Peroxisome proliferator-activated receptor delta facilitates lipid secretion and catabolism of fatty acids in dairy goat mammary epithelial cells

In rodents, peroxisome proliferator-activated receptor delta (PPARD) is associated primarily with catabolism of fatty acids. However, the role of PPARD in regulating lipid metabolism in ruminant mammary gland remains unknown. In the present study, we assessed the mRNA abundance of PPARD at 3 stages of lactation in goat mammary tissue. Results revealed that PPARD had lower expression at peak lactation than in the nonlactating period. Luciferase assays revealed that GW0742 (GW), a specific PPARD ligand, enhanced the activity of the PPARD response element in goat mammary epithelial cells. Activation of PPARD by GW selectively upregulated the expression of genes related to fatty acid activation (ACSL1), lipid droplet formation (PLIN2), and transport (FABP4), and had no effect on genes involved in de novo fatty acid synthesis (ACACA and FASN), desaturation (SCD), hydrolysis and oxidation (PNPLA2 and CPT1A), transport and uptake (FABP3 and CD36), or triacylglycerol synthesis (DGAT1 and AGPAT6) in goat mammary epithelial cells. In contrast, knockdown of PPARD using small interfering RNA dramatically decreased the expression of genes related to fatty acid activation (ACSL1) and lipid formation (PLIN2) and increased the expression of genes related to fatty acid transport (FABP3) and triacylglycerol synthesis (AGPAT6 and DGAT1). The expression of genes related to fatty acid synthesis (FASN), hydrolysis (PNPLA2), and fatty acid oxidation (CPT1A) was downregulated significantly only after knockdown of PPARD in cells incubated with GW. We observed no significant change in fatty acid profiles. However, the total cellular triacylglycerol increased after knockdown of PPARD in goat mammary epithelial cells plus GW. Collectively, these results highlight an important role for PPARD in the homeostasis of ruminant mammary cells by facilitating fatty acid activation and lipid droplet formation and secretion.     

Combined effects of trans-10,cis-12 conjugated linoleic acid, propionate, and acetate on milk fat yield and composition in dairy cows

Diets inducing milk fat depression (MFD) are known to alter ruminal lipid metabolism, leading to the formation of specific isomers [such as trans-10,cis-12 conjugated linoleic acid (CLA)] that inhibit milk fat synthesis in lactating dairy cows. However, ruminal outflow of these isomers does not fully account for the decreases in milk fat synthesis observed during diet-induced MFD. The high-concentrate diets inducing MFD also induce a greater production of propionate, suggesting a possible inhibition of milk fat by propionate associated with trans-10,cis-12-CLA during MFD. The present experiment aimed to study the combined effects of propionate and trans-10,cis-12-CLA (both inhibitors of milk fat synthesis) on milk fat secretion and the effects of the combination of 2 nutrients with opposite effects (acetate and propionate). Six Holstein cows were used in a 6 × 6 Latin square design with 21-d periods (14 d of nutrient infusion). The treatments were control; ruminal infusion of 1,500 g/d of acetate (A); ruminal infusion of 800 g/d of propionate (P); duodenal infusion of 1.60 g/d of trans-10,cis-12-CLA (CLA); ruminal infusion of 750 g/d of acetate + 400 g/d of propionate (A+P); and duodenal infusion of 1.60 g/d of trans-10,cis-12-CLA + ruminal infusion of 800 g/d of propionate (CLA+P). The amounts of nutrients infused were chosen to induce a similar variation in milk fat content. Treatments A and P decreased dry matter intake. Compared with the control, P and CLA treatments decreased milk fat content and yield by 9% and 15% on average. Treatment A increased milk fat content by 6.5% but did not modify milk fat yield (because of a decrease in milk yield). The effects of A and P, and CLA and P on milk fat and fatty acid percentages and yield were additive (A+P and CLA+P treatments). With a same dose of trans-10,cis-12-CLA, the additional supply of propionate induced a decrease in milk fat 40% higher than that induced by trans-10,cis-12-CLA alone. The milk fatty acid profile obtained with CLA+P was similar to those observed with high-concentrate diets inducing MFD. In conclusion, under our experimental conditions, the effects of the 3 nutrients were additive on mammary lipogenesis, regardless of their separate effects. We also show that propionate could contribute to the milk fat reductions unaccounted for by trans-10,cis-12-CLA during MFD induced by high-concentrate diets.     

