Glucose activates free fatty acid receptor 1 gene transcription via phosphatidylinositol-3-kinase-dependent O-GlcNAcylation of pancreas-duodenum homeobox-1

Abstract

The G protein-coupled free fatty acid receptor-1 (FFA1/GPR40) plays a major role in the regulation of insulin secretion by fatty acids. GPR40 is considered a potential therapeutic target to enhance insulin secretion in type 2 diabetes; however, its mode of regulation is essentially unknown. The aims of this study were to test the hypothesis that glucose regulates GPR40 gene expression in pancreatic β-cells and to determine the mechanisms of this regulation. We observed that glucose stimulates GPR40 gene transcription in pancreatic β-cells via increased binding of pancreas-duodenum homeobox-1 (Pdx-1) to the A-box in the HR2 region of the GPR40 promoter. Mutation of the Pdx-1 binding site within the HR2 abolishes glucose activation of GPR40 promoter activity. The stimulation of GPR40 expression and Pdx-1 binding to the HR2 in response to glucose are mimicked by N-acetyl glucosamine, an intermediate of the hexosamine biosynthesis pathway, and involve PI3K-dependent O-GlcNAcylation of Pdx-1 in the nucleus. We demonstrate that O-GlcNAc transferase (OGT) interacts with the product of the PI3K reaction, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), in the nucleus. This interaction enables OGT to catalyze O-GlcNAcylation of nuclear proteins, including Pdx-1. We conclude that glucose stimulates GPR40 gene expression at the transcriptional level through Pdx-1 binding to the HR2 region and via a signaling cascade that involves an interaction between OGT and PIP(3) at the nuclear membrane. These observations reveal a unique mechanism by which glucose metabolism regulates the function of transcription factors in the nucleus to induce gene expression.

Fig. 1.

Isolated mouse (A; n = 17) or human (B; n = 8) islets were treated with 2.8 mmol/L low glucose (LG) or 16.7 mmol/L high glucose (HG) for 6 (mouse islets) or 24 (human islets) h, and GPR40 mRNA levels measured by RT-PCR. *P < 0.05; **P < 0.01. (C) Representative immunoblots for GPR40 in human islets exposed to 2.8 or 16.7 mmol/L glucose for 24 h (n = 3). The p85 subunit of PI3K was used as a loading control. (D) Mouse islets were treated with 2.8 or 16.7 mmol/L glucose for 6 h. Insulin secretion was assessed in 1-h static incubations in response to 2.8 or 16.7 mmol/L glucose ± 0.5 mmol/L oleate. Data are expressed as insulin secretion and normalized to insulin content (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2.

(A) GPR40 mRNA levels in islets exposed to 2.8 (LG) or 16.7 (HG) mmol/L glucose ± 5 μg/mL of actinomycin-D for 0, 2, 4, and 6 h (n = 3). (B) MIN6 cells were cotransfected with 1 μg of a pTK-Luc control, pGL3-2095-Luc, pTK-HR2-Luc, pTK-HR3-Luc, or a pTK-HR2-Luc plasmid in which the Pdx-1 site is mutated (pTK-HR2M-Luc) and 0.1 μg of internal control pGL4-74-RLuc. Transfected cells were treated with either 1 (LG) or 11.1 (HG) mmol/L glucose for 24 h (n = 8). **P < 0.01. (C) Chromatin immunoprecipitation analysis showing the effect of 16.7 (HG) mmol/L glucose on the recruitment of Pdx-1 to the GPR40 promoter compared with islets treated with 2.8 (LG) mmol/L glucose (n = 3). **P < 0.01.

Fig. 3.

(A) GPR40 mRNA levels in mouse islets treated with 2.8 (LG) or 16.7 (HG) mmol/L glucose ± 10 μmol/L U0126, 10 μmol/L wortmannin (WRT), or 20 μmol/L LY294002 (LY) for 6 h (n = 3). **P < 0.01, *P < 0.05. (B) GPR40 mRNA levels in βIRKO and MIN6 cells treated with 1 (LG) or 11.1 (HG) mmol/L glucose for 6 h. (C) GPR40 mRNA levels in mouse islets treated with 2.8 (LG) or 16.7 (HG) mmol/L glucose ± 625 μmol/L diazoxide. (D) GPR40 and L-type pyruvate kinase (l-PK) mRNA levels in mouse islets treated with 2.8 (LG) mmol/L glucose ± insulin (n = 3). *P < 0.01.

