| Insulin is an important peptide hormone that regulates blood sugar in humans and animals.It regulates the lipid metabolism in animal liver mainly through promoting lipid anabolism（lipogenesis）and inhibiting lipid catabolism.Insulin has been confirmed to improve the m RNA expression level and enzyme activity of FADS1 and FADS2,two key enzymes of long-chain polyunsaturated fatty acids（LC-PUFA）biosynthesis in mammals,but there are few relevant reports in teleost.The rabbitfish Siganus canaliculatus is an herbivorous marine teleost,it has the ability to convert C18PUFA into LC-PUFA,and all of the LC-PUFA biosynthesis key enzyme genes（includingΔ6Δ5 fads2,Δ4 fads2,elovl5 and elovl4）had already been cloned.Thus,rabbitfish is an ideal model to study the regulation mechanism of LC-PUFA biosynthesis in fish.In this study,we used S.canaliculatus hepatocyte line（SCHL）as material to study the effects of insulin to LC-PUFA biosynthesis,and explored its regulation mechanism in the direction of transcription level and cellular signaling pathway,with the help of transcriptome sequencing technology.The main research contents,results and conclusions are as follows:1.Insulin can promote the expression of LC-PUFA biosynthesis key enzyme genes in SCHL cellsSCHL cells were treated with different doses of insulin（0,40,80,120,200 nM）in serum-free medium for different treatment time（6 h,12 h）.The results showed that insulin could increase the m RNA expression levels ofΔ6Δ5 fads2 and elovl5 in time-and dose-depended manners.The promoting effects of insulin at 12 h were more obvious than 6 h.And the relative m RNA expression levels ofΔ6Δ5 fads2 and elovl5first increased then decreased following with the increase of insulin dose,and according to the trend lines added by Origin software,when the insulin dose was 65 nM（treated cells for 12 h）,the promoting effects toΔ6Δ5 fads2 and elovl5 were the most significant.Then used 65 nM insulin and treated the SCHL cells for 12 h,the results of fatty acid composition analysis showed that the proportion of arachidonic acid（ARA,20:4n-6）in insulin-treated group was significantly higher than that in the control group,and EPA（20:5n-3）and DHA（22:6n-3）had increasing trends.The conversion rates of18:2n-6 to 18:3n-6 and 18:3n-3 to 18:4n-3,catalyzed byΔ6Δ5 Fads2,were significantly increased（P<0.05）;and the conversion rates of 18:3n-3 to 20:3n-3 and 20:5n-3 to22:5n-3,catalyzed by Elovl5,had increasing trends.These results indicated that insulin could improve the m RNA expression level and enzyme activity of LC-PUFA biosynthesis key enzymes in SCHL cells,and then promoted LC-PUFA biosynthesis.2.The transcriptome sequencing results and analysis of insulin-treated SCHL cellsTranscriptome analysis were performed using SCHL cells treated with 65 nM insulin for 12 h,a total of 69,116 unigenes with mean length of 1383 bp were assembled and clustered,and 63.72%of unigenes were annotated.Compared with the control group,250 unigenes were up-regulated and 277 down-regulated by insulin.A total of130 differentially expressed genes（DEGs）were enriched in 157 KEGG pathways,among them,10 DEGs were enriched in 8 lipid metabolism related signaling pathways.Δ6 fads2,one of the key enzyme genes in LC-PUFA biosynthesis,was significantly up-regulated by insulin in RNA-seq results,and elovl5 had an upward trend.The m RNA expression levels of two transcription factors srebp1 and lxrα,several key genes in PI3K pathway（pik3ca、pdk1、prkci、akt2 and mtor）had upward trends.Based on the transcriptomic data,other genes which may be involved in the regulation of insulin inspiring rabbitfish LC-PUFA biosynthesis were screened.Among them,the m RNA expression levels of ampkαhad a downward trend and calm had an upward trend,and creb1,plcb3 and hsp90a were significantly up-regulated by insulin.The RT-q PCR experiments