饲料脂肪对黄颡鱼卵巢脂类代谢以及PI3KCa甲基化和转录水平的影响

Effects of dietary lipid on lipid metabolism, methylation and expression of PI3KCa in the ovary of yellow catfish (Pelteobagrus fulvidraco)

为了探究饲料不同脂肪水平对黄颡鱼卵巢脂肪代谢潜在的影响,本研究设计了活体与离体两个实验。活体实验中,分别投喂3组不同脂肪水平的饲料(脂肪含量分别为6.98%、11.34%和15.41%)8周。离体实验中,分离原代黄颡鱼卵母细胞,采用(0、0.2和0.5 mmol/L)3组不同脂肪酸浓度孵育48 h。活体实验结果显示:与6.98%和15.41%脂肪水平组相比,11.34%脂肪水平显著增加黄颡鱼卵巢甘油三酯水平(TG),并上调FAS、G6PD、6PGD和ME的酶活性水平,以及LPL和CD36基因表达水平。与6.98%和11.34%脂肪水平组相比,15.41%脂肪水平组显著升高黄颡鱼的性腺指数(GSI),并上调CPTIA和DNMT3b的基因表达水平,以及PI3KCa启动子-64和-52 CpG位点的甲基化水平,而显著降低PI3KCa的基因表达水平。体外细胞实验显示:与对照组(0)相比,0.5 mmol/L脂肪酸孵育显著增加卵母细胞TG含量,并上调FAS、G6PD和ME酶活水平,以及G6PD和PI3KCa的基因表达水平。同时,与对照组相比,脂肪酸孵育组显著降低CPTIA、ACCb、LPL、CD36、DNMT1以及DNMT3b基因的表达水平。然而,脂肪酸孵育对卵母细胞PI3KCa的启动子甲基化水平没有影响。研究表明,饲料不同脂肪水平影响卵巢TG的合成,可能主要是通过脂肪转运相关基因,并且高脂肪很可能是通过影响黄颡鱼卵巢PI3KCa启动子甲基化水平来影响PI3KCa的表达。而离体条件下,脂肪酸促进卵母细胞TG的合成很可能是通过升高脂肪合成相关基因、降低脂肪分解和转运相关基因来实现,但脂肪酸孵育不通过影响黄颡鱼卵母细胞PI3KCa甲基化水平来影响PI3KCa基因表达。

The present study was conducted to determine the potential mechanisms of dietary lipid level influencing lipid deposition in the ovary of yellow catfish Pelteobagrus fulvidraco. For this purpose, two experiments were designed. In vivo, yellow catfish were fed with three diets containing 6.98%, 11.34% and 15.41% of lipid levels. In vitro, the primary oocytes from yellow catfish were incubated with three fatty acid levels (0, 0.2 and 0.5 mmol/L), respectively. Triacylglyceride (TG) contents, methylation level of PI3KCa, and enzymes activities of FAS, G6PD, 6PGD, and ME, as well as the mRNA level of PI3KCa, DNMTs and other genes involved in lipid metabolism were determined in the ovary and oocytes of yellow catfish. GSI increased with increasing dietary lipid levels. Among three lipid treatments, TG content and the enzyme activities of FAS, G6PD, 6PGD, and ME, and mRNA expression of LPL and CD36 were highest in the 11.34% of dietary lipid level in the ovary;CPT IA mRNA expression was the highest for fish fed 15.41% dietary lipid;the lowest mRNA level of PI3KCa and the highest methylation percentage of PI3KCa promoter at -64 and -52 CpG sites were found in the 15.41% of dietary lipid group;Dietary lipid levels influenced the mRNA level of DNMT3b, but not the expression of DNMT1 and DNMT3a. DNMT3b mRNA expression was the highest in 15.41% of dietary lipid group, indicating that DNMT3b was more sensitive than DNMT1 and DNMT3a in response to dietary lipid level. In contrast, the in vitro study indicated that TG content, enzyme activities of FAS, G6PD and ME, and G6PD mRNA expression were the highest in the 0.5 mmol/L FA group, and mRNA expression levels of CPT IA, ACCb, LPL and CD36 were the highest in the control;FA incubation significantly increased the mRNA level of PI3KCa, reduced mRNA levels of DNMT1 and DNMT3b, but showed no significant effect on the methylation of PI3KCa promoter and on mRNA expression of DNMT3a.