Quantitative analysis of islet prohormone convertase 1/3 expression in human pancreas donors with diabetes

Abstract

Aims/hypothesis: Islet prohormone-processing enzymes convert peptide hormone precursors to mature hormones. Defective beta cell prohormone processing and the release of incompletely processed peptide hormones are observed prior to the onset of diabetes, yet molecular mechanisms underlying impaired prohormone processing during the development of diabetes remains largely unknown. Previous studies have shown that prohormone convertase 1/3 (PC1/3) protein and mRNA expression levels are reduced in whole islets from donors with type 1 diabetes, although whether PC1/3-mediated prohormone processing in alpha and beta cells is disrupted in type 1 diabetes remained to be explored. Herein, we aimed to analyse the expression of PC1/3 in islets from non-diabetic donors, autoantibody-positive donors and donors diagnosed with type 1 diabetes or type 2 diabetes.

Methods: Immunostaining and high-dimensional image analysis were performed on pancreatic sections from a cross-sectional cohort of 54 donors obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD) repository, to evaluate PC1/3 expression patterns in islet alpha, beta and delta cells at different stages of diabetes.

Results: Alpha and beta cell morphology were altered in donors with type 1 diabetes, including decreased alpha and beta cell size. As expected, the insulin-positive and PC1/3-positive areas in the islets were both reduced, and this was accompanied by a reduced percentage of PC1/3-positive and insulin-positive/PC1/3-positive cells in islets. PC1/3 and insulin co-localisation was also reduced. The glucagon-positive area, as well as the percentage of glucagon-positive and glucagon-positive/PC1/3-positive cells in islets, was increased. PC1/3 and glucagon co-localisation was also increased in donors with type 1 diabetes. The somatostatin-positive cell area and somatostatin staining intensity were elevated in islets from donors with recent-onset type 1 diabetes.

Conclusions/interpretation: Our high-resolution histomorphological analysis of human pancreatic islets from donors with and without diabetes has uncovered details of the cellular origin of islet prohormone peptide processing defects. Reduced beta cell PC1/3 and increased alpha cell PC1/3 in islets from donors with type 1 diabetes pinpointed the functional deterioration of beta cells and the concomitant potential increase in PC1/3 usage for prohormone processing in alpha cells during the pathogenesis of type 1 diabetes. Our finding of PC1/3 loss in beta cells may inform the discovery of new prohormone biomarkers as indicators of beta cell dysfunction, and the finding of elevated PC1/3 expression in alpha cells may encourage the design of therapeutic targets via leveraging alpha cell adaptation in diabetes.

Keywords: Beta cell dysfunction; Prohormone convertase 1/3; Proinsulin; Quantitative image analysis.

Fig. 1

Schematic illustration of the confocal image analysis workflow using QuPath, version 0.4.3. Islets were detected using a pixel classifier. After islet detection, cells were identified using a StarDist plug-in. The single measurement classifier tool was employed to detect positive cells for the marker of interest. Cells were identified as areas of staining above the background level by applying optimised cell mean intensity thresholds. Single measurement classifiers were combined to identify negative, single-positive, double-positive or triple-positive cells and a colour code was used to differentiate different beta and alpha cell populations. INS, insulin; ProINS, proinsulin

Fig. 2

Morphological analysis of alpha and beta cells. Mean number of cells per islet (a), median islet area (b), mean size of individual insulin-positive cells (c), median size of individual nucleus of insulin-positive cells (d), median nucleus/cytoplasm ratio of insulin-positive cells (e), median size of individual glucagon-positive cells (f), mean size of individual nucleus of glucagon-positive cells (g) and mean nucleus/cytoplasm ratio of glucagon-positive cells (h) were analysed in pancreatic sections from donors. Data are shown for pancreases from non-diabetic donors (filled circles, older adult; empty circles, young adult), AAb⁺ donors (filled circles, double AAb⁺; empty circles, single AAb⁺), donors with recent-onset type 1 diabetes (duration <5 years), donors with long-duration type 1 diabetes (>15 years) and donors with type 2 diabetes. Each data point represents the average value from one donor. Normally distributed data (a, c, g, h) are expressed as mean ± SEM, non-normal data (b, d, e, f) are expressed as median ± IQR, *p<0.05, **p<0.01 and ***p<0.001. GCG, glucagon; INS, insulin; N/C, nucleus/cytoplasm; ND, non-diabetic; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years

