A pro-endocrine pancreatic transcriptional program established during development is retained in human gallbladder epithelial cells

Abstract

Objective: Pancreatic islet β-cells are factories for insulin production; however ectopic expression of insulin is also well recognized. The gallbladder is a next-door neighbour to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells. Design: We compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, ELISAs, RNA-sequencing, real time PCR, chromatin immunoprecipitation and functional studies. Results: We demonstrate that the epithelial lining of developing, as well as adult mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show for the first time that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a NOD mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Conclusion: In summary, our biochemical, transcriptomic, and functional data in human gallbladder epithelial cells collectively demonstrate their potential for insulin-production under pathophysiological conditions, opening newer areas for diabetes research and therapy.

Identification and functional validation of human islet microRNAs associated with donor trait

“…Total RNA isolation and quantification for concentration and RNA integrity number (RIN) was performed as described previously [65][66][67]. RNA was quantified using the Nanodrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA).…”

“…Details of PCR results analysis are described in our previous study [66]. MicroRNA transcript abundance was then log2 transformed for statistical analyses Bulk RNA-sequencing and analysis RNA-sequencing on human islet samples (GSE152111) was performed on the HiSeq4000 platform at 150 paired-end reads as detailed in previous study [67]. RNA-sequencing analysis was carried out using the Strand Next-generation sequencing (NGS) version 2.5 software as described previously [67].…”

“…MicroRNA transcript abundance was then log2 transformed for statistical analyses Bulk RNA-sequencing and analysis RNA-sequencing on human islet samples (GSE152111) was performed on the HiSeq4000 platform at 150 paired-end reads as detailed in previous study [67]. RNA-sequencing analysis was carried out using the Strand Next-generation sequencing (NGS) version 2.5 software as described previously [67]. Samples were aligned to the human hg38 transcriptome and genome together (with novel splice variants) with the Ensembl genes and transcript model.…”

Analysis of half a billion datapoints across ten machine-learning algorithms identify key determinants of insulin gene transcription

“…Human pancreatic islet RNA-seq dataset was obtained from GSE152111 [41]. RNA-seq dataset contains n=66 human islet samples, across 65 different donors with no diabetes.…”

Machine learning workflows identify a microRNA signature of insulin transcription in human tissues

“…Studies comparing microRNA profiles of cells/tissues that naturally transcribe the (pro-)insulin gene (e.g. thymus, gallbladder, brain (Choi et al, 2019;Devaskar et al, 1994;Heller et al, 2010;Mehran et al, 2012;Sahu et al, 2009;Dutton et al, 2007;Joglekar et al, 2021) are also lacking. Here, we sought to identify microRNAs that fine-tune the expression of the human (pro-)insulin gene using an unbiased, machine learning discovery approach, followed by wet-lab validation.…”