Inhibition of glycosaminoglycan synthesis and protein glycosylation with WAS-406 and azaserine result in reduced islet amyloid formation in vitro

Abstract

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in approximately 90% of individuals with Type 2 diabetes and is associated with decreased islet beta-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet beta-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-alpha-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 7 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 1,000 microM 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 beta-cell area in hIAPP transgenic islets (55.0 +/- 2.6 vs. 60.6 +/- 2.2% islet area for 0 vs. 100 microM inhibitor, P = 0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and nontransgenic islets (the latter is a control that does not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in nontransgenic 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.

Small Interfering RNA–Mediated Suppression of Proislet Amyloid Polypeptide Expression Inhibits Islet Amyloid Formation and Enhances Survival of Human Islets in Culture

“…Thioflavine S staining revealed the presence of clearly detectable amyloid deposits after 10-day culture of untreated human islets (Fig. 3A, top right), similar to those observed in type 2 diabetic human pancreas, suggesting that amyloid formation occurs very rapidly in cultured human islets, as in transgenic human IAPP-expressing mouse islets (23,(32)(33)(34)(35). Islets transduced with AdhProIAPP-siRNA to inhibit proIAPP expression had a marked reduction in visible amyloid (Fig.…”

“…Islet amyloid may take years to form in the type 2 diabetic pancreas, but recent reports have described rapid amyloid formation in islets of human IAPP transgenic mice after prolonged culture or after transplantation (23,34,43) and after transplantation of human islets into immunedeficient mice (43). Here, we show that human islets, when cultured in moderately elevated glucose concentrations, rapidly develop amyloid associated with cell death.…”

“…In the present study, we tested the hypothesis that suppression of human IAPP expression would inhibit amyloid formation and enhance survival and/or function of human islets. We and others have found that islet amyloid forms rapidly in islets from transgenic mice with ␤-cell expression of human IAPP, and its formation is potentiated by elevated glucose concentrations (23,(32)(33)(34)(35). We used cultured human islets as an ex vivo model of amyloid formation to investigate whether short interfering RNA (siRNA)-mediated suppression of endogenously expressed human proIAPP will enhance ␤-cell survival in human islets.…”

Molecular mechanisms of Diabetic Retinopathy, general preventive strategies and novel therapeutic targets

“…The use of appropriate compounds has been described that can potentially alleviate the metabolic and functional abnormalities in diabetic retinopathy. WAS-406 (2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy--D-xylo-hexopyranose) and Azaserine reduce cardiovascular effects caused by hyperglycemia as antioxidants rather than by inhibiting only the hexosamine pathway [123,124]. Rhein, an anthraquinone compound isolated from rhubarb, decreases hexosamine pathway and is helpful in treatment of experimental diabetic nephropathy [125].…”

Novel Pharmacological Approaches to the Treatment of Type 2 Diabetes

“…Understanding this mechanism precisely could be the basis of a novel therapeutic approach. Azaserine reduces islet amyloid formation (Hull et al, 2007) (see also section X.E with respect to this compound). NC-503 (fibrillex; phase II) is an amyloid precursor protein antagonist.…”