氨基酸对奶牛乳腺蛋白质合成调控及其分子机制的研究进展

乳蛋白中含有大量人体所需的必需氨基酸,其组成平衡、含量丰富,是一种具有极高营养价值的蛋白质,而乳中 90%以上的蛋白质是乳腺利用氨基酸从头合成的,因此氨基酸对奶牛乳蛋白合成发挥着重要的作用。此外,氨基酸不仅是合成乳蛋白不可或缺的前体物质,而且还是重要的信号调控因子,通过哺乳动物雷帕霉素靶蛋白（mTOR） 信号通路调控乳蛋白的合成。基于此,作者就影响奶牛乳腺氨基酸供应、摄取、利用的因素及氨基酸的信号传导作用的研究进展进行综述,以期为提高乳蛋白的合成提供一定的理论基础。     

乳腺内氨基酸调控乳蛋白合成的分子作用机制

作为维持哺乳动物生命活动重要的"生物工厂",乳腺利用从流经血液中摄取的氨基酸等营养物质为底物合成乳蛋白。研究证实,氨基酸还可作为一种信号因子,通过乳腺内多种信号级联传导通路,调控乳蛋白基因的转录及翻译过程,从而影响乳腺中乳蛋白的合成。酪氨酸蛋白激酶-信号转导子和转录激活子(JAK-STAT)信号通路和哺乳动物雷帕霉素靶蛋白(m TOR)信号通路是乳蛋白基因转录和翻译过程中的主要调控路径。本文综述了乳腺JAKSTAT和m TOR信号通路的分子机制及氨基酸通过这些通路调控乳蛋白合成的研究进展,旨在进一步阐明氨基酸调控乳蛋白合成的作用机理。     

Amino acid utilisation by dairy cows. II. Concept of amino acid requirementsUtilisation des acides aminés par les vaches laitières. II. Concept des besoins en acides aminésAminosäureverwertung bei milchkühen. II. Zum konzept des aminosäurenbedarfs

In dairy cows amino acids may be required for four processes: maintenance; as precursors for the synthesis of glucose (gluconeogenesis); for protein deposition in muscle or associated with foetal growth; and for the synthesis of milk protein.Estimates of the protein requirements for maintenance are often conflicting and seem far from accurate. Estimates of the ratio in which essential amino acids are required for maintenance are not yet available.Requirements of amino acids for gluconeogenesis are difficult to estimate. It seems rather unlikely that essential amino acids will be used in significant quantities for gluconeogenesis, even at high milk yields.Protein requirements for pregnancy and muscle growth in dairy cows are relatively low. The ratio in which essential amino acids are supplied in the blood seems adequate for both processes.Amino acid requirements for milk protein synthesis are somewhat higher than the net protein output. This is particularly true for essential amino acids of which, in the mammary gland, a surplus of some 50% needs to be extracted from the blood. The ratio in which essential amino acids are supplied to the mammary gland seems reasonably adequate for milk protein synthesis.     

乳腺对氨基酸的摄取及其调控机制研究进展

乳蛋白是乳中重要的营养成分之一,超过90％的乳蛋白是乳腺利用从血液中摄取的氨基酸从头合成,因此在保证氨基酸充足供给的前提下,乳腺对氨基酸摄取率的高低是影响乳蛋白产量的关键因素.血液中的氨基酸不能自由扩散进出乳腺,需要由乳腺上皮细胞膜上特异的氨基酸转运载体(AAT)协助完成.而乳腺AAT活性受到营养物质和激素水平的调节,...     

小肽对反刍动物乳蛋白合成影响的研究进展

小肽能被乳腺组织有效利用,用于弥补乳蛋白合成过程中游离氨基酸供应的不足,因此有必要研究小肽在反刍动物体内的代谢及其在乳蛋白合成过程中的作用,加深人们对反刍动物利用氨基酸的了解。作者从肽的概念、乳蛋白对小肽的利用、乳腺对小肽的吸收机制、影响乳腺对小肽利用的因素及其研究展望等方面对反刍动物乳腺的小肽代谢进行了综述。     

