Research Resource: RNA-Seq Reveals Unique Features of the Pancreatic β-Cell Transcriptome

Ku, Gregory; Kim, Hail; Vaughn, Ian W.; Hangauer, Matthew J.; Oh, Chang‐Myung; German, Michael S.; McManus, Michael T.

doi:10.1210/me.2012-1176

Cited by 98 publications

(96 citation statements)

References 30 publications

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“…Pancreatic β cells express mRNA encoding multiple members of the RAS, RAF, and RASSF protein families (ref. 16 and Supplemental Figure 9). B-RAF and RASSF1 were broadly detected by immunostaining in the pancreas at P5, with relatively high expression in islets ( Figure 3, E and F).…”

Section: Resultsmentioning

confidence: 99%

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Menin determines K-RAS proliferative outputs in endocrine cells

et al. 2014

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R e s e a R c h a R t i c l e4 0 9 4 jci.org Volume 124 Number 9 September 2014 het; Figure 1B). We discovered that pancreata from E13.5 Kras-het embryos contained more endocrine cells, as determined by combined glucagon and insulin staining, than did WT pancreata, while overall pancreatic size remained unchanged (Figure 1, C-E, and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI69004DS1). By P5, Kras-het animals had a relative expansion of both the glucagon-expressing α cell and insulin-expressing β cell populations without any increase in the exocrine pancreas (Figure 1, F-H, and Supplemental Figure 2). Further, Kras-het adults had improved glucose tolerance compared with that of WT controls (Supplemental Figure 3), consistent with an increase in functional β cell capacity. Next, we sought to determine the source of additional endocrine cells in Kras-het animals. The fetal pancreas generates endocrine cells by neogenesis, followed by rapid expansion of those cells by proliferation during perinatal and postnatal growth (Figure 2A and ref. 13). Using expression of the transcription factor NEUROG3 to identify the transient precursor cells generated susceptible to MEN1 gene mutation, menin normally prevents the MAPK effector pathway from driving proliferation, while leaving inhibitory effector pathways such as RASSF1A intact. In this model, loss of menin causes proliferation in susceptible cells due to removal of the blockage of MAPK-driven proliferation downstream of K-RAS, while loss of K-RAS signaling increases proliferation by decreasing unopposed RASSF1A activity. Our data explain the absence of activating KRAS mutations and the high frequency of MEN1 and RASSF1A inactivation in pancreatic endocrine tumors. Our study also suggests potential antiproliferative strategies for treating these tumors and proproliferative therapies for diseases that result from a deficiency of endocrine cell types, such as β cells, in both type 1 and type 2 diabetes. ResultsTo test K-RAS function in pancreatic endocrine cell growth, we used mice carrying the Kras G12D allele, which is a null allele in the absence of Cre recombinase (12), in place of 1 WT allele (Kras- G12D-knock-in allele, which contains the G12D mutation in exon 2 and an upstream LoxP-Stop-LoxP sequence (12), to an active allele expressing constitutively active K-RAS G12D after Cre-mediated recombination. (C and D) Projection images of the dorsal pancreas were collected by whole-mount confocal microscopy from E13.5 mouse embryos of the indicated genotypes and immunostained for E-cadherin (blue) and glucagon and insulin (green) (n = 6 Kras WT, n = 6 Kras-het). (E) Quantification of immunostained areas in the epithelial pancreas (E-cadherin) and endocrine pancreas (glucagon and insulin) relative to WT embryos. (F-H) Pancreatic sections from P5 WT (F) and Kras-het (G) neonates were immunostained for glucagon (red) and insulin (green), with quantification of the relative insulin and glucagon areas and pancreatic mass (H...

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Section: Resultsmentioning

confidence: 99%

“…Gene expression from adult β cells (Supplemental Figure 9) was derived from reanalysis of the data from ref. 16.…”

Section: Glucose Tolerance Testsmentioning

confidence: 99%

Menin determines K-RAS proliferative outputs in endocrine cells

et al. 2014

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“…Pancreatic fragments were pelleted for 2 min at 400 × g at 4°C in a microcentrifuge and then were resuspended in 5 mL of TRIzol and were homogenized with a pellet pestle motor using an RNase-free pestle (Fisher Scientific). GFP + β cells were separated by FACS from digested pancreata, and RNA was purified as previously described (62).…”

Section: Methodsmentioning

confidence: 99%

“…Adult islet and β-cell RNA-sequencing data were derived from published data from massively parallel sequencing of cDNA purified from isolated 4-mo-old adult mouse islets and sorted β cells (62) and were expressed as reads per kilobase of exon model per million mapped reads (RPKM).…”

Section: Methodsmentioning

confidence: 99%

Gα _i/o -coupled receptor signaling restricts pancreatic β-cell expansion

Berger

Scheel

Macias

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic β cells, and variants in genes encoding several Gi-GPCRs—including the α-2a adrenergic receptor, ADRA2A—increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total β-cell mass, and the role of Gi-GPCRs in establishing β-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates β-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic β cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal β cells decreased β-cell proliferation, reduced adult β-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal β-cell proliferation, increased adult β-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult β cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of β-cell replication. These studies link Gi-GPCR signaling to β-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase β-cell mass in patients with diabetes.

