INHIBITION OF GLYCOSAMINOGLYCAN SYNTHESIS AND PROTEIN GLYCOSYLATION WITH WAS-406 AND AZASERINE RESULT IN REDUCED ISLET AMYLOID FORMATION IN VITRO

doi:10.1152/ajpcell.00208.2007

Am J Physiol Cell Physiol. Author manuscript; available in PMC 2008 November 1.

Published in final edited form as:

Am J Physiol Cell Physiol. 2007 November; 293(5): C1586–C1593.

Published online 2007 September 5. doi: 10.1152/ajpcell.00208.2007

PMCID: PMC2365901

NIHMSID: NIHMS44854

INHIBITION OF GLYCOSAMINOGLYCAN SYNTHESIS AND PROTEIN GLYCOSYLATION WITH WAS-406 AND AZASERINE RESULT IN REDUCED ISLET AMYLOID FORMATION IN VITRO

Rebecca L. Hull,¹ Sakeneh Zraika,¹ Jayalakshmi Udayasankar,¹ Robert Kisilevsky,² Walter A. Szarek,³ Thomas N. Wight,⁴ and Steven E. Kahn¹

¹Division of Metabolism, Endocrinology and Nutrition, VA Puget Sound Health Care System and University of Washington, Seattle, WA

²Department of Pathology and Molecular Medicine and Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada

³Department of Chemistry, Queen’s University, Kingston, Ontario, Canada

⁴Hope Heart Program, Benaroya Research Institute at Virginia Mason and Department of Pathology, University of Washington, Seattle, WA

Correspondence to: Rebecca L. Hull, Ph.D., VA Puget Sound Health Care System (151), 1660 S. Columbian Way, Seattle, WA 98108, Email: rhull/at/u.washington.edu, Tel: 206-764-2897, Fax: 206-764-2164

The publisher's final edited version of this article is available free at Am J Physiol Cell Physiol

See other articles in PMC that cite the published article.

Keywords: islet amyloid, IAPP, heparan sulfate, glycosaminoglycan, proteoglycan, protein glycosylation, ß-cell mass, ß-cell dysfunction

SUMMARY

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in ~90% of individuals with type 2 diabetes and is associated with decreased islet ß-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet ß cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs). The small molecule 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-α-D-xylo-hexopyranose (WAS-406) inhibits HSPG synthesis in hepatocytes and blocks systemic amyloid A deposition in vivo.

To determine whether WAS-406 inhibits localized amyloid formation in the islet, we incubated hIAPP transgenic mouse islets for up to seven days in 16.7 mM glucose (conditions that result in amyloid deposition), plus increasing concentrations of the inhibitor. WAS-406, at doses of 0, 10, 100 and 1000 μM resulted in a dose-dependent decrease in amyloid deposition (% islet area occupied by amyloid: 0.66±0.14, 0.10±0.06, 0.09±0.07 and 0.004±0.003%, p<0.001) and an increase in ß-cell area in hIAPP transgenic islets (55.0±2.6 vs. 60.6±2.2 % islet area for 0 vs. 100 μM inhibitor, p=0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and non-transgenic islets (the latter a control that do not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in non-transgenic islets. Azaserine, an inhibitor of the rate limiting step of the hexosamine biosynthesis pathway, replicated the effects of WAS-406, resulting in reduction of O-linked protein glycosylation and glycosaminoglycan synthesis and inhibition of islet amyloid formation. In summary, interventions that decrease both glycosaminoglycan synthesis and O-linked protein glycosylation are effective in reducing islet amyloid formation, but their utility as pharmacological agents may be limited due to adverse effects on the islet.

Islet amyloid deposition is a pathogenic hallmark of the islet in type 2 diabetes, occurring in the vast majority of individuals with the disease (33), and is associated with decreased ß-cell mass and function (5, 32). The unique amyloidogenic component of islet amyloid is the ß-cell peptide islet amyloid polypeptide (IAPP, amylin) (6, 35). Human IAPP (hIAPP) is amyloidogenic and in vitro studies have shown that early aggregates or oligomers of hIAPP are cytotoxic, leading to ß-cell death via apoptosis (12, 22). In contrast, the rodent (rat and mouse) forms of IAPP differ from hIAPP in a number of critical amino acids, rendering rodent IAPP non-amyloidogenic and non-toxic (34). Due to these species-specific differences, several groups have produced transgenic mice expressing hIAPP in their pancreatic islet ß-cells in order to create models of islet amyloid deposition. In our colony of hIAPP transgenic mice, male mice develop islet amyloid deposits in vivo following one year of high fat feeding (29). We have also recently developed a rapid in vitro model of islet amyloid deposition by culturing isolated islets from our hIAPP transgenic mice in high glucose for seven days (10, 38).

Besides the amyloidogenic peptide IAPP, islet amyloid contains other components that are common to all amyloidoses, including those formed in Alzheimer’s disease (Aß amyloid) and chronic inflammation (AA amyloid). These include apolipoprotein E (4), serum amyloid P component (24) and heparan sulfate proteoglycans (HSPGs) (36), all of which may contribute to hIAPP amyloidogenesis and its related cytotoxicity.