Fig. 4.

(A) GPR40 mRNA levels in mouse islets treated with 2.8 (LG) ± 13.9 mmol/L mannitol (MAN), 3-O-methyl-d-glucose (3MG), 2-deoxy-d-glucose (2DG), 5 mmol/L methyl-pyruvate (MPY), or 20 mmol/L N-acetylglucosamine (GlcNAc). (B) Effect of GlcNAc on GPR40 mRNA levels of islets cultured with 2.8 (LG) mmol/L glucose for 6 h (n = 2–5). **P < 0.01. (C) GPR40 mRNA levels in MIN6 cells treated with 1 (LG) or 11.1 (HG) mmol/L glucose ± 10 mmol/L GlcNAc and ± 20 μmol/L DON for 6 h (n = 3). *P < 0.05, **P < 0.01. (D) GPR40 mRNA levels in nontransfected MIN6 cells or in MIN6 cells transfected with either control or GFAT-1 siRNA. Twenty-four hours after transient transfection, cells were treated with 1 (LG) or 11.1 (HG) mmol/L glucose ± 10 mmol/L GlcNAc for 6 h (n = 3). *P < 0.05, **P < 0.01. (E) GPR40 mRNA levels in mouse islets treated with 2.8 mmol/L glucose ± 20 mmol/L GlcNAc ± 20 μmol/L LY294002 for 6 h (n = 3). *P < 0.05. (F) Chromatin immunoprecipitation analysis showing the effect of 20 mmol/L GlcNAc on the recruitment of Pdx-1 to the GPR40 promoter (n = 3). *P < 0.05. (G) MIN6 cells were cotransfected with 1 μg of either a pTK-Luc control, pGL3-2095-Luc, pTK-HR2-Luc, pTK-HR3-Luc, or a pTK-HR2-Luc plasmid in which the Pdx-1 site is mutated (pTK-HR2M-Luc) and 300 ng of internal control pCMV-β-gal. Transfected cells were treated with either 1 mmol/L glucose (LG) ± 10 mmol/L GlcNAc or 11.1 (HG) mmol/L glucose for 24 h. Luciferase activity was normalized to β-gal activity of the internal control (n = 3). *P < 0.05, **P < 0.01.

Fig. 5.

(A) MIN6 cells were treated with 1 (LG) or 11.1 (HG) mmol/L glucose ± 20 μmol/L LY294002 for 6 h. Cytoplasmic and nuclear proteins were immunoblotted using anti–RL-2 antibody. (B) Quantification of blots presented in A (n = 3). *P < 0.05. (C) MIN6 and βIRKO cells were treated with 1 (LG) or 11.1 (HG) mmol/L glucose ± 20 μmol/L LY294002 for 6 h. Nuclear proteins were pulled down by using sWGA-conjugated beads, and analyzed by immunoblotting. Input proteins were immunoblotted with anti-TFIID antibody as a loading control. (D) Quantification of the blots presented in C (n = 3). *P < 0.05. Quantification of the RL-2 blot is shown in Inset (n = 3). (E) MIN6 cells were treated with 1 (LG) or 11.1 (HG) mmol/L glucose for 6 h. Nuclear proteins were immunoprecipitated by using an anti–Pdx-1 or anti-CTD11.6 antibody and immunoblotted using anti–RL-2, anti–Pdx-1, or anti-BETA2 antibody. Input proteins were immunoblotted with anti-Pdx1, anti-BETA2, or anti–RL-2 antibody. (F) Quantification of Pdx-1 blots presented in E (n = 3). *P < 0.05.

Fig. 6.

(A) Nuclear extracts from MIN6 cells were pulled down by phosphoinositide-conjugated affinity beads followed by immunoblotting with an anti-OGT antibody (n = 2). (B) MIN6 cells cultured at HG were treated ± 20 μmol/L LY294002 for 24 h. Nuclear proteins were immunoprecipitated with either an anti-PIP₃ or anti-OGT antibody and immunoblotted with anti-OGT, anti-PIP₃, or anti–Pdx-1 antibodies. Input protein was immunoblotted with an anti-OGT, anti-PIP₃, anti–Pdx-1, or anti-TFIID antibody as loading controls (n = 3). (C) A model for glucose regulation of GPR40 gene expression in pancreatic β-cells.