Characterization Of Zinc Homeostasis And Its Mechanisms In Zinc Regulating Lipid Metabolism In Yellow Catfish Pelteobagrus Fulvidraco

Zinc(Zn)is an essential trace element for aquatic economic animals,and it is widely involved in the physiological and metabolic processes in fish,and the metabolic disorder of Zn has an adverse effect on fish.Zn homeostasis in fish is co-regulated by some key proteins,including Zn transporters(Zn Ts and ZIPs family),metallothioneins(MTs)and metal response element-binding transcription factor 1(MTF-1).In the present study,using yellow catfish Pelteobagrus fulvidraco as the research object,we cloned and characterized key genes and promoters of the regulation of Zn homeostasis of yellow catfish,and explored inorganic Zn(Zn SO₄.7H₂O)and ZnO nanoparticle(ZnO NP)in diets were used to explore the effect and molecular mechanism of Zn homeostasis disorder influencing the lipid metabolism and lipid deposition in yellow catfish.The main results in the present study are as follows:1.Cloning and functional analysis of key genes involved in Zn homeostasis in yellow catfishTo preliminarily explore the regulation mechanism of zinc homeostasis in fish,the full-length cDNA sequences of six Zn transport-relevant genes(2816 bp znt1,2792 bp znt5,2017 bp znt7,3664 bp zip4,2921 bp zip5 and 4904 bp mtf-1)from yellow catfish P.fulvidraco were cloned,the structural domains were identified and the evolutionary trees were constructed.Their m RNA amounts were widely existent across 8 tissues(intestine,liver,brain,heart,gill,muscle,spleen and mesenteric fat),but the expression level variesd in different tissues.The gene expression of these genes showed concentration-and time-dependent manners.Our study built good basis for further investigating their physiological functions of these genes,and provided new insights into the regulatory mechanisms of Zn homeostasis for fish.2.Cloning and functional analysis of MT and MT promoters involved in Zn homeostasis in yellow catfishTo further explore the regulation mechanism of zinc homeostasis in fish,the structure and functions of 1845 bp mtf-1 and 802 bp mt promoters in yellow catfish P.fulvidraco were cloned characterized.Zn induced the increase of mtf-1 promoter activity and HSE site of mtf-1 promoter was the functional binding locus responsible for Zn-induced mtf-1 transcriptional activation;Zn induced the increase of mtf-1 promoter activity and MRE sites of mt promoter were the functional binding locus responsible for Zn-induced mtf-1 transcriptional activation.Using primary hepatocytes of yellow catfish,we found that Zn induced the mt expression,and also up-regulated MTF-1 nuclear protein expression,which in turn increased the mt expression.Taken together,these findings delineated the transcriptional regulation of MT and MTF-1 under Zn treatments,and provided some mechanisms for the regulation of Zn homeostasis in fish.3.Effect of dietary Zn supplementation on intestinal Zn metabolism and lipid metabolism of yellow catfishThe present study explored the mechanisms of dietary Zn influencing Zn and lipid deposition of the fore-and mid-intestine in fish.For that purpose,yellow catfish were fed three diets containing Zn levels of 8.83,19.20,and 146.65 mg Zn kg^-1 for 8 weeks,respectively.Growth performance,intestinal TG and Zn contents,activities and m RNA expression of enzymes and genes involved in Zn transport and lipid metabolism from the fore-and mid-intestine were analyzed.Dietary Zn supplementation increased Zn accumulation but reduced TG content in both the fore-and mid-intestine of yellow catfish.Increased Zn accumulation was attributable to the changes in enzymatic activities and m RNA expression of genes involved in Zn absorption and transporter.The reduced TG content in both the fore-and mid-intestine of yellow catfish were attributable to the up-regulated lipolysis and down-regulated lipogenesis.All observations revealed the similar regulatory mechanisms of Zn and lipid metabolism between the fore-and mid-intestine in response to dietborne Zn addition of yellow catfish.Our study provided the reference for the precise regulation of zinc nutrition in fish.4.Mitochondrial ROS mediates the mechanism of ZnO NP-induced hepatic lipid metabolism in yellow catfishThe present study explored the mechanisms of dietary ZnO NP influencing Zn and lipid metabolism in fish.For that purpose,yellow catfish were fed four diets containing ZnO NP content of 0(Control),10 mg/kg ZnO NP(Low ZnO NP),100 mg/kg ZnO NP(High ZnO NP)and 100 mg/kg ZnO NP+10 mg/kg TPEN(high ZnO NP+TPEN group)for 10 weeks,respectively.We found that dietary ZnO NP disturbed hepatic Zn metabolism,and significantly increased hepatic Zn and lipid accumulation,induced oxidative stress and activated mitophagy;dietary TPEN addition alleviated high ZnO NP-induced Zn and lipid accumulation.Using primary hepatocytes of yellow catfish,we found that TPEN attenuated ZnO NP-induced Zn and lipid accumulation.Further investigation found that the suppression of mitochondrial ROS attenuated ZnO NP-activated mitophagy and ZnO NP-induced lipid accumulation in hepatocytes.Meanwhile,ZnO NP directly regulated cpt1a1b expression,which in turn induced transcriptional suppression of genes and ultimately lipid deposition.Our study provided innovative evidence for the direct relationship between mineral elements and lipid metabolism.