HSPGs in particular may play a role in islet amyloidogenesis. The HSPG perlecan has been shown to be present in human ß cells from individuals with and without type 2 diabetes (13) and the ß cell synthesizes several HSPGs that are capable of binding amyloidogenic hIAPP but not non-amyloidogenic rodent IAPP (25). Further, binding of amyloidogenic peptides, including IAPP, to HSPGs via their heparan sulfate (HS) glycosaminoglycan (GAG) chains has been shown to stimulate amyloid fibril formation (2, 3). Thus, HSPGs may play a critical role in islet amyloid formation and evidence that decreasing GAG synthesis reduces islet amyloid formation would provide further evidence to support this hypothesis.

We have generated a series of N-acetylglucosamine analogs that act as small molecule inhibitors of GAG synthesis (17-20). These compounds are effective in reducing amyloid formation in a mouse model of AA amyloidosis (20), and in a transgenic mouse model of CNS Aβ amyloid (16). In the present study we examined the effect of one of these compounds, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-α-D-xylo-hexopyranose (WAS-406), on ß-cell GAG synthesis and on islet amyloid formation in vitro.

Precursors for GAG synthesis are synthesized via the hexosamine biosynthesis pathway (HBP). The HBP is a nutrient sensing pathway that has many additional effects in the cell including regulation of O-linked protein glycosylation. Azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of the HBP, has previously been shown to have no effect on GAG synthesis in arterial smooth muscle cells (28). Therefore, we compared the effects of azaserine and WAS-406 on GAG synthesis and islet amyloid formation in vitro.

RESEARCH DESIGN AND METHODS

Isolation and Culture of Mouse Islets

Hemizygous hIAPP transgenic and non-transgenic mice on a F1 C57BL/6 × DBA/2 background were bred and utilized under protocols approved by the Institutional Animal Care and Use Committee of the VA Puget Sound Health Care System. Eight to ten-week old male and female mice were anesthetized with sodium pentobarbital (100 mg/kg i.p.), and pancreatic islets were isolated by collagenase digestion (Collagenase P, 0.5 mg/ml, Roche Applied Science, Indianapolis, IN) via bile duct cannulation, followed by pancreas excision. Islets were purified on a Histopaque gradient, hand picked and cultured overnight in RPMI-1640 containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 11.1 mM glucose. Islets were then cultured for up to seven days in medium containing 16.7 mM glucose alone or together with WAS-406 (10 - 1000 μM for dose response studies, 100 μM thereafter). In a subset of experiments, islets were also cultured in the presence of the HBP inhibitor azaserine (20 μM). Stock solutions of WAS-406 (100 mM) and azaserine (20 mM) were prepared in sterile water, and then diluted 1:1000 in culture medium. Culture medium was changed, and thus new compound was supplied to islets, every 48 hours.

Histological Determination of Islet Amyloid and ß-cell Area

At the end of each experiment, islets were fixed in 4% (w/v) phosphate-buffered paraformaldehyde. Islets were embedded in agar, re-fixed in 4% paraformaldehyde, embedded in paraffin and processed for histology. Five-μm sections were cut throughout the islet pellet and sections at 100 μm intervals were stained for amyloid with thioflavin S and for insulin as previously described (9). Islet area was determined by circumscribing each islet image with a video cursor; the outline of islets is clearly visible when viewed in the thioflavin S channel. Islet-, thioflavin S-positive- and insulin-positive areas were determined for each islet cross-section in an average of 25 islets per experimental condition. From these data, the following measures were determined: islet amyloid prevalence (% islets containing thioflavin S positive staining), islet amyloid severity (Σ thioflavin S area / Σ islet area × 100%) and ß-cell area (Σ insulin area / Σ islet area × 100%).

Insulin Secretion and Content

Insulin secretion was determined following seven days of culture. Islets (100-150 per condition) were loaded into a perifusion chamber and were pre-incubated in Krebs-Ringer bicarbonate buffer containing basal (1.67 mM) glucose for one hour, followed by measurement of basal insulin secretion at 1.67 mM glucose every 2 minutes for 8 minutes. Glucose-stimulated insulin secretion was then assessed by perifusion of islets with Krebs-Ringer bicarbonate buffer containing 16.7 mM glucose for 30 minutes (fractions collected every 2-5 minutes). Glucose-stimulated insulin secretion was expressed as the incremental area under the curve. Secretion data from these studies using only the hIAPP transgenic and non-transgenic islets cultured for seven days in 16.7 mM alone have been previously published (38).

For determination of insulin content islets were solubilized in acid ethanol (0.2 M HCl, 48 % (v/v) ethanol). Total protein was determined (Coomassie Plus Protein Assay; Pierce Biotechnology, Rockford, IL) and insulin content was measured by radioimmunoassay as described previously (10).

Islet Cell Viability

Islet cell viability after seven days of culture was assessed using the Cell Proliferation Kit I (Roche) according to the manufacturer’s instructions. Twenty islets/well (triplicates for each condition) were incubated for four hours in culture medium containing 0.5mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Islets were solubilized by overnight incubation in solubilization buffer and solubilized formazan was quantified spectrophotometrically (590 nm).

