目的 从基因启动子区DNA甲基化环节, 探讨n-3多不饱和脂肪酸(n-3 polyunsaturated fatty acids, n-3 PUFAs)对肥胖状态下瘦素表达的表观遗传调控机制。方法 3~4周龄C57BL/6J雄性小鼠30只, 随机被分为3组(每组10只), 分别给予高脂饲料、鱼油n-3 PUFAs高脂饲料(脂肪含量均为34.9%, 供能比为40%)以及正常脂饲料(脂肪含量为4.3%, 供能比为10%)喂养4个月以诱导肥胖。喂养结束时取性腺周围脂肪组织, 应用RT-PCR检测脂肪组织瘦素mRNA水平;应用亚硫酸盐修饰直接测序(bisulfite sequencing-PCR, BSP)法检测瘦素基因启动子区CpG甲基化程度;应用染色质免疫共沉淀-荧光定量PCR(CHIP-RT-PCR)技术测定瘦素基因启动子区结合的DNA甲基化转移酶(DNMTs)、甲基化CpG结合蛋白2(MECP-2)以及RNA聚合酶2(Poly II)的变化。结果 与正常脂饲料组小鼠相比, 脂肪组织中瘦素mRNA 表达在高脂饲料诱导肥胖小鼠明显增加, 而在鱼油高脂饲料组肥胖小鼠无变化。基因启动子区甲基化结果显示, 高脂肥胖小鼠瘦素基因启动子区CpG位点甲基化程度、DNMT1、DNMT3a、DNMT3b和MECP-2含量均较正常饲料组显著升高, 并伴随Poly II含量减少;而鱼油高脂饲料组瘦素基因启动子区结合DNMT3a、DNMT3b and MeCP-2增加, 但启动子区MECP-2含量与高脂饲料组比缺有显著降低, DNMT1和Poly II以及CpG位点甲基化程度无改变。结论 肥胖状态下瘦素表达变化可能与基因启动子区DNA甲基化以及结合的相关调控蛋白、RNA聚合酶等有关;n-3 PUFAs对瘦素基因表达的调节也可能是通过对这些蛋白的影响而实现的。
Abstract
Obejective To investigate the epigenetic mechanisms linking n-3 polyunsaturated fatty acids (n-3 PUFAs) and the increased leptin expression in obesity. Methods Thirty 3~4 week-old male C57BL/6J mice were randomly divided into 3 groups and fed 4 months with two different high-fat diets (34.9% of fat providing 40% of total energy), high-fat diets (lard oil and sunflower oil) and fish oil high-fat diet (lard oil and fish oil), and one lean control diet (lard oil and sunflower oil, 4.3% of fat providing 10% of total energy).After fasted for 12 hours, mice were sacrificed and epididymal fat was collected.The mRNA expression of leptin in fat was assessed by real-time PCR (RT-PCR).Bisulfite sequencing-PCR (BSP) was used to determine the methylation level of CpG sites in the leptin promoter.Chromatin immunoprecipitation (ChIP) and RT-PCR were used to determine the amount of DNA methyltransferase (DNMTs), methyl CpG binding protein 2 (MeCP-2) and RNA polymerase II (Poly II) associated with the leptin promoter. Results As compared to the lean control diet group, the leptin mRNA expression in fat from the high-fat diet group was increased, whereas no change was found in the fish oil high-fat diet group.The methylation level of CpG sites in the leptin promoter and the associated proteins, DNMT1, DNMT3a, DNMT3b, MeCP-2 were higher than the lean control diet group with reduced binding of Poly II.In the fish oil high-fat group, increased DNMT3a, DNMT3b and MeCP-2 binding to the leptin promoter were found and the extent of MeCP-2 increase was less compared to the high-fat diet group.No changes were found in the associated DNMT1 and Poly II, and methylation of the leptin promoter in the fish oil high-fat diet group. Conclusion Our findings suggest that the leptin expression may be associated with the promoter methylation of the gene and the binding proteins and RNA polymerase in obesity, and n-3 PUFAs regulate the leptin expression through these pathways.
