α Cell dysfunction in islets from nondiabetic, glutamic acid decarboxylase autoantibody-positive individuals

Abstract

BACKGROUNDMultiple islet autoantibodies (AAbs) predict the development of type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in only approximately 15% of individuals who are positive for single AAbs (generally against glutamic acid decarboxylase [GADA]); hence, the single GADA+ state may represent an early stage of T1D.METHODSHere, we functionally, histologically, and molecularly phenotyped human islets from nondiabetic GADA+ and T1D donors.RESULTSSimilar to the few remaining β cells in the T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, α cell glucagon secretion was dysregulated in both GADA+ and T1D islets, with impaired glucose suppression of glucagon secretion. Single-cell RNA-Seq of GADA+ α cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of cAMP-dependent protein kinase inhibitor β (PKIB), providing a molecular basis for the loss of glucose suppression and the increased effect of 3-isobutyl-1-methylxanthine (IBMX) observed in GADA+ donor islets.CONCLUSIONWe found that α cell dysfunction was present during the early stages of islet autoimmunity at a time when β cell mass was still normal, raising important questions about the role of early α cell dysfunction in the progression of T1D.FUNDINGThis work was supported by grants from the NIH (3UC4DK112217-01S1, U01DK123594-02, UC4DK112217, UC4DK112232, U01DK123716, and P30 DK019525) and the Vanderbilt Diabetes Research and Training Center (DK20593).

Keywords: Autoimmune diseases; Diabetes; Endocrinology; Islet cells.

Figure 1. Study design and workflow.

HPAP, working with Network for Pancreatic Organ Donors with Diabetes (nPOD), identifies organ donors of interest (recent-onset T1D, antibody⁺ donors, and control participants). For organ donors younger than 30 years of age without diabetes, the organ procurement organization (OPO), using HPAP/nPOD protocols and reagents, screens for the presence of AAbs (GADA, IA-2, mIAA, ZnT8). If a suitable organ donor is identified, pancreatic and immune tissues are shipped to Penn for processing. The tissue and islets are then analyzed for hormone secretion, multiplex imaging, molecular phenotyping, transcriptomics, and immunofluorescence staining at Vanderbilt and UPenn. All data are coregistered and integrated into a publicly accessible database (PANC-DB; https://hpap.pmacs.upenn.edu).

Figure 2. Insulin and glucagon secretion in islets from healthy and T1D donors.

(A) The dynamics of insulin secretion in response to different stimuli. (B) Total insulin secretion during 16 mM glucose stimulation. (C) Total insulin secretion during IBMX potentiation. The basal and maximal insulin secretion in T1D islets was 1/60 that of normal islets. Notably, T1D islets had similar insulin response patterns at low glucose, high glucose, and with IBMX treatment compared with control islets. (D) Glucagon secretion profiles. (E) Magnified view of a selected section (53–100 min) of the experiment presented in D to highlight the difference in glucose suppression of glucagon secretion between normal and T1D islets. (F) Total glucagon secretion during AAM stimulation. (G) Total glucagon secretion during IBMX potentiation. *P < 0.05 and ***P < 0.001. Ctrl, control.

Figure 3. Insulin and glucagon secretion in islets from healthy and GADA⁺ donors.

(A) The dynamics of insulin secretion during different interventions. (B) The dynamics of glucagon secretion. (C) Magnified view of a selected section (53–100 min) of the data from B highlights the difference in glucose suppression of glucagon secretion between normal and GADA⁺ islets. (D–G) Total glucagon secretion during 3 mM glucose (D), 16.7 mM glucose (E), G16.7 plus IBMX treatment (F), and KCl treatment (G) calculated as the AUC. (H–K) Islets from the same preparations were assessed by perifusion assay at Vanderbilt (see Supplemental Figure 2, A and B). AUC analysis of glucagon responses to high glucose (H), c-AMP–mediated secretion in response to IBMX (I), and epinephrine (J), and an unaltered KCl response (K). *P < 0.05 and **P < 0.01, by unpaired two-tailed t test. EQs, islet equivalents.

Figure 4. Composition of control and GADA⁺ donor islets.

(A–C) Endocrine cell type proportions were determined by flow CyTOF of single-cell suspensions of islets. (A) Cell type fraction plotted for the indicated HPAP cases. (B and C) Average α (B) and β (C) cell percentage as determined by flow CyTOF. (D) Endocrine cell type proportions determined by IMC. (E) β Cell percentage of endocrine cells determined from IMC for islets from controls, GADA⁺, and T1D individuals. (F) α Cell percentage of endocrine cells determined from IMC for islets from controls, GADA⁺, and T1D individuals. ***P < 0.001 and ****P < 0.0001, by unpaired two-tailed t test. (G and F) Representative examples of IMC images of control (G) and GADA⁺ (H) pancreas with 6 channels shown (cyan, insulin-peptide; blue, glucagon; yellow, somatostatin; red, PECAM; magenta, pancreatic polypeptide; green, ghrelin). Scale bars: 100 μm.

Figure 5. Single-cell transcriptome analysis and immunofluorescence staining of control and GADA⁺ α cells.

Heatmap of hierarchical clustering of differentially expressed genes obtained by comparing pseudobulk α cell gene expression of 9 control and 6 GADA⁺ organ donors. This heatmap was generated with the R package heatmap.

Figure 6. GSEA of glycolysis and gluconeogenesis genes in GADA⁺ donors compared with controls.

(A) Genes annotated to the “glycolysis_gluconeogenesis” pathway are highly enriched among the genes downregulated in α cells from GADA⁺ organ donors. up, upregulated; down, downregulated. (B) Expression of the top 15 genes in the leading edge of the “glycolysis_gluconeogensis” pathway. Triangles indicate the mean.

Figure 7. GSEA of oxidative phosphorylation genes in GADA⁺ donors compared with controls.

(A) Genes annotated to the oxidative phosphorylation pathway were highly enriched among the genes downregulated in α cells from GADA⁺ organ donors. (B) Expression of the genes in the leading edge of the “oxidative_phosphorylation” pathway. Triangles indicate the mean.

Figure 8. p-CREB staining in pancreatic sections from control and GADA⁺ donors.

(A) Immunofluorescence staining of pancreatic sections with antisera against p-CREB and glucagon. Scale bars: 60 μm. (B) Nuclear p-CREB⁺ α cells were increased in frequency in GADA⁺ islets (n = 7 for control islets and n = 6 for GADA⁺ islets). *P < 0.05, by unpaired two-tailed t test.