Assessment of Glycosaminoglycan Synthesis in Islets and ß-TC3 cells

Islets were isolated from hIAPP transgenic and non-transgenic mice and recovered overnight as described above. The immortalized islet ß-cell line ß-TC3 was plated at 1.2×10⁶ cells/ml and cultured for 5 days prior to study in DMEM containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 5.5 mM glucose. Islets (450 per plate) and ß-TC3 cells were then metabolically labeled for 48 hours with [³⁵S]-Na₂SO₄ (100 μCi/ml; MP Biomedicals, Costa Mesa, CA) in medium containing 16.7 mM glucose alone or together with WAS-406 (100 μM for islets and 10 - 1000 μM for ß-TC3 cells) or azaserine (20 μM; in a subset of experiments). This approach labels newly synthesized GAG chains on proteoglycans.

Incorporation of [³⁵S]-Na₂SO₄ into GAGs was assessed in combined medium + cell preparations by cetylpyridinium chloride precipitation (31). Labeled media were collected in the presence of protease inhibitors (5 mM benzamidine, 100 mM 6-aminohexanoic acid, and 0.1 mM phenylmethylsulfonyl fluoride) and labeled islets/cells were solubilized in 8 M urea buffer (8 M urea, 2 mM EDTA, 0.25 M NaCl, 50 mM Tris-HCl, and 0.5% Triton-X 100 detergent, pH 7.4) containing protease inhibitors.

To determine the effects of WAS-406 to reduce GAG synthesis and its specificity for HS versus chondroitin/dermatan sulfate (CS/DS), labeled glycosaminoglycans were purified from ß-TC3 cells. [³⁵S]-Na₂SO₄ labeling was performed as described above and cells solubilized in phosphate-buffered saline (PBS, pH 7.2) containing 1% (v/v) Triton X-100, pooled together with medium and incubated with pronase (Streptomyces griseus 100 μg/ml, Roche) overnight at 37°C. GAGs were isolated by application to DEAE Sephacel equilibrated in PBS and eluted over a 0.15 - 0.8 M sodium chloride gradient. Residual core protein fragments were removed by alkaline elimination and borohydride reduction and the resulting glycosaminoglycan preparations digested with no enzyme (control), heparinase I, II + III (0.8, 0.4 and 0.8 U per digestion respectively, Sigma St. Louis, MO), chondroitinase ABC (0.03 U per digestion, Seikagaku USA, Cape Cod, MA) or a combination of heparinase I, II, III + chondroitinase ABC for three hours at 37°C. Reaction products were analyzed by molecular sieve chromatography (Sepharose CL-6B, equilibrated in 0.2 M Tris, 0.2 M NaCl, pH 7.0).

Western Blotting

Islet lysates (75 islets per culture condition) were prepared by sonication on ice in 20 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Nonidet P-40, pH 7.5 followed by centrifugation for 20 minutes at 12,000g. Total protein concentration was determined using the Coomassie Plus Protein Assay (Pierce Biotechnology) and from this equal protein (15 μg) was loaded for each sample. Samples were separated by SDS-PAGE, transferred to polyvinylidene fluoride membrane and non-specific binding blocked by incubation in 50 mM Tris-HCl, 150 mM NaCl, 0.1% (v/v) Tween-20 and 5% (w/v) non-fat dry milk, pH 7.5. Membranes were probed with antisera directed against O-linked N-acetylglucosamine (RL2, Affinity BioReagents, Golden, CO; 1:100 dilution). Primary antibody binding was detected using peroxidase-conjugated anti-mouse IgGs followed by enhanced chemiluminescence (PerkinElmer, Wellesley, MA).

Data Analysis

Data are expressed as mean ± SEM. For MTT islet viability, data are presented as % of non-transgenic islets cultured in 16.7 mM glucose due to variability among experiments resulting from the use of different lots of MTT kits. For metabolic labeling experiments n=2-3 proteoglycan preparations; for all other experiments n=3-8 islet preparations. Dose response data were compared by analysis of variance with Bonferroni post-hoc analysis. Two group comparisons were made by T-test or Mann Whitney U non-parametric test if the data were not normally distributed. A p≤0.05 was considered statistically significant.

RESULTS

Dose-Dependent Inhibition of Islet Amyloid Formation by WAS-406

Incubation of isolated hIAPP transgenic islets for seven days in 16.7 mM glucose and increasing concentrations of WAS-406 was associated with a marked, dose-dependent decrease in both the prevalence (Figure 1A) and severity (Figure 1B) of islet amyloid formation.

Figure 1

Dose-dependent decrease in islet amyloid prevalence (% islets with amyloid; panel A) and severity (% islet area occupied by amyloid; panel B) in hIAPP transgenic islets cultured for 7 days in the presence of increasing concentrations of WAS-406. n=4-8 (more ...)

Effect of WAS-406 on ß-cell Area, Insulin Secretion, Insulin Content and Islet Viability