氨基酸影响奶牛乳腺内乳脂合成的机理

乳蛋白前体物主要有游离氨基酸和小肽等。氨基酸不仅能影响乳腺内乳蛋白的合成,而且对乳脂的合成起一定的调控作用。本文主要阐述了氨基酸在乳脂合成过程中的调节作用,并从乳腺对乳脂前体物的摄取规律、乳脂合成相关基因表达、哺乳动物雷帕霉素靶蛋白和腺苷酸活化蛋白激酶信号通路的角度综述了氨基酸对乳脂合成的可能机理,为进一步研究乳脂合成机理及改进牛奶营养品质提供理论依据。     

Recent progress of porcine milk components and mammary gland function

As the only nutritional source for newborn piglets,porcine colostrum and milk contain critical nutritional and immunological components including carbohydrates,lipids,and proteins(immunoglobulins).However,porcine milk composition is more complex than these three components.Recently,scientists identified additional and novel components of sow colostrum and milk,including exosomes,oligosaccharides,and bacteria,which possibly act as biological signals and modulate the intestinal environment and immune status in piglets and later in life.Evaluation of these nutritional and non-nutritional components in porcine milk will help better understand the nutritional and biological function of porcine colostrum and milk.Furthermore,some important functions of the porcine mammary gland have been reported in recent published literature.These preliminary studies hypothesized how glucose,amino acids,and fatty acids are transported from maternal blood to the porcine mammary gland for milk synthesis.Therefore,we summarized recent reports on sow milk composition and porcine mammary gland function in this review,with particular emphasis on macronutrient transfer and synthesis mechanisms,which might offer a possible approach for regulation of milk synthesis in the future.     

Effects of phenylalanine and threonine oligopeptides on milk protein synthesis in cultured bovine mammary epithelial cells

This study was conducted to investigate the effects of phenylalanine (Phe) and threonine (Thr) oligopeptides on α_s1 casein gene expression and milk protein synthesis in bovine mammary epithelial cells. Primary mammary epithelial cells were obtained from Holstein dairy cows and incubated in Dulbecco's modified Eagle's medium‐F12 medium (DMEM/F12) containing lactogenic hormones (prolactin and glucocorticoids). Free Phe (117 μg/ml) was substituted partly with peptide‐bound Phe (phenylalanylphenylalanine, phenylalanyl threonine, threonyl‐phenylalanyl‐phenylalanine) in the experimental media. After incubation with experimental medium, cells were collected for gene expression analysis and medium was collected for milk protein or amino acid determination. The results showed that peptide‐bound Phe at 10% (11.7 μg/ml) significantly enhanced α_s1 casein gene expression and milk protein synthesis as compared with equivalent amount of free Phe. When 10% Phe was replaced by phenylalanylphenylalanine, the disappearance of most essential amino acids increased significantly, and gene expression of peptide transporter 2 and some amino acid transporters was significantly enhanced. These results indicate that the Phe and Thr oligopeptides are important for milk protein synthesis, and peptide‐bound amino acids could be utilised more efficiently in milk protein synthesis than the equivalent amount of free amino acids.     

奶牛乳蛋白源前体物生成与利用的相关影响因素

乳蛋白的高低是衡量乳品质的质量的一个重要指标。作者阐述了乳腺、肝脏、小肠和瘤胃中氨基酸合成、转运和利用方面对乳蛋白合成的影响,旨在为提高乳蛋白含量和产量提供理论参考。     

牛乳腺上皮细胞SNAT2对氨基酸调节乳合成的影响

试验旨在研究氨基酸转运体钠离子依赖的中性氨基酸转运蛋白（the sodium-dependent neutral amino acid transporter 2,SNAT2）在牛乳腺上皮细胞（bovine mammary epithelial cell,BMEC）中对氨基酸调节乳合成的影响。利用组织块法成功培养原代BMEC,添加不同氨基酸（蛋氨酸（Met）、赖氨酸（Lys）和亮氨酸（Leu）刺激BMEC后,通过实时荧光定量PCR、Western blotting技术和甘油三酯试剂盒检测SNAT2、酪蛋白（β-casein）基因的表达量和BMEC培养液上清甘油三酯的分泌量;将N-flag-SNAT2真核表达载体及SNAT2 siRNA分别转入细胞中进行SNAT2基因的过表达和敲低试验;通过Western blotting和甘油三酯试剂盒分别检测SNAT2、哺乳动物雷帕霉素靶蛋白（the mammalian target of rapamycin,mTOR）、β-casein蛋白表达量和BMEC培养液上清的甘油三酯含量。结果显示,3种氨基酸（Met、Lys、Leu）均能显著促进BMEC分泌乳蛋白和乳脂,并激活mTOR信号途径,其中Met、Lys还能够显著上调SNAT2基因表达;SNAT2能够正向调节BMEC乳蛋白和乳脂肪的合成,并激活mTOR信号通路,说明氨基酸激活mTOR信号通路是通过SNAT2基因介导完成的,进而调节了BMEC乳蛋白和乳脂肪合成。     

