Increase in Pancreatic Proinsulin and Preservation of β-Cell Mass in Autoantibody-Positive Donors Prior to Type 1 Diabetes Onset

Abstract

Type 1 diabetes is characterized by the loss of insulin production caused by β-cell dysfunction and/or destruction. The hypothesis that β-cell loss occurs early during the prediabetic phase has recently been challenged. Here we show, for the first time in situ, that in pancreas sections from autoantibody-positive (Ab+) donors, insulin area and β-cell mass are maintained before disease onset and that production of proinsulin increases. This suggests that β-cell destruction occurs more precipitously than previously assumed. Indeed, the pancreatic proinsulin-to-insulin area ratio was also increased in these donors with prediabetes. Using high-resolution confocal microscopy, we found a high accumulation of vesicles containing proinsulin in β-cells from Ab+ donors, suggesting a defect in proinsulin conversion or an accumulation of immature vesicles caused by an increase in insulin demand and/or a dysfunction in vesicular trafficking. In addition, islets from Ab+ donors were larger and contained a higher number of β-cells per islet. Our data indicate that β-cell mass (and function) is maintained until shortly before diagnosis and declines rapidly at the time of clinical onset of disease. This suggests that secondary prevention before onset, when β-cell mass is still intact, could be a successful therapeutic strategy.

Figure 1

Proinsulin area but not insulin area is significantly increased in the pancreas of Ab+ donors compared with control donors without diabetes. Insulin (left panel), proinsulin (middle panel), and glucagon (right panel) areas expressed as percentage of positive area were measured in whole-tissue sections from the head (A), body (B), and tail (C) region of the pancreas obtained from control donors without diabetes (n = 9) and from single Ab+ (n = 8) and double Ab+ (n = 5) cadaveric organ donors without diabetes. *P ≤ 0.05; **P ≤ 0.01.

Figure 2

Proinsulin accumulates in the cytoplasmic compartment in β-cells from Ab+ donors. Pancreatic sections from control (upper rows), single Ab+ (middle rows), and double Ab+ (lower rows) cadaveric organ donors without diabetes were stained for insulin (green), proinsulin (red), glucagon (white), and DAPI (blue) after a standard immunofluorescence staining protocol. The merged image can be seen on the right panel. Images were taken using a Zeiss LSM 880 confocal microscope with Airyscan and a ×63 objective. Scale bar: 10 μm.

Figure 3

β-Cell mass is not reduced in single or double Ab+ donors. β-Cell mass was calculated as total pancreas weight multiplied by insulin area (A) and as total pancreas weight multiplied by proinsulin area (B), and α-cell mass was calculated as total pancreas weight multiplied by glucagon area (C) in control donors without diabetes (n = 9) and in single Ab+ (n = 8) and double Ab+ (n = 5) donors without diabetes. *P ≤ 0.05.

Figure 4

The PI-to-INS area ratio is increased in Ab+ donors and constitutes a potential indicator of β-cell dysfunction. A: The PI-to-INS area ratio was calculated for head (left panel), body (middle panel), and tail (right panel) regions of the pancreas from control donors (n = 9) and single Ab+ (n = 8) and double Ab+ (n = 5) cadaveric organ donors without diabetes. B: Proinsulin area vs. insulin area xy plot: a theoretical reference value of 1:1 (proinsulin-to-insulin) was chosen and graphically represented as a line capable of separating control from “at-risk” donors. The area under this line represents a ratio smaller than 1 and vice versa. The distance from the donor’s area values to the line was used as a score to estimate the risk of developing disease and to classify and distinguish control donors from Ab+ donors (single and double combined). C: Receiver operating characteristic curve for the head (left panel), body (middle panel), and tail (right panel) regions of the pancreas. The AUC was calculated for the classifier described in panel B. The P values show the significance of the logistic regression model, including the predictor, when compared with a model with just the intercept. **P ≤ 0.01; ***P ≤ 0.001.

Figure 5

Higher glucagon and lower insulin and proinsulin areas but increased PI-to-INS area ratio in patients with recent-onset type 1 diabetes. A: Box plots represent islet size distribution for control donors without diabetes (HC, n = 5,109), single Ab+ (SingleAb, n = 4,692) and double Ab+ (DoubleAb, n = 2,859) donors, and donors with type 1 diabetes (T1D, n = 1,748) in the tail region of the pancreas. B: Islet density was calculated as the total number of islets per section divided by the total area of the tissue for the tail region of the pancreas. C: Representative image from whole-tissue section of donor #6362, with type 1 diabetes, at onset. Insulin is shown in green, glucagon in red, and DAPI in blue. Note the presence of insulin-deficient and insulin-containing islets scattered across the pancreas parenchyma. Scale bar: 500 µm. D: Insulin (left panel), proinsulin (middle panel), and glucagon (right panel) areas expressed as a percentage of the positive area were measured in whole-tissue sections from the tail region of the pancreas. E: The PI-to-INS area ratio (left panel) and the β-cell–to–α-cell ratio (right panel) were calculated for the pancreas tail region. F: Representative image of an islet from a recent-onset donor (DiViD study). Insulin is shown in green, proinsulin in red, and glucagon in blue. Scale bar: 50 µm. G: The percentage of islets containing only β-cells (left panel) and only α-cells (right panel) is shown. All panels: control donors without diabetes (n = 9), single Ab+ (n = 8) and double Ab+ (n = 5) donors without diabetes, and donors with type 1 diabetes (n = 7). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Figure 6

Systematic analysis of islet distribution reveals heterogeneity within the pancreas and subtle differences between Ab+ donors and control donors. A: Islet density was calculated as the total number of islets per section divided by the total area of the tissue for the head, body, and tail regions of the pancreas of control donors (n = 9), single Ab+ (SingleAb) donors (n = 8), and double Ab+ (DoubleAb) donors (n = 5). B: Box plots represent islet size distribution for healthy control (HC) donors, single Ab+ donors, and double Ab+ donors in the head (left panel), body (middle panel), and tail (right panel) region of the pancreas. C: Box plots represent islet size distribution for head, body, and tail regions in HC donors (left panel), single Ab+ (middle panel) donors, and double Ab+ (right panel) donors. Number of islets: head (HC, n = 4,390; SingleAb, n = 4,021; DoubleAb, n = 2,067), body (HC, n = 3,446; SingleAb, n = 4,052; DoubleAb, n = 2,043), and tail (HC, n = 5,109; SingleAb, n = 4,692; DoubleAb, n = 2859). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.