Based on the dose-response study, a dose of 100 μM of WAS-406 was selected for further assessments of islet morphology. The reduction in amyloid formation observed in hIAPP transgenic islets in the presence of WAS-406 was associated with an increase in ß-cell area as a proportion of islet area (Figure 2A). In contrast, the proportion of islet area comprised of ß-cells was not significantly altered in non-transgenic islets cultured in the presence or absence of 100 μM WAS-406 (Figure 2A). Islet area did not differ among treatment conditions or between hIAPP transgenic and non-transgenic islets (data not shown). Islet insulin content was not different in hIAPP transgenic islets compared to non-transgenic islets cultured in 16.7 mM glucose alone (Figure 2B). However, WAS-406 treatment resulted in a significant increase in insulin content in both hIAPP transgenic and non-transgenic islets (Figure 2B). Glucose-stimulated insulin secretion (n=4 per condition) was not significantly different with WAS-406 treatment in hIAPP transgenic islets (11.1±2.8 vs. 18.0±6.9 μU/islet.30 minutes for untreated vs. WAS-406) or non-transgenic islets (15.2±1.3 vs. 22.2±3.2 μU/islet.30 minutes for untreated vs. WAS-406, p=0.1). Islet viability was significantly lower in hIAPP transgenic islets compared to non-transgenic islets cultured in 16.7 mM glucose alone (Figure 2C) as we have previously shown (38), and WAS-406 treatment was associated with a tendency towards a reduction in viability in non-transgenic islets (p=0.065) but no change in viability in hIAPP transgenic islets.

Figure 2

Incubation of hIAPP transgenic (hIAPP) islets for seven days in the presence of 100 μM WAS-406 resulted in a significant increase in ß-cell area as a proportion of islet area compared to hIAPP transgenic islets cultured in the absence (more ...)

WAS-406 Decreases Glycosaminoglycan Synthesis in ß-TC3 Cells and Islets

WAS-406 decreased incorporation of [³⁵S]-sulfate into newly synthesized GAGs in a dose-dependent manner in immortalized ß-TC3 cells (Figure 3A). GAG synthesis was reduced by 62% in ß-TC3 cells incubated with 100 μM WAS-406 (p<0.001 vs. untreated cells). GAG synthesis during a 48-hour label was similarly reduced with WAS-406 (100 μM) by 66% in both hIAPP transgenic and non-transgenic islets (Figure 3B). Ion exchange chromatography showed that ß-TC3 cell GAGs synthesized in the presence and absence of WAS-406 eluted from DEAE Sephacel at a similar salt concentration (0.45 M NaCl; Figure 3C), although, as expected, the abundance of GAGs synthesized in the presence of WAS-406 was much reduced compared to untreated cells.

Figure 3

Analysis of glycosaminoglycan synthesis during a 48-hour incubation with WAS-406. Incorporation of [³⁵S]-sulfate into newly synthesized GAGs was assessed by cetylpyridinium chloride precipitation (panels A and B). WAS-406 treatment resulted in a dose-dependent (more ...)

WAS-406 Inhibits Heparan and Chondroitin/Dermatan Sulfate Synthesis in ß-TC3 Cells

To determine whether WAS-406 was specific for inhibition of heparan sulfate synthesis, equal numbers of counts of ³⁵S-sulfate-labeled GAGs synthesized by ß-TC3 cells in the presence or absence of WAS-406 were digested with no enzyme (intact GAG control), heparinases I, II and III (to digest HS), chondroitinase ABC (to digest CS/DS) or the combination of heparinases I, II, III + chondroitinase ABC. If WAS-406 were specific for inhibition of HS synthesis, the residual GAGs synthesized in the presence of this compound would be expected to be insensitive to heparinase digestion, but sensitive to chondroitinase ABC treatment.

Analysis of reaction products by CL-6B Sepharose size exclusion chromatography showed that intact GAGs were of a similar size in control (Figure 4A) and WAS-406 (Figure 4E)-treated ß-TC3 cells. As we showed previously, ß-TC3 cells synthesize predominantly HS (25); thus a greater proportion of GAGs synthesized in control cells (without WAS-406) were sensitive to heparinase I, II and III digestion (Figure 4B; shaded area denotes digested material, in this case heparinase-sensitive material) than to chondroitinase ABC digestion (Figure 4C). As expected, no intact GAGs were present following digestion with the combination of heparinase I, II and III and chondroitinase ABC (Figure 4D). ß-TC3 GAGs synthesized in the presence of WAS-406 contained a similar proportion of heparinase I, II and III sensitive material (Figure 4F) and chondroitinase ABC sensitive material (Figure 4G) compared to control cells, and all material was sensitive to the combination of heparinase I, II and III and chondroitinase ABC treatment (Figure 4H).

Figure 4

Sepharose CL-6B size-exclusion analysis of [³⁵S]-sulfate-labeled ß-TC3 GAGs synthesized in 16.7 mM glucose alone (panels A-D) or together with WAS-406 (100 μM, panels E-H). GAGs were treated with no enzyme (panels A and E), heparinase (more ...)

WAS-406 Decreases O-linked Protein Glycosylation

Since WAS-406 is an analog of N-acetylglucosamine, an intermediate of the HBP, we analyzed flux through the HBP by measuring O-linked protein glycosylation in islets treated for seven days with 16.7 mM glucose alone or with WAS-406 (100 μM) or the HBP inhibitor azaserine (20 μM) as a positive control. WAS-406 and azaserine treatment in both non-transgenic islets (Figure 5) and hIAPP transgenic islets (not shown) resulted in a decrease in O-linked protein glycosylation as demonstrated by a reduced number of glycosylated proteins detected using an anti-O-linked N-acetylglucosamine antibody. The magnitude of the effect to decrease O-linked protein glycosylation was greater with WAS-406 than with azaserine.

Figure 5

Western blot showing O-linked protein glycosylation in non-transgenic islets cultured for seven days in the presence or absence of 100 μM WAS-406 or 20 μM azaserine. Several O-linked N-acetylglucosamine-modified proteins are detected in (more ...)

