Sex differences in islet stress responses support female ß cell resilience.

OBJECTIVE: Pancreatic ß cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in ß cells, but few studies have examined male-female differences in ß cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in ß cell and islet gene expression and function using both human and rodent samples. METHODS: In humans, we compared ß cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity. Our unbiased gene expression analysis pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we hypothesized female islets would be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and ß cell insulin production and secretion. Transcriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress. RESULTS: Our single-cell analysis of human ß cells revealed sex-specific changes to gene expression and function in T2D, correlating with more robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes linked with protein synthesis, folding, and processing. This differential expression was physiologically significant, as islets isolated from female mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-stimulated insulin production and secretion during ER stress compared with males. CONCLUSIONS: Our data demonstrate sex differences in ß cell gene expression in both humans and mice, and that female ß cells show a greater ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.

Dynamic Ins2 Gene Activity Defines ß-Cell Maturity States.

Transcriptional and functional cellular specialization has been described for insulin-secreting ß-cells of the endocrine pancreas. However, it is not clear whether ß-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in ß-cells from Ins2GFP knockin mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ∼25% of ß-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single ß-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 h. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single-cell RNA sequencing showed that Ins2(GFP)HIGH ß-cells were enriched for markers of ß-cell maturity. Ins2(GFP)HIGH ß-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human ß-cells, can be accounted for by dynamic states of insulin gene activity.