Functional Analysis of Promoters from Three Subtypes of the PI3K Family and Their Roles in the Regulation of Lipid Metabolism by Insulin in Yellow Catfish Pelteobagrus fulvidraco

Abstract

In the present study, the length of 360, 1848 and 367 bp sequences of promoters from three subtypes of PI3K family (PI3KCa, PI3KC2b and PI3KC3) of yellow catfish Pelteobagrus fulvidraco were cloned and characterized. Bioinformatics analysis revealed that PI3KCa, PI3KC2b and PI3KC3 had different structures in their core promoter regions. The promoter regions of PI3KCa and PI3KC2b had CpG islands but no CAAT and TATA box. In contrast, the promoter of PI3KC3 had the canonical TATA and CAAT box but no CpG island. The binding sites of several transcription factors, such as HNF1, STAT and NF-κB, were predicted on PI3KCa promoter. The binding sites of transcription factors, such as FOXO1, PPAR-RXR, STAT, IK1, HNF6 and HNF3, were predicted on PI3KC2b promoter and the binding sites of FOXO1 and STAT transcription factors were predicted on PI3KC3 promoter. Deletion analysis indicated that these transcriptional factors were the potential regulators to mediate the activities of their promoters. Subsequent mutation analysis and electrophoretic mobility-shift assay (EMSA) demonstrated that HNF1 and IK1 directly bound with PI3KCa and PI3KC2b promoters and negatively regulated the activities of PI3KCa and PI3KC2b promoters, respectively. Conversely, FOXO1 directly bound with the PI3KC2b and PI3KC3 promoters and positively regulated their promoter activities. In addition, AS1842856 (AS, a potential FOXO1 inhibitor) incubation significantly reduced the relative luciferase activities of several plasmids of PI3KC2b and PI3KC3 but did not significantly influence the relative luciferase activities of the PI3KCa plasmids. Moreover, by using primary hepatocytes from yellow catfish, AS incubation significantly down-regulated the mRNA levels of PI3KCa, PI3KC2b and PI3KC3 and reduced triacylglyceride (TG) accumulation and insulin-induced TG accumulation, as well as the activities and the mRNA levels of several genes involved in lipid metabolism. Thus, the present study offers new insights into the mechanisms for transcriptional regulation of PI3Ks and for PI3Ks-mediated regulation of lipid metabolism by insulin in fish.

Transcriptional control by HNF-1: Emerging evidence showing its role in lipid metabolism and lipid metabolism disorders

“… 36 , 44 HNF-1 countered STAT phosphorylation and the expression of DPP4/NOX1, ameliorating the oxidative function of mitochondria and hepatocellular lipid decomposition, which hindered hepatic lipidosis and the progression of NAFLD. 1 , 2 , 6 , 56 , 57 Moreover, the deletion of HNF-1α/HNF-1β accelerated the synthesis of fatty acids and hepatic fat accumulation to boost the development of NAFLD in C57BL/6J mice. HNF-1β overexpression remarkably decreased hepatic lipid accumulation in both HF diet-fed mice and db/db mice.…”

Novel insights for PI3KC3 in mediating lipid accumulation in yellow catfish Pelteobagrus fulvidraco

“…At present, the underlying transcriptional mechanism of PI3KC3 was still little known in sh. Our previous study have isolated partial promoter sequence of pi3kc3 from yellow cat sh and have founded that FOXO1 positively regulated the transcription of pi3kc3 (Zhuo et al 2018). However, whether the transcription of pi3kc3 could be regulated by the transcript factors of STAT3, PPARα and PPARγ was not studied due to the short length of pi3kc3 promoter sequence we have obtained.…”

“…Hepatocytes were isolated from yellow cat sh according to our previous studies and were cultured in M199 medium containing 1 mmol/L L-glutamine, 5% (v/v) FBS, penicillin (100 IU/mL) and streptomycin (100 g/mL) in a humidi ed atmosphere with 5% CO 2 at 28°C (Zhuo et al 2018). Hepatocytes were counted using a hemocytometer based on the trypan blue exclusion method and only more than 95% cell viability were used for the present experiment.…”

Screening of anti‑chronic nonbacterial prostatitis activity of different extractions of the aerial part of Glycyrrhiza uralensis, and network pharmacology research

“…Zhuo et al ( 55 ) reported that oxidative stress is the primary cause of cancer and inflammation, amongst other diseases. In prostatitis, numerous inflammatory factors are released in response to local inflammatory reactions.…”