Glucose- and time-dependence of islet amyloid formation in vitro

Abstract

Islet amyloid contributes to the loss of β-cell mass in type 2 diabetes. To examine the roles of glucose and time on amyloid formation, we developed a rapid in vitro model using isolated islets from human islet amyloid polypeptide (hIAPP) transgenic mice. Islets from hIAPP transgenic and non-transgenic mice were cultured for up to 7 days with either 5.5, 11.1, 16.7 or 33.3 mmol/l glucose. At various time-points throughout the culture period, islets were harvested for determination of amyloid and β-cell areas, and for measures of cell viability, insulin content and secretion. Following culture of hIAPP transgenic islets in 16.7 or 33.3 mmol/l glucose, amyloid formation was significantly increased compared to 5.5 or 11.1 mmol/l glucose culture. Amyloid was detected as early as day 2 and increased in a time-dependent manner so that by day 7, a decrease in the proportion of β-cell area in hIAPP transgenic islets was evident. When compared to non-transgenic islets after 7-day culture in 16.7 mmol/l glucose, hIAPP transgenic islets were 24% less viable, had decreased β-cell area and insulin content, but displayed no change in insulin secretion. Thus, we have developed a rapid in vitro model of light microscopy-visible islet amyloid formation that is both glucose-and time-dependent. Formation of amyloid in this model is associated with reduced cell viability and β-cell loss but adequate functional adaptation. It thus enables studies investigating the mechanism(s) underlying the amyloid-associated loss of β-cell mass in type 2 diabetes. Keywords islet amyloid; human islet amyloid polypeptide; islets; ß-cell; insulin secretion; cell viability; amylin; mouse model; diabetes Islet amyloid polypeptide (IAPP) is a normal product of the pancreatic β-cell. Human, but not rodent, IAPP is capable of forming amyloid deposits, which are associated with loss of both β-cell mass and normal islet function [1,2]. Models have been developed to elucidate the underlying mechanisms responsible for the deleterious effects of amyloidogenesis. Initially, these involved the extracellular application of human IAPP (hIAPP) to immortalized cells [3], or the transfection of cells that lack the sophisticated secretory machinery that is typical of the β-cell [4]. While cell death and/or impaired insulin secretion were demonstrated, these models do not depict what is occurring in humans.

Differences between amyloid toxicity in alpha and beta cells in human and mouse islets and the role of caspase-3

“…Islets from hIAPP-expressing mice with or without caspase-3 expression had comparable levels of amyloid formation both in vitro and in vivo, confirming that the absence of caspase-3 per se does not have any detectable effect on amyloid formation in hIAPP-expressing mouse islets. As expected [16,49], hIAPP aggregates were not detectable in freshly isolated hIAPP-expressing mouse islets but were present in cultured islets. Interestingly, hIAPP-expressing islets lacking caspase-3 had markedly lower apoptotic beta cells, higher insulin content and glucose response compared with islets expressing hIAPP and caspase-3, suggesting that deletion of caspase-3 protects islet beta cells from endogenously produced hIAPP aggregates and improves their survival and function.…”

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.…”

“…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.…”

Resident Macrophages Mediate Islet Amyloid Polypeptide–Induced Islet IL-1β Production and β-Cell Dysfunction

“…Although we cannot rule out effects on the islet secondary to macrophage depletion in other tissues, wild-type mice displayed no change in glucose tolerance in response to clodronate liposomes, suggesting a unique role for macrophages in the setting of hIAPP aggregation. We observed improved glucose-stimulated insulin secretion in isolated islets from clodronate liposome-treated hIAPP Tg/o mice and in clodronate liposome-treated hIAPP Tg/o islets cultured in high glucose, conditions demonstrated by others to promote amyloid formation (54). Thus, improved b-cell function caused by macrophage depletion in vivo may be largely independent of peripheral effects.…”