Studies on molecular mechanisms of lipid metabolism in Japanese flounder Paralichthys olivaceus and red seabream Pagrus major

Lipid stored tissue plays a major role in free fatty acid supply for whole body energy homeostasis.The lipid in the form of triacylglycerol (TAG) is hydrolyzed by catalytic enzymes into free fatty acid (FFA) and glycerol.Hormone-sensitive lipase (HSL) is one of those catalytic enzymes which have an important role in lipolysis in mammalian adipose tissue.In fish, lipids are mainly deposited in three organs, adipose tissue, liver and skeletal muscle.Different fish species are likely to deposit lipid in the different types of organs, known as "tissueand species-specific" manners.However, the process of HSL-mediated lipolysis in fish is poorly documented and remains unclear, which leads to misunderstanding and contradictory in our knowledge of lipid metabolism.In order to clarify the function of fish HSLs, HSL cDNAs were cloned from two different species: Japanese flounder Paralichthys olivaceus, mainly stored their lipid in inclinator muscle of fin, and red seabream Pagrus major, mainly in visceral adipose tissue.The full-length cDNAs of two HSL genes were determined and designated as HSL1 and HSL2.In Japanese flounder, HSL1 and HSL2 consisted of 2,922 bp and 2,832 bp, respectively, while 2,955 for HSL1 and 2,723 bp for HSL2 were observed in red seabream.Additionally, proteins encoding HSLs were 702 and 837 amino acids in Japanese flounder, and 710 and 874 amino acids in red seabream, respectively.The molecular mass of Japanese flounder showed that the HSLs protein migrated on SDS-PAGE exhibited an apparent molecular mass of approximately 96 kDa of HSL1 and 125 kDa of HSL2.On the other hand, HSL1 protein of red seabream has molecular mass of 98 kDa.However, HSL2 protein was not observed in red seabream.The deduced amino acid sequences of HSL1 and HSL2 genes shared 58.7% identity in Japanese flounder and 55% identity in red seabream.The identity of HSL1 and HSL2 in both of Japanese founder and red seabream showed about 57-89%, and 60-73% with HSLs from rainbow trout Oncorhynchus mykiss.The multiple amino acid alignment with other species clearly showed that HSLs of the present study were comprised of two major domains; N-terminal and C-terminal, which were separated by glutamine and aspartate (Gln316 and Asp317 in Japanese flounder HSLs and, Gln322 and Asp323 for HSL1, and Gln355 and Asp356 for HSL2 of red seabream, respectively).Three amino acid residues comprising the catalytic triad were conserved in all HSLs.The multiple alignments also showed that both Japanese flounder and red seabream HSLs also have several serine residues aligned with the potential phosphorylation sites of rainbow trout HSLs, but these were misaligned with the rat and human HSL of phosphorylation sites.Several phosphorylation motifs of mammalian PKA (R/K-R/K-X-pS/T or R/K-R/K-X-X-pS/T) were also conserved to both species.Tissue distribution of HSLs performed by RT-PCR found that the HSL transcripts of the Japanese flounder genes were abundant in the inclinator muscle of fin, liver, and skeletal muscle, whereas the highest transcripts were observed in adipose tissue and gonad in red seabream.The relative mRNA levels revealed that the transcripts of HSL1 and HSL2 genes were broadly expressed in all tested tissues.The relative mRNA levels of HSL2 were lower than HSL1 in both Japanese flounder and red seabream, suggesting that HSL2 has a minor function in the hydrolysis of stored lipid.In addition, the relative mRNA levels