关键词
n-3多不饱和脂肪酸 /
肥胖 /
DNA甲基化 /
DNA甲基化转移酶 /
甲基化CpG结合蛋白
Key words
n-3 polyunsaturated fatty acids /
obesity /
DNA methylation /
DNA methyltransferase /
methyl CpG binding protein
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参考文献
[1] Marti A, Ordovas J.Epigenetics lights up the obesity field[J].Obes Facts, 2011, 4(3):187-190.
[2] Milagro FI, Campión J, García-Díaz DF, et al.High fat diet-induced obesity modifies the methylation pattern of leptin promoter in rats[J].J Physiol Biochem, 2009, 65(1):1-9.
[3] Uriarte G, Paternain L, Milagro FI, et al.Shifting to a control diet after a high-fat, high-sucrose diet intake induces epigenetic changes in retroperitoneal adipocytes of Wistar rats[J].J Physiol Biochem, 2013, 69 (3):601-611.
[4] Buckley JD, Howe PR.Anti-obesity effects of long-chain omega-3 polyunsaturated fatty acids[J].Obes Rev, 2009, 10(6):648-659.
[5] Ailhaud G, Massiera F, Weill P, et al.Temporal changes in dietary fats:role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity[J].Prog Lipid Res, 2006, 45(3):203-236.
[6] Fan C, Liu X, Shen W, et al.The regulation of leptin, leptin receptor and pro-opiomelanocortin expression by N-3 PUFAs in diet-induced obese mice is not related to the methylation of their promoters[J].Nutri Metab (Lond), 2011, 8:31-40.
[7] Stoger R.In vivo methylation patterns of the leptin promoter in human and mouse[J].Epigenetics, 2006, 1(4):155-162.
[8] Yang M, Sun JZ, Sun YL, et al.Association between leptin gene promoter methylation and type 2 diabetes mellitus[J].Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 2012, 29(4):474-477.
[9] Okada Y, Sakaue H, Nagare T, et al.Diet-induced up-regulation of gene expression in adipocytes without changes in DNA methylation[J].Kobe J Med Sci, 2009, 54(5):e241-249.
[10] Cordero P, Campion J, Milagro FI, et al.Leptin and TNF-alpha promoter methylation levels measured by MSP could predict the response to a low-calorie diet[J].J Physiol Biochem, 2011, 67(3):463-470.
[11] 申文雯, 郑东旖, 刘兆秋, 等.肥胖儿童食欲调节因子基因启动子区DNA甲基化探讨[J].中国儿童保健杂志, 2013, 21(11):1132-1134.
[12] Blackledge NP, Klose R.CpG island chromatin:a platform for gene regulation[J].Epigenetics, 2011, 6(2):147-152.
[13] Tobi EW, Lumey LH, Talens RP, et al.DNA methylation differences after exposure to prenatal famine are common and timing-and sex-specific[J].Hum Mol Genet, 2009, 18:4046-4053.
[14] Attig L, Gabory A, Junien C.Nutritional developmental epigenomics:immediate and long-lasting effects[J].Proc Nutr Soc, 2010, 69(2):221-231.
[15] Vel Szic KS, Ndlovu MN, Haegeman G, et al.Nature or nurture:let food be your epigenetic medicine in chronic inflammatory disorders[J].Biochem Pharmacol, 2010, 80(12):1816-1832.
[16] Kulkarni A, Dangat K, Kale A, et al.Effects of altered maternal folic acid, vitamin B12 and docosahexaenoic acid on placental global DNA methylation patterns in Wistar rats[J].PLoS One, 2011, 6:e17706.
[17] Hubertus J, Zitzmann F, Trippel F, et al.Selective methylation of CpGs at regulatory binding sites controls NNAT expression in Wilms tumors[J].PLoS One, 2013, 8(6):e67605.
基金
荷兰Nutricia Research Foundation(2009-15, 2010-E4);北京市科委行业定额经费自主项目(2012-bjsekyjs-2)
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