Effect of short‐chain fatty acids on triacylglycerol accumulation,lipid droplet formation and lipogenic gene expression in goat mammary epithelial cells

Short‐chain fatty acids (SCFAs) are the major energy sources for ruminants and are known to regulate various physiological functions in other species. However, their roles in ruminant milk fat metabolism are still unclear. In this study, goat mammary gland epithelial cells (GMECs) were treated with 3 mmol/L acetate, propionate or butyrate for 24 h to assess their effects on lipogenesis. Data revealed that the content of triacylglycerol (TAG) and lipid droplet formation were significantly stimulated by propionate and butyrate. The expression of FABP3, SCD1, PPARG, SREBP1, DGAT1, AGPAT6 and ADRP were upregulated by propionate and butyrate treatment. In contrast, the messenger RNA (mRNA) expression of FASN and LXRα was not affected by propionate, but reduced by butyrate. Acetate had no obvious effect on the content of TAG and lipid droplets but increased the mRNA expression of SCD1 and FABP3 in GMECs. Additionally, it was observed that propionate significantly increased the relative content of mono‐unsaturated fatty acids (C18:1 and C16:1) at the expense of decreased saturated fatty acids (C16:0 and C18:0). Butyrate and acetate had no significant effect on fatty acid composition. Overall, the results from this work help enhance our understanding of the regulatory role of SCFAs on goat mammary cell lipid metabolism.     

牛乳腺脂肪合成关键酶基因在乳汁体细胞中的表达研究

为了阐明乳脂合成的影响因素及其内在分子机理,为反刍动物原料乳的优化,特别是为乳脂肪的营养调控和遗传改良提供理论依据。本试验以奶牛初乳、常乳和末乳中的乳汁体细胞为研究对象,以看家基因GAPDH为内参,对初乳、常乳和末乳中LPL、CD36、VLDLR、ACSS2、ACSL1、FABP3、ACC、FASN、SCD、ADFP、XDH和BTN1A1 mRNA进行半定量RT-PCR分析。结果发现,LPL、CD36、VLDLR、ACSS2、ACSL1、FABP3、SCD、AD-FP、XDH和BTN1A1 mRNA在初乳、常乳和末乳中均有表达,而ACC和FASNmRNA只在初乳中表达,常乳和末乳中均不表达;半定量结果表明,与初乳相比,常乳和末乳中LPL、CD36、VLDLR、ACSS2、ACSL1、FABP3、SCD、ADFP、XDH和BTN1A1 mRNA转录水平显著降低(P<0.05),且常乳与末乳间差异不显著(P>0.05)。研究结果提示初乳期乳腺脂肪合成能力明显高于常乳和末乳期乳腺,且脂肪合成关键酶基因的表达与细胞内脂转运和代谢的生理变化有关。     

牦牛乳脂合成关键酶基因全泌乳期的表达研究

试验以川西北高原的乳用麦洼牦牛为研究对象,应用实时荧光定量PCR技术对麦洼牦牛乳腺组织中的氨酰-CoA合成酶长链家族成员1(acyl-CoA synthetase long-chain family member 1,ACSL1)、氨酰-CoA合成酶短链家族成员1、2(acyl-CoA synthetase short-chain family member 1、2,ACSS1、ACSS2)和脂肪酸结合蛋白家族成员3(fatty acid binding protein 3,FABP3)等乳脂代谢相关基因全泌乳期转录水平进行了分析。结果表明,4个基因在整个泌乳期均持续表达;分娩后30 d(30 d )ACSL1、ACSS1、ACSS2和FABP3基因的转录水平均比分娩前15 d(-15 d)显著增加(P<0.05),且达到最大值后逐步下降。产乳量测定结果表明,麦洼牦牛产乳量在120 d达到峰值,比乳脂代谢相关基因峰值表达水平晚90 d,其后产乳量逐步下降。结果表明,牦牛脂肪合成关键酶基因在全泌乳期的表达规律可能与牦牛对青藏高原生存环境的适应有关。     

