Abstract
Aims/hypothesisA strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM.MethodsTwo groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements.ResultsPAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death.Conclusions/interpretationThe coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3864-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.
Highlights
The islet of Langerhans is the core unit of the endocrine pancreas, which regulates blood glucose homeostasis
Microarray profiling revealed that paired box 4 (PAX4) but not PAX4R129W targeted expression of genes implicated in cell cycle and endoplasmic reticulum (ER) homeostasis
Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose
Summary
The islet of Langerhans is the core unit of the endocrine pancreas, which regulates blood glucose homeostasis. Regulation is achieved by the release of insulin from beta cells in response to increasing levels of glucose and by the secretion of glucagon from alpha cells under fasting conditions. Imbalance in this circuitry leads to either hyperglycaemia, the hallmark of diabetes mellitus, or hypoglycaemia. MODY genes such as Pdx and Hnf1a regulate UPR-associated genes [7, 8]. These clinical conditions suggest that islet-enriched transcription factors involved in insulin biosynthesis and secretion preserve the BCM by limiting ER stress
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