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DiabetologiaPathologyPeer-reviewed journal article

Increased Inflammation as well as Decreased Endoplasmic Reticulum Stress and Translation Differentiate Pancreatic Islets of Pre-symptomatic Stage 1 Type 1 Diabetes and Non-diabetic Cases

A. C. SwenseniD, Paul PiehowskiiD, Jing Chen, X’avia Y. Chan, Shane S. KellyiD, Vladislav PetyukiD, Ronald MooreiD, Lith H. NasifiD, Elizabeth Ashley ButterworthiD, Mark A. AtkinsoniD, Rohit KulkarniiD, Martha Campbell‐ThompsoniD, Clayton E. MathewsiD, Weijun QianiD

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Abstract

Aims/hypothesisProgression to type 1 diabetes (T1D) is associated with genetic factors, the presence of autoantibodies, and a decline in {beta} cell insulin secretion in response to glucose. Very little is known regarding the molecular changes that occur in human insulin-secreting {beta}-cells prior to the onset of T1D. Herein, we applied an unbiased proteomics approach to identify changes in proteins and potential mechanisms of islet dysfunction in islet autoantibody-positive organ donors with pre-symptomatic stage 1 T1D (HbA1c [≤] 6). We aimed to identify pathways in islets that are indicative of {beta}-cell dysfunction. MethodsMultiple islet sections were collected through laser microdissection of frozen pancreatic tissues of organ donors positive for islet autoantibodies (AAb+, n=5), compared to age/sex-matched nondiabetic controls (ND, n=5) obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD). Islet sections were subjected to mass spectrometry-based proteomics and analyzed with label-free quantification followed by pathway and functional annotations. ResultsAnalyses resulted in [~]4,500 proteins identified with low false discovery rate (FDR) <1%, with 2,165 proteins reliably quantified in every islet sample. We observed large inter-donor variations that presented a challenge for statistical analysis of proteome changes between donor groups. We therefore focused on the three multiple AAb+ cases (mAAb+) with high genetic risk and their three matched controls for a final statistical analysis. Approximately 10% of the proteins (n=202) were significantly different between mAAb+ cases versus ND. The significant alterations clustered around major functions for upregulation in the immune response and glycolysis, and downregulation in endoplasmic reticulum (ER) stress response as well as protein translation and synthesis. The observed proteome changes were further supported by several independent published datasets, including proteomics dataset from in vitro proinflammatory cytokine-treated human islets and single cell RNA-seq data sets from AAb+ cases. Conclusion/interpretationIn-situ human islet proteome alterations at the stage 1 of AAb+ T1D centered around several major functional categories, including an expected increase in immune response genes (elevated antigen presentation / HLA), with decreases in protein synthesis and ER stress response, as well as compensatory metabolic response. The dataset serves as a proteomics resource for future studies on {beta} cell changes during T1D progression and pathogenesis.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (19 Sept 2024) · CC BY 4.0

Laser-microdissected islets of 3 multiple-autoantibody-positive stage 1 type 1 diabetes donors versus matched controls show 202 altered proteins: immune and glycolytic up, translation and ER-stress proteins down.

Molecular events inside human beta cells before clinical type 1 diabetes are poorly defined, because insulitis is rare and beta-cell mass appears preserved in autoantibody-positive donors, and pre-diabetic pancreas tissue is scarce. Combining laser microdissection and mass spectrometry, it delivers what the authors call the first in situ islet proteome from autoantibody-positive donors, nominating pathways and proteins for studying early beta-cell dysfunction.

Key results

  1. SuggestedRestricting the analysis to the three multiple-autoantibody-positive (mAAb+) donors and their matched controls gave 202 differentially abundant islet proteins that separated the groups by clustering. Comparing all autoantibody-positive donors with all controls showed no clear separation.

    Mixed-effects model test, n=3 per group, FDR < 0.15 and p < 0.025: 202 proteins (about 10% of quantified proteins) changed. A hierarchical heatmap clustered mAAb+ islets apart from non-diabetic (ND) controls. PCA of all AAb+ (n=5) vs ND (n=5) showed no significant group differences (data not shown). · Abstract; Comparative Proteome Profiling of Islets from…; Figure legends

  2. ShownLaser-microdissected islet pools gave broad, replicable proteome coverage from small amounts of in situ tissue.

    About 4,500 proteins were identified at <1% FDR, and 2,165 were quantified in every islet sample using MaxQuant match-between-runs label-free quantification. Each donor had two pools of ~100 sections (about 1-2 islet equivalents per slide) from adjacent 10 µm slides, used as replicates. Reproducibility is described as relatively high, but no metric is given. · Abstract; Laser microdissection (LMD) of human Islet…; Comparative Proteome Profiling of Islets from…

  3. SuggestedmAAb+ islets had less translation machinery and fewer ER protein-folding and quality-control proteins. The authors read this as a dampened, not heightened, unfolded protein response (UPR) at stage 1.

    IPA: decreased EIF2 signaling, translation and tRNA charging. Lower 40S (RPS6, RPS11, RPS14, RPS19, RPS25, FAU) and 60S (e.g., RPL7, RPL8, RPL26, RPL38) ribosomal proteins. Lower P4HB, PDIA2, CALR, CANX, DNAJA1, DNAJB9, HSPA5 (BiP) and CTH. DAVID/REVIGO GO analysis separately found reduced translation and protein folding. · Altered Pathways and Functions Potentially…; Deficiency in Translation and Protein Synthesis; Reduced ER Stress Response; DISCUSSION

  4. SuggestedImmune and metabolic proteins rose in mAAb+ islets, including MHC class I antigen presentation and most glycolytic enzymes, while fructose-1,6-bisphosphatase 1 (FBP1) fell. Several proteins involved in insulin processing and secretion were also altered.

