Pax6 controls the expression of critical genes involved in pancreatic {alpha} cell differentiation and function.

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased ß and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

EIF4A2 is a positional candidate gene at the 3q27 locus linked to type 2 diabetes in French families.

One of the most replicated loci influencing type 2 diabetes-related quantitative traits (quantitative trait loci [QTL]) is on chromosome 3q27 and modulates both type 2 diabetes-and metabolic syndrome-associated phenotypes. A QTL for type 2 diabetes age of onset (logarithm of odds [LOD] score = 3.01 at D3S3686, P = 0.0001) was identified in a set of French families. To assess genetic variation underlying both age-of-onset QTL and our previous type 2 diabetes linkage in a 3.87-Mb interval, we explored 36 single nucleotide polymorphisms (SNPs) in two biologically relevant candidate genes for glucose homeostasis, kininogen (KNG1), and eukaryotic translation initiation factor 4alpha2 (EIF4A2). Analysis of 148 families showed significant association of a frequent SNP, rs266714, located 2.47 kb upstream of EIF4A2, with familial type 2 diabetes (family-based association test, P = 0.0008) and early age of onset (P = 0.0008). This SNP also contributes to both age-of-onset QTL (1.13 LOD score decrease P = 0.02) and type 2 diabetes linkage (genotype identical-by-descent sharing test, P = 0.02). However, no association was observed in three independent European diabetic cohorts. EIF4A2 controls specific mRNA translation and protein synthesis rate in pancreatic beta-cells, and our data indicates that EIF4A2 is downregulated by high glucose in rat beta-INS832/13 cells. The potential role of EIF4A2 in glucose homeostasis and its putative contribution to type 2 diabetes in the presence of metabolic stress will require further investigation.

Analysis of common PTPN1 gene variants in type 2 diabetes, obesity and associated phenotypes in the French population.

BACKGROUND: The protein tyrosine phosphatase-1B, a negative regulator for insulin and leptin signalling, potentially modulates glucose and energy homeostasis. PTP1B is encoded by the PTPN1 gene located on chromosome 20q13 showing linkage with type 2 diabetes (T2D) in several populations. PTPN1 gene variants have been inconsistently associated with T2D, and the aim of our study was to investigate the effect of PTPN1 genetic variations on the risk of T2D, obesity and on the variability of metabolic phenotypes in the French population. METHODS: Fourteen single nucleotide polymorphisms (SNPs) spanning the PTPN1 locus were selected from previous association reports and from HapMap linkage disequilibrium data. SNPs were evaluated for association with T2D in two case-control groups with 1227 cases and 1047 controls. Association with moderate and severe obesity was also tested in a case-control study design. Association with metabolic traits was evaluated in 736 normoglycaemic, non-obese subjects from a general population. Five SNPs showing a trend towards association with T2D, obesity or metabolic parameters were investigated for familial association. RESULTS: From 14 SNPs investigated, only SNP rs914458, located 10 kb downstream of the PTPN1 gene significantly associated with T2D (p = 0.02 under a dominant model; OR = 1.43 [1.06-1.94]) in the combined sample set. SNP rs914458 also showed association with moderate obesity (allelic p = 0.04; OR = 1.2 [1.01-1.43]). When testing for association with metabolic traits, two strongly correlated SNPs, rs941798 and rs2426159, present multiple consistent associations. SNP rs2426159 exhibited evidence of association under a dominant model with glucose homeostasis related traits (p = 0.04 for fasting insulin and HOMA-B) and with lipid markers (0.02 = p = 0.04). Moreover, risk allele homozygotes for this SNP had an increased systolic blood pressure (p = 0.03). No preferential transmission of alleles was observed for the SNPs tested in the family sample. CONCLUSION: In our study, PTPN1 variants showed moderate association with T2D and obesity. However, consistent associations with metabolic variables reflecting insulin resistance and dyslipidemia are found for two intronic SNPs as previously reported. Thus, our data indicate that PTPN1 variants may modulate the lipid profile, thereby influencing susceptibility to metabolic disease.

Pax6 is a key component of regulated glucagon secretion.

The Pax6 transcription factor is crucial for pancreatic α-cells. Indeed, Pax6-deficient mouse models are characterized by markedly altered α-cell differentiation. Our objective was to investigate the role of Pax6 in glucagon secretion process. We used a Pax6-deficient model in rat primary enriched-α cells with specific small interfering RNA leading to a 70% knockdown of Pax6 expression. We first showed that Pax6 knockdown decreases glucagon biosynthesis as well as glucagon release. Through physiological assays, we demonstrated that the decrease of Pax6 affects specifically acute glucagon secretion in primary α-cell in response to glucose, palmitate, and glucose-dependent insulinotropic peptide (GIP) but not the response to arginine and epinephrine. We identified in Pax6 knockdown model that genes involved in glucagon secretion such as the glucokinase (GCK), G protein-coupled receptor (GPR40), and GIP receptor (GIPR) as well as the corresponding proteins were significantly decreased whereas the insulin receptor (IR) Kir6.2/Sur1, and glucose transporter 1 genes were not affected. We demonstrated that Pax6 directly binds and activates specific elements on the promoter region of the GPR40, GCK, and GIPR genes. Finally, through site-directed mutagenesis experiments, we showed that disruption of Pax6 binding on the GCK, GPR40, and GIPR gene promoters led to specific decreases of their activities in the αTC1.9 glucagon-producing cell line. Hence our results indicate that Pax6 acts on the regulation of glucagon secretion at least through the transcriptional control of GCK, GPR40, and GIPR. We propose that Pax6 is not only critical for glucagon biosynthesis but also for glucagon secretion particularly in response to nutrients.

Pax6 is crucial for ß-cell function, insulin biosynthesis, and glucose-induced insulin secretion.

The Pax6 transcription factor is crucial for endocrine cell differentiation and function. Indeed, mutations of Pax6 are associated with a diabetic phenotype and a drastic decrease of insulin-positive cell number. Our aim was to better define the ß-cell Pax6 transcriptional network and thus provide further information concerning the role of Pax6 in ß-cell function. We developed a Pax6-deficient model in rat primary ß-cells with specific small interfering RNA leading to a 75% knockdown of Pax6 expression. Through candidate gene approach, we confirmed that Pax6 controls the mRNA levels of the insulin 1 and 2, Pdx1, MafA, GLUT2, and PC1/3 genes in ß-cells. Importantly, we identified new Pax6 target genes coding for GK, Nkx6.1, cMaf, PC2, GLP-1R and GIPR which are all involved in ß-cell function. Furthermore, we demonstrated that Pax6 directly binds and activates specific elements on the promoter region of these genes. We also demonstrated that Pax6 knockdown led to decreases in insulin cell content, in insulin processing, and a specific defect of glucose-induced insulin secretion as well as a significant reduction of GLP-1 action in primary ß-cells. Our results strongly suggest that Pax6 is crucial for ß-cells through transcriptional control of key genes coding for proteins that are involved in insulin biosynthesis and secretion as well as glucose and incretin actions on ß-cells. We provide further evidence that Pax6 represents a key element of mature ß-cell function.