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.