• Le 26 février 2018
  • à 16h

« Multiple Type 2 diabetes genes and functional genetic networks identified using single-cell RNA-sequencing of human pancreatic islets »

Nils Wierup
Lund University Diabetes Centre, Malmö, Sweden

Deranged islet function is a culprit in type 2 diabetes (T2D). Here we used Smart-seq2 single-cell transcriptomics to sequence human pancreatic islet cells from 6 human control donors and 4 T2D donors, obtaining the transcriptome of 3075 cells. Cell identity was determined using the two-way unsupervised clustering algorithm BackSPIN and t-SNE technique, uncovering 12 distinct cell populations, including alpha-, beta-, delta-, PP- and ghrelin cells. Clustering was verified manually by comprehensive analysis of differential expression of established markers for each cell type. Furthermore, we have identified numerous genes that are differentially regulated in alpha- (857 genes), beta- (972 genes) and delta cells (43 genes) in T2D donors. Most of these genes have not previously been described in islet function and functional validation in INS-1 832/13 cells revealed important roles for several of the top hits in regulation of insulin transcription and secretion. Furthermore, weighted gene co-expression network analysis (WGCNA) showed that the top listed genes were co-expressed with important T2D-associated islet genes, e.g. PDX1, INS, GCGR, and GLP1R. These predicted networks were functionally tested using RNAseq of cells silenced for selected candidate genes. WGCNA was also used to create a network map of differentially regulated genes by T2D for each cell type, showing connections between them and with important islet genes based on co-expression levels. In this network, highly interconnected clusters of upregulated and/or downregulated genes by T2D for every specific cell type could give us a global perspective of the genetic mechanisms underlying islet dysfunction in T2D.

Acknowledgments : The single-cell transcriptome data was generated at the Eukaryotic Single Cell Genomics Facility and the National Genomics Infrastructure at Science for Life Laboratory in Stockholm, Sweden.
Funding : The Novo Nordisk, EFSD-AZN, and Crafoord Foundations.

The main strengths of Neuroendocrine Cell Biology, Lund University Diabetes Centre, Sweden is islet and gut biology, and we master a broad range of techniques for functional studies, ranging from molecular biology to in vivo physiology in small and large animals, as well as humans. We are also experts on histological analyses of most tissues and have a freezer packed with well-characterized antibodies. We have three main lines of research.
1) The function of novel peptide hormones in the islets and gut. Currently a major focus is on the CART-peptide that has several “antidiabetic” effects.
2) Mechanisms behind T2D remission induced by gastric bypass surgery (GBP), i.e. the acute effect that is evident within days after surgery. We are currently performing 3 clinical studies and have developed animal models of GBP in pigs and rats. The overall aim is to identify mechanism that could replace surgery. A major focus is the effect of GBP on splanchnic blood flow and the role of GIP. Another is to understand the effect of GBP on the islets. 3) Understanding islet pathophysiology and identify targetable disease mechanism by studying human islets with single cell RNAseq.