Research Article: Changes of MODY signal pathway genes in the endoplasmic reticulum stress in INS-1-3 cells

Date Published: June 7, 2018

Publisher: Public Library of Science

Author(s): Yanan Dong, Shirui Li, Wenhui Zhao, Yanlei Wang, Tingting Ge, Jianzhong Xiao, Yukun Li, Herve Le Stunff.

http://doi.org/10.1371/journal.pone.0198614

Abstract

Metabolic disturbances induce endoplasmic reticulum stress (ERS) in pancreatic beta cells. This study aims to investigate whether a common pathway exists in the ERS induced by various chemicals, including high levels of glucose and palmitate in INS-1-3 cells.

ERS in INS-1-3 cells was induced by exposure cells to thapsigargin (TG), tunicamycin (TM) or palmitic acid (PA) +high glucose (HG). Digital gene expression (DGE) profiling technique was used to detect differentially expressed genes. The profile of gene expression was detected by gene oncology (GO) function and pathway enrichment analysis. Nkx6.1 over-expression was established in INS-1-3 cell lines by lentivirus infection to revert the inhibition of Nkx6.1 expression found in the situation of ERS. Real time reverse transcription polymerase chain reaction (RT-PCR) was used to verify the expression changes of key genes. Cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The apoptosis was determined by flow cytometry. INS-1-3 cell function was measured by glucose stimulated insulin secretion test(GSIS).

As compared to control, DGE demonstrated that there were 135, 57 and 74 differentially expressed genes in TM, TG and HG+PA groups, respectively. Those differentially expressed genes were enriched to ERS, antigen processing and presentation, protein export pathways, and interestingly, the maturity onset diabetes of the young (MODY) pathway. Nkx6.1 is one of common down-regulated gene in MODY signaling pathway among TM, TG and HG+PA groups. Over-expression of Nkx6.1 ameliorated glucolipotoxicity induced apoptosis rate by 45.4%, and increased proliferation by 40.9%. At the same time, GSIS increased by 1.82 folds.

MODY pathway genes expression was changed in the state of ERS. Over-expression of Nkx6.1 protected the INS-1-3 cells from glucolipotoxicity.

Partial Text

Ample evidences indicate that β-cell dysfunction determines the development and progression of type 2 diabetes (T2DM). By using homeostasis model assessment (HOMA), UK Prospective Diabetes Study (UKPDS) finds that pancreatic β-cell function have already lost by 50% at the time of diagnosis and declined by ∽5% annually[1]. The function failure of β-cells is associated with worsening of metabolic control, which is the major underlying mechanism for the acute and chronic complications in type 2 diabetes[2,3]. The preservation and even regaining of β-cell function is a critical therapeutic strategy for T2DM.

It is well known that ER stress of pancreatic β cells often occurs in the development of diabetes. Little is known about the profile of gene expression to various ER stress. DGE helps to screen the differentially expressed genes and pathway-based analysis helps to further understand genes biological functions. In this study, we used pathway enrichment analysis to identify significantly enriched metabolic pathways or signal transduction pathways under the whole genome background.

The endoplasmic reticulum is the intracellular organelle of protein synthesis, folding and transport. ERS is defined as an imbalance between the folding capacity of the ER and the protein load, resulting in the accumulation of misfolded and unfolded protein. ERS can be induced by chemicals, high level of glucose and palmitate and other stress conditions in pancreatic β cells. Recent evidences suggest that ER stress may responsible for the molecular mechanism of glucolipotoxicity of β cells in type 2 diabetes[10–12]. Whether ERS induced by various conditions share common signal pathway remains to be clarified. In the context of association of metabolic disturbance in T2DM, gaining a better understanding of molecular mechanisms in ERS of beta cells induced by high levels of glucose and fatty acid is therefore of great relevance for development of new diabetes therapies.

 

Source:

http://doi.org/10.1371/journal.pone.0198614

 

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