Abstract
Newly differentiated pancreatic β cells lack proper insulin secretion profiles of mature functional β cells. The global gene expression differences between paired immature and mature β cells have been studied, but the dynamics of transcriptional events, correlating with temporal development of glucose-stimulated insulin secretion (GSIS), remain to be fully defined. This aspect is important to identify which genes and pathways are necessary for β-cell development or for maturation, as defective insulin secretion is linked with diseases such as diabetes. In this study, we assayed through RNA sequencing the global gene expression across six β-cell developmental stages in mice, spanning from β-cell progenitor to mature β cells. A computational pipeline then selected genes differentially expressed with respect to progenitors and clustered them into groups with distinct temporal patterns associated with biological functions and pathways. These patterns were finally correlated with experimental GSIS, calcium influx, and insulin granule formation data. Gene expression temporal profiling revealed the timing of important biological processes across β-cell maturation, such as the deregulation of β-cell developmental pathways and the activation of molecular machineries for vesicle biosynthesis and transport, signal transduction of transmembrane receptors, and glucose-induced Ca2+ influx, which were established over a week before β-cell maturation completes. In particular, β cells developed robust insulin secretion at high glucose several days after birth, coincident with the establishment of glucose-induced calcium influx. Yet the neonatal β cells displayed high basal insulin secretion, which decreased to the low levels found in mature β cells only a week later. Different genes associated with calcium-mediated processes, whose alterations are linked with insulin resistance and deregulation of glucose homeostasis, showed increased expression across β-cell stages, in accordance with the temporal acquisition of proper GSIS. Our temporal gene expression pattern analysis provided a comprehensive database of the underlying molecular components and biological mechanisms driving β-cell maturation at different temporal stages, which are fundamental for better control of the in vitro production of functional β cells from human embryonic stem/induced pluripotent cell for transplantation-based type 1 diabetes therapy.
Highlights
Pancreatic β cells are functionally defined by their capacity for insulin secretion, stimulated by glucose and other nutrients
Before applying the temporal analysis on both embryonic and postnatal stages, we first checked the utility of the MipeGFP cell– based gene expression for studying β-cell maturation at postnatal stages
As further validation of using MipeGFP cell–based gene expression for monitoring β-cell maturation, we examined the expression of 60 genes in RipmCherry β cells with Realtime polymerase chain reaction (RT-PCR) and determined whether the results consistently match the RNA sequencing (RNA-seq) results in MipeGFPβ cells
Summary
Pancreatic β cells are functionally defined by their capacity for insulin secretion, stimulated by glucose and other nutrients. Type 1 diabetes results from autoimmune destruction of β cells in the pancreatic islet, whereas the more common type 2 diabetes results from peripheral tissue insulin resistance and β-cell dysfunction. Diabetic patients, those suffering from type 1 diabetes, could potentially be cured through transplantation of new β cells. Those suffering from type 1 diabetes, could potentially be cured through transplantation of new β cells To this end, several protocols have allowed the production of glucose responsive β-like cells from human embryonic stem/induced pluripotent cells (Kushner et al, 2014; Schiesser and Wells, 2014; Tabar and Studer, 2014). This low efficiency is partly due to our lack of understanding the signaling pathways that direct β-cell maturation (Kieffer, 2016)
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