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Mutations in the gene encoding the transcription factor regulatory factor X-box binding 6 (RFX6) are associated with human diabetes. Within pancreatic islets, RFX6 expression is most abundant in islet α-cells, and α-cell RFX6 expression is altered in diabetes. However, the roles of RFX6 in regulating gene expression, glucagon output, and other crucial human adult α-cell functions are not yet understood. We developed a method for selective genetic targeting of human α-cells and assessed RFX6 -dependent α-cell function. RFX6 suppression with RNA interference led to impaired α-cell exocytosis and dysregulated glucagon secretion in vitro and in vivo. By contrast, these phenotypes were not observed with RFX6 suppression across all islet cells. Transcriptomics in α-cells revealed RFX6 -dependent expression of genes governing nutrient sensing, hormone processing, and secretion, with some of these exclusively expressed in human α-cells. Mapping of RFX6 DNA-binding sites in primary human islet cells identified a subset of direct RFX6 target genes. Together, these data unveil RFX6-dependent genetic targets and mechanisms crucial for regulating adult human α-cell function. Article Highlights: RFX6 is expressed in all islet endocrine cell types and is dysregulated in multiple forms of diabetes, but its function has not yet been delineated in α-cells. We used specific targeting of shRNA-mediated suppression of RFX6 in primary human α-cells to unveil glucagon secretion phenotypes. RFX6 is required in adult human α-cells to maintain gene regulation and hallmark functions, including regulated glucagon secretion. RNA-sequencing and cleavage under targets and release using nuclease studies reveal distinct RFX6 genetic targets in adult human α- and β-cells. [ABSTRACT FROM AUTHOR]

Reliance on rodents for understanding pancreatic genetics, development and islet function could limit progress in developing interventions for human diseases such as diabetes mellitus. Similarities of pancreas morphology and function suggest that porcine and human pancreas developmental biology may have useful homologies. However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α- and δ-cells followed by transcriptome analysis (RNA-seq) and immunohistology identified celland stage-specific regulation, and revealed that pig and human islet cells share characteristic features that are not observed in mice. Morphometric analysis also revealed endocrine cell allocation and architectural similarities between pig and human islets. Our analysis unveiled scores of signaling pathways linked to native islet β-cell functional maturation, including evidence of fetal α-cell GLP-1 production and signaling to β-cells. Thus, the findings and resources detailed here show how pig pancreatic islet studies complement other systems for understanding the developmental programs that generate functional islet cells, and that are relevant to human pancreatic diseases. [ABSTRACT FROM AUTHOR]

The islets of Langerhans are endocrine organs characteristically dispersed throughout the pancreas. During development, endocrine progenitors delaminate, migrate radially and cluster to form islets. Despite the distinctive distribution of islets, spatially localized signals that control islet morphogenesis have not been discovered. Here, we identify a radial signaling axis that instructs developing islet cells to disperse throughout the pancreas. A screen of pancreatic extracellular signals identified factors that stimulated islet cell development. These included semaphorin 3a, a guidance cue in neural development without known functions in the pancreas. In the fetal pancreas, peripheral mesenchymal cells expressed Sema3a, while central nascent islet cells produced the semaphorin receptor neuropilin 2 (Nrp2). Nrp2 mutant islet cells developed in proper numbers, but had defects in migration and were unresponsive to purified Sema3a. Mutant Nrp2 islets aggregated centrally and failed to disperse radially. Thus, Sema3a-Nrp2 signaling along an unrecognized pancreatic developmental axis constitutes a chemoattractant system essential for generating the hallmark morphogenetic properties of pancreatic islets. Unexpectedly, Sema3a- and Nrp2-mediated control of islet morphogenesis is strikingly homologous to mechanisms that regulate radial neuronal migration and cortical lamination in the developing mammalian brain. [ABSTRACT FROM AUTHOR]

Defects in primary cilia result in syndromes collectively called "ciliopathies", which often present with obesity and diabetes. Although glucose is the primary mediator of GSIS, circulating factors including free fatty acids, amino acids, and various hormones also play critical roles. At the interface between these circulating factors and GSIS, GPCR signaling is often central. The primary cilium is a membrane and microtubule-based sensory organelle and is highly enriched with specialized GPCRs. Cilia are present on pancreatic β-cells, but the role of cilia in β-cell function remains unclear. To discover ciliary GPCRs important for β-cell function, we compiled a list of candidate β-cell specific or highly expressed GPCRs based on human pancreatic gene expression profiles. We identified 66 β-cell specific GPCRs comparing with α-cells and acinar-cells. These 66 candidate GPCRs was expressed as C-terminally tagged GFP fusion proteins and screened for ciliary localization in a mouse pancreatic β-cell line, MIN6. To date, we have identified the following GPCRs localize to cilia in MIN6 cells: FFAR4, PTGER4, ADRB2, KISS1R, and P2RY14. To validate ciliary localization of GPCRs, we also used antibodies against the endogenous GPCRs. We have confirmed that the free fatty acid receptor 4 (Ffar4) and the prostaglandin E receptor 4 (Ptger4) are localized to the primary cilium of mouse and human β-cells. In addition, the treatment of each of agonists of these receptors promote glucose-stimulated insulin secretion (GSIS) in mouse and human islet. Tulp3 is required for transporting GPCRs into the primary cilium. We used shRNA targeting Tulp3 delivered by lentivirus to knock-down Tulp3 in mouse and human islet cells. Tulp3 depletion leaves ciliary structure intact but impairs the localization of Ffar4 and Ptger4 in cilia and disrupts agonist-stimulated GSIS in mouse and human pseudo-islets. Together, these data suggest that primary cilia and ciliary GPCRs are important for the circulating factors-regulated GSIS in β-cell. Disclosure: C. Wu: None. K. Hilgendorf: None. Y. Hang: None. R.J. Bevacqua: None. C.T. Johnson: None. C.A. Chang: None. S. Park: None. K. Tellez: None. S. Kim: None. P.K. Jackson: None. Funding: Stanford Diabetes Research Center [ABSTRACT FROM AUTHOR]