Modelling the effects of human SUR1 R1420H variation on insulin secretory function using isogenic iPSC-derived pancreatic islets

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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 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 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 . Graphical
Funding Information
  • Intramural Research Program of NIH, The National Institute of Diabetes and Digestive and Kidney Diseases
  • National Institutes of Health

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