FADS2基因在奶牛乳腺细胞中的过表达和干扰研究

为了研究脂肪酸脱氢酶2(fatty acid desaturases 2,FADS2)基因在奶牛乳腺细胞脂肪酸代谢中的作用,本研究在奶牛乳腺上皮细胞中对FADS2基因进行过表达和干扰,研究FADS2基因表达对脂肪酸合成相关基因的调控及对奶牛乳腺上皮细胞中甘油三酯含量的影响。针对FADS2基因的CDS序列设计siRNA和过表达载体pcDNA3.1-FADS2-EGFP,转染奶牛乳腺细胞检测FADS2基因过表达和干扰对脂肪酸代谢相关基因表达的影响及细胞中甘油三酯含量的变化。结果显示,试验成功获得过表达载体pcDNA3.1-FADS2-EGFP和干扰片段,转染细胞后具有良好的过表达和干扰效果。FADS2基因过表达后,1-酰基甘油磷酸酰基转移酶(AGPAT1)、固醇调节元件结合蛋白裂解激活蛋白(SCAP)、3-磷酸甘油转移酶(GPAM)、脂肪酸延长链5(ELOVL5)、乙酰辅酶A酰基转移酶1(ACAA1)、脂肪酸脱氢酶1(FADS1)、二酰基甘油转酰基酶1(DGAT1)和过氧化物酶体增殖激活受体α(PPARα)基因显著下调(P<0.05),脂滴蛋白2(PLIN2)基因极显著上调(P<0.01)。FADS2基因干扰过后可引起AGPAT1、GPAM、ELOVL5、ACAA1、PLIN2和FADS1基因显著上调(P<0.05),脂肪酸合成胰岛素诱导基因1(INSIG1)极显著上调(P<0.01),DGAT1和PPARα基因显著下调(P<0.05)。甘油三酯检测结果显示,FADS2基因过表达和干扰均可降低奶牛乳腺上皮细胞中甘油三酯的含量。综上所述,在奶牛乳腺上皮细胞中,FADS2基因能调控脂质合成相关基因的表达,对乳腺脂质合成具有调控作用。     

Genetic mapping using 1.4M SNP array refined loci for fatty acid composition traits in Chinese Erhualian and Bamaxiang pigs

Chinese indigenous pigs display marked genetic and phenotypic differences compared with western commercial pigs. In this study, we tested the association between 660K SNPs and longissimus muscle fatty acid composition traits in Chinese Erhualian (n = 331) and Bamaxiang (n = 315) pigs based on a customized 1.4 million SNP array. We identified a total of 64 significant associations for 20 fatty acid composition traits at the p‐value threshold of 1 × 10^?6 among which 42 associations in low linkage disequilibrium (r² < .2) with previously reported loci were considered novel. We substantially improved the strength and precision of the associations at four previously detected loci near FADS2, ELOVL7, ELOVL6 and FASN genes, facilitating follow‐up candidate gene studies. Moreover, we also identified loci near ABCD2, ACSBG1, ELOVL5, HPGDS, DAGT2, ACAD10 and ACSL1 genes with function relevant to metabolism of fatty acids. In this study, valuable genetic variants and candidate genes associated with fatty acid composition traits were identified in Erhualian and Bamaxiang pigs. Some identified loci could be used to improve pork nutrition in pig breeding practice. Using the SNP array with higher marker density and less ascertainment bias improved QTL detection power and precision in Chinese indigenous pigs.     

小尾寒羊前体脂肪细胞分化过程相关基因表达规律的研究

为了探究小尾寒羊脂肪细胞分化过程中相关基因的变化规律,试验采集2月龄小尾寒羊腹股沟白色脂肪组织,通过酶消化法体外分离小尾寒羊前体脂肪细胞。培养前体脂肪细胞布满细胞板后,分别用诱导Ⅰ液、诱导Ⅱ液对细胞进行诱导分化,使其成为成熟的脂肪细胞。利用油红O染色法验证成熟脂肪细胞并检测脂滴含量。分别在增殖期细胞增殖70%、90%及分化期诱导Ⅰ液处理48 h、诱导Ⅱ液处理48 h、完全培养液处理48 h时（2、4、6、8、10 d）提取细胞总RNA,反转录成cDNA。采用实时荧光定量PCR检测PPARγ、C/EBPα、LPL、SREBP1、KLF5、KLF6、FABP4、STAT5、ACSS2、IGF1、ADD1、FOXO1、ACACA、DGAT1、CPT1A基因的表达规律。结果表明,试验成功分离并诱导前体脂肪细胞变为成熟的脂肪细胞,细胞内部具有明显脂滴;实时荧光定量PCR结果表明,上述基因在细胞分化阶段具有明显波动,峰值出现的时间均不相同;C/EBPα、FOXO1基因表达峰值出现在第6天,可能在细胞分化早期发挥作用;PPARγ、LPL、SREBP1、KLF5、KLF6、FABP4、STAT5、ADD1、ACSS2基因表达峰值出现在第8天,但表达倍数与趋势均不相同;ACACA基因表达量出现上下波动;IGF1、DGAT1基因表达峰值出现在第10天;CPT1A基因表达量则一直下降;FABP4基因表达倍数显著高于其他基因。本研究全面检测了小尾寒羊前体脂肪细胞在分化过程中关键基因的表达规律,可为探究小尾寒羊脂肪分化过程分子机制、挖掘参与脂肪分化新的关键基因、提高小尾寒羊肌间脂肪含量等研究提供一定的理论参考。     