Fig. 3

PC1/3 expression in beta cells. (a) Representative images showing PC1/3, insulin and proinsulin expression in islets from donors with or without diabetes. nPOD case numbers are indicated in the merged images. Scale bar, 100 μm. (b) Proportional insulin-positive area in islets. (c) RFU of insulin in donor islets. (d, e, f) Percentage of proinsulin- (d), insulin- (e) and PC1/3- (f) positive cells in donor islets. (g) Proinsulin/insulin RFU ratio in individual beta cells in islets from donors with or without diabetes. (h, i) Percentage of proinsulin, insulin and PC1/3 triple-positive cells (h) and PC1/3 and insulin co-localisation (i) in beta cells from donors with or without diabetes. Data are shown for islets from non-diabetic donors (filled circles, older adults; empty circles, young adults), AAb⁺ donors (filled circles, double AAb⁺; empty circles, single AAb⁺), donors with recent-onset type 1 diabetes (duration <5 years), donors with long-duration type 1 diabetes (>15 years) and donors with type 2 diabetes. Each data point represents the average value from one donor. Hatched bars represent data from images analysed at the islet level; non-hatched bars represent data from image analysed at the single-cell level. Normally distributed data (c) are expressed as mean ± SEM, non-normal data (b, d–i) are expressed as median ± IQR, *p<0.05, **p<0.01 and ***p<0.001. INS, insulin; ND, non-diabetic; ProINS, proinsulin; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years

Fig. 4

Trajectory of islet PC1/3 expression in diabetes. (a) Venn diagram showing the dynamic of cells positive for insulin (pink circle), proinsulin (green circle), PC1/3 (blue circle), both insulin and proinsulin (orange circle), or positive (grey circles) or negative (white circles) for insulin, proinsulin and PC1/3, in the progression of type 1 diabetes. Non-diabetic donors, n=20; single AAb⁺ donors (1AAb⁺), n=4; double AAb⁺ donors (2AAb⁺), n=3; donors with recent-onset type 1 diabetes (duration <5 years), n=8; donors with long-duration type 1 diabetes (>15 years), n=8; donors with type 2 diabetes, n=6. (b–d) RFU of insulin (b), proinsulin (c) and PC1/3 (d) in insulin-, proinsulin- and PC1/3-positive cells in donor islets. (e) RFU of PC1/3 in insulin-negative, proinsulin-negative and PC1/3-positive cells from donors with or without diabetes. (f) Percentage of PC1/3-positive area in islets from donors with or without diabetes. Data are shown for islets from non-diabetic donors (filled circles, older adults; empty circles, young adults), AAb⁺ donors (filled circles, double AAb⁺; empty circles, single AAb⁺), donors with recent-onset type 1 diabetes, donors with long-duration type 1 diabetes and donors with type 2 diabetes. The average read-out of all cells from each donor was represented as one data point. Hatched bars indicate data from images analysed at the islet level; non-hatched bars represent images analysed at the single-cell level. Normally distributed data (d) are expressed as mean ± SEM, non-normal data (b, c, e, f) are expressed as median ± IQR, *p<0.05, **p<0.01 and ***p<0.001. INS, insulin; ND, non-diabetic; ProINS, proinsulin; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years

Fig. 5

PC1/3 expression in alpha cells. (a) Representative images showing PC1/3, glucagon and somatostatin expression in islets from donors with or without diabetes. nPOD case numbers are indicated in the merged images. Scale bar, 100 μm. (b) Proportional glucagon-positive area in islets. (c) Median RFU of glucagon in islets. (d–f) Percentage of glucagon-positive, PC1/3-positive (d), glucagon-positive, PC1/3-negative (e) and glucagon-positive islet cells (f) in islets. In (f) the darker columns represent PC1/3-negative cells, the lighter columns represent PC1/3-positive cells. (g) Mean RFU of glucagon in glucagon-positive alpha cells. (h) Mean RFU of glucagon in PC1/3-positive vs PC1/3-negative cells. (i) Median RFU of PC1/3 in PC1/3-positive, glucagon-positive alpha cells. (j) Mean PC1/3 and glucagon co-localisation in islets. Data are shown for islets from non-diabetic donors (filled circles, older adults; empty circles, young adults), AAb⁺ donors (filled circles, double AAb⁺; empty circles, single AAb⁺), donors with recent-onset type 1 diabetes (duration <5 years), donors with long-duration type 1 diabetes (>15 years) and donors with type 2 diabetes. Each data point represents the average value from one donor. Hatched bars show data for images analysed at the islet level; non-hatched bars show data for images analysed at the single-cell level. Normally distributed data (b, g, h, j) are expressed as mean ± SEM, non-normal data (c, d, e, f, i) are expressed as median ± IQR. *p<0.05, **p<0.01 and ***p<0.001. GCG, glucagon; ND, non-diabetic; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years

Fig. 6

Glucagon and somatostatin expression pattern in islets. (a) Representative images showing PC1/3, glucagon and somatostatin expressions in islets from a donor without diabetes. Yellow arrows indicate the cytoplasmic processes of delta cells. Scale bar, 100 μm. (b–d) Proportional somatostatin-positive area (b), median RFU of somatostatin (c) and co-localisation of PC1/3 and somatostatin (d) in islets from non-diabetic donors (filled circles, older adults; empty circles, young adults), AAb⁺ donors (filled circled, double AAb⁺; empty circles, single AAb⁺), donors with recent-onset type 1 diabetes (duration <5 years), donors with long-duration type 1 diabetes (>15 years) and donors with type 2 diabetes. Each data point represents the average value from one donor. Data are expressed as median ± IQR. *p<0.05 and **p<0.01. GCG, glucagon; ND, non-diabetic; SST, somatostatin; T1D, type 1 diabetes; T2D, type 2 diabetes; y, years