Nuclear factor-κB subunit p65 is involved in lipopolysaccharide-induced lipid accumulation via regulating DGAT1b in Ctenopharyngodon idellus kidney cells.

Lipopolysaccharide (LPS) can promote the accumulation of triglycerides (TGs) in CIK (Ctenopharyngodon idellus kidney) cells, but the underlying mechanism is unclear. In this study, two genes involved TG synthesis, DGAT1a and DGAT1b, were isolated and characterized from grass carp Ctenopharyngodon idella, which encode peptides of 498 and 501 amino acids, respectively. Phylogenetic and synteny analyses indicated that DGAT1a and DGAT1b could have originated from the teleost-specific genome duplication event. Analysis of the exon-intron structures clarified that genomic structures of all DGAT1 proteins are conserved in vertebrates. DGAT1a mRNA was highly expressed in gut, adipose tissue and heart, while DGAT1b mRNA was highly expressed in liver and kidney. After LPS treatment, only expression of DGAT1b was up-regulated and knockdown of DGAT1b reduced the content of TG, suggesting that DGAT1b is involved in LPS-induced lipid accumulation. To explore the mechanism underlying the transcriptional regulation of DGAT1b in response to LPS, we cloned DGAT1b promoter sequence. Its promoter sequence consists of IRF7, RelA (p65) and RelB binding elements. Dual luciferase assay and q-PCR suggested that the promoter of DGAT1b can be activated by the overexpression of p65, but cannot be triggered by IRF7 and RelB. Mutational analysis shows that the potential p65 binding sites may locate in the region -111/-100 bp of the DGAT1b promoter. These results indicated that DGAT1b is the target gene of NF-κB p65. Finally, inhibiting p65 effectively decreased LPS-induced lipid accumulation. Taken together, we demonstrate that NF-κB p65 takes part in the lipid accumulation by regulating DGAT1b-induced TG synthesis in LPS signalling in CIK cells. The finding that NF-κB p65 links LPS signalling and TG synthesis adds to our growing appreciation of the interplay between immunity and lipid metabolism in fish.