Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid on Mitochondrial DNA Replication and PGC-1α Gene Expression in C2C12 Muscle Cells

Abstract

Mitochondrial biogenesis is a complex process requiring coordinated expression of nuclear and mitochondrial genomes. The peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis, and it controls mitochondrial DNA (mtDNA) replication within diverse tissues, including muscle tissue. The aim of this study was to investigate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number and PGC-1α promoter activity in C₂C₁₂ muscle cells. mtDNA copy number and mRNA levels of genes related to mitochondrial biogenesis such as PGC-1α, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (Tfam) were assayed by quantitative real-time PCR. The PGC-1α promoter from -970 to +412 bp was subcloned into the pGL3-basic vector, which includes a luciferase reporter gene. Both EPA and DHA significantly increased mtDNA copy number, dose and time dependently, and up-regulated mRNA levels of PGC-1α, NRF1, and Tfam. Furthermore, EPA and DHA stimulated PGC-1α promoter activity in a dose-dependent manner. These results suggest that EPA and DHA may modulate mitochondrial biogenesis, which was partially associated with increased mtDNA replication and PGC-1α gene expression in C₂C₁₂ muscle cells.

Keywords: DHA; EPA; PGC-1α; mtDNA; muscle cells.

Fig. 1

Effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mtDNA copy number in C₂C₁₂ muscle cells. Differentiated C₂C₁₂ muscle cells were treated with 1% bovine serum albumin serum-free medium alone (control), 50 μM palmitate (PA) or 1, 10, and 50 μM EPA (A) or DHA (B) for 24 h. Cells were time-dependently exposed to 50 μM PA, EPA, or DHA for 6, 16, and 24 h (C). mtDNA copy number was measured by real-time quantitative PCR. Values are expressed as mean±SD (n=3) of three independent experiments. *P<0.05 and **P<0.01 compared to control.

Fig. 2

Effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the mRNA levels of genes involved in mitochondrial biogenesis in C₂C₁₂ muscle cells. Differentiated C₂C₁₂ muscle cells were treated in 1% bovine serum albumin serum-free medium with 50 μM of palmitate (PA), EPA, or DHA for 24 h. The mRNA levels were measured by quantitative real-time RT-PCR. Values are means±SD (n=3) of three independent experiments. *P<0.05 and **P<0.01 compared to control.

Fig. 3

Effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) promoter activity in C₂C₁₂ muscle cells. Differentiated cells were transfected with the PGC-1α (−1790/+52 bp)/luc reporter gene and pCMV-β galactosidase and were incubated 1% bovine serum albumin serum-free medium with 0 (control) to 50 μM EPA (A) and DHA (B) for 40 h. Promoter activities measured by luciferase activity were calculated in relative light units (RLU) and normalized to β-galactosidase activity. Values are expressed as mean±SD (n=3) of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 compared to control.