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“…Characterization of βLINC1 lncRNAs expressed in the pancreatic islet are often highly tissue-specific, associated with clusters of open chromatin, and located in the genomic vicinity of transcription factors involved in β-cell development and/or function (Ku et al 2012;Moran et al 2012). We analyzed βLINC1 (formally HI-LNC15), a 6.8-kb post-transcriptionally processed human islet-specific transcript without coding potential (Moran et al 2012) that is located in a region of open chromatin ∼20 kb upstream of NKX2.2, an essential islet homeobox transcription factor gene (Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

βlinc1 encodes a long noncoding RNA that regulates islet β-cell formation and function

et al. 2016

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Pancreatic β cells are responsible for maintaining glucose homeostasis; their absence or malfunction results in diabetes mellitus. Although there is evidence that long noncoding RNAs (lncRNAs) play important roles in development and disease, none have been investigated in vivo in the context of pancreas development. In this study, we demonstrate that βlinc1 (β-cell long intergenic noncoding RNA 1), a conserved lncRNA, is necessary for the specification and function of insulin-producing β cells through the coordinated regulation of a number of isletspecific transcription factors located in the genomic vicinity of βlinc1. Furthermore, deletion of βlinc1 results in defective islet development and disruption of glucose homeostasis in adult mice. Type 2 diabetes (T2D) arises when pancreatic islets are no longer able to compensate for the increasing demand of insulin to maintain glucose homeostatic conditions. Despite the improvement in current therapies, >300 million people live with T2D, and those numbers are expected to almost double in the next 20 years. Accordingly, it is necessary to increase our understanding of the underlying biology of islet dysfunctions associated with T2D and identify novel druggable targets.Studies in pancreas development have identified numerous transcription factors and lineage-specific regulatory networks that are required for the specification of the different pancreatic cell types, including acini, ducts, and endocrine cells (Arda et al. 2013). This functionally diverse group of cells arises from a common pool of pancreatic progenitors in two major waves of differentiation termed the primary and secondary transitions. Differentiation of the pancreatic β cells occurs during the secondary transition, and the signaling pathways and regulatory factors involved in this important developmental process have been well characterized (for review, see Pan and Wright 2011). These studies have provided the platform to generate functional β cells in vitro for regenerative medicine and suggested novel therapeutic approaches to prevent and treat β-cell dysfunction.The study of β-cell biology has been recently complemented by genome-wide transcriptome analysis and characterization of epigenetic modifications, suggesting that non-protein-coding regions of the genome are integral to the transcriptional network regulating development and function of β cells (for review, see Arnes and Sussel 2015). Furthermore, a large number of T2D-associated common variants that were identified in genome-wide association studies map to noncoding genomic regions (Pasquali et al. 2014). Although there is increasing evidence that long noncoding RNAs (lncRNAs) play an important role in development and disease (Batista and Chang 2013), none have been investigated in vivo in the context of pancreas development and β-cell function. In this study, we describe the functional characterization of a newly identified conserved lncRNA termed βlinc1 (β-cell long intergenic noncoding RNA 1). We demonstrated that βlinc1 is required for ...

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Research Resource: RNA-Seq Reveals Unique Features of the Pancreatic β-Cell Transcriptome

Cited by 98 publications

References 30 publications

Menin determines K-RAS proliferative outputs in endocrine cells

Menin determines K-RAS proliferative outputs in endocrine cells

Gα _i/o -coupled receptor signaling restricts pancreatic β-cell expansion

βlinc1 encodes a long noncoding RNA that regulates islet β-cell formation and function

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Research Resource: RNA-Seq Reveals Unique Features of the Pancreatic β-Cell Transcriptome

Cited by 98 publications

References 30 publications

Menin determines K-RAS proliferative outputs in endocrine cells

Menin determines K-RAS proliferative outputs in endocrine cells

Gα i/o -coupled receptor signaling restricts pancreatic β-cell expansion

βlinc1 encodes a long noncoding RNA that regulates islet β-cell formation and function

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Gα _i/o -coupled receptor signaling restricts pancreatic β-cell expansion