Abstract
Changes to islet cell identity in response to type 2 diabetes (T2D) have been reported in rodent models, but are less well characterized in humans. We assessed the effects of aspects of the diabetic microenvironment on hormone staining, total gene expression, splicing regulation and the alternative splicing patterns of key genes in EndoC-βH1 human beta cells. Genes encoding islet hormones [somatostatin (SST), insulin (INS), Glucagon (GCG)], differentiation markers [Forkhead box O1 (FOXO1), Paired box 6, SRY box 9, NK6 Homeobox 1, NK6 Homeobox 2] and cell stress markers (DNA damage inducible transcript 3, FOXO1) were dysregulated in stressed EndoC-βH1 cells, as were some serine arginine rich splicing factor splicing activator and heterogeneous ribonucleoprotein particle inhibitor genes. Whole transcriptome analysis of primary T2D islets and matched controls demonstrated dysregulated splicing for ~25% of splicing events, of which genes themselves involved in messenger ribonucleic acid processing and regulation of gene expression comprised the largest group. Approximately 5% of EndoC-βH1 cells exposed to these factors gained SST positivity in vitro. An increased area of SST staining was also observed ex vivo in pancreas sections recovered at autopsy from donors with type 1 diabetes (T1D) or T2D (9.3% for T1D and 3% for T2D, respectively compared with 1% in controls). Removal of the stressful stimulus or treatment with the AKT Serine/Threonine kinase inhibitor SH-6 restored splicing factor expression and reversed both hormone staining effects and patterns of gene expression. This suggests that reversible changes in hormone expression may occur during exposure to diabetomimetic cellular stressors, which may be mediated by changes in splicing regulation.
Highlights
A reduction in beta cell mass occurs during the progression of type 2 diabetes (T2D) and has been attributed to net enhancement of the rate of beta cell death [1,2]
The cellular microenvironment induced by diabetes is stressful to beta cells, which may respond by either undergoing apoptosis [1,2] or by altering their differentiation state [10,28,29] resulting in an overall loss of beta cell mass
We report here that human EndoC-βH1 beta cells treated with diabetes-related cellular stressors, such as altered glucose, fatty acids, cytokines or hypoxia or with the ER stress inducer tunicamycin, displayed altered expression of genes involved in splicing regulation, as well as altered expression of important genes for cell fate or function in vitro
Summary
A reduction in beta cell mass occurs during the progression of type 2 diabetes (T2D) and has been attributed to net enhancement of the rate of beta cell death [1,2]. It is increasingly apparent, that changes in the differentiation status of beta cells may be a contributory factor [3,4]. Beta to delta cell transdifferentiation has been reported by lineage tracing in mouse islets in response to immunological stimuli [8]. Data from human pancreas are scarce, but the limited information available suggests that similar changes in differentiation status may occur in humans [9,10].
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