植物乳杆菌p-8转化亚油酸为共轭亚油酸的分析

研究植物乳杆菌（Lactobacillus plantarum）p-8的菌体、菌体破碎液和重组亚油酸异构酶系转化亚油酸（linoleic acid,LA）为共轭亚油酸（conjugated linoleic acid,CLA）的能力和机制。结果表明：L. plantarum p-8在含有LA的MRS上清液和菌体破碎液体外催化LA时,都可以低效产生cis9,trans11-CLA（c9,t11-CLA）、trans10,cis12-CLA（t10,c12-CLA）和trans9,trans11-CLA（t9,t11-CLA）,但菌体中只有很少的t10,c12-CLA。实时聚合酶链式反应结果表明,亚油酸异构酶系的表达水平较低可能是CLA产量较低的原因。独立表达的重组亚油酸异构酶系成员、黄素腺嘌呤二核苷酸（flavin denine dinucleotide,FAD）和烟酰胺腺嘌呤二核苷酸都存在才可完成LA转化为c9,t11-CLA、t10,c12-CLA和t9,t11-CLA,转化途径与L. plantarum AUK1009一致。L. plantarum p-8的亚油酸水合酶经同源建模后有3 个结构域,底物结合位点与FAD位点位于3 个结构域连接处的疏水空腔中,M76和Y180是2 个必需基团。     

c9t11- 和t10c12- 共轭亚油酸抗癌和影响脂质代谢的异同

共轭亚油酸(CLA)是一组位置和构象异构体的总称,异构体c9t11-CLA 和t10c12-CLA 或二者的协同作用赋予了CLA 的许多生理功能,比如抗癌、降低体脂含量、预防糖尿病的发生、降低血脂抑制动脉粥样硬化等;异构体c9t11-CLA 和t10c12-CLA 在结构及来源上存在一定差别--由于双键位置的不同,t10c12-CLA 异构体比c9t11-CLA 异构体更容易氧化;而在生理功能上二者也有差异-- c9t11-CLA 异构体的主要作用是抗癌,而 t10c12-CLA 则是降低体脂、血脂等,大量单一异构体的体外实验研究表明二者在抗癌及脂肪代谢的调节上作用机制也不尽相同。     

n-3多不饱和脂肪酸对小鼠血液外泌体中miRNA的调节和抑制肥胖作用

目的：研究体内n-3多不饱和脂肪酸（n-3 polyunsaturated fatty acids,n-3 PUFAs）含量的增加对小鼠体质量和血液中外泌体miRNAs表达的影响,探讨n-3 PUFAs通过外泌体抑制肥胖的作用机制。方法：利用能自发生成n-3 PUFAs的fat-1转基因小鼠和同窝野生型小鼠（对照）,通过高脂饮食（high-fat diet,HFD）建立肥胖动物实验模型,测定小鼠体质量。提取小鼠血浆中的外泌体并鉴定;分离外泌体内的RNA,构建文库并进行miRNA高通量测序。根据测序结果通过生物信息学方法分析找到其调控的靶基因和相关联的通路,发现miRNA-靶基因互作关系。验证miRNA与肥胖的关联度以及在肥胖中所发挥的作用。结果：fat-1转基因小鼠体质量明显低于野生型;外泌体提取鉴定成功;miRNA高通量测序结果显示,不同小鼠组间进行对比时,差异表达显著（P＜0.05且差异倍数（fold change,FC）≠1）的miRNA有46 个;生物信息学分析发现6 个重要miRNA（mmu-miR-665-3p、mmu-miR-122-5p、mmu-miR-122-3p、mmu-miR-194-5p、mmu-miR-34c-5p、mmu-miR-223-3p）落在脂肪酸代谢通路以及内吞通路关键位置,功能与脂质代谢和肥胖相关,所对应的靶基因分别为Fads1、Elovl2、Elov6、Hadha、Scad1、Scad2、Hsd17b12、Acot2、Acot4和Arf6、H2-T-ps、Arrb1、Ist1、H2-T10、Wwp1、Snx4、IL2rb、Mvb12b、Rab11、fip3、Kif5a、Nedd4l。结论：n-3 PUFAs含量的增加能够有效降低小鼠的体质量,抑制肥胖。n-3 PUFAs能够调节血液中外泌体内miRNA的表达,其中具有显著差异性的miRNA与肥胖有关,其相关的靶基因集中在脂质代谢相关的分子通路中,提示可能是n-3 PUFAs降低体质量抑制肥胖机制之一。     

