The Effects Of CPT1A And MTP Interference On Lipidosis Induced By Three Kinds Of Sugar In Goose Primary Hepatocytes

Objective: The liver maintains the balance of the body glucolipid metabolism plays an important role.This research preliminary experiments confirmed in different abilities of lipid deposition of hepatocytes induced by glucose and fructose,prompt lipid deposition ability induced differ in different types of sugar.So,in this study,the aim is to cloning the full-length coding sequences of CPT1 A,MTP gene of Sichuan White Goose;using goose primary hepatocytes as experimental material to explore the role of CPT1 A,MTP gene in lipid deposition induced by different types of sugar,through transfection CPT1 A,MTP interference plasmid,adding glucose,fructose and sucrose separately,fatty acid synthesis,fatty acid oxidation in VLDL-TG secretion were detected to reflect the lipid metabolism change with the RT-PCR,oil red O staining method and MTT,which can be more clear to understand liver lipid deposition mechanism induced different types of sugar.Main obtained results are as follows:1.Cloning and bioinformatics analysis of goose CPT1 A,MTP gene:In the current study,the full-length coding sequence of CPT1 A,MTP encoding 770,893 amino acids respectively was cloned using segmented primers to polymerasechain reaction(PCR),and PCR sequence products were spliced to gain CPT1 A,MTP coding sequences.2.After 24 hours of transfection CPT1 A,MTP RNAi plasmid respectively,fluorescent quanfitation PCR detection showed CPT1 A,MTP interference efficiency were51.3%,62.6% respectively,which showed CPT1 A,MTP were inhibited in goose liver cells.3.After transfection CPT1 A RNAi plasmid,FAS,ACCα mRNA expression showed no significant change,SREBP1 mRNA expression was significantly decreased(P < 0.05),FAS,ACCα kinase expression significantly increased(P < 0.05);CPT1A,ACOX1,PPARαmRNA expression showed significantly decreased(P < 0.05),CPT1 A kinase expression decreased significantly(P < 0.05);MTP,APOB,DGAT1 mRNA expression was significantly decreased(P < 0.05),no significant change in MTP,APOB kinase expression.Oil red O staining showed goose hepatocytes CPT1 A interference multiply increased lipid droplets and lipid deposition,meanwhile,increased intracellular and extracellular TG content,decreased extracellular VLDL content.4.After transfection MTP RNAi plasmid,FAS,ACCα mRNA expression showed significantly increased(P < 0.05),SREBP1 mRNA expression significantly decreased(P <0.05),FAS,ACCα kinase expression significantly increased(P < 0.05);CPT1A,ACOX1,PPARα mRNA expression and CPT1 A kinase expression showed no significant changes;MTP,APOB mRNA expression was significantly decreased(P < 0.05),DGAT1 mRNA expression had no significant changes,MTP kinase expression decreased significantly(P <0.05),APOB kinase expression had no significant change.Oil red O staining showed goose hepatocytes CPT1 A interference multiply increased lipid droplets and lipid deposition,meanwhile,increased intracellular and extracellular TG content,decreased extracellular VLDL content.5.Compared with single glucose or sucrose treatment separately,after glucose or sucrose treatment separately with CPT1 A interference,FAS、ACCα、SREBP-1 mRNA and FAS、ACCα kinase expression increased,CPT1A、ACOX1 mRNA expression decreased,MTP、APOB mRNA and MTP kinase expression decreased.Compared with single fructose treatment,after fructose treatment with CPT1 A interference,FAS 、 ACCα 、 SREBP-1mRNA and FAS、ACCα kinase expression decreased,CPT1A、ACOX1 mRNA expression decreased,MTP、APOB mRNA and MTP kinase expression decreased.Oil red O staining showed three kinds of sugar and CPT1 A interference respectively of goose hepatocytes multiply increased lipid droplets and lipid deposition,meanwhile,increased intracellular TG content and intracellular VLDL,decreased extracellular VLDL content.6.Compared with single glucose or sucrose treatment separately,after glucose or sucrose treatment separately with MTP interference,FAS、ACCα、SREBP-1 mRNA and FAS、ACCα kinase expression increased,mRNA and kinase expression expression of key gene of fatty acid oxidation had no significant change,MTP、APOB mRNA and MTP kinase expression decreased.Compared with single fructose treatment,after fructose treatment with MTP interference,FAS、ACCα、SREBP-1 mRNA and FAS、ACCα kinase expression decreased,CPT1A、ACOX1 mRNA expression increased,MTP、APOB mRNA and MTP kinase expression decreased.Oil red O staining showed three kinds of sugar and MTP interference respectively of goose hepatocytes multiply increased lipid droplets and lipid deposition,meanwhile,increased intracellular and extracellular TG content and intracellular VLDL.