Effect of the HBP Inhibitor Azaserine on Proteoglycan Synthesis and Islet Amyloid Deposition

Given the similar effects of WAS-406 and azaserine to decrease O-linked protein glycosylation, but the reported lack of an effect of azaserine to reduce GAG synthesis in arterial smooth muscle cells (28), we sought to determine the effect of azaserine on ß-cell GAG synthesis and on islet amyloid formation. Unexpectedly, azaserine treatment in ß-TC3 cells was associated with a significant reduction in GAG synthesis as determined by cetylpyridinium chloride precipitation (Figure 6A) and elution from DEAE Sephacel over a 0.15-0.8 M NaCl gradient (Figure 6B). Furthermore, incubation of hIAPP transgenic islets for seven days in 16.7 mM glucose and in the presence of azaserine (20 μM) was associated with a marked reduction in islet amyloid prevalence (78±5 vs 10±6 % in untreated vs treated islets, p<0.01) and islet amyloid severity (Figure 6C) and with an increase in the proportion of ß-cell area to islet area (Figure 6D).

Figure 6

Analysis of glycosaminoglycan synthesis in ß-TC3 cells during a 48-hour incubation with or without azaserine (20 μM). Azaserine reduced incorporation of [³⁵S]-sulfate into newly synthesized proteoglycans from ß-TC3 cells, assessed (more ...)

DISCUSSION

We have shown, for the first time that interventions that decrease GAG synthesis and protein glycosylation in islets are capable of inhibiting islet amyloid deposition and preventing the amyloid-induced loss of ß-cell area in vitro. In the present study, WAS-406 and azaserine reduced both GAG synthesis and flux through the HBP, measured as O-linked protein glycosylation, resulting in a marked decrease in islet amyloid deposition.

While WAS-406 has been shown to be effective in reducing other amyloidoses (16, 20), it was not clear that the same would hold true for islet amyloid formation. In the present study, we found that incubation of hIAPP transgenic islets with this compound resulted in a marked, dose-dependent decrease in both the prevalence and severity of islet amyloid; and this was associated with an increase in ß-cell area. We also found that WAS-406 was effective in reducing [³⁵S]-sulfate incorporation into GAGs synthesized both by intact primary islets and by immortalized ß-TC3 cells, in line with previous data that this compound also decreased GAG synthesis in hepatocytes; reducing both [³⁵S]-sulfate and [³H]-glucosamine incorporation into glycosaminoglycans (20). In ß-TC3 cells, residual GAGs synthesized in the presence of WAS-406 were sensitive to both heparinase and chondroitinase treatment, with the proportions of HS and CS/DS being similar to control cells. These data suggest that WAS-406 is not specific for HS synthesis, which is not unexpected, given that the CS/DS disaccharide constituent N-acetylgalactosamine is formed by the epimerization of N-acetyglucosamine (a component of the repeating disaccharide in HS) and that levels of these two compounds exist in equilibrium, such that any intervention that perturbs the levels of one compound would also be expected to affect levels of the other. Since HS and CS/DS are important components of the cell and extracellular matrix, it is possible that the effect of decreased GAG synthesis with WAS-406 may have confounded the results of the present study. The inclusion of non-transgenic islets treated with WAS-406 allowed us to distinguish the effects of these compounds to decrease amyloid formation from other effects due to decreased GAG synthesis, but the potential adverse effects of long term use of WAS-406 may limit its utility as a pharmacological agent, as described in more detail below.

Previously, we have shown that a radiolabeled analog of N-acetylglucosamine, similar to WAS-406, can be incorporated into growing GAG chains, blocking their extension (20). It is possible that WAS-406 acts via this mechanism in ß cells, although in the present study we did not evaluate whether it was directly incorporated into GAGs. Alternatively, WAS-406 may reduce islet GAG synthesis via down-regulation of the HBP, the biosynthetic pathway responsible for N-acetyglucosamine and N-acetylgalactosamine synthesis. The observed marked decrease in O-linked protein glycosylation following incubation of islets for seven days with WAS-406 is consistent with down-regulation of the HBP.

Unexpectedly, incubation of islets with azaserine also resulted in a reduction of GAG synthesis in ß-TC3 cells. This differs from a previous study showing no effect of azaserine in the presence of high glucose to alter GAG synthesis in arterial smooth muscle cells (28). This may be due to the fact that, in that cell type, substrate supply through the HBP was not rate limiting for GAG synthesis at high glucose. However, increasing glucose concentrations have been associated with increased HBP activity in other cell types (7) and this is likely also the case in ß cells, since ß-cell glucose metabolism is regulated by the high Km enzyme glucokinase. Thus, as our data suggest, azaserine would be predicted to be more effective at inhibiting GAG synthesis at high glucose in ß cells than in smooth muscle cells. The decreases in GAG synthesis and O-linked protein glycosylation with azaserine were smaller in magnitude than those seen with WAS-406, which is likely a result of the lower dose of azaserine (20 μM vs. 100 μM for WAS-406). This lower dose of azaserine was chosen based on previous studies in mouse islets (37), with a higher dose being avoided due to concerns of toxicity. However, this lower dose of azaserine was extremely effective in inhibiting islet amyloid formation, suggesting that modulation of flux through the HBP is important for islet amyloid formation.