Acetate regulates milk fat synthesis through the mammalian target of rapamycin/eukaryotic initiation factor 4E signaling pathway in bovine mammary epithelial cells

Acetate is a short-chain fatty acid (SFA) that is the major substrate for de novo fatty acid synthesis. The mammalian target of rapamycin/eukaryotic initiation factor 4E (mTOR/eIF4E) signaling pathway is involved in fat synthesis. However, the effect and mechanism of acetate on fatty acid synthesis by the mTOR/eIF4E signaling pathway is unclear in bovine mammary epithelial cells (BMECs). The objectives of this study were to investigate the effect of acetate on cell viability, triacylglycerol (TG), and mRNA expression of the genes related to lipid synthesis. The mechanism of acetate regulation milk fat synthesis through the mTOR/eIF4E signaling pathway was assessed by blocking the mTOR signaling pathway and silencing eIF4E in BMECs. Third-passage BMECs were allocated to 6 treatments including 0, 4, 6, 8, 10, and 12 mM acetate to evaluate the effect of acetate on lipid synthesis; the optimum concentration in the first study was selected for the subsequent study. Subsequently, cells were randomly allocated to 4 treatments, 1 control group and 3 treated groups, consisting of acetate (6 mM), rapamycin (100 nM), and acetate + rapamycin to test the role of mTOR signaling pathway response to acetate in milk lipid synthesis. Finally, eIF4E was silenced by small interfering RNA (siRNA) to detect the role of eIF4E in milk lipid synthesis. Treatments included control, eIF4E siRNA, acetate (6 mM), and acetate+ eIF4E siRNA. Results showed that acetate increased TG accumulation and the relative expression of fatty acid synthase (FASN), acetyl-coenzyme A carboxylase α (ACACA), fatty acid-binding protein 3 (FABP3), sterol regulatory element binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma (PPARG), mTOR, eIF4E, P70 ribosomal protein S6 kinase-1 (S6K1), and 4E-binding protein-1 (4EBP1) in a dose-dependent manner. Rapamycin effectively inhibited the positive effect of acetate on the relative expression of mTOR, eIF4E, S6K1, 4EBP1, FASN, ACACA, FABP3, stearoyl-CoA desaturase (SCD1), SREBP1, and PPARG. The upregulation of acetate on the relative expressions of FASN, ACACA, SCD1, and SREBP1 was suppressed when eIF4E was knocked down. It suggested that acetate regulated milk fat synthesis through mTOR/eIF4E signaling pathway in BMECs.     

共轭亚油酸对小鼠肥胖的抑制作用

采用小鼠营养性肥胖模型法,以昆明小鼠为实验动物,设置基础饲料对照组、肥胖模型对照组、共轭亚油酸（conjugated linoleic acid,CLA）高、中、低剂量组,分别连续灌胃6 周,考察小鼠体质量、体内脂肪质量、血脂水平、肝脏脂肪酸合成酶（fatty acid synthase,FAS）含量及脏器的变化,研究CLA对小鼠肥胖的抑制作用。结果表明：CLA各剂量组小鼠的Lee’s指数、脂肪系数和血清甘油三酯（triglyceride,TG）、总胆固醇（totalcholesterol,TC）、低密度脂蛋白胆固醇（low density lipoprotein cholesterol,LDL-C）水平均显著或极显著低于肥胖模型对照组（P＜0.05或P＜0.01）,高密度脂蛋白胆固醇（high density lipoprotein cholesterol,HDL-C）水平均极显著高于肥胖模型对照组（P＜0.01）,各剂量的CLA对小鼠除肝脏以外的其他脏器无显著影响（P＞0.05）,高剂量（0.15 mL/10 g）CLA可使喂食营养饲料小鼠的各项肥胖指标均处于喂食基础饲料小鼠的水平,表明CLA能有效抑制小鼠肥胖,同时对小鼠生长无毒副作用;CLA可极显著降低小鼠肝脏FAS含量（P＜0.01）,降低脂肪酸的合成,从而抑制小鼠肥胖。     

