Novel insights for SREBP-1 as a key transcription factor in regulating lipogenesis in a freshwater teleost, grass carp Ctenopharyngodon idella

Abstract

Disturbances in lipid metabolism are at the core of several health issues facing modern society, including fatty liver and obesity. The sterol regulatory element-binding protein 1 (SREBP-1) is one important transcription factor regulating lipid metabolism, but the relevant mechanism still remains unknown. The present study determined the transcriptional regulation of SREBP-1 and its target genes (including acetyl-CoA carboxylase α (accα), fatty acid synthase (fas) and stearoyl-CoA desaturase 1 (scd1)) in a freshwater teleost, grass carp Ctenopharyngodon idella. We cloned and characterised the 1988 bp, 2043 bp, 1632 bp and 1889 bp sequences of srebp-1, accα, scd1 and fas promoters, respectively. A cluster of putative binding sites of transcription factors, such as specific protein, yin yang 1, nuclear factor Y, sterol response elements (SRE) and enhancer box (E-box) element, were predicted on their promoter regions. Overexpression of nSREBP-1 reduced srebp-1 promoter activity, increased scd1 and fas promoter activity but did not influence accα promoter activity. The site-mutation and electrophoretic mobility shift assay analysis indicated that srebp-1, fas and scd1 promoters, but not accα promoter, possessed SRE. In Ctenopharyngodon idella kidney (CIK) cells of grass carp, nSREBP-1 overexpression significantly reduced srebp-1 mRNA expression and up-regulated miR-29 mRNA expression. The 3′UTR of srebp-1 possessed the potential miR-29 binding site and miR-29 up-regulated the luciferase activity of srebp-1 3′UTR and srebp-1 mRNA expression, implying a self-activating loop of SREBP-1 and miR-29 in grass carp. Based on the above-mentioned results, we found two novel transcriptional mechanisms for SREBP-1 in grass carp: (1) the auto-regulation sited on the SREBP-1 promoter regions was suppressive and (2) there was a self-activating loop of SREBP-1 and miR-29.

Dietary Phosphorus Reduced Hepatic Lipid Deposition by Activating Ampk Pathway and Beclin1 Phosphorylation Levels to Activate Lipophagy in Tilapia Oreochromis niloticus

“…To identify the phosphorylation sites of Beclin1 and AMPKα1, we constructed the vectors of Beclin1 and AMPKα1 expression according to our publication ( 46 ). The open reading frames (ORFs) of Beclin1 and AMPKα1 sequences were subcloned into the pcDNA3.1 (+) vector with the GFP-tag and the His-tag, respectively.…”

“…The siRNA sequences were given in Supplementary Table 3 . Cell transfections was operated based on the protocols ( 46 ). Cells were treated for 48h to investigate the potential mechanism that AMPK and Beclin1 mediated phosphorus-induced lipolysis and lipophagy.…”

Effects of dietary carbohydrate sources on lipid metabolism and SUMOylation modification in the liver tissues of yellow catfish

“…Plasmid construction. We constructed SUMO1-6 × His, UBC9-6 × His and SREBP1-Flag plasmids based on the methods described previously (25) . Mutants with the K145R, K254R, K264R, K392R and K419R mutations were obtained from wild-type SREBP1 after the site-directed mutagenesis.…”

Phosphorus Overload Promotes Hepatic Lipolysis by Suppressing GSK3β-Dependent Phosphorylation of PPARα at Ser84 and Thr265 in a Freshwater Teleost

“…The promoter sequences and functional analyses of adipose triglyceride lipase (atgl) and very long-chain-specific acyl-CoA dehydrogenase (acadvl) were based on the methods described in our previous study. 24 The primers are provided in Table S5. The activities of promoters were assessed by a dual luciferase reporter assay system (#E1910, Promega).…”