Multi-Omic Mapping of Human Pancreatic Islet Endoplasmic Reticulum and Cytokine Stress Responses Provides Genetic Insights into Type 2 Diabetes

Background and Research Motivation

Globally, Type 2 Diabetes (T2D) is a common metabolic disease characterized by the combined effects of genetic and environmental factors leading to dysfunction and/or cell death of pancreatic β-cells, resulting in insufficient insulin secretion. Based on findings from Genome-Wide Association Studies (GWAS), over 600 loci in the human genome have been associated with T2D risk, many of which are located in non-coding regions. Studies suggest that variations in these non-coding regions may lead to pancreatic dysfunction by regulating the functions of islet-specific cis-regulatory elements (CREs) and the expression of their effector genes. However, the roles of these genetic variations in pathological endoplasmic reticulum (ER) stress and inflammation induced by pro-inflammatory cytokines remain largely unknown. The research team of this paper comes from the Jackson Laboratory for Genomic Medicine in the United States and the University of Connecticut, and the study is published in the journal “Cell Metabolism” (November 5, 2024). The research aims to fill this knowledge gap by analyzing the transcriptional regulatory networks in human islets under ER stress and pro-inflammatory cytokine conditions through multi-omics approaches, in hopes of revealing the potential pathogenic mechanisms of these variations and providing new perspectives for T2D drug targets.

Research Source

This study was jointly completed by Eishani K. Sokolowski, Romy Kursawe, Vijay Selvam, and others, from institutions including Jackson Laboratory for Genomic Medicine and University of Connecticut Health Center. The leading authors are Duygu Ucar and Michael L. Stitzel. The paper is published in the journal “Cell Metabolism,” and the research was funded by the U.S. Department of Defense and the American Diabetes Association.

Research Process

This study used multi-omics technologies, including RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), and Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq), to systematically analyze stress responses in human islets from non-diabetic donors under ER stress and cytokine treatments.

1. Experimental Design and Islet Sample Processing
   The study subjects were islet cells from multiple non-diabetic donors, divided into two groups based on experimental conditions: one treated with the ER stress inducer thapsigargin, and the other with pro-inflammatory cytokines (IL-1β and IFN-γ), with control groups for each. Each treatment condition lasted for 24 hours, and comprehensive RNA sequencing was conducted on the islet cells under each condition to analyze the genomic expression changes.

2. Transcriptome Analysis
   RNA sequencing results indicated that around 30% of expressed genes and 14% of islet CREs responded to ER stress and cytokine treatments. The study found that these stress-responsive genes and CREs exhibited stress specificity. For example, 85% of genes displayed distinct response characteristics under different stress treatments. ER stress significantly induced genes associated with the Unfolded Protein Response (UPR) and ER protein handling (e.g., ATF4, DDIT3), while cytokine treatments primarily activated NF-κB signaling pathways related to pro-inflammatory responses.

3. Single-Cell RNA Sequencing and Cell-Type Specific Responses
   To further investigate the stress responses of different cell types, single-cell RNA sequencing was performed. Results showed that β-cells had a stronger response to ER stress compared to α-cells, with β-cells exhibiting heterogeneity in stress response, forming two distinct transcriptional states, one of which was more prone to apoptosis. α-cells had a relatively weak response to cytokine treatments, indicating specific response patterns of different islet cell types under pathological stress conditions related to T2D.

4. ATAC-seq Analysis and CREs Dynamic Regulation
   Measuring the openness of CREs through ATAC-seq revealed that about 14% of CREs were significantly reconfigured under stress conditions, with 7171 and 8819 stress-specific CREs responding to ER stress and cytokines, respectively. Most responsive CREs were located in distal non-coding regions, suggesting that these enhancers might be crucial regulatory factors mediating islet cell stress responses. Additionally, the openness of CREs was significantly correlated with the expression of specific genes induced by stress.

5. Overlap Analysis of T2D Risk Variants in CREs
   The study further overlapped GWAS-mapped T2D-related variants with stress-responsive CREs, identifying 161 T2D risk variants overlapping with stress-responsive CREs. These variants might impact islet cell survival under stress by regulating the transcription levels of associated genes.

Research Results

1. Specificity of Gene and CREs Responses to ER and Cytokine Treatments
   The study demonstrated specific gene and CREs responses of islet cells under different stress conditions, with ER stress inducing pathways related to UPR and protein synthesis, while cytokine treatments activated various pro-inflammatory signaling pathways.

2. Heterogeneity of β-Cell Stress Responses
   Single-cell RNA sequencing revealed two distinct transcriptional states of β-cells under ER stress, with one state showing higher susceptibility to apoptosis, potentially contributing to β-cell loss related to T2D.

3. Reconfiguration of CREs and TF Binding Specificity Under Stress Conditions
   ATAC-seq analysis uncovered changes in the openness of numerous distal CREs under stress conditions, enriched with binding sites for crucial transcription factors. ER stress-related CREs were rich in binding sites for transcription factors like ATF4 and CHOP, while cytokine-induced CREs were rich in binding sites for pro-inflammatory factors like IRF8 and NF-κB-p65.

4. Regulatory Functions of Specific T2D Variants in Stress-Responsive CREs
   The study used the example of rs6917676, demonstrating how its T2D risk allele promotes MAP3K5 expression by enhancing CRE openness under ER stress, thereby increasing β-cell apoptosis. This suggests that rs6917676 may play a role in T2D pathological progression through MAP3K5 regulation.

Conclusion and Significance

This study systematically revealed the transcriptional regulatory networks of human islets under ER stress and inflammation induced by pro-inflammatory cytokines through multi-omics approaches, highlighting the functions of T2D risk variants under these stress conditions. The study particularly points out that alleles like rs6917676 may lead to β-cell apoptosis by increasing MAP3K5 expression, providing a theoretical basis for developing MAP3K5-based therapeutic targets for T2D. For example, the MAP3K5 inhibitor selonsertib has shown potential to mitigate diabetes-related cell apoptosis in animal models, suggesting it could be an effective drug option for protecting β-cell function. Furthermore, the study underscores the research value of T2D-related genetic variants in stress responses of disease-specific cell states.

Innovation and Outlook

This study is the first to systematically compare gene expression and CREs dynamic changes of islets under ER stress and pro-inflammatory cytokine stress, revealing the regulatory functions of specific T2D variants under stress conditions. Future research might explore the interactions between other stress factors related to T2D (such as oxidative stress) and genetic variations, thereby providing more targets for T2D mechanism research and drug development.

The findings provide important insights into the genomics and transcriptional regulatory network aspects of T2D pathogenesis, recommending stress-specific interventions based on genetic backgrounds in T2D treatment to improve islet function and delay disease progression.