Functional Analysis Of PI3Ks And Its Related Signal Pathway In Insulin Regulating Lipid Metabolism In Yellow Catfish Pelteobagrus Fulvidraco

PI3K provided a critical signal for cell survival,proliferation and nutrient metabolism.In mammals,widely studies indicated that insulin regulation nutrition metabolism by unsing IRS-PI3K-AKT signal pathway,and insulin stimulated glycogenesis,lipogenesis,and protein synthesis in the periphery tissues,such as liver,muscle,and adipose.In fish,studies on the regulation of insulin on lipid metabolism is limited,and the underlying mechanism of IRS-PI3K-AKT signal pathway in insulin regulation lipid metabolism is poorly known.In the present study,we studied the structure and functional of PI3 K family in yellow catfish Pelteobagrus fulvidraco,and investigated the roles and mechanisms of PI3 K pathway involved in insulin-induced alteration in hepatic lipid metabolism.The main results are as follows:1.Molecular cloning and tissue mRNA levels of phosphoinositide 3-kinases(PI3Ks)family in yellow catfish Pelteobagrus fulvidracoIn the present study,seven phosphoinositide 3-kiase(PI3K)members(pi3kca,pi3 kcb,pi3kcg,pi3 kcd,pi3kc2a,pi3kc2 b and pi3kc3,respectively)were isolated and characterized from yellow catfish Pelteobagrus fulvidraco.Their full-length c DNA sequences ranged from 3,352 bp to 6,350 bp,with ORFs(open reading frames)of 2,534-5,013 bp,encoding 877-1,670 amino acids.These seven PI3 Ks can be divided into three classes,class I(including pi3 kca,pi3kcb,pi3 kcd and pi3kcg),class II(including pi3kc2 a,pi3kc2b)and class III(only including pi3kc3).All of pi3 ks contain,a N-C2 domain,a PIK domain and a catalytic domain.The catalytic domain,which possessed the ATP-binding sites and catalytic function,showed the highest identity among all pi3 ks.The phylogenetic tree showed that seven pi3 k members sequences were clearly separated into three separate clades,indicating that PI3 Ks were divided into three clustered.Furthermore,pi3 kcb showed more closely to pi3 kcd,and the yellow catfish PI3 Ks were closer to cave fish and zebrafish and more distant from human and rat.The mRNA levels of all of pi3 ks were the highest in the ovary.Among other tissues,the mRNAs of pi3 kca,pi3kcb,pi3kc2 a,pi3kc2b and pi3kc3 were widely distributed in the tested tissues,but at different levels.pi3 kcd mRNA was predominant in muscle,intermediate in fat and spleen,and was low at liver,brain,kidney,gill,heart and intestine. In contrast,pi3 kcg mRNA level was very low in muscle and fat,and almost undetectable in other tested tissues.2.Molecular cloning and functional analysis of pi3 kca,pi3kc2b and pi3kc3 promoters in yellow catfish Pelteobagrus fulvidracoIn the present study,the length of 360,1848 and 367 bp sequences of promoters from three subtypes of PI3 K family(pi3kca,pi3kc2 b and pi3kc3)of yellow catfish were cloned and characterized,respectively.Bioinformatics analysis revealed that pi3 kca,pi3kc2b and pi3kc3 had different structures in their core promoter regions.The promoter regions of pi3 kca and pi3kc2 b had Cp G islands but no CAAT and TATA box.In contrast,the promoter of pi3kc3 had the canonical TATA and CAAT box but no Cp G island.The binding sites of several transcription factors,such as HNF1,STAT and NF-k B,were predicted on pi3 kca promoter.The binding sites of transcription factors,such as FOXO1,PPAR-RXR,STAT,IK1,HNF6 and HNF3,were predicted on pi3kc2 b promoter,and the binding sites of FOXO1 and STAT transcription factors were predicted on pi3kc3 promoter.Deletion analysis indicated that some promoter regions could mediate the activities of their promoters.Subsequent mutation analysis and EMSA assay demonstrated that HNF1 and IK1 directly bound with pi3 kca and pi3kc2 b promoters,and negatively regulated the activities of pi3 kca and pi3kc2 b promoters,respectively.Conversely,FOXO1 directly bound with the pi3kc2 b 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 pi3kc2 b and pi3kc3,but did not significantly influence the relative luciferase activities of the pi3 kca plasmids.3.Methylation characteristics of pi3 kca promoter in different tissues of yellow catfish Pelteobagrus fulvidracoThe present study was conducted to determine methylation and expression of pi3 kca in different tissues of yellow catfish.The DNA methylation status of pi3 kca promoters were determined by the BSP(bisulfite sequencing PCR)method.The mRNA expressions of pi3 kca and dnmts were assay by the Q-PCR method.The sequencing of individual bisulfite-converted genomic DNAs revealed that methylation was present in the-71,-64,-52 and +8 Cp G loci of pi3 kca promoter.There is no correlation between the mRNA level and the methylation level of pi3 kca in different tissues of yellow catfish.Further,a hypomethylation of pi3 kca promoter(0.88%–3.82%)was present in different tissues which may contribute to the widely high expression of pi3 kca in different tissues.In addition,the highest methylation percentages of pi3 kca were