were performed using the same batch cell samples with those for RNA-seq,and the results of RT-q PCR were approximately consistent with RNA-seq results,that is,insulin increased the m RNA expression levels ofΔ6 fads2,elovl5,srebp1,lxrα,pdk1,prkci,akt2,mtor,calm,creb1 and plcb3,and pik3ca had an upward trend,ampkαhad a downward trend.These results suggested that the transcription factors Lxrαand Srebp1,as well as PI3K signaling pathway key genes are likely to participate in the regulation of insulin on LC-PUFA biosynthesis.In addition,other target genes screened based on transcriptomic data,such as ampk,calm,creb1,plcb3 and hsp90a,may also be involved in the regulation of insulin on rabbitfish LC-PUFA biosynthesis.3.The role validation of PI3K signaling pathway in the regulation of insulin on rabbitfish LC-PUFA biosynthesisFirstly,the open reading frame（ORF）of pik3ca,akt2 and prkci（encoding PI3Kα,Akt2 and Pkcλ,respectively）were cloned from rabbitfish,their nucleotide length and number of encoded amino acids were 3207 bp and 1068,1449 bp and 482,1767 bp and588,respectively.The results of multi-sequence alignment and similarity comparison showed that the amino acid sequences of PI3Kα,Akt2 and Pkcλhad typical and conservative domains,and had high homology with the corresponding amino acid sequences from other species（similarity>87%）.Phylogenetic tree analysis revealed that Larimichthys crocea,Seriola dumerili,Collichthys lucidus and Labrus bergylta are the most closely related with rabbitfish.Tissue specific analysis showed that pik3ca,akt2 and prkci were all expressed in rabbitfish eye,brain,gills,spleen,heart,muscle,intestine and liver.The m RNA expression level of pik3ca was the highest in brain and the lowest in muscle;akt2 was the highest in liver,and other tissues had no significant difference;and prkci was highest in liver and gill.Secondly,in order to verify the role of PI3K signaling pathway in insulin regulating LC-PUFA biosynthesis in rabbitfish,SCHL cells were treated with the agonists and inhibitors of PI3Kα,Akt2 and Pkcλcombined with insulin,and performed the RT-q PCR experiments.The results showed that PI3Kαagonist 740 Y-P and IGF-1could increase the m RNA expression levels of the key genes in PI3K pathway（pik3ca,prkci,akt2,mtor）,transcription factors srebp1,and key knase genes in LC-PUFA biosynthesis（Δ6Δ5 fads2,Δ4 fads2,elovl5,elovl4）.While the PI3K inhibitors LY294002 and Wortmannin had the opposite results,and impaired the inspiring effects of insulin on the transcription of these genes.The Akt2 agonist（SC-79）promoted the m RNA expression of akt2,mtor,srebp1,Δ6Δ5 fads2,Δ4 fads2 and elovl5.While the inhibitors of Akt2（AKT inhibitor VIII）and Pkcλ（Rottlerin）both attenuated the promoting effects of insulin onΔ6Δ5 fads2 and elovl5 transcription.Finally,the fatty acids composition analysis was performed,the results showed that the addition of inhibitor LY294002,AKT inhibitor VIII and Rottlerin all reduced the content of EPA and DHA in SCHL cells,and inhibited the promoting effects of insulin on DHA biosynthesis.The addition of agonists IGF-1 and SC-79 both increased the content of EPA in cells.These results showed that PI3K signaling pathway genes pik3ca,akt2 and prkci were involved in the regulation of insulin on LC-PUFA biosynthesis in SCHL cells.In conclusion,the results of this study suggested that insulin can regulate LC-PUFA biosynthesis through the PI3K pathway and its downstream transcription factor Srebp1 in rabbitfish hepatocytes.It is the first time to explore the regulation mechanism of insulin on LC-PUFA biosynthesis in teleost fish.The research results will provide new knowledge in the regulation mechanism of LC-PUFA biosynthesis in fish,and provide a new approach for developing methods to improve the ability of LC-PUFA biosynthesis in marine fish. |
