PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets.

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.

Transient Pax8 expression was reported in mouse islets during gestation, whereas a genome-wide linkage and admixture mapping study highlighted PAX8 as a candidate gene for diabetes mellitus (DM). We sought the significance of PAX8 expression in mouse and human islet biology. PAX8 was induced in gestating mouse islets and in human islets treated with recombinant prolactin. Global gene expression profiling of human and mouse islets overexpressing the corresponding speciesspecific PAX8 revealed the modulation of distinct genetic pathways that converge on cell survival. Accordingly, apoptosis was reduced in PAX8-overexpressing islets. These findings support that PAX8 could be a candidate gene for the study of gestational DM (GDM). PAX8 was genotyped in patients with GDM and gestational thyroid dysfunction (GTD), a pathology commonly found in patients with mutations on PAX8. A novel missense PAX8 mutation (p.T356M, c.1067C>T) was identified in a female diagnosed with GDM and GTD as well as in her father with type 2 DM but was absent in control patients. The p.T356M variant did not alter protein stability or cellular localization, whereas its transactivation activity was hindered. In parallel, a retrospective clinical analysis uncovered that a pregnant female harboring a second PAX8 mutation (p.P25R, c.74C>G) previously reported to cause congenital hypothyroidism also developed GDM. These data indicate that increased expression of PAX8 affects islet viability and that PAX8 could be considered as a candidate gene for the study of GDM. During gestation, maternal metabolic adaptations are essential to ensure the health of the mother and the viability of the fetus. One of the organs that must undergo a profound adaptation during pregnancy is the endocrine pancreas. The islet b-cell mass expands as an adaptive response to the progressive insulin resistance that develops in the pregnant female to favor nutrient accessibility to the fetus (1). This mechanism of b-cell mass expansion includes b-cell hypertrophy, increased b-cell proliferation, and increased prosurvival/antiapoptotic signaling (2). Failure to adapt may result in the development of gestational diabetes mellitus (GDM), with concomitant health issues not only for the mother but also for the fetus. The underlying molecular mechanisms triggering GDM remain largely uncharted (3). Toward identifying these molecular pathways, two independent studies have analyzed the transcriptome landscape of islets isolated from gestating mice (2,4). Of note, one of the most upregulated genes was the transcription factor paired box 8 (PAX8) (2). Historically, PAX8 is known for its essential role in the