Effect of high or low protein ration combined or not with rumen protected conjugated linoleic acid (CLA) on meat CLA content and quality traits of double-muscled Piemontese bulls

A trial was carried out on double-muscled Piemontese bulls to evaluate the effects of two rations differing in crude protein density (HP=14.5% DM and LP=10.8% DM) and top dressed or not with 80 g/d of rumen protected CLA (rpCLA) for a long period (336 d) on meat quality traits and CLA content. Forty-eight bulls were fed one of the four experimental diets based on corn silage and cereals and were slaughtered at an average age and body weight (BW) of 562 ± 18 d and 668 ± 56 kg, respectively. After slaughter the 5th rib cut was dissected into Longissimus thoracis (LT), other muscles (OM), inter-muscular fat (IF), cover fat (CF), and bones. Muscles and fatty tissues were analyzed for proximate composition and fatty acid (FA) profiles. Rib was composed by 81.1, 3.7, 1.6 and 13.6% of muscles, IF, FC and bone, respectively; LT and OM contained only 0.8 and 1.4% of lipid, respectively. The treatments did not influence these values, but rpCLA increased, compared to control, both c9,t11-CLA and t10,c12-CLA concentrations in all the tissues (P<0.01); t10,c12-CLA concentration was increased much more in muscles (+20 times) than in fatty tissues (from +0.2 to +0.9 times). This suggests that in the muscle this isomer is preferentially stored and/or less combusted with respect to other fatty acids. Low protein rations did not exert any influence on carcass and meat quality, as on growth performance, but reduced nitrogen excretion, their use for improving the environmental impact (process quality) of this meat production system is recommended.     

Fatty acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat mammary epithelial cells

In nonruminants, it is well established that elongation of very long-chain fatty acid-like fatty acid elongase 6 (ELOVL6) catalyzes the synthesis of C18:0 from C16:0 in lipogenic tissues like adipose and liver. However, the role of ELOVL6 in regulating lipid metabolism in ruminant mammary gland remains unknown. In the present study, ELOVL6 was overexpressed or knocked down via adenoviral transfection to assess its role in goat mammary epithelial cells. Results revealed that ELOVL6 overexpression had a weak effect on the expression of genes related to triacylglycerol (TAG) synthesis and desaturation. Overexpression of ELOVL6 increased the content of C18:0 at the expense of C16:0, and increased the elongation index of C16:0. Overexpression of ELOVL6 had no significant effect on the elongation index of C16:1n-7 and the desaturation indices of C16:0 and C18:0. Knockdown of ELOVL6 had a negative effect on mRNA expression of the esterification genes GPAM and diacylglycerolacyltransferase 2 (DGAT2) and TAG concentration; however, it increased the concentration of C16:0 and decreased C18:1n-7 and C18:1n-9 in goat mammary epithelial cells. Accordingly, downregulation of ELOVL6 significantly decreased the elongation indices of C16:0 and C16:1n-7. The lack of change in the desaturation indices of C16:0 and C18:0 upon knockdown of ELOVL6 was consistent with the minor change in SCD1 expression. In conclusion, these are the first results highlighting an important role of ELOVL6 in long-chain fatty elongation and TAG synthesis in ruminant mammary cells.     

Synthesis of the suspected trans-11,cis-13 conjugated linoleic acid isomer in ruminant mammary tissue by FADS3-catalyzed Δ13-desaturation of vaccenic acid

