Abstract
AIMS/HYPOTHESIS: An R1420H variation in sulfonylurea receptor 1 (SUR1), a subunit of the K(ATP) channel, was previously identified in an Indigenous community in Arizona where a homozygous carrier (1420HH) had hyperinsulinaemic hypoglycaemia during infancy (HHI), suggestive of a K(ATP) channel loss of function (LoF). Interestingly, heterozygous carriers of this variation (1420RH, occurring in 3% of the community), had a twofold increased risk of type 2 diabetes. We aimed to create an isogenic induced pluripotent stem cell (iPSC)-derived pancreatic islet (SC-islet)-based platform to test whether the R1420H variant results in K(ATP) channel LoF, and to examine the distinct temporal effects of SUR1 1420HH and 1420RH K(ATP) channel variations on insulin secretion from developing and mature SC-islets. METHODS: Using CRISPR-Cas9, isogenic iPSCs with all three genotypes (SUR1 1420RR, 1420RH and 1420HH) were generated from two different parental Indigenous American iPSC lines (IS1, isogenic cell lines derived from parental cell line 1; and IS2, isogenic cell lines derived from parental cell line 2). These isogenic cell lines were used to generate immature SC-islets (resembling fetal islets) and mature SC-islets (resembling adult islets), which were used to assess insulin secretion dynamics during different stages of development and identify differences in gene expression by single-cell RNA-seq. This study was consistent with the CONSIDER statement for research studies among Indigenous American communities. RESULTS: Immature SUR1 1420HH SC-islets secreted 3.4-fold (IS1, p<0.001) and 4.2-fold (IS2, p=0.001) more insulin under basal conditions than normal (SUR1 1420RR) SC-islets. Modest hyperinsulinaemia was also seen in immature SUR1 1420RH SC-islets (2.2-fold [IS1] and 2.3-fold [IS2]) but the results were not statistically significant. After maturation, the 1420HH SC-islets failed to achieve glucose responsiveness whereas the 1420RH SC-islets achieved biphasic insulin secretion but had significantly lower glucose responsiveness than normal SC-islets (AUC for insulin secretion [as a % of total insulin] under high glucose challenge: 1.04 vs 0.56 in normal vs 1420RH SC-islets, p<0.001). Diazoxide reduced hyperinsulinaemia in SUR1 1420RH and 1420HH immature SC-islets, while tolbutamide elicited a greatly diminished or undetectable insulin secretory response from mature SUR1 1420RH SC-islets (13.2-fold increase in insulin secretion) and 1420HH SC-islets (1.9-fold increase) compared with normal SC-islets (31.5-fold increase). Results were directionally comparable for both IS1 and IS2 SC-islets. SUR1 1420RH SC-islets also responded to the glucokinase activator dorzagliatin with improvement in first-phase insulin secretory response (first-phase stimulation index: 3.9-fold vs 7.3-fold, p=0.01 [IS1, 11 mmol/l glucose ± dorzagliatin]; 5.5-fold vs 9.0-fold, p=0.13 [IS1, 20 mmol/l glucose ± dorzagliatin]). Single-cell RNA-seq identified dysregulated genes in SUR1 1420RH SC-beta cells, including lower expression of glycolytic genes and upregulation of G6PC2, which could explain the lower insulin secretory response to glucose. CONCLUSIONS/INTERPRETATION: A SUR1 R1420H variant identified in an Indigenous American population causes hyperinsulinaemia in homozygous immature SC-islets during basal conditions and these SC-islets fail to achieve glucose responsiveness after maturation. In the heterozygous state, modest hyperinsulinaemia is observed in immature SC-islets, which after maturation have significantly lower glucose responsiveness. These results demonstrate that SUR1 R1420H is a K(ATP) channel LoF variation and suggest that a lower insulin secretory response during adulthood is the cause of the higher type 2 diabetes risk in individuals heterozygous for this variation. We also show that the isogenic iPSC-based platform can be used to test therapeutic agents to treat HHI in infants homozygous for LoF K(ATP) channels and screen for drugs that can improve glucose-responsive insulin secretion in adult heterozygous carriers. DATA AVAILABILITY: See the database of Genotypes and Phenotypes (dbGaP; dbgap.ncbi.nlm.nih.gov/home; accession no.: phs002490.v1.p1) for details concerning data requests. All source codes can be found in the GitHub repository under https://github.com/Koushik-Cheranda/SC-islet-scRNAseq-analysis-R-codes .