赖氨酸对奶牛乳腺上皮细胞内乳脂肪合成相关基因和蛋白表达的影响

本试验旨在研究赖氨酸(Lys)对奶牛乳腺上皮细胞(BMECs)内乳脂肪合成相关基因和蛋白表达的影响,探讨Lys影响乳脂肪合成的机理。将第3代BMECs随机分为6组,每组6个重复,每个重复1个培养孔。各组培养基中Lys的浓度分别为0.5(基础培养基,对照)、1.0、2.0、4.0、8.0和16.0mmol/L,37℃、5%CO2培养48h后测定BMECs甘油三酯(TAG)含量、乳脂肪合成相关基因和蛋白的表达量。结果表明:BMECs内TAG含量(P=0.013)以及脂肪酸结合蛋白3(FABP3,P=0.001)、脂蛋白脂酶(LPL,P=0.096)、脂肪酸合成酶(FASN,P=0.003)、乙酰甘油磷酸脂酰转移酶6(AGPAT6,P=0.038)和甘油-3-磷酸酰基转移酶(GPAM,P=0.022)基因表达量对Lys呈显著或趋于显著的浓度依赖效应。FABP3基因表达量以2.0、4.0、8.0、16.0mmol/L组和LPL基因表达量以1.0、2.0、4.0、8.0、16.0 mmol/L组显著高于0.5mmol/L组(P0.05);FASN基因表达量以2.0mmol/L组最高,显著高于16.0mmol/L组(P0.05);硬脂酰辅酶A去饱和酶1(SCD1)基因表达量以2.0、4.0mmol/L组显著高于其他组(P0.05);磷脂酸磷酸酯酶1(LPIN1)、嗜乳脂蛋白亚家族1成员1(BTN1 A1)和黄嘌呤脱氢酶(XDH)基因表达量均以1.0、2.0、4.0、8.0mmol/L组显著高于0.5mmol/L组(P0.05);过氧化物酶体增殖物激活受体γ(PPARγ)基因及蛋白表达量均以2.0、4.0mmol/L组显著高于0.5和8.0、16.0mmol/L组(P0.05);固醇调节元件结合蛋白1(SREBP1)基因表达量以1.0、2.0、4.0mmol/L组显著高于其他组(P0.05),蛋白表达量以1.0 mmol/L组显著高于其他组(P0.05)。但高浓度Lys抑制AGPAT6和GPAM的基因表达,AGPAT6基因表达量以2.0、4.0、8.0、16.0mmol/L组显著低于0.5、1.0mmol/L组(P0.05),GPAM基因表达量以16.0mmol/L组显著低于0.5、1.0、2.0、4.0mmol/L组(P0.05)。可见,Lys对BMECs的乳脂肪合成具有显著的促进效果,但高浓度的Lys抑制了乳脂肪合成相关基因的表达。本试验条件下,培养基中Lys适宜浓度为2.0~4.0mmol/L。     

DGAT1, GH, GHR, PRL and PRLR polymorphism in water buffalo (Bubalus bubalis)

The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1‐exon8 K232A, GH‐intron3 MspI, GH‐exon5 AluI, GHR‐exon8 F279Y, PRL‐exon3 RsaI and PRLR‐exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili‐ravi, Murrah‐Nili‐Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR–Single‐strand conformation polymorphism and sequencing. Twenty‐eight inter‐specific single‐nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra‐specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1‐exon8, MspI⁺ restriction site(c nucleotide) and AluI⁺ site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI^? restriction site at PRL‐exon3/exon4 and N allele genotype of S18N mutation at PRLR‐exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra‐specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL‐exon3/exon4 and 43rd bp (c/t) in PRLR‐exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances.     