While it is possible that the effects of WAS-406 and azaserine to downregulate HBP flux in general, rather than a specific effect on GAG synthesis, may explain their ability to reduce islet amyloid formation, other lines of evidence support our hypothesis that decreased GAG, and particularly HS, synthesis is critical for the deposition of amyloid. First, several in vitro studies have shown that the HSPG perlecan, HS and the related glycosaminoglycan heparin can bind amyloidogenic peptides including hIAPP (2, 3, 25) and that this interaction leads to increased amyloid fibril formation (2, 3). HS is more effective in increasing hIAPP fibril formation than CS/DS (2), suggesting a specific effect for HS in fibril formation. More direct evidence comes from studies using a murine model of AA amyloidosis together with over-expression of the enzyme heparanase that results in in vivo fragmentation of HS, but does not target CS/DS (21). Transgenic mice overexpressing heparanase showed marked resistance to hepatic and renal amyloid deposition but developed amyloid deposition in the spleen in a model of AA amyloidosis. The differential organ sensitivity in this transgenic mouse was inversely correlated with the organ expression of the transgene. That is, the heparanase transgene and its protein were expressed in kidney and liver but not the spleen. Thus, in the same animal fragmentation of HS was observed in kidney and liver but not the spleen rendering only the spleen sensitive to AA amyloid deposition. This argues for a clear role of HS in amyloidogenesis.

Our observation that decreasing glycosaminoglycan synthesis with an N-acetylglucosamine analog results in reduced islet amyloid formation is in keeping with findings that these compounds reduce amyloid formation in mouse models of AA and CNS amyloidosis (16, 20). However, while these effects were similar and amyloid deposits derived from different amyloidogenic peptides contain several invariant components, it is important to recognize that the role of these components in amyloid formation is not equivalent among different amyloidoses. For example, apolipoprotein E is important in the development of Aß amyloid, as shown by delayed and reduced amyloid deposition in a mouse model of Aß deposition lacking one or both apolipoprotein E alleles (1). In contrast, we found that while apolipoprotein E is a component of islet amyloid, apolipoprotein E deficiency had no effect on islet amyloid formation in human IAPP transgenic mice (30). Further, we found that apolipoprotein E is not synthesized by islet ß-cells (30), suggesting that it may be trapped during the deposition of islet amyloid, perhaps by binding to proteoglycans, but that it appears to have no causative role in islet amyloidogenesis.

While we have observed that WAS-406 and azaserine are capable of decreasing amyloid formation, an important aspect of our findings is that they highlight the need for specificity in the development of amyloid inhibitors. While increased flux through the HBP has been shown to be detrimental to the ß cell (14, 27), protein glycosylation is critical for normal cellular function, such that decreased protein glycosylation is associated with impaired insulin secretion (37). Thus, the effects of chronic down-regulation of the HBP by WAS-406 or azaserine may have effects on the cell that are unrelated to their ability to reduce islet amyloid formation and may be detrimental to the ß cell. Islet viability tended to be decreased in non-transgenic islets treated with WAS-406, and islet viability did not improve when islet amyloid was inhibited with WAS-406 in contrast to our findings with other amyloid inhibitors (Zraika S et al, unpublished observation), consistent with a detrimental effect of WAS-406 on islet viability. In contrast, insulin content was significantly increased in both hIAPP transgenic and non-transgenic islets with WAS-406 treatment, a finding seemingly at odds with the viability data. However, glucose-stimulated insulin secretion was not significantly altered (in islets from either genotype) in the presence of WAS-406, despite this significant increase in insulin content. Thus, we speculate that the increase in insulin content may occur, at least in part, due to a failure to adequately increase insulin secretion following WAS-406 treatment, consistent with a role of WAS-406 to decrease ß-cell secretory function and impair islet viability.

Even in the absence of down-regulation of the HBP, systemic down-regulation of GAG synthesis would also be expected to have detrimental effects on the cell, as GAGs at the cell surface and in the extracellular matrix are important for sequestration and signaling of growth factors and chemokines (11). Thus, alternative approaches to reduce islet amyloid deposition have to be pursued. To date these have mainly targeted the amyloidogenic peptide IAPP. Several groups have described peptide-based inhibitors that are effective at reducing fibril formation and/or cytotoxicity of synthetic hIAPP (15, 26). However, the bioavailability and thus applicability of these compounds have not been studied in models of de novo islet amyloid formation. Similarly, small molecules such as Congo red and rifampicin have also been shown to reduce hIAPP fibril formation (8, 23), but further work is required to investigate their toxicity and/or efficacy in long-term studies.

In summary, our data demonstrate that small molecules such as WAS-406 and azaserine are highly effective inhibitors of islet amyloid formation. However, further studies with more specific interventions will be required to definitively prove that HS is required for islet amyloid deposition. Understanding of the actions of compounds that are capable of inhibiting islet amyloid will be useful in developing therapeutic interventions to reduce amyloid formation that occurs as part of the islet lesion in type 2 diabetes.

