Abstract
AIMS/HYPOTHESIS: Residual pancreatic beta cells in type 1 diabetes show reduced insulin production but the mechanisms remain unclear. Beta cells undergo stress responses during type 1 diabetes, including endoplasmic reticulum (ER) stress and DNA damage-associated senescence, which may affect insulin production. ER stress reduces insulin production but whether senescence disrupts insulin production in human beta cells has not been investigated.
METHODS: DNA damage-mediated senescence was induced using bleomycin in human donor islets. Relative levels of prohormone convertase 1/3 (PC1/3), prohormone convertase 2 (PC2), carboxypeptidase E (CPE) and the endogenous PC1/3 inhibitor, proprotein convertase subtilisin/kexin type 1 inhibitor (proSAAS), were quantified by western blot. Levels of proinsulin and insulin were measured by ELISA. Flow cytometry was used to measure insulin expression in islet cells. FACS was used to sort endogenous senescent beta cells from islets for analysis of insulin content. Proinsulin immunofluorescence staining was quantified in endogenous senescent vs non-senescent beta cells in pancreas tissue from control donors and donors with type 1 diabetes. Publicly available datasets were used to interrogate relationships between senescence effectors, proinsulin-processing genes and insulin content. DNA damage was induced with bleomycin in the non-proliferative female-fetus-derived EndoC-βH5 human beta cell model to study the impact of the DNA damage response on insulin production in clonal cells growth-arrested due to p16INK4A expression.
RESULTS: DNA damage-mediated senescence led to increased PC1/3 without changes in levels of PC2, CPE or proSAAS in human islets. Consistent with these changes, no significant differences in proinsulin or insulin content were observed, compared with control islets. Flow cytometry confirmed maintenance of insulin content in DNA damage-mediated senescent beta cells vs control cells and sorted endogenous senescent beta cells had similar insulin content to non-senescent beta cells. Proinsulin staining was similar in endogenous senescent vs non-senescent beta cells from a control donor and donor with type 1 diabetes. Analysis of proteomics datasets from Humanislets.com and single-cell RNA-seq datasets from the Human Pancreas Analysis Program corroborated these findings. In EndoC-βH5 beta cells, which are growth-arrested, DNA damage led to decreased levels of CPE and proSAAS, and reduced levels of insulin.
CONCLUSIONS/INTERPRETATION: Our findings suggest that the expression of proinsulin-processing enzymes and the production of insulin are sustained in both chemically induced DNA damage-related senescence and in endogenous senescent adult human beta cells. Collectively, these findings suggest that senescent beta cells may be a source of insulin production among residual beta cells in type 1 diabetes.