Isolation, sequence characterization, and tissue transcription profiles of two novel buffalo genes: INSIG1 and INSIG2

Insulin-induced genes (INSIGs) are recently discovered genes that are involved in the metabolism of cholesterol and lipogenesis in animal tissues. In this study, two INSIG genes (INSIG1 and INSIG2) were isolated and characterized in 11 buffalo. The full-length coding sequence (CDS) of the buffalo INSIG1 consists of 831 bp which encodes a 276 amino acid protein with molecular mass 29.55 kD. And the INSIG2 CDS is 678 bp in length which encodes a 225 amino acid protein with molecular mass 24.87 kD. No polymorphisms were found in the CDSs of the buffalo INSIGs, but seven and two nucleotide differences were found in the CDSs between buffalo and other bovine species. Phylogenetic analyses based on the INSIG amino acid sequences showed that buffalo was grouped with other members in the Bovidae family. Four types of putative modification sites were detected in buffalo INSIG proteins. And two predicted microRNA target sites were found respectively in the CDSs of buffalo INSIG1 and INSIG2. The tissue expression analyses by quantitative PCR (qPCR) revealed that the buffalo INSIG1 was expressed in ten tissues tested. Among these tissues, the liver and mammary gland showed high expression levels. And the INSIG2 was only expressed in the brain, mammary glands, pituitary, abomasum, heart, and liver. Among these tissues, the mammary gland, brain, and pituitary demonstrated a high expression levels. These data provide the primary foundation for further insights into the buffalo INSIG genes.     

Interactions of bovine oocytes with follicular elements with respect to lipid metabolism

Among many factors, lipid metabolism within the follicular environment emerges as an important indicator of oocyte quality. In the literature a crucial significance is described concerning follicular fluid (FF) composition as well as messenger RNA (mRNA) expression in follicular cells. The aim of this study was to describe the relationship between oocyte, FF and follicular cells with regard to lipid metabolism. The set of data originating from individual follicles comprised: lipid droplets (LD) number in oocytes (BODIPY staining), mRNA expression of seven genes in cumulus and granulosa cells (SCD, FADS2, ELOVL2, ELOVL5, GLUT1, GLUT3, GLUT8; real time polymerase chain reaction) and fatty acid (FA) composition in FF (gas chromatography). Obtained results demonstrate significant correlation between oocyte lipid droplets number and FA composition in FF. However, gene expression studies show significant correlation between LD number and GLUT1 gene only. Moreover, the present experiment revealed correlations between FA content in FF and expression of several genes (SCD, FADS2, ELOVL5, GLUT8) in granulosa cells, whereas only the SCD gene in cumulus cells. We suggest that the results of our experiment indicate the importance of glucose : lipid metabolism balance, which contributes to better understanding of energy metabolism conversion between oocytes and the maternal environment.     

Expression of Mitochondria‐Associated Genes (PPARGC1A,NRF‐1, BCL‐2 and BAX) in Follicular Development and Atresia of Goat Ovaries

Most follicles undergo atresia during the developmental process. Follicular atresia is predominantly regulated by apoptosis of granulosa cells, but the mechanism underlying apoptosis via the mitochondria‐dependent apoptotic pathway is unclear. We aimed to investigate whether the mitochondria‐associated genes peroxisome proliferator‐activated receptor‐gamma, coactivator1‐alpha (PPARGC1A), nuclear respiratory factor‐1 (NRF‐1), B‐cell CLL/lymphoma 2 (BCL‐2) and BCL2‐associated X protein (BAX) played a role in follicular atresia through this pathway. The four mitochondria‐associated proteins (PGC‐1α, which are encoded by the PPARGC1A gene, NRF‐1, BCL‐2 and BAX) mainly expressed in granulosa cells. The mRNA and protein levels of PPARGC1A/PGC‐1α and NRF‐1 in granulosa cells increased with the follicular development. These results showed that these genes may play a role in the regulation of the follicular development. In addition, compared with healthy follicles, the granulosa cell in atretic follicles had a reduced expression of NRF‐1, increased BAX expression and increased ratio of BAX to BCL‐2 expression. These results suggested that changes of the mitochondria‐associated gene expression patterns in granulosa cells may lead to follicular atresia during goat follicle development.     