Cellular uptake of valine by lactating porcine mammary tissue

The cellular uptake of branched-chain amino acids in mammary tissue is important for understanding their role in milk synthesis in the sow. This study characterized the kinetic properties and substrate specificity of the valine uptake system in the porcine mammary gland. Mammary tissue was collected from lactating sows at slaughter and tissue explants were incubated in media containing isosmotic salt and amino acids of interest, plus [3H]valine tracer. Valine uptake was time-dependent and was dependent on the presence of sodium, as indicated by a reduction in uptake when sodium in the medium was replaced by choline. The valine transport system in porcine mammary tissue had a Km of 0.64 mM, a Vmax of 1.84 mmol-kg cell water(-1) 30 min(-l), and a Kd (diffusion constant) of 1.16 L x kg cell water(-1) x 30 min(-1). Valine uptake was inhibited by leucine and alpha-aminoisobutyric acid and by high concentrations of L-alanine, L-lysine, cycloleucine, L-glutamine, and L-methionine, but not by 2-(methyl-amino)-isobutyric acid. This transport system is the primary system responsible for uptake of valine, and probably other branched-chain amino acids, in lactating sow mammary tissue. Physiological concentrations of valine in the blood are below the Km of the specific valine transport system and well below the diffusion uptake capabilities. The kinetic parameters of this valine transport system should not be limiting to valine uptake for milk protein synthesis. However, competition of valine uptake with branched-chain amino acids, as well as with other amino acids, may affect valine uptake in lactating tissue.     

Dynamic ideal protein and limiting amino acids for lactating sows: the impact of amino acid mobilization

The limiting amino acids for lactating sows were determined using 28 primiparous sows that were intentionally underfed both energy and protein during a 21-d lactation. Groups of four sows were allotted to litter-size treatments of 6, 7, 8, 9, 10, 11, or 12 by cross-fostering as needed within 48 h postpartum. Sows were killed on d 21 of lactation. The carcass, liver, gastrointestinal tract, reproductive tract, mammary gland, and other viscera were separated, weighed, ground, and analyzed for dry matter, crude protein, and amino acids. Simple linear equations were obtained for each amino acid within tissues as a function of litter size. The mobilization of amino acids from carcass, liver, gastrointestinal tract, reproductive tract, and other viscera increased as litter size increased. Amino acids were accreted to mammary glands as litter size increased (2.65 g lysine/21 d for each one-pig increase in litter size). Milk production needs were estimated (49.9 g lysine/21 d for each one-pig increase in litter size). The quantity of each amino acid required additionally as litter size increased was obtained from the difference between amino acid needs for milk production and mammary gland growth and those provided from tissue mobilization. The relative ratio among amino acids that are required additionally (ideal amino acid pattern) was compared with the relative ratio of amino acids that can be provided from a corn-soybean meal lactation diet. From the comparison, it was shown that threonine and lysine are the first-limiting amino acids, followed by valine, when tissue mobilization occurs during lactation. Lysine is the first-limiting amino acid, and valine becomes second-limiting followed by threonine, when sows do not mobilize body tissues during lactation. Thus, the limiting order of essential amino acids changes depending on feed intake and tissue mobilization of sows during lactation. Proper feeding of lactating sows should consider the expected degree of tissue mobilization during lactation.     

