Type 2 Diabetes Susceptibility Gene Expression in Normal or Diabetic Sorted Human Alpha and Beta Cells: Correlations with Age or BMI of Islet Donors

Abstract

BackgroundGenome-wide association studies have identified susceptibility genes for development of type 2 diabetes. We aimed to examine whether a subset of these (comprising FTO, IDE, KCNJ11, PPARG and TCF7L2) were transcriptionally restricted to or enriched in human beta cells by sorting islet cells into alpha and beta – specific fractions. We also aimed to correlate expression of these transcripts in both alpha and beta cell types with phenotypic traits of the islet donors and to compare diabetic and non-diabetic cells.Methodology/Principal FindingsIslet cells were sorted using a previously published method and RNA was extracted, reverse transcribed and used as the template for quantitative PCR. Sorted cells were also analysed for insulin and glucagon immunostaining and insulin secretion from the beta cells as well as insulin, glucagon and GLP-1 content. All five genes were expressed in both alpha and beta cells, with significant enrichment of KCNJ11 in the beta cells and of TCF7L2 in the alpha cells. The ratio of KCNJ11 in beta to alpha cells was negatively correlated with BMI, while KCNJ11 expression in alpha cells was negatively correlated with age but not associated with BMI. Beta cell expression of glucagon, TCF7L2 and IDE was increased in cells from islets that had spent more time in culture prior to cell sorting. In beta cells, KCNJ11, FTO and insulin were positively correlated with each other. Diabetic alpha and beta cells had decreased expression of insulin, glucagon and FTO.Conclusions/SignificanceThis study has identified novel patterns of expression of type 2 diabetes susceptibility genes within sorted islet cells and suggested interactions of gene expression with age or BMI of the islet donors. However, expression of these genes in islets is less associated with BMI than has been found for other tissues.

A local glucagon-like peptide 1 (GLP-1) system in human pancreatic islets

“…This is in line with protein abundance data previously reported by our group [26]. Beta cells expressed PC1/3 (also known as PCSK1) and PC2 (also known as PCSK2) (Fig.…”

“…Glucose-stimulated insulin release was higher than that at the basal (3.3 mmol/l glucose) condition, with a further significant potentiation by the conditioned medium, which was prevented by exendin other major incretin [35]. Therefore, although cell fractions from isolated islets usually show some degree of contamination, as shown here and by others [25,26], the available evidence suggests that alpha cells are the source of islet GLP-1. However, the quantification of GLP-1 and glucagon release from human islet cells has yielded conflicting results.…”

“…The corresponding spectra of the GLP-1(7-37) and GLP-1 (7-36) tryptic sequences were matched with the putative digested sequences of the peptides using the sequence editor implemented on BIOTOOLS. GLP-1(7-37) and GLP-1(7-36) peptides were identified by the species at m/z 1105 (aa [21][22][23][24][25][26][27][28] and at m/z 2098 (aa [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20].…”

“…Preparation of alpha and beta cells Methods previously developed and validated were used [25,26]. The dissociation of islets into single-cell suspensions was achieved by incubation with constant agitation for 3 min at 37°C in 0.05% (wt/vol.)…”

The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes

“…Apoptotic cells were accounted for (7-AAD staining) and islet cells were sorted using BD Biosciences FACSAria (BD Biosciences, San Jose, CA, USA). Beta cell enrichment was verified by immunohistochemistry and insulin-positive cells accounted for 90±5% of all cells in the beta cell fractions, comparable with previous observations [22].…”