Korean J Physiol Pharmacol.  2015 Mar;19(2):141-149. 10.4196/kjpp.2015.19.2.141.

Differential Gene Expression in GPR40-Overexpressing Pancreatic beta-cells Treated with Linoleic Acid

• Kim IS
• Yang SY
• Han JH
• Jung SH
• Park HS
• Myung CS

• KMID: 2285566
• DOI: http://doi.org/10.4196/kjpp.2015.19.2.141

Abstract

"G protein-coupled receptor 40" (GPR40), a receptor for long-chain fatty acids, mediates the stimulation of glucose-induced insulin secretion. We examined the profiles of differential gene expression in GPR40-activated cells treated with linoleic acid, and finally predicted the integral pathways of the cellular mechanism of GPR40-mediated insulinotropic effects. After constructing a GPR40-overexpressing stable cell line (RIN-40) from the rat pancreatic beta-cell line RIN-5f, we determined the gene expression profiles of RIN-5f and RIN-40. In total, 1004 genes, the expression of which was altered at least twofold, were selected in RIN-5f versus RIN-40. Moreover, the differential genetic profiles were investigated in RIN-40 cells treated with 30 microM linoleic acid, which resulted in selection of 93 genes in RIN-40 versus RIN-40 treated with linoleic acid. Based on the Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG, http://www.genome.jp/kegg/), sets of genes induced differentially by treatment with linoleic acid in RIN-40 cells were found to be related to mitogen-activated protein (MAP) kinase- and neuroactive ligand-receptor interaction pathways. A gene ontology (GO) study revealed that more than 30% of the genes were associated with signal transduction and cell proliferation. Thus, this study elucidated a gene expression pattern relevant to the signal pathways that are regulated by GPR40 activation during the acute period. Together, these findings increase our mechanistic understanding of endogenous molecules associated with GPR40 function, and provide information useful for identification of a target for the management of type 2 diabetes mellitus.

Keyword

G protein-coupled receptor 40; Linoleic acid; Insulin secretion; Pancreatic beta-cell; Type 2 diabetes mellitus

MeSH Terms

Animals
Cell Line
Cell Proliferation
Diabetes Mellitus, Type 2
Fatty Acids
Gene Expression*
Genes, vif
Genome
Insulin
Linoleic Acid*
Rats
Signal Transduction
Transcriptome
Fatty Acids
Insulin
Linoleic Acid

Figure

• 

  Fig. 1 RIN-40, a stable cell line highly and constitutively expressing GPR40. RIN-40 was constructed from rat insulinoma cell line, RIN-5f by transfection with pCMV6-Neo-GPR40, as described in the Materials and Methods. (A) Comparison of human GPR40 (1, GPR40) and rat GPR40 (2, Gpr40) mRNA expression in RIN-40 with RIN-5f. GPR40 and Gpr40 mRNA expression was measured using quantitative real-time RT-PCR. (B) Comparison of human GPR40 protein expression in RIN-40 with RIN-5f by immunoblotting. (C) Concentration-response relationship of linoleic acid on glucose-stimulated insulin secretion in RIN-5f and RIN-40. Results are the means±SEM of four similar independent experiments, each performed in triplicate. ***p<0.001, vs. basal glucose-stimulated insulin secretion in RIN-40 (no linoleic acid). (D) Fluorescence detection of intracellular Ca2+ in both RIN-5f and RIN-40.

• 

  Fig. 2 Effect of linoleic acid on ERK activation and glucose-stimulated insulin secretion in RIN-40 cell line. (A) Incubation time course of linoleic acid treatment with ERK phosphorylation. RIN-40 cells were treated with 30 µM linoleic acid for 0, 3, 5, 10, 20, 30, 60, 90, and 120 min and an immunoblot was performed using total and phospho-ERK antibodies. (B) Correlation of insulin secretion via GPR40 activation induced by linoleic acid treatment with ERK activation. RIN-40 and RIN-40 cells transfected with siRNA for GPR40 (RIN-40-siGPR40) were treated with 0, 10, 30, 50, and 100 µM linoleic acid, and ERK phosphorylation and insulin secretion were measured. All values in the bar and line graphs are means±SEM of three similar, independent experiments and gel images are representative of three independent experiments. **p<0.01 and ***p<0.001, vs. vehicle (DMSO)-treated glucose-stimulated insulin secretion in RIN-40 and #p<0.05 and ###p<0.001, vs. glucose-stimulated insulin secretion in RIN-40 at the indicated concentration of linoleic acid.

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