derterminded in the ovary,whicih proable due to the highest expression of all dnmts in the ovay of yellow catfish.4.Effect of insulin on hepatic lipid metabolism by activation IRS-PI3K-AKT pathway in yellow catfish Pelteobagrus fulvidracoIn oreder to study the regulatory effect of insulin on lipid metabolism,two experiment were conducted.freshly hepatocytes were isolated from yellow catfish,cultured and subjected to different insulin levels(0,10 n M,100 n M and 1000 n M)for 48 h.Triglyceride(TG)content,activity and expression of several key enzymes involved in lipid metabolism,as well as mRNA levels of different subtypes of IRS-PI3K-AKT,transcription factors related to lipid metabolism were assessed.AS a result,insulin incubation significantly stimulated the mRNA level of irs1,irs2,pi3 kcb,pi3kc2a,akt1 and akt2 at different level,but it showed no changes on the expression of pi3 kca,pi3kcd,pi3 kcg,pi3kc2b,pi3kc3,akt3 a and akt3 b.Insulin incubation tended to increase TG accumulation and the activities and expression of several lipogenic enzymes(such as FAS,G6 PD,and 6PGD),especially with 100 n M insulin incubation.However,insulin reduced cpt 1a gene expression was observed in hepatocytes following incubation treatment.Insulin administration also tended to down-regulate ppar? mRNA levels.The results indicated that insulin promotes lipid storage in freshly isolated hepatocytes of yellow catfish,possibly through up-regulating lipogenesis and down-regulating lipolysis.In order to study the way in which insulin influences lipid metabolism in vivo,yellow catfish intraperitoneally injected with 1 ?l/g fish body weight(BW)of PBS(control)or with different doses of insulin(0.01 ?g/g,0.1 ?g/g and 1 ?g/g BW,respectively),sapmpling acccured at 48 h.Hepatic lipid and intracellular triglceride(TG)content,the activities and/or expression level of several enzymes(CPT I,6PGD,G6 PD,and FAS)as well as the mRNA expression of transcription factors(PPAR? and PPAR?)involved in lipid metabolism were determined.As a result,insulin tended to increase hepatic lipid accumulation,activities of lipogenic enzymes(6PGD,G6 PD and FAS)as well as mRNA levels of genes fas,g6 pd,6pgd,and ppar?,but down-regulated mRNA level of ppar?.Oil red O staining indicated that hepatic lipid content tended to increase with the higher insulin treatment.Overall,the present study indicated that insulin tended to increase hepatic lipid deposition,the activities and expressions of FAS,G6 PD and 6PGD,as well as the expression of ppar? both in vivo and in vitro.However,there are still some differences between the in vivo and in vitro study on the effect of insulin on the activities and expression of CPT I,indicating that regulation of insulin on CPT I was rather complication in fish.5.Effects of PI3 K and FOXO1 pathway on the inuslin-induced the changes of lipid metabolism in the primary hepatocytes from yellow catfishIn the present study,wortmannin(PI3K specific blocker)and AS1842856(FOXO1 pecific blocker)were used to explore the signaling pathways of insulin influencing lipid metabolism.Firstly,hepatocytes were isolated from yellow catfish,cultured and incubated with 10 m M PBS(control),1?M wortmannin,100 n M insulin,and 1?M wortmannin + 100 n M insulin,sampling accured at 48 h.As a result,compared to the control,single wortmannin incubation reduced TG accumulation.Pretreatment with wortmannin significantly reduced insulin-induced TG accumulation.Compared to the control,single wortmannin treatment did not significantly influence the mRNA levels of fas,g6 pd,6pgd,ppar? and ppar?,but down-regulated cpt 1a mRNA expression.Furthermore,wortmannin pre-treatment significantly reduced the insulin-induced up-regulation of g6 pd,6pgd and ppar? expression,but did not significantly influence the mRNA levels of fas and cpt 1a.The results showed that insulin-induced TG accumulation and the expression of gene involved in lipogenesis could be reversed by wortmannin,indicating that PI3 K pathway were involved in the insulin-induced alteration of lipid metabolism.Secondly,hepatocytes were isolated from yellow catfish,cultured and incubated with 10 m M PBS(control),1?M AS1842856,100 n M insulin,and 1?M AS1842856 + 100 n M insulin,sampling accured at 48 h.As a result,AS1842856 incubation significantly down-regulated the mRNA levels of all pi3 ks,and pi3 kcb and pi3kc3 are more sensitively to AS1842856 than other pi3 ks.Furthermore,compared with the control,AS1842856 alone significantly reduced TG accumulation and the mRNA expression of g6 pd,ppar?,ppar? and cpt 1a.Compared with single insulin incubation,pre-treatment with AS1842856 also significantly decreased insulin-induced TG accumulation and the up-regulation of g6 pd,6pgd and ppar? expressions.The reslut showed that insulin-induced TG accumulation and the expression of gene involved in lipogenesis could be also reversed by AS1842856,indicating that insulin-induced alteration of lipid metabolism could be mediated by both FOXO1 and PI3 K pathway.