Effect of the gene polymorphism of peroxisome proliferator-activated receptor γ coactivator-1α on transcription of gluconeogenesis gene

Abstract

Objective To investigate the effect of the glycine to serine mutations in codon 482 (Gly482Ser)gene polymorphism of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene on the transcription of gluconeogenesis key gene phosphoenolpyruvate carboxykinase (PEPCK). Methods (1) We designed to construct PGC-1α G1444A polymorphism expression plasmid using oligonucleotide based site directed mutagenesis and polymerase chain reaction and PEPCK promoter gene luciferase reporter plasmid PGL3-hPCK-luc using common PCR and restrictive enzyme digestion method first. And then wild-type plasmid with glycine in codon 482 pcDNA3.1-PGC-1α (G) and mutated plasmid with serine in codon 482 pcDNA3.1-PGC-1α (S) were co-transfected respectively with transcription factor expression plasmid pcDNA3.0-HNF4α into cultured HepG2 cell and L02 cell. (2) The mRNA and protein levels of PEPCK were detected after transfection 48 hours. Then PGL3-hPCK-luc and the reference plasmid PRL-SV40 were co-transfected according to different combinations. After 48 hours of culture the relative activity of luciferase was detected by the dual luciferase assay kit.One way ANOVA and t-test of independent samples were used for data analysis. Results PGC-1α G1444A mutation plasmid and recombinant PGL3-hPCK-luc reporter plasmid were constructed successfuly. After being transiently transfected into liver cells, both the mRNA and protein levels of PGC-1α (G) and PGC-1α (S) were increased significantly, but there was no statistical significant difference between the two groups(P>0.05). The PEPCK mRNA and protein levels were increased significantly in both co-transfecting PGC-1α and HNF4α group(10.4±0.7 and 4.5±0.5) when compared with the sigle-transfection(0.86±0.18 and 0.99±0.09, both P<0.05). Compared with PGC-1α (S) plus HNF4α group, the PEPCK mRNA and protein levels of PGC-1α (G) plus HNF4α group increased 1.83-fold(10.4±0.7 vs 5.35±0.23) and 1.4-fold(4.5±0.5 vs 3.0±0.4) in PGC-1α(G) plus HNF4α group (both P<0.05). Moreover, Co-transfected PGC-1α plus HNF4α group had a significant promotion of PEPCK promoter activity compared with the single transfection PGL3-hPCK-luc group(28±5 vs 2.4±0.4, F=23.41, P<0.05); PEPCK promoter activity was increased 2-fold(83±10 vs 41±5, F=23.41, P<0.001)while being transfected with PGC-1α (G) into HepG2 cells than with PGC-1α (S). When transfected into L02 it was increased 2.25-fold(28±5 vs 13.0±1.5, F=60.75, P<0.001). Conclusions PGC-1α can coactivate hepatocyte nuclear factor 4α in promoting the transcription of PEPCK, while PGC-1α gene Gly482 has a more significant promotion by the activation of HNF4α than Ser 482. Accordingly, it is possible that the gene polymorphism can express protein with different structures, which would affect the protein-protein interactions. Key words: Diabetes mellitus, type 2; Gluconeogenesis; Phosphoenolpyruvate carboxykinase; Peroxisome proliferator-activated receptor γ coactivator-1α; Gene polymorphism