Abstract
Aim: To identify the influence of pancreatic stellate cell (PSCs) secretions on gene expression profiles of Min6 cells by whole transcriptome sequencing. Methods: Pancreatic stellate cells (PSCs) were isolated from C57BL6J mice and propagated in vitro to acquire the activated phenotype. Total RNA was isolated from monocultured (MC) and PSC cocultured (CC) Min6 cells to prepare cDNA libraries, which were subjected to whole transcriptome sequencing for identifying differential expression of β-cell transcription factors (Pdx-1, Rfx6 and NeuroD1) related to insulin gene transcription and GSIS related genes such as Glut2, Gck, Abcc8, Kcnj11 and L-type Ca2+ channels (Cacnb2, Cacna1c). qRT-PCR was used to validate the gene expression. GSIS of Min6 cells was examined by estimating insulin levels in response to high glucose challenge. Results: Transcriptome analysis of discovery set revealed that coculture of Min6 cells with PSCs caused increased expression of β-cell specific genes (Ins1, Rfx6 and NeuroD1) concomitant with decreased expression of Pdx-1, MafA and Nkx2-2. Expression of GSIS associated genes (Glut2, Gck, Abcc8, Kcnj11 and Cacnb2) was decreased in such conditions. Validation by qRT-PCR in Min6 cells cocultured with PSCs revealed increased significant expression of Ins1 (2.1 ± 0.22 folds; p ≤ 0.001), Rfx6 (1.68 ± 0.23 folds; p ≤ 0.002) and NeuroD1 (0.96 ± 0.11 folds; p ≤ 0.01), accompanied by downregulation of Cacnb2 (-0.93 ± 0.57 folds; p ≤ 0.05). PSC secretions did not restore the GSIS from glucose unresponsive higher passage Min6 cells (MC: 1.33 ± 0.42; CC: 1.55 ± 0.72 pmol/mg protein; p = ns) upon high glucose stimulation. However, glucose responsive higher passage Min6 cells cocultured with PSCs presented increased insulin secretion (MC: 7.025 ± 0.64; CC: 14.84 ± 1.01 pmol/mg protein; p ≤ 0.04) concomitant with marginal increase of insulin contents. Conclusion: PSC secretions increase Ins1, Rfx6 and NeuroD1 gene expression, GSIS from glucose responsive Min6 cells, but do not restore the GSIS from glucose unresponsive Min6 cells.
Highlights
The functioning of pancreatic β-cells, which perform a pivotal role in maintaining glucose homeostasis, is influenced by various factors including nutritional [1] [2] [3], metabolic [4] [5] and hormonal factors [6] [7] [8], as well as pancreatic inflammatory microenvironment [9] [10] [11] [12]
The discovery set of cDNA libraries, with an average size of 248 and 269 bp length respectively, were prepared from Min6 cells cultured in presence or in absence of pancreatic stellate cells (PSCs) and subjected to whole transcriptome sequencing
Of all the genes subjected to transcriptome analysis in the discovery set, important changes could be noted with regard to increased expression of β-cell specific genes such Ins1 (1.68 folds), Rfx6 (2.33 folds) and NeuroD1 (1.31 folds) accompanied with decreased expression of Pdx1 (−1.92 folds), MafA (−2.32 folds), Nkx2-2 (−1.46 folds) and Glucose Stimulated Insulin Secretion (GSIS) associated genes such as Glut2 (−3.28 folds), Gck (−1.10), Abcc8 (−1.61 folds), Kcnj11 (−2.36 folds), Cacnb2 (−2.97 folds) and Cacna1c (−1.30 folds)
Summary
The functioning of pancreatic β-cells, which perform a pivotal role in maintaining glucose homeostasis, is influenced by various factors including nutritional [1] [2] [3], metabolic [4] [5] and hormonal factors [6] [7] [8], as well as pancreatic inflammatory microenvironment [9] [10] [11] [12]. Availability of adequate information regarding gene expression in β-cells in response to the presence of PSCs in coculture conditions, would resolve these observations and comprehensively elucidate the influence of PSCs on β-cell functions. In view of these considerations, the present study involving indirect coculture of Min cells with activated PSCs was aimed to 1) study the transcriptome profile alterations and the accompanying 2) expression of β-cell specific transcription factors and GSIS related genes in Min cells
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