126/science.1099196.

    • Search Google Scholar
    • Export Citation
  • ConsidineRVCookseyRCWilliamsLBFawcettRLZhangPAmbrosiusWTWhitfieldRMJonesRInmanMHuseJ2000Hexosamines regulate leptin production in human subcutaneous adipocytes. Journal of Clinical Endocrinology and Metabolism8535513556doi:10.1210/jc.85.10.3551.

    • Search Google Scholar
    • Export Citation
  • D'AlessandrisCAndreozziFFedericiMCardelliniMBrunettiARanalliMDel GuerraSLauroDDel PratoSMarchettiP2004Increased O-glycosylation of insulin signaling proteins results in their impaired activation and enhanced susceptibility to apoptosis in pancreatic beta-cells. FASEB Journal18959961doi:10.1096/fj.03-0725fje.

    • Search Google Scholar
    • Export Citation
  • DanielsMCCiaraldiTPNikoulinaSHenryRRMcClainDA1996Glutamine:fructose-6-phosphate amidotransferase activity in cultured human skeletal muscle cells: relationship to glucose disposal rate in control and non-insulin-dependent diabetes mellitus subjects and regulation by glucose and insulin. Journal of Clinical Investigation9712351241doi:10.1172/JCI118538.

    • Search Google Scholar
    • Export Citation
  • DaugaardMRohdeMJaattelaM2007The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Letters58137023710doi:10.1016/j.febslet.2007.05.039.

    • Search Google Scholar
    • Export Citation
  • DioumEMChenRAlexanderMSZhangQHoggRTGerardRDGarciaJA2009Regulation of hypoxia-inducible factor 2alpha signaling by the stress–responsive deacetylase sirtuin 1. Science32412891293doi:10.1126/science.1169956.

    • Search Google Scholar
    • Export Citation
  • EinsteinFHFishmanSBaumanJThompsonRFHuffmanDMAtzmonGBarzilaiNMuzumdarRH2008Enhanced activation of a "nutrient-sensing" pathway with age contributes to insulin resistance. FASEB Journal2234503457doi:10.1096/fj.08-109041.

    • Search Google Scholar
    • Export Citation
  • GagarinaVGabayODvir-GinzbergMLeeEJBradyJKQuonMJHallDJ2010SirT1 enhances survival of human osteoarthritic chondrocytes by repressing PTP1B and activating the IGF receptor pathway. Arthritis and Rheumatism6213831392doi:10.1002/art.27369.

    • Search Google Scholar
    • Export Citation
  • GaoYMiyazakiJHartGW2003The transcription factor PDX-1 is post-translationally modified by O-linked N-acetylglucosamine and this modification is correlated with its DNA binding activity and insulin secretion in min6 beta-cells. Archives of Biochemistry and Biophysics415155163doi:10.1016/S0003-9861(03)00234-0.

    • Search Google Scholar
    • Export Citation
  • GuarenteL2008Mitochondria – a nexus for aging, calorie restriction, and sirtuins?Cell132171176doi:10.1016/j.cell.2008.01.007.

  • GuinezCMirAMDehennautVCacanRHarduin-LepersAMichalskiJCLefebvreT2008Protein ubiquitination is modulated by O-GlcNAc glycosylation. FASEB Journal2229012911doi:10.1096/fj.07-102509.

    • Search Google Scholar
    • Export Citation
  • HanleySCAustinEAssouline-ThomasBKapelutoJBlaichmanJMoosaviMPetropavlovskaiaMRosenbergL2010{beta}-Cell mass dynamics and islet cell plasticity in human type 2 diabetes. Endocrinology15114621472doi:10.1210/en.2009-1277.

    • Search Google Scholar
    • Export Citation
  • HebertLFJrDanielsMCZhouJCrookEDTurnerRLSimmonsSTNeidighJLZhuJSBaronADMcClainDA1996Overexpression of glutamine:fructose-6-phosphate amidotransferase in transgenic mice leads to insulin resistance. Journal of Clinical Investigation98930936doi:10.1172/JCI118876.

    • Search Google Scholar
    • Export Citation
  • KanetoHXuGSongKHSuzumaKBonner-WeirSSharmaAWeirGC2001Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress. Journal of Biological Chemistry2763109931104doi:10.1074/jbc.M104115200.

    • Search Google Scholar
    • Export Citation
  • KanfiYPeshtiVGozlanYMRathausMGilRCohenHY2008Regulation of SIRT1 protein levels by nutrient availability. FEBS Letters58224172423doi:10.1016/j.febslet.2008.06.005.

    • Search Google Scholar
    • Export Citation
  • KimHSKimEMLeeJYangWHParkTYKimYMChoJW2006Heat shock protein 60 modified with O-linked N-acetylglucosamine is involved in pancreatic beta-cell death under hyperglycemic conditions. FEBS Letters58023112316doi:10.1016/j.febslet.2006.03.043.

    • Search Google Scholar
    • Export Citation
  • KonradRJMikolaenkoITolarJFLiuKKudlowJE2001The potential mechanism of the diabetogenic action of streptozotocin: inhibition of pancreatic beta-cell O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase. Biochemical Journal3563141doi:10.1042/0264-6021:3560031.

    • Search Google Scholar
    • Export Citation
  • LeeJHSongMYSongEKKimEKMoonWSHanMKParkJWKwonKBParkBH2009Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway. Diabetes58344351doi:10.2337/db07-1795.

    • Search Google Scholar
    • Export Citation
  • LinSJDefossezPAGuarenteL2000Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science28921262128doi:10.1126/science.289.5487.2126.

    • Search Google Scholar
    • Export Citation
  • MonauniTZentiMGCrettiADanielsMCTargherGCarusoBCaputoMMcClainDDel PratoSGiaccariA2000Effects of glucosamine infusion on insulin secretion and insulin action in humans. Diabetes49926935doi:10.2337/diabetes.49.6.926.

    • Search Google Scholar
    • Export Citation
  • MoynihanKAGrimmAAPluegerMMBernal-MizrachiEFordECras-MeneurCPermuttMAImaiS2005Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice. Cell Metabolism2105117doi:10.1016/j.cmet.2005.07.001.

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