高精料对泌乳期山羊肝脏氨基酸分配与重分配及乳蛋白的影响

为了探讨饲喂不同精粗比日粮对肝脏氨基酸分配与重分配的影响及可能的机制,本研究选择6只安装了肝脏多血管瘘的健康泌乳奶山羊,分别饲喂精粗比为40∶60(对照组)和60∶40(高精料组)的饲料,2×2拉丁方设计,饲喂期16周。期间取乳样,测定乳蛋白含量,并通过肝门静脉、肝静脉和股动脉血管瘘取血, RP-HPLC法测定血浆中游离氨基酸含量。结果表明,高精料组山羊乳中乳蛋白含量显著低于低精料组(P<0.05);肝门静脉血液中各游离氨基酸含量均高于低精料组,而肝静脉和股动脉血液中各游离氨基酸含量均低于低精料组,即高精料组各游离氨基酸在肝脏中的消耗量均多于低精料组,通过股动脉进入乳腺组织的氨基酸含量也少于低精料组。本研究结果显示,长期饲喂高精料日粮,乳蛋白含量降低。其机制与肝脏中氨基酸分配与重分配发生改变,氨基酸在肝脏中消耗量增大,进入乳腺组织合成乳蛋白的前体物氨基酸减少有关。     

必需氨基酸上调乳腺腺泡中L型氨基酸转运载体1的表达

To investigate the effect of essential amino acids on LAT1 expression in lactation mammary gland, the lactation mammary acini were cultured and LAT1 and 4F2hc expression were detected by Western blotting. The results showed that essential amino acids upregulated LAT1 and 4F2hc expression significantly in lactation mammary acini of dairy cow. It revealed that LAT1 was the mainly amino acid transporter in lactation mammary gland. The expression of LAT1 and 4F2hc was induced by essential amino acids.     

十二指肠大豆小肽梯度灌注对山羊乳腺氨基酸吸收与氨肽酶N基因表达的影响

旨在研究乳腺对不同水平大豆小肽的吸收利用情况.试验选用8只带有十二指肠瘘管和颈动脉、乳静脉血插管的泌乳奶山羊(体质量38 kg±k2 kg),采用交叉设计,分别向十二指肠灌注大豆小肽0、60、120和180 g·d~(-1),连续灌注14 d.结果表明:通过小肽的灌注,(1)提高了乳蛋白产量与乳蛋白含量,而且乳蛋白产量显著提高(P＜0.05).乳脂产量与乳脂含量随着灌注量的升高呈显著下降趋势(P＜0.05).(2)乳腺血浆流星有轻微的提高,但不显著(P＞0.05).与对照组相比,各处理组血浆流量/产奶量出现下降,但只有120 g·~(-1)灌注组下降显著(P＜0.05).(3)与对照组相比,大部分游离氨基酸在60和120 g·d~(-1)灌注组吸收量增加,而在180 g·d~(-1)灌注组出现下降趋势.除了PB-Ile,大豆小肽的灌注增加了所有肽结合必需氨基酸的乳腺吸收.但PB-Val、PB-Leu、PB-Phe、PB-Thr、PB-Met和PB-Lys均在120 g·d~(-1)灌注组吸收率最高.非必需氨基酸中,小肽的灌注提高了PB-Ser、PBTyr、PB-Pro的吸收(P＜0.05),而PB-Gly的吸收却出现了下降(P＞0.05).(4)除了Lys,处理组中所有必需氨基酸的乳中分泌量都高于对照组,从0～120 g·d~(-1)试验组,这些氨基酸乳中分泌量随着小肽灌注量的升高而呈增加趋势(P＜0.05);在180 g·d~(-1)灌注组,大部分必需氨基酸的分泌量增加不显著(P＞0.05),且低于120 g·d~(-1)灌注组.(5)小肽的灌注明显促进了APN基因的表达.灌注60、120和180 g·d~(-1)小肽后,氨肽酶N(APN)的表达分别是对照组的13.55、18.69和10.01倍.结论:乳腺组织能够吸收动脉血液中的小肽,大豆小肽的灌注提高了乳蛋白产量显示乳腺组织可以将吸收的小肽用于乳蛋白的合成.但当乳腺所需的氨基酸达到饱和以后,再增加氨基酸的浓度会出现吸收抑制.APN表达变化与乳腺小肽吸收增加相一致,这可能因为APN是调控小肽乳腺吸收利用的主要酶类之一.     