ACKNOWLEDGEMENTS

We thank Jeanette Teague, Rebekah Koltz, Rahat Bhatti, Robin Vogel, Shani Wilbur and Michael Peters for excellent technical support. We are grateful to Michael Kinsella for helpful discussion in the preparation of this manuscript. This work was supported by the Department of Veterans Affairs (SEK), NIH grants DK17047 (RLH), DK74404 (RLH) and RR16066 (SEK) and the American Diabetes Association (SEK). Preparation and development of WAS-406 was funded by the Canadian Institutes for Health Research grant MOP-3153 (RK), the Natural Sciences and Engineering Research Council of Canada (WAS) and the Institute for the Study of Aging (RK and WAS)

Abbreviations


CS	chondroitin sulfate

DS	dermatan sulfate

DEAE	diethylaminoethyl

GAG	glycosaminoglycan

hIAPP	human islet amyloid polypeptide

HBP	hexosamine biosynthesis pathway

HS	heparan sulfate

HSPG	heparan sulfate proteoglycan

IAPP	islet amyloid polypeptide

MTT	3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

WAS-406	2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-α-D-xylo-hexopyranose

REFERENCES

1. Bales KR, Verina T, Dodel RC, Du Y, Altstiel L, Bender M, Hyslop P, Johnstone EM, Little SP, Cummins DJ, Piccardo P, Ghetti B, Paul SM. Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition. Nat Genet. 1997;17:263–264. [PubMed]

2. Castillo GM, Cummings JA, Yang WH, Judge ME, Sheardown MJ, Rimvall K, Hansen JB, Snow AD. Sulfate content and specific glycosaminoglycan backbone of perlecan are critical for perlecan’s enhancement of islet amyloid polypeptide (amylin) fibril formation. Diabetes. 1998;47:612–620. [PubMed]

3. Castillo GM, Ngo C, Cummings J, Wight TN, Snow AD. Perlecan binds to the beta-amyloid proteins (A beta) of Alzheimer’s disease, accelerates A beta fibril formation, and maintains A beta fibril stability. J Neurochem. 1997;69:2452–2465. [PubMed]

4. Chargé SBP, Esiri MM, Bethune CA, Hansen BC, Clark A. Apolipoprotein-E is associated with islet amyloid and other amyloidoses - implications for Alzheimer’s disease. J Pathol. 1996;179:443–447. [PubMed]

5. Clark A, Wells CA, Buley ID, Cruickshank JK, Vanhegan RI, Matthews DR, Cooper GJS, Holman RR, Turner RC. Islet amyloid, increased A-cells, reduced B-cells and exocrine fibrosis - quantitative changes in the pancreas in type-2 diabetes. Diab Res Clin Exptl. 1988;9:151–159.

6. Cooper GJS, Willis AC, Clark A, Turner RC, Sim RB, Reid KBM. Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients. Proc Natl Acad Sci USA. 1987;84:8628–8632. [PubMed]

7. Du XL, Edelstein D, Rossetti L, Fantus IG, Goldberg H, Ziyadeh F, Wu J, Brownlee M. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation. Proc Natl Acad Sci USA. 2000;97:12222–12226. [PubMed]

8. Harroun TA, Bradshaw JP, Ashley RH. Inhibitors can arrest the membrane activity of human islet amyloid polypeptide independently of amyloid formation. FEBS Lett. 2001;507:200–204. [PubMed]

9. Hull RL, Andrikopoulos S, Verchere CB, Vidal J, Wang F, Cnop M, Prigeon RL, Kahn SE. Increased dietary fat promotes islet amyloid formation and ß-cell secretory dysfunction in a transgenic mouse model of islet amyloid. Diabetes. 2003;52:372–379. [PubMed]

10. Hull RL, Shen Z, Watts MR, Kodama K, Carr DB, Utzschneider KM, Zraika S, Wang F, Kahn SE. Long term treatment with rosiglitazone and metformin reduce the extent of, but do not prevent, islet amyloid deposition in mice expressing the gene for human islet amyloid polypeptide. Diabetes. 2005;54:2235–2244. [PubMed]

11. Iozzo RV. Basement membrane proteoglycans: from cellar to ceiling. Nat Rev Mol Cell Biol. 2005;6:646–656. [PubMed]

12. Janson J, Ashley RH, Harrison D, McIntyre S, Butler PC. The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles. Diabetes. 1999;48:491–498. [PubMed]

13. Kahn SE, Andrikopoulos S, Verchere CB. Islet amyloid: A long-recognized but underappreciated pathological feature of type 2 diabetes. Diabetes. 1999;48:241–253. [PubMed]

14. Kaneto H, Xu G, Song KH, Suzuma K, Bonner-Weir S, Sharma A, Weir GC. Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress. J Biol Chem. 2001;276:31099–31104. [PubMed]

15. Kapurniotu A, Schmauder A, Tenidis K. Structure-based design and study of non-amyloidogenic, double N- methylated IAPP amyloid core sequences as inhibitors of IAPP amyloid formation and cytotoxicity. J Mol Biol. 2002;315:339–350. [PubMed]

16. Kisilevsky R, Ancsin JB, Szarek WA, Petanceska S. Heparan sulfate as a therapeutic target in amyloidogenesis: prospects and possible complications. Amyloid. 2007;14:21–32. [PubMed]

17. Kisilevsky R, Szarek WA. Novel glycosaminoglycan precursors as anti-amyloid agents. In: Fillit HM, O’Connell AW, editors. Drug Discovery and Development for Alzheimer’s Disease 2000. Springer; New York: 2002. pp. 98–105.