Effect of DNA polymorphisms related to fatty acid composition in adipose tissue of Holstein cattle

Fatty acid composition of adipose tissue has been recognized as an important carcass trait because of its relationship with eating quality such as favorable beef flavor and tenderness. Therefore, we investigated the effects of genetic polymorphisms of liver X receptor, alpha (LXR), stearoyl‐CoA desaturase (SCD), Fatty acid synthase (FASN), and Fatty acid binding protein 4 (FABP4) on fatty acid composition in intramuscular fat tissue of Holstein steers. The major allele frequencies were 0.705 in SCD, 0.518 in FABP4, 0.888 in FASN, and 0.984 in LXR. Genotyping of SCD showed significant effect on C14:0, C14:1, C18:0 and saturated fatty acid (P < 0.05). In addition, the result suggested that SCD genotype possibly had effect on composition of C18:1 and monounsaturated fatty acid. Genotype of FABP4 had significant effect on composition of C16:0. Effect of LXR genotypes could not be analyze because of extremely biased genotype frequencies. Our results suggest that genotypes of SCD and FABP4 may in part affect meat quality in Holstein.     

Expression of fat deposition and fat removal genes is associated with intramuscular fat content in longissimus dorsi muscle of Korean cattle steers

Intramuscular fat (IMF) in cattle is an important component of traits that influence meat quality. We measured carcass characteristics and gene expression in Korean steers to clarify the molecular mechanism(s) underlying IMF deposition in LM tissue by determining the correlation between IMF content and gene expression abundance and by developing models to predict IMF content using gene expression abundance. The deposition of IMF is determined by a balance between fat deposition and fat removal in the LM. We measured mRNA abundance of lipid metabolic genes including lipogenesis [acetyl CoA carboxylase (ACC), fatty acid synthase (FASN)], fatty lipid uptake [lipoprotein lipase (LPL), fatty acid translocase (CD36), fatty acid transport protein 1 (FATP1)], fatty acid esterification [glycerol-3-phosphate acyltransferase 1 (GPAT1), acylglycerol phosphate acyltransferase 1 (AGPAT1), diacylglycerol acyltransferase 1 (DGAT1), DGAT2], lipolysis [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), monoglyceride lipase (MGL)], and fatty acid oxidation [carnitine palmitoyl transferase 1B, very long-chain acyl-CoA dehydrogenase (VLCAD), medium-chain acyl-CoA dehydrogenase (MCAD)] in the LM. The mRNA abundance of the GPAT1 gene showed the greatest correlation (r = 0.74; P < 0.001) with IMF content among 9 fat deposition genes. The gene expression abundance of other fat deposition genes including ACC, FASN, LPL, CD36, FATP1, AGPAT1, DGAT1, and DGAT2 also exhibited significant positive correlations (P < 0.05) with IMF content in the LM. Conversely, ATGL mRNA abundance showed the greatest negative correlation (r = -0.68; P < 0.001) with IMF content in the LM among 6 fat removal genes. The expression of other fat removal genes including MGL, VLCAD, and MCAD showed significant negative correlations (P < 0.05) with IMF content. Our findings show that the combined effects of increases in lipogenesis, fatty acid uptake, fatty acid esterification, and of decreases in lipolysis and fatty acid oxidation contribute to increasing IMF deposition in Korean steers. The multiple regression analysis revealed that the mRNA abundance of the GPAT1 gene in the LM was the first major variable predicting IMF content (54%) among 15 lipid metabolic genes. The second was mRNA abundance of ATGL (11%). In conclusion, these results suggest that GPAT1 and ATGL genes could be used as genetic markers to predict IMF deposition in the LM.     

Microarray analysis of Longissimus thoracis muscle gene expressions in vitamin A‐restricted Japanese Black steers in middle fattening stage

Vitamin A (VA) restriction in beef cattle improves meat marbling; however, the underlying molecular mechanisms remain incompletely understood. We performed microarray analysis to clarify the effect of VA restriction on Longissimus thoracis gene expressions in Japanese Black steers. Six Japanese Black steers 13–14 months of age were divided into two groups: S group (n = 3), which received VA supplementation, and R group (n = 3), in which dietary VA intake was restricted. Steers were fattened for 7 months, following which tissue samples were obtained. Extracted RNA samples were analyzed by Affymetrix Genechip Bovine Genome Array. Lists of genes highly expressed in the R and S groups were obtained. The lists were functionally interpreted using functional annotation software, DAVID. In the R and S groups, 48 and 40 genes were significantly highly expressed, respectively. The gene list of the R group included CD36, LPL, GPAM, DGAT2, and SCD and additional genes annotated ‘PPAR signaling pathway,’ ‘lipid biosynthesis’ and ‘mitochondrion,’ whereas that of the S group included COL1A2, FN1 and DCN and additional genes annotated ‘extracellular matrix.’ Changes in the expression of these genes are possibly involved in marbling improvement in beef cattle by VA restriction.     

