The S20G substitution in hIAPP is more amyloidogenic and cytotoxic than wild-type hIAPP in mouse islets

Abstract

Aims/hypothesis: The S20G human islet amyloid polypeptide (hIAPP) substitution is associated with an earlier onset of type 2 diabetes in humans. Studies of synthetic S20G hIAPP in cell-free systems and immortalised beta cells have suggested that this may be due to increased hIAPP amyloidogenicity and cytotoxicity. Thus, using primary islets from mice with endogenous S20G hIAPP expression, we sought to determine whether the S20G gene mutation leads to increased amyloid-induced toxicity, beta cell loss and reduced beta cell function.

Methods: Islets from mice in which mouse Iapp was replaced with human wild-type or S20G hIAPP were isolated and cultured in vitro under amyloid-forming conditions. Levels of insulin and hIAPP mRNA and protein, amyloid deposition and beta cell apoptosis and area, as well as glucose-stimulated insulin and hIAPP secretion, were quantified.

Results: Islets expressing S20G hIAPP cultured in 16.7 mmol/l glucose demonstrated increased amyloid deposition and beta cell apoptosis, reduced beta cell area, decreased insulin content and diminished glucose-stimulated insulin secretion, compared with islets expressing wild-type hIAPP. Amyloid deposition and beta cell apoptosis were also increased when S20G islets were cultured in 11.1 mmol/l glucose (the concentration that is thought to be physiological for mouse islets).

Conclusions/interpretation: S20G hIAPP reduces beta cell number and function, thereby possibly explaining the earlier onset of type 2 diabetes in individuals carrying this gene mutation.

Keywords: Amylin; Amyloid; Beta cell; Insulin secretion; Islet; Islet amyloid polypeptide; Toxicity; Type 2 diabetes.

Fig. 1

Histological analysis of wild-type and S20G hIAPP islets after in vitro culture for 144 h. (a–d) Representative images of single islets stained for insulin (red), amyloid (green) and nuclei (blue) of wild-type hIAPP islets cultured in 11.1 mmol/l glucose with amyloid severity 0.1% and insulin area/islet area 63.8% (a), wild-type hIAPP islets cultured in 16.7 mmol/l glucose with amyloid severity 8.5% and insulin area/islet area 52.8% (b), S20G hIAPP islets cultured in 11.1 mmol/l glucose with amyloid severity 1.0% and insulin area/islet area 60.3% (c) and S20G hIAPP islets cultured in 16.7 mmol/l glucose with amyloid severity 14.9% and insulin area/islet area 48.5% (d). (e) Islets were also stained with propidium iodide (red) and for insulin (green) and a representative image of a S20G islet cultured in 16.7 mmol/l glucose for 144 h indicating an apoptotic nucleus (arrow head) is shown. Scale bars, 50 μm. Magnification ×20, except for (e), where magnification is ×40

Fig. 2

Morphological assessment of isolated wild-type and S20G hIAPP islets. Islets were cultured for 144 h in media containing either 11.1 mmol/l (n=10–12) or 16.7 mmol/l (n=10–16) glucose. (a) Amyloid prevalence (proportion of islets with amyloid). (b) Amyloid severity (proportion of islet area occupied by amyloid). (c) Rate of beta cell apoptosis. (d) Fractional islet beta cell area. Black bars, 11.1 mmol/l glucose; white bars, 16.7 mmol/l glucose. *p≤0.05 vs wild-type islets cultured in 11.1 mmol/l glucose; ^†p≤0.05 vs S20G islets cultured in 11.1 mmol/l glucose; ^‡p≤0.05 vs wild-type islets cultured in 16.7 mmol/l glucose; ND, not detected

Fig. 3

Insulin assessments of isolated wild-type and S20G hIAPP islets. (a, b) Insulin mRNA (a) and insulin content (b) of islets measured after 144 h culture in media containing either 11.1 or 16.7 mmol/l glucose followed by 2.5 h in 2.8 mmol/l glucose. Black bars, 11.1 mmol/l glucose; white bars, 16.7 mmol/l glucose. (c, d) GSIS in response to 1 h of 2.8 or 20.0 mmol/l glucose following 144 h culture in media containing either 11.1 mmol/l glucose (c) or 16.7 mmol/l glucose (d). Light grey bars, 2.8 mmol/l glucose; dark grey bars, 20.0 mmol/l glucose. (e) GSIS normalised to insulin content. Black bars, 11.1 mmol/l; white bars, 16.7 mmol/l. n=5 experiments, except mRNA n=7. *p≤0.05 vs wild-type islets cultured in 11.1 mmol/l glucose; ^†p≤0.05 vs S20G islets cultured in 11.1 mmol/l glucose; ^‡p≤0.05 vs wild-type islets cultured in 16.7 mmol/l glucose. Secretion experiments: *p≤0.05 vs wild-type islets exposed to 2.8 mmol/l glucose; ^†p≤0.05 vs S20G islets exposed to 2.8 mmol/l glucose; ^‡p≤0.05 vs wild-type islets exposed to 20.0 mmol/l glucose

Fig. 4

hIAPP assessments of isolated wild-type and S20G hIAPP islets. (a, b) hIAPP mRNA (a) and hIAPP content (b) after 144 h culture in media containing either 11.1 or 16.7 mmol/l glucose. (c) Glucose-stimulated hIAPP secretion in response to 1 h of 20 mmol/l glucose following 144 h culture in media containing either 11.1 or 16.7 mmol/l glucose. (d) Glucose-stimulated hIAPP secretion normalised to hIAPP content. (e) Ratio of hIAPP insulin released in response to 1 h of 20 mmol/l glucose following 144 h culture in media containing either 11.1 or 16.7 mmol/l glucose. Black bars, 11.1 mmol/l glucose; white bars, 16.7 mmol/l glucose. n=5 experiments, except mRNA n=7. *p≤0.05 vs wild-type cultured in 11.1 mmol/l glucose; ^†p≤0.05 vs S20G islets cultured in 11.1 mmol/l glucose; ^‡p≤0.05 vs wild-type islets cultured in 16.7 mmol/l glucose