猪CRTC3基因表达的品种差异及forskolin对其表达的影响

Breed Difference of Porcine CRTC3 Expression and Its Regulation by Forskolin

本试验旨在探究糖脂代谢通路关键基因CRTC3在不同品种猪肌肉和脂肪组织中的表达情况,并通过forskolin处理猪皮下脂肪前体细胞,研究forskolin对脂肪前体细胞分化聚酯和CRTC3基因表达的影响,阐明猪CRTC3基因表达与脂肪沉积的关系。试验选取杜长大猪和莱芜猪各5头,检测肌肉、脂肪组织中CRTC3的mRNA和蛋白表达水平以及脂肪代谢相关基因的mRNA表达水平;选取2头3日龄的杜长大仔猪,分离猪皮下脂肪前体细胞,待完全融合后用MDI诱导培养基诱导4 d,然后用分化培养基继续诱导4 d,完成诱导分化。Forskolin组在诱导分化的第1天即加入forskolin,使其终浓度为10μmol/L,对照组则加入同浓度的二甲基亚砜(DMSO)进行诱导分化。结果表明:在莱芜猪的背最长肌和腰大肌中,CRTC3的蛋白表达水平高于杜长大猪;在莱芜猪的皮下和内脏脂肪组织中,CRTC3及脂肪沉积相关基因过氧化物酶体增殖剂激活受体γ(PPARγ)、脂肪酸结合蛋白4(FABP4)、CCAAT/增强子结合蛋白α(C/EBPα)、围脂滴蛋白(PLIN)和瘦素(LEP)的mRNA表达水平显著或极显著高于杜长大猪(P<0.05或P<0.01),而脂肪棕色化相关基因NF-E2相关因子1(NRF1)、过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC⁃1α)、PRDM16、解偶联蛋白2(UCP2)、解偶联蛋白3(UCP3)的mRNA表达水平则显著或极显著低于杜长大猪(P<0.05或P<0.01)。进一步的研究发现,猪皮下脂肪前体细胞分化后CRTC3和脂肪沉积相关基因的mRNA表达水平极显著提高(P<0.01),脂肪棕色化相关基因的mRNA表达水平也均极显著升高(P<0.01)。10μmol/L forsko⁃lin处理能抑制猪皮下脂肪前体细胞分化,极显著升高环磷腺苷效应元件结合蛋白(CREB)和脂肪棕色化相关基因的mRNA表达水平(P<0.01),促进CRTC3的进核,极显著降低CRTC3和脂肪沉积相关基因的mRNA表达水平(P<0.01)。上述研究结果表明,CRTC3基因与猪脂肪沉积密切相关,forskolin处理可以调控猪CRTC3及脂质代谢相关基因表达,调控猪皮下脂肪前体细胞分化聚酯。

CRTC3 is a key gene in glucose and lipid metabolism pathway.In this study,we aimed to study the expression pattern of porcine CRTC3 in muscle and adipose tissues of different breeds,and the effects of fors⁃kolin treatment on expression of CRTC3 gene and differentiation in porcine subcutaneous preadipocytes.Five Laiwu pigs and five Duroc×Landrace×Yorkshire(DLY)pigs were selected to detect the mRNA and protein expression levels of CRTC3 and lipid metabolism related genes in muscle and adipose tissues.Before differenti⁃ation,the porcine subcutaneous preadipocytes were separated from 3⁃day⁃old DLY piglets.The above medium was replaced by DMEM containing MDI induction 4 days.Then the medium was replaced by DMEM contai⁃ning insulin until day 8.10μmol/L forskolin was added at the 1st day of differentiation and induction in forsko⁃lin group,and the dimethyl sulfoxide(DMSO)with the same concentration was added in control group.The results showed as follows:compared with DLY pigs,Laiwu pig had a higher CRTC3 protein expression level in longissimus dorsi muscle and psoas major muscle.The mRNA expression levels of CRTC3 and fat deposition related genes peroxisome proliferators activated receptorγ(PPARγ),fatty acid binding protein 4(FABP4),CCAAT/enhancer binding proteinα(C/EBPα),perilipin(PLIN)and leptin(LEP)in subcutaneous and vis⁃ceral adipose tissues of Laiwu pigs were significantly higher than those of DLY pigs(P<0.05 or P<0.01).However,the mRNA expression levels of adipose browning related genes NF⁃E2 related factor 1(NRF1),PPARγcoactivator 1α(PGC⁃1α),PRD1⁃BF1⁃RIZ1 homologous domain containing 16(PRDM16),uncoup⁃ling protein 2(UCP2)and uncoupling protein 3(UCP3)in subcutaneous and visceral adipose tissues of Laiwu pigs were significantly or extremely significantly lower than those of DLY pigs(P<0.05 or P<0.01).Further⁃more,the mRNA expression levels of CRTC3,adipose deposition related genes and adipose browning related genes in porcine subcutaneous preadipocytes were extremely significantly increased after differentiation(P<0.01).Treatment with 10μmol/L forskolin inhibited the differentiation of porcine subcutaneous preadipo⁃cytes,upregulated the mRNA expression levels of cAMP⁃response element binding protein(CREB)and adi⁃pose browning related genes,promoted the entry of CRTC3 into the nucleus.Moreover,it reduced the mRNA expression levels of CRTC3(P<0.01),as well as adipose deposition related genes(P<0.01).Therefore,CRTC3 is a adipose deposition related gene for selecting bioactive or nutrient factors,which should be applica⁃ble to regulation of fat deposition.