    Upregulated: HLA-A and other MHC class I variants, plus PRKAR2B, ILF2 and TIMP2 (immune response heatmap), and the glycolytic enzymes GPI, ALDOA, ALDOC, GAPDH, PGK1, PKM and ENO. Glycolysis/gluconeogenesis ranked as the second most significant IPA pathway. FBP1 was significantly reduced; PCSK1, CPE, PPP1R1A and GNAI2 increased. · Altered Pathways and Functions Potentially…; Increased Glycolysis; Elevated Immune Response; DISCUSSION

  5. SuggestedNPTX2 was upregulated and found mainly in beta cells. The differential proteome partly matched independent proteomics of cytokine-treated islets and Human Pancreas Analysis Program (HPAP) single-cell RNA-seq of AAb+ donors.

    Multiplex immunofluorescence (INS, GCG, SST, NPTX2) of a non-diabetic donor islet placed NPTX2 in beta cells. HPAP scRNA-seq showed NPTX2 upregulation in the beta-2 subpopulation of AAb+ cases. Overlap with IFN-γ plus IL-1β-treated islets was assessed by pathway-level enrichment comparison, and the authors note important differences from the in vitro model. · Multiplex immunofluorescence imaging; DISCUSSION; Figure legends

Methods, data and reproducibility

Two pools of ~100 laser-microdissected islet sections per donor (frozen nPOD pancreas) were analyzed by online immobilized-trypsin digestion and 5-hour LC-MS/MS on an Orbitrap Q-Exactive Plus. MaxQuant label-free intensities then went through mixed-effects testing and IPA/DAVID enrichment, and NPTX2 was localized by multiplex immunofluorescence.

  • Frozen pancreas sections from islet autoantibody-positive organ donors, Network for Pancreatic Organ donors with Diabetes (nPOD) · human · n = 5 AAb+ (3 mAAb+, 2 single AAb+)
  • Age/sex-matched non-diabetic, autoantibody-negative control donors from nPOD · human · n = 5 ND (3 matched to mAAb+ in final analysis)
  • LC-MS/MS raw data from laser-microdissected islet section pools · human · n = two pools of ~100 islet sections per donor · MassIVE MSV000090212
  • FFPE nPOD pancreas sections for multiplex immunofluorescence of INS, GCG, SST and NPTX2 · human
  • Published proteomics of isolated human islets treated in vitro with IFN-γ plus IL-1β (comparison dataset) · human
  • Human Pancreas Analysis Program (HPAP) scRNA-seq of beta-cell subpopulations from AAb+ and control donors (comparison dataset) · human
Methods & tools
Leica LMD7000 laser microdissection, Simplified nanoproteomics platform with online immobilized trypsin reactor (IMER), Orbitrap Q-Exactive Plus mass spectrometer, MaxQuant 1.5.3.30 (label-free quantification, match between runs), Mixed-effects model differential testing, Qiagen Ingenuity Pathway Analysis (IPA), DAVID Gene Functional Classification, REVIGO, Multiplex immunofluorescence (anti-NPTX2 HPA049799), Zeiss 710 confocal microscope
Reagents & environment
MaxQuant 1.5.3.30 (protein FDR 0.01, match between runs, default LFQ, 20 and 4.5 ppm tolerances); IPA with default parameters; REVIGO small (0.5) list setting.
Reproducibility gaps
No analysis code or scripts (e.g., for mixed-effects testing) are referenced. Quantification tables sit in supplementary tables not included here, and the PCA comparing all AAb+ donors with all controls is reported as data not shown.
Limitations (5)
  • Only 3 multiple-autoantibody-positive stage 1 donors with risk HLA were available from nPOD for the final comparison. The authors therefore call the study exploratory and say larger mAAb+ and recent-onset cohorts are needed. · stated by the authors
  • Large differences between donors made statistical testing hard. With all 5 AAb+ donors vs 5 controls, PCA showed no group separation, which the authors partly attribute to classifying pre-diabetic cases by autoantibody positivity alone. · stated by the authors
  • Low-abundance unfolded protein response markers (CHOP, PERK, IRE1) were not detected. The conclusion that the ER stress response is dampened therefore rests on lower BiP and ER chaperone levels rather than on direct pathway sensors. · stated by the authors
  • The significance cutoff is permissive (FDR < 0.15, p < 0.025), and the mAAb+ subgroup was chosen after the full comparison showed no separation. With n=3 per group and no independent proteomic validation cohort, some of the 202 proteins may be false positives. · noted in this summary
  • Whole-islet sections mix endocrine and other cell types, so changes attributed to beta cells may partly reflect altered cell composition or immune infiltrate. Imaging confirmed only where NPTX2 is located, shown in a non-diabetic donor, not its abundance change. · noted in this summary

Read this if you study pre-symptomatic type 1 diabetes or beta-cell stress and want an in situ islet proteome and candidate protein list from autoantibody-positive donors, bearing in mind the very small cohort.

Citations
9
FWCI
4.02
Field percentile
top 5%

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