18. Kisilevsky R, Szarek WA. Novel glycosaminoglycan precursors as anti-amyloid agents part II. J Mol Neurosci. 2002;19:45–50. [PubMed]

19. Kisilevsky R, Szarek WA, Ancsin J, Bhat S, Li Z, Marone S. Novel glycosaminoglycan precursors as anti-amyloid agents, part III. J Mol Neurosci. 2003;20:291–297. [PubMed]

20. Kisilevsky R, Szarek WA, Ancsin JB, Elimova E, Marone S, Bhat S, Berkin A. Inhibition of amyloid A amyloidogenesis in vivo and in tissue culture by 4-deoxy analogues of peracetylated 2-acetamido-2-deoxy-alpha- and beta-d-glucose: implications for the treatment of various amyloidoses. Am J Pathol. 2004;164:2127–2137. [PubMed]

21. Li JP, Galvis ML, Gong F, Zhang X, Zcharia E, Metzger S, Vlodavsky I, Kisilevsky R, Lindahl U. In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis. Proc Natl Acad Sci USA. 2005;102:6473–6477. [PubMed]

22. Lorenzo A, Razzaboni B, Weir GC, Yankner BA. Pancreatic islet cell toxicity of amylin associated with type 2 diabetes mellitus. Nature. 1994;368:756–760. [PubMed]

23. Meier JJ, Kayed R, Lin CY, Gurlo T, Haataja L, Jayasinghe S, Langen R, Glabe CG, Butler PC. Inhibition of human IAPP fibril formation does not prevent beta-cell death: evidence for distinct actions of oligomers and fibrils of human IAPP. Am J Physiol Endocrinol Metab. 2006;291:E1317–E1324. [PubMed]

24. Ohsawa H, Kanatsuka A, Tokuyama Y, Yamaguchi T, Makino H, Yoshida S, Horie H, Mikata A, Kohen Y. Amyloid protein in somatostatinoma differs from human islet amyloid polypeptide. Acta Endocrinologica. 1991;124:45–53. [PubMed]

25. Potter-Perigo S, Hull RL, Tsoi C, Braun KR, Andrikopoulos S, Teague JC, Verchere CB, Kahn SE, Wight TN. Proteoglycans synthesized and secreted by pancreatic islet ß-cells bind amylin. Arch Biochem Biophys. 2003;413:182–190. [PubMed]

26. Scrocchi LA, Chen Y, Waschuk S, Wang F, Cheung S, Darabie AA, McLaurin J, Fraser PE. Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis. J Mol Biol. 2002;318:697–706. [PubMed]

27. Tang J, Neidigh JL, Cooksey RC, McClain DA. Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance. Diabetes. 2000;49:1492–1499. [PubMed]

28. Tannock LR, Little PJ, Wight TN, Chait A. Arterial smooth muscle cell proteoglycans synthesized in the presence of glucosamine demonstrate reduced binding to LDL. J Lipid Res. 2002;43:149–157. [PubMed]

29. Verchere CB, D’Alessio DA, Palmiter RD, Weir GC, Bonner-Weir S, Baskin DG, Kahn SE. Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide. Proc Natl Acad Sci USA. 1996;93:3492–3496. [PubMed]

30. Vidal J, Verchere CB, Andrikopoulos S, Wang F, Hull RL, Cnop M, Olin KL, LeBoeuf RC, O’Brien KD, Chait A, Kahn SE. The effect of apolipoprotein E deficiency on islet amyloid deposition in human islet amyloid polypeptide transgenic mice. Diabetologia. 2003;46:71–79. [PubMed]

31. Wasteson A, Uthne K, Westermark B. A novel assay for the biosynthesis of sulphated polysaccharide and its application to studies on the effects of somatomedin on cultured cells. Biochem J. 1973;136:1069–1074. [PubMed]

32. Westermark P. Amyloid and polypeptide hormones - what is their interrelationship? Amyloid. 1994;1:47–60.

33. Westermark P. Quantitative studies on amyloid in the islets of Langerhans. Ups J Med Sci. 1972;77:91–94. [PubMed]

34. Westermark P, Engström U, Johnson KH, Westermark GT, Betsholtz C. Islet amyloid polypeptide - pinpointing amino acid residues linked to amyloid fibril formation. Proc Natl Acad Sci USA. 1990;87:5036–5040. [PubMed]

35. Westermark P, Wernstedt C, Wilander E, Hayden DW, O’Brien TD, Johnson KH. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc Natl Acad Sci USA. 1987;84:3881–3885. [PubMed]

36. Young ID, Ailles L, Narindrasorasak S, Tan R, Kisilevsky R. Localization of the basement membrane heparan sulfate proteoglycan in islet amyloid deposits in Type-II diabetes mellitus. Arch Path Lab Med. 1992;116:951–954. [PubMed]

37. Zraika S, Dunlop M, Proietto J, Andrikopoulos S. The hexosamine biosynthesis pathway regulates insulin secretion via protein glycosylation in mouse islets. Arch Biochem Biophys. 2002;405:275–279. [PubMed]

38. Zraika S, Hull RL, Udayasankar J, Utzschneider KM, Tong J, Gerchman F, Kahn SE. Glucose- and time-dependence of islet amyloid formation in vitro. Biochem Biophys Res Commun. 2007;354:234–239. [PMC free article] [PubMed]