保护性蛋氨酸与赖氨酸在奶牛生产中的应用

乳蛋白是体现牛奶质量的重要指标,乳蛋白氨基酸来源于日粮氨基酸。乳蛋白中酪蛋白、乳白蛋白和乳球蛋白除了满足人体基本营养需求,还对人体生长、发育和健康发挥重要作用,是动物源优质蛋白质来源。蛋氨酸与赖氨酸是奶牛日粮限制性氨基酸,不仅是乳蛋白合成的重要底物,同时还作为信号分子调控乳蛋白基因表达。本文综述了奶牛氨基酸需要与来源、必需氨基酸、限制性氨基酸、保护性蛋氨酸和赖氨酸在日粮中的应用,阐述了氨基酸营养在蛋白质合成代谢中的核心作用,为指导奶牛日粮配方提供参考。     

影响牛奶trans11C18∶1和CLA合成的因素

乳脂肪受日粮因素的调控的可塑性大，CLA是乳脂肪中的一种微量不饱和脂肪酸，影响乳脂肪的物理和生物学特性。CLA最初发现于奶牛瘤胃，但牛奶中CLA主要通过乳腺组织Δ9去饱和酶作用于trans11C18∶1而内源合成。研究结果表明，日粮因素、动物个体间的差异和动物组织间SCD酶活性的差异是影响乳脂CLA合成的关键因素。作者针对影响trans11C18∶1和Δ9去饱和酶的因素对CLA合成调控因素作了简要论述。     

Lysine uptake by mammary gland tissue from lactating sows

Kinetic properties and substrate specificity of the lysine transport system in porcine mammary gland were studied using mammary tissue explants from nine lactating sows. Sodium dependence of lysine uptake was determined by replacing sodium in the medium with choline. Kinetic parameters of lysine uptake were determined using lysine concentrations from 5 microM to 5.12 mM. Competition of lysine uptake by other amino acids was determined using the cationic amino acids, arginine and ornithine, and using other essential amino acids. Transport of lysine was time-dependent and was unaffected by replacing sodium with choline. Lysine uptake occurred by a transport mechanism with a Km of approximately 1.4 mM and a Vmax of 7.9 mmol x kg cell water(-1) x 30 min(-1). Lysine uptake was inhibited by arginine and ornithine and by high concentrations of L-alanine, L-methionine, L-leucine, cycloleucine, and D-lysine, but not by 2-(methylamino)-isobutyric acid. This transport mechanism is the primary system responsible for uptake of cationic amino acids in lactating sow mammary tissue. The relatively high Km, compared with physiological blood concentrations of lysine, indicates that the kinetic properties of the lysine transport system should not be limiting to milk protein synthesis. Transmembrane transport of lysine by lactating sow mammary tissue should be a direct function of plasma concentrations. However, interactions of other amino acids with the uptake system may affect lysine uptake.     

Proteomic analysis to unravel the effect of heat stress on gene expression and milk synthesis in bovine mammary epithelial cells

Heat stress can play a negative effect on milk yield and composition of dairy cattle, leading to immeasurable economic loss. The basic components of the mammary gland are the alveoli; these alveolar mammary epithelial cells reflect the milk producing ability of dairy cows. In this study, we exposed bovine mammary epithelial cells to heat stress and compared them to a control group using isobaric tags for relative and absolute quantitation combined with liquid chromatography coupled with tandem mass spectrometry. Compared with a control group, 104 differentially elevated proteins (>1.3‐fold) and 167 decreased proteins (<0.77‐fold) were identified in the heat treatment group. Gene Ontology analysis identified a majority of the differentially expressed proteins are associated in cell‐substrate junction assembly, catabolic processes and metabolic processes. Some of these significantly regulated proteins were related to the synthesis and secretion of milk, such as milk protein and fat. This finding was further supported by the results obtained from the reduced β‐casein expression through the system of plasminogen activator – plasminogen – plasmin and decreased fatty acid synthase could partly explain why milk fat synthesis ability of dairy cows decreased under heat stress. Our results highlight the effects of heat stress on synthesis of milk protein and fat, thus providing additional clues for further studies of heat stress on dairy milk production.