山羊DGAT1基因的克隆及对前体脂肪细胞脂质沉积的调控作用研究

旨在克隆山羊DGAT1基因序列,明确DGAT1基因在山羊不同组织中的表达模式,并进一步揭示过表达DGAT1基因对山羊肌内前体脂肪细胞脂质代谢的影响。本试验以10月龄健康简州大耳公羊（n=7）为试验动物。采用RT-PCR法克隆山羊DGAT1基因序列,并对序列进行生物信息学分析;利用实时荧光定量PCR （real-time quantitative PCR,RT-qPCR）检测DGAT1在山羊不同组织中的相对表达水平;采用双酶切法构建pcDNA3.1-DGAT1真核表达载体并转染至山羊肌内前体脂肪细胞;使用RT-qPCR检测DGAT1过表达效率及脂质代谢相关基因的表达情况;通过油红O染色法观察过表达DGAT1对脂滴形成的影响,利用GPO-Trinder酶学反应检测甘油三酯含量。结果显示,获得山羊DGAT1基因序列全长1 651 bp （GenBank登录号：MT221183）,包含5'UTR 125 bp,CDS 1 470 bp,3'UTR 56 bp,编码489个氨基酸残基;山羊DGAT1基因在小肠中的表达量最高,在脾中表达量最低;RT-qPCR检测结果显示,DGAT1在细胞中过表达极显著（P<0.01）,GPAM基因的相对表达水平显著上调（P<0.05）,ADRP和ACOX1基因极显著上调（P<0.01）,而AGPAT6基因相对表达水平显著下调（P<0.05）,MLYCD和HSL基因极显著下调（P<0.01）;油红O染色结果显示,过表达DGAT1后脂滴聚积相较于对照组极显著增多（P<0.01）,甘油三酯测定结果显示,过表达DGAT1基因可极显著增加山羊肌内脂肪细胞甘油三酯含量（P<0.01）。本研究成功获得山羊DGAT1基因CDS区序列并构建了pcDNA3.1-DGAT1真核表达载体,过表达DGAT1可显著促进山羊肌内前体脂肪细胞脂质沉积,并显著影响脂质代谢相关基因的表达,这些结果为进一步阐明DGAT1对调控山羊肌内脂肪代谢的作用机制提供了重要数据。     

亮氨酸对奶牛乳腺上皮细胞内乳脂合成相关基因和蛋白表达的影响

本试验旨在研究亮氨酸(Leu)对泌乳奶牛乳腺上皮细胞(BMECs)内乳脂合成相关基因和蛋白表达的影响,以探讨Leu对乳脂合成的影响机理。将第3代BMECs随机分为6个处理,每个处理6个重复。6个处理培养液中Leu浓度分别为0.45、0.90、1.80、2.70、3.60和7.20 mmol/L,37℃、5%CO2培养48 h后测定BMECs内甘油三酯(TG)的含量及乳脂合成相关基因和过氧化物酶体增殖物激活受体γ(PPARγ)与固醇调节元件结合蛋白(SREBP1)蛋白的相对表达量。结果显示:Leu浓度对BMECs内TG含量无显著影响(P0.05)。适宜浓度的Leu显著促进脂肪酸合成酶(FASN)和乙酰辅酶A羧化酶A(ACACA)基因的表达(P0.05),FASN基因的相对表达量以1.80~2.70 mmol/L Leu处理、ACACA基因的相对表达量以1.80~7.20 mmol/L Leu处理较高。Leu浓度显著影响BMECs内SREBP1基因及蛋白表达(P0.05),以1.80 mmol/L Leu的促进效果最好。虽然Leu显著抑制BMECs内脂肪酸结合蛋白3(FABP3)、脂蛋白脂酶(LPL)、乙酰甘油磷酸脂酰转移酶6(AGPAT6)、线粒体甘油-3-磷酸酰基转移酶(GPAM)和嗜乳脂蛋白亚家族1成员1(BTN1A1)基因的表达(P0.05),但只有高浓度(3.60~7.20 mmol/L)的Leu抑制作用较大。综合来看,Leu浓度影响BMECs乳脂合成相关基因及PPARγ和SREBP1蛋白的表达。Leu浓度为1.80~2.70 mmol/L时,对脂肪酸从头合成相关基因及调控因子SREBP1蛋白表达的促进效果较好,对TG合成及脂滴形成相关基因表达的抑制作用较小。