Project description:RNA-Seq to investigate significant transcriptional differences underlying the defective glucose-stimulated insulin secretion of Pax6 knockout mice in comparison to floxed littermate controls.
Project description:Recent advances in the understanding of the genetics of type 2 diabetes (T2D) susceptibility have focused attention on the regulation of transcriptional activity within the pancreatic beta-cell. MicroRNAs (miRNAs) represent an important component of regulatory control, and have proven roles in the development of human disease and control of glucose homeostasis. We set out to establish the miRNA profile of human pancreatic islets and of enriched beta-cell populations, and to explore their potential involvement in T2D susceptibility. We used Illumina small RNA sequencing to profile the miRNA fraction in three preparations each of primary human islets and of enriched beta-cells generated by fluorescence-activated cell sorting. In total, 366 miRNAs were found to be expressed (i.e. >100 cumulative reads) in islets and 346 in beta-cells; of the total of 384 unique miRNAs, 328 were shared. A comparison of the islet-cell miRNA profile with those of 15 other human tissues identified 40 miRNAs predominantly expressed (i.e. >50% of all reads seen across the tissues) in islets. Several highly-expressed islet miRNAs, such as miR-375, have established roles in the regulation of islet function, but others (e.g. miR-27b-3p, miR-192-5p) have not previously been described in the context of islet biology. As a first step towards exploring the role of islet-expressed miRNAs and their predicted mRNA targets in T2D pathogenesis, we looked at published T2D association signals across these sites. We found evidence that predicted mRNA targets of islet-expressed miRNAs were globally enriched for signals of T2D association (p-values <0.01, q-values <0.1). At six loci with genome-wide evidence for T2D association (AP3S2, KCNK16, NOTCH2, SCL30A8, VPS26A, and WFS1) predicted mRNA target sites for islet-expressed miRNAs overlapped potentially causal variants. In conclusion, we have described the miRNA profile of human islets and beta-cells and provide evidence linking islet miRNAs to T2D pathogenesis. Examination of the miRNA profiles in 3 preparations of isolated pancreatic islets and 3 preparations of FACS-enriched pancreatic beta-cells
Project description:To explore the underlying mechanism for the regulatory role of SIRT3 in pancreatic islets under standard and high fat diet feeding, we conducted RNA sequencing on the isolated islets from standard diet and high fat diet-fed wild type and pancreatic beta cell selective Sirt3 knockout mice (four groups in total). Three biological replicates were performed for each group.
Project description:The zinc finger factor Insm1 is known to regulate differentiation of pancreatic β cells during development, Here we show that Insm1 is essential for the maintenance of functionally mature pancreatic β cells in mice. We used microarrays to analyse the global gene expression after deletion of insm1 in adult pancreatic β cells and identified functional important genes and immature islets releated genes deregulated in the mutatant islets.
Project description:Pathways that stimulate β-cell regeneration remain of great clinical interest, yet effective therapeutic avenues that promote survival or reconstitution of β-cell mass remain elusive. Utilizing a mouse model with inducible β-cell apoptosis followed by adiponectin-mediated regeneration, we aimed to identify key molecules boosting β-cell viability. Within the regenerating pancreatic islets, we examined changes within the transcriptome, and observed an extensive upregulation of genes encoding proteins involved in lipid transport and metabolism. The most prominent targets were further confirmed by quantitative PCR and immunofluorescence. Among the upstream regulators predicted by pathway analysis of the transcriptome, we detected enhanced levels of two key transcription factors, HNF4α and PPARα. Enhanced leptin levels in circulation may also contribute to the anti-lipotoxic program in islets. In summary, our data suggest that improving local lipid metabolism as an important anti-lipotoxic phenomenon to boost β-cell regeneration, primarily mediated by adiponectin’s action on the β-cells directly as well as on the adipocyte. RNA profiles of pancreatic islets isolated from PANIC-ATTAT mice crossed with adiponectin wild-type (P-Adn+/+) or the overexpressing transgene (P-AdnTg/+) at 5 weeks after initial dimerizer administration.
Project description:The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep-wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6Leca2 mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite β cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for β cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6.
Project description:Transcriptome profiling using RNA-seq of β-TC3 cell, a mouse pancreatic cell line used in the study of novel Cis-regulatory elements for the Pax6 gene .
Project description:The short chain fatty acid (SCFA) receptor (free fatty acid receptor-3; FFAR3) is expressed in pancreatic beta cells; however, its role in insulin secretion is not clearly defined. Here, we examined the role of FFAR3 in insulin secretion. Using islets from global knockout FFAR3 (Ffar3-/-) mice, we explored the role of FFAR3 and ligand-induced FFAR3 signaling on glucose stimulated insulin secretion. RNA sequencing was also performed to gain greater insight into the impact of FFAR3 deletion on the islet transcriptome. First exploring insulin secretion, it was determined that Ffar3-/- islets secrete more insulin in a glucose-dependent manner as compared to wildtype (WT) islets. Next, exploring its primary endogenous ligand, propionate, and a specific agonist for FFAR3, signaling by FFAR3 inhibited glucose-dependent insulin secretion, which occurred through a Gαi/o pathway. To help understand these results, transcriptome analyses by RNA-sequencing of Ffar3-/- and WT islets observed multiple genes with well known roles in islet biology to be altered by genetic knockout of FFAR3. Our data shows that FFAR3 signaling mediates glucose stimulated insulin secretion through Gαi/o sensitive pathway. Future studies are needed to more rigorously define the role of FFAR3 by in vivo approaches. Analysis of total RNA from 3 biological replicates of pancreatic islets isolated from free fatty acid receptor 3 knockout (Ffar3 KO) and wildtype (Ffar3 WT) male mice
Project description:In this study, we achieved integrated transcriptomic and proteomic profiles of GK islets in a time-course fashion at different stages of T2D. Subsequent bioinformatics analysis revealed the chronological order of T2D-related molecular events during the deterioration of pancreatic islets. Our large quantitative dataset provide a valuable resource to obtain a comprehensive picture of the mechanisms responsible for islet dysfunction and to identify potential interventions to prevent beta-cell failure in human T2D.