COUP-TFII Controls Mouse Postnatal Pancreatic β-Cell Mass through GLP-1 β-Catenin

ABSTRACT Background: The control of the functional pancreatic b-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how b-cell mass is determined. Methodology/principal findings: Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal b-cell proliferation and apoptosis, this suggests decreased b-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured b-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the b-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via b-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced b-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat b-cells providing a feedback loop. Conclusions/significance: Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases b-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the b-catenin-dependent pathwayand its expression is under the control of TCF7L2. We determined the RNA profile of COUP-TFII knockdown β-cells (832/13 INS-1 cells transfected with COUP-TFII specific siRNA) with respect to control cells (832/13 INS-1 cells transfected with scrambled siRNA) using Affymetrix expression analysis technical manual 701025Rev.5 (Affymetrix, Santa Clara, California, USA). Briefly, 1 mg of total cellular mRNA was reverse transcribed into cDNA (SuperScript Choice System Invitrogen, Carlsbad, CA) using oligo-dT primers and a T7 RNA polymerase promoter site. The cDNA was in vitro transcribed and biotin-labeled for microarray analysis using the Affymetrix IVT labeling kit. The concentration of labelled cRNA was measured using a NanoDrop ND-1000 spectrophotometer. Labeled cRNA was fragmented in a fragmentation buffer for 35 min at 94°C. The quality of labeled and fragmented cRNA was analyzed using the Agilent bioanalyzer 2100 (Van Lommel et al, 2006). Fragmented cRNA was hybridized to the rat 230 2.0 array (Affymetrix) during 16h at 45°C. Arrays were washed and stained in a fluidics station (Affymetrix) and scanned using the Affymetrix 3000 GeneScanner.