Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia-associated insulin resistance.

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of 'hyperinsulinemia' and 'starvation' programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

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.