β-cell loss and β-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition

Abstract

Amyloid deposition and reduced β-cell mass are pathological hallmarks of the pancreatic islet in type 2 diabetes; however, whether the extent of amyloid deposition is associated with decreased β-cell mass is debated. We investigated the possible relationship and, for the first time, determined whether increased islet amyloid and/or decreased β-cell area quantified on histological sections is correlated with increased β-cell apoptosis. Formalin-fixed, paraffin-embedded human pancreas sections from subjects with (n = 29) and without (n = 39) diabetes were obtained at autopsy (64 ± 2 and 70 ± 4 islets/subject, respectively). Amyloid and β cells were visualized by thioflavin S and insulin immunolabeling. Apoptotic β cells were detected by colabeling for insulin and by TUNEL. Diabetes was associated with increased amyloid deposition, decreased β-cell area, and increased β-cell apoptosis, as expected. There was a strong inverse correlation between β-cell area and amyloid deposition (r = -0.42, P < 0.001). β-Cell area was selectively reduced in individual amyloid-containing islets from diabetic subjects, compared with control subjects, but amyloid-free islets had β-cell area equivalent to islets from control subjects. Increased amyloid deposition was associated with β-cell apoptosis (r = 0.56, P < 0.01). Thus, islet amyloid is associated with decreased β-cell area and increased β-cell apoptosis, suggesting that islet amyloid deposition contributes to the decreased β-cell mass that characterizes type 2 diabetes.

Figure 1

Islet amyloid deposition in individuals with diabetes (n = 29) and control subjects (n = 39). A: Representative images of islets stained for insulin (red) and thioflavin S (green). The left and middle images show islets from individuals with diabetes, with amyloid severities of 6% and 32%, respectively. The image at the right shows an islet from a control subject, amyloid negative. Scale bar = 50 μm. B–E: Quantitative analysis. The proportion of subjects with any detectable islet amyloid (B), the mean islet amyloid prevalence expressed as % amyloid-positive islets (C), and mean islet amyloid severity expressed as amyloid positive area/islet area × 100% (D) were all significantly greater in diabetic subjects than control subjects. Individual points represent data for each subject, with mean ± SEM for the two groups also depicted (C and D). Islet amyloid prevalence and severity were correlated in a linear manner (E); solid circles indicate individuals with diabetes, and open triangles indicate control subjects (r = 0.87). *P < 0.001 versus control subjects.

Figure 2

Measurements of cross-sectional islet and β-cell areas in individuals with diabetes (n = 29) and control subjects (n = 39). Islet size distribution was determined in individuals with diabetes (A) and in control subjects (B). Median islet area (C) was identical between groups. Median β-cell area (D) and mean β-cell area normalized to islet area, expressed as insulin positive area/islet area × 100% (E), were significantly lower in individuals with diabetes; individual points represent data for each subject, with mean ± SEM for the two groups also depicted. For box plots, boxes denote the 25th to 75th percentiles and whiskers denote 5th and 95th percentiles; data that fall outside the range of the 5th to 95th percentiles are outliers and are shown as circles (C and D). *P < 0.05 versus control subjects.

Figure 3

Increased islet amyloid deposition was associated with reduced β-cell area. Correlation is shown among all subjects between amyloid severity and absolute β-cell area (r = −0.42 and P < 0.001) (A), or β-cell area/islet area (r = −0.76 and P < 0.001) (B), in individuals with diabetes (solid circles) and in control subjects (open triangles).

Figure 4

Median islet (A) and β-cell area/islet area (B) in amyloid-positive islets from individuals with diabetes (n = 859 islets), amyloid-negative islets from individuals with diabetes (n = 985 islets), and islets from control subjects (n = 2726 islets). Amyloid-laden islets were significantly larger (A), but had significantly lower β-cell area/islet area (B). Islet area and β-cell area/islet area were equivalent between amyloid-negative islets from individuals with diabetes and control subjects. For box plots, boxes denote the 25th to 75th percentiles and whiskers denote 5th and 95th percentiles. *P < 0.001 versus control subjects.

Figure 5

Representative examples of staining for β-cell apoptosis (A) with TUNEL (green), insulin (red), and Hoechst 33258 (blue) and for β-cell replication (B) with Ki-67 (red), insulin (green), and Hoechst 33258 (blue). Arrows indicate TUNEL- or KI-67-positive β cells; arrowheads indicate TUNEL-positive non-β cells. Mean β-cell apoptosis (C) was significantly higher in islets from individuals with diabetes (n = 13), compared with control subjects (n = 16). Mean β-cell replication (D) did not differ between islets from individuals with diabetes (n = 15) and control subjects (n = 17); P = 0.63. β-cell apoptosis was significantly associated with increased islet amyloid severity (E), r = 0.56 and P < 0.01, and tended to be associated with decreased absolute β-cell area (F), r = −0.33 and P = 0.07. Solid circles: diabetes; open triangles, controls. *P < 0.05 versus control subjects; scale bar = 50 μm.