Molecular Characterization of Peroxisome Proliferator-Activated Receptor-Gamma Coactivator-1α (PGC1α) and Its Role in Mitochondrial Biogenesis in Blunt Snout Bream (Megalobrama amblycephala)

摘要

PGC1α is a transcriptional coactivator that plays key roles in mitochondrial biogenesis, so exploring its molecular characterization contributes to the understanding of mitochondrial function in cultured fish. In the present study, a full-length cDNA coding PGC1α was cloned from the liver of blunt snout bream (Megalobrama amblycephala) which covered 3741 bp with an open reading frame of 2646 bp encoding 881 amino acids. Sequence alignment and phylogenetic analysis revealed high conservation with other fish species, as well as other higher vertebrates. Comparison of the derived amino acid sequences indicates that, as with other fish, there is a proline at position 176 (RIRP) compared to a Thr in the mammalian sequences (RIRT). To investigate PGC1α function, three in vitro tests were carried out using primary hepatocytes of blunt snout bream. The effect of AMPK activity on the expression of PGC1α was determined by the culture of the hepatocytes with an activator (Metformin) or inhibitor (Compound C) of AMPK. Neither AMPK activation nor inhibition altered PGC1α expression. Knockdown of PGC1α expression in hepatocytes using small interfering RNA (si-RNA) was used to determine the role of PGC1α in mitochondrial biogenesis. No significant differences in the expression of NRF1 and TFAM, and mtDNA copy number were found between control and si-RNA groups. Also, hepatocytes were cultured with oleic acid, and the findings showed the significant reduction of mtDNA copy number in oleic acid group compared to control. Moreover, oleic acid down-regulated the expression of NRF1 and TFAM genes, while PGC1α expression remained unchanged. Our findings support the proposal that PGC1α may not play a role in mitochondrial biogenesis in blunt snout bream hepatocytes.