The octadecadienoic conjugated linoleic acid (CLA) isomer with trans-11 and cis-13 double bonds (trans-11,cis-13 CLA) has been described in ruminant milk. For now, this specific CLA is suspected to derive exclusively from ruminal biohydrogenation of dietary α-linolenic acid. However, in rodents, the fatty acid desaturase 3 (FADS3) gene was recently shown to code for an enzyme able to catalyze the unexpected Δ13-desaturation of vaccenic acid, producing a Δ11,13-CLA with all the structural characteristics of the trans-11,cis-13 isomer, although no commercial standard exists for complete conclusive identification. Because the FADS3 gene has already been reported in bovine animals, we hypothesized in the present study that an alternative direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue may therefore co-exist with α-linolenic acid biohydrogenation to explain the final ruminant milk trans-11,cis-13 CLA presence. Here, we first confirm that the FADS3 gene is present in ruminant mammal genomic sequence databases. Second, we demonstrate that the Δ11,13-CLA found in milk fat and the highly probable trans-11,cis-13 CLA isomer produced by rodent FADS3 possess exactly the same structural characteristics. Then, we show that bovine mammary MAC-T and BME-UV epithelial cells express both FADS3 and stearoyl-CoA desaturase 1 (SCD1) mRNA and are able to synthesize both the suspected trans-11,cis-13 CLA and cis-9,trans-11CLA (rumenic acid) isomers when incubated with vaccenic acid. Finally, the concomitant presence of the suspected trans-11,cis-13 CLA isomer with FADS3 mRNA was shown in goat mammary tissue, whereas both were conversely very low or even absent in goat liver. Therefore, this study provides several lines of evidence that, by analogy with rumenic acid, trans-11,cis-13 CLA may originate both from ruminal biohydrogenation and from direct FADS3-catalyzed Δ13-desaturation of vaccenic acid in mammary tissue.     

Expression of genes related to energy metabolism and the unfolded protein response in dairy cow mammary cells is affected differently during dietary supplementation with energy from protein and fat

Secretory capacity of bovine mammary glands is enabled by a high number of secretory cells and their ability to use a range of metabolites to produce milk components. We isolated RNA from milk fat to measure expression of genes involved in energy-yielding pathways and the unfolded protein response in mammary glands of lactating cows given supplemental energy from protein (PT) and fat (FT) tested in a 2 × 2 factorial arrangement. We hypothesized that PT and FT would affect expression of genes in the branched-chain AA catabolic pathway and tricarboxylic acid (TCA) cycle based on the different energy types (aminogenic versus lipogenic) used to synthesize milk components. We also hypothesized that the response of genes related to endoplasmic reticulum (ER) homeostasis via the unfolded protein response would reflect the increase in milk production stimulated by PT and FT. Fifty-six multiparous Holstein-Friesian dairy cows were fed a basal total mixed ration (34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter basis) for a 28-d control period. Experimental rations were then fed for 28 d, consisting of (1) low protein, low fat (LP/LF); (2) high protein, low fat (HP/LF); (3) low protein, high fat (LP/HF); or (4) high protein and high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on dry matter basis) and 0.68 kg/d hydrogenated palm fatty acids on a dry matter basis. RNA from milk fat samples collected on d 27 of each period underwent real-time quantitative PCR. Energy from protein increased expression of BCAT1 (branched-chain amino acid transferase 1) mRNA, but only at the LF level, and tended to decrease expression of mRNA encoding the main subunit of the branched-chain keto-acid dehydrogenase complex. mRNA expression of malic enzyme, a proposed channeling route for AA though the TCA cycle, was decreased by PT, but only at the LF level. Expression of genes associated with de novo fatty acid synthesis was not affected by PT or FT. Energy from fat had no independent effect on genes related to ER homeostasis. At the LF level, PT activated XBP1 (X-box binding protein 1) mRNA. At the HF level, PT increased mRNA expression of the gene encoding GADD34 (growth arrest and DNA damage-inducible 34). These findings support our hypothesis that mammary cells use aminogenic and lipogenic precursors differently for milk component production when dietary intervention alters AA and fatty acid supply. They also suggest that mammary cells respond to increased AA supply through mechanisms of ER homeostasis, dependent on the presence of FT.     

瘤胃细菌生物合成共轭亚油酸的累积特性

为了解瘤胃细菌生物合成共轭亚油酸(CLA)的特性,通过毛细管电泳(CE)分析,发现瘤胃细菌生物合成CLA的主要异构体有c9,t11-CLA、t10,c12-CLA和t9,t11-CLA 3种。在厌氧和有氧条件下,瘤胃细菌均能合成CLA,且氧气有利于CLA的累积,随反应时间的延长,CLA的量呈现先增加后减少的变化趋势。结果表明瘤胃细菌参与了反刍动物体内CLA异构体的生物合成与代谢,瘤胃细菌生物合成CLA异构体的特异性还有待更深入的研究。