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 β-cell Pax6 transcriptional network and thus provide further information concerning the role of Pax6 in β-cell function. We developed a Pax6-deficient model in rat primary β-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 β-cells. Importantly, we identified new Pax6 target genes coding for GK, Nkx6.1, cMaf, PC2, GLP-1R and GIPR which are all involved in β-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 β-cells. Our results strongly suggest that Pax6 is crucial for β-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 β-cells. We provide further evidence that Pax6 represents a key element of mature β-cell function.

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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 ␤ and ␦ cells and absent ␣ cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat ␣ cells. To study the transcriptional network of Pax6 in adult and differentiated ␣ cells, we generated Pax6-deficient primary rat ␣ cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominantnegative form of Pax6. In primary rat ␣ 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 ␣ 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 ␣ cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for ␣ cell development. We conclude that Pax6 is critical for ␣ cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and ␣ cell differentiation.

The glucagon gene is expressed in α-cells of the pancreas, L cells of the intestine and the hypothalamus. The determinants of the α-cell-specific expression of the glucagon gene are not fully characterized, although Arx, Pax6 and Foxa2 are critical for α-cell differentiation and glucagon gene expression; in addition, the absence of the β-cell-specific transcription factors Pdx1, Pax4 and Nkx6.1 may allow for the glucagon gene to be expressed. Pax6, along with cMaf and MafB, binds to the DNA control element G(1) which confers α-cell specificity to the promoter and to G(3) and potently activates glucagon gene transcription. In addition, to its direct role on the transcription of the glucagon gene, Pax6 controls several transcription factors involved in the activation of the glucagon gene such as cMaf, MafB and NeuroD1/Beta2 as well as different steps of glucagon biosynthesis and secretion. We conclude that Pax6 independently of Arx and Foxa2 is critical for α-cell function by coordinating glucagon gene expression as well as glucagon biosynthesis and secretion.