PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus

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.

PAX4 R192H and P321H polymorphisms in type 2 diabetes and their functional defects

“…In contrast, their expression levels were decreased in the cells overexpressing PAX4 R192H but not PAX4 P321H variant protein (Figure 4). In consistence with the previous report, 24 we also observed that PAX4 R121W, a diabetes-linked variant reported in Japanese with T2D, 12 was less efficient to activate the expression of Lgals9 and Calr genes than the wild-type PAX4 protein ( Figure 4). These data illustrated that PAX4 R192H variant protein impaired transcription activation on target genes that required to maintain β-cell function, proliferation and survival.…”

The Diabetes-Linked Transcription Factor PAX4: From Gene to Functional Consequences

“…The pro-proliferative and anti-apoptotic role of PAX4 has been validated in vivo in a mouse model that conditionally over-express PAX4 specifically in β-cells. Over-expression of PAX4 in vivo protects β-cells from apoptosis induced by streptozotocin (STZ) and in a mouse model of experimental autoimmune diabetes (RIPB7.1 mice) [37,39]. Remarkably, in the latter model, PAX4 overexpression decreases islet immune cell infiltration (insulitis), suggesting a novel immunomodulatory function of PAX4 [39].…”

“…Moreover, long-term in vivo ectopic expression of PAX4 increases c-Myc and Cdk4 mRNA levels, correlating with the increase in proliferation of PDX1 + /insulin − β-cells [37]. Remarkably, transcriptome analysis of islet cells after PAX4 overexpression in vivo revealed the downregulation of cyclin-dependent kinase inhibitor 2A, which strongly inhibits Cdk4 , [39]. Further analysis or these microarray data revealed a functional enrichment in cell cycle pathway after PAX4 overexpression.…”

Levothyroxine enhances glucose clearance and blunts the onset of experimental type 1 diabetes mellitus in mice

“…Given the effects of T4 supplementation in the promotion of beta cell proliferation and the increase of circulating insulin levels in healthy C57BL/6 mice, we next sought to determine whether T4 supplementation could blunt the onset of T1DM in the RIP-B7.1 model of EAD (Harlan et al, 1994;Mellado-Gil et al, 2016). To this end, RIP-B7.1 mice were treated with T4, (5 μg·mL À1 in drinking water) starting at 6 weeks of age.…”

“…Female mice were treated or not with T4 starting at birth (s.c. injection of 18 ng·g À1 of body weight daily until weaning). After weaning, mice were supplemented with T4 in drinking water (50 μg·mL À1 ) (Spencer and West, 1961;Weinstein et al, 1991;Weinstein et al, 1994 Buras et al, 2014 For experiments related to EAD, transgenic RIP-B7.1 mice were used (Harlan et al, 1994;Mellado-Gil et al, 2016); these mice were supplied by Dr Bernhard. O. Boehm (Ulm University Medical Centre, Ulm, Germany).…”