孫志洪 李 貌 許慶慶 印遇龍 朱偉云 江青艷 黃飛若

(1. 西南大學(xué)生物飼料與分子營(yíng)養(yǎng)實(shí)驗(yàn)室，重慶400715；2.中國(guó)科學(xué)院亞熱帶農(nóng)業(yè)生態(tài)研究所，長(zhǎng)沙410125；3.南京農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，南京210095；4.華南農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，廣州510642；5.華中農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，武漢430070)

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豬氨基酸代謝節(jié)儉機(jī)制新假說(shuō)

孫志洪1李 貌1許慶慶1印遇龍2朱偉云3江青艷4黃飛若5

(1. 西南大學(xué)生物飼料與分子營(yíng)養(yǎng)實(shí)驗(yàn)室，重慶400715；2.中國(guó)科學(xué)院亞熱帶農(nóng)業(yè)生態(tài)研究所，長(zhǎng)沙410125；3.南京農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，南京210095；4.華南農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，廣州510642；5.華中農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，武漢430070)

豬尿氮排放量為總氮排放量的60%～70%，而尿素是尿液中的主要含氮物，其合成速率在很大程度上決定著尿氮以及總氮的排放量。因此，降低豬肝臟尿素合成速率是減少氮排放量的根本途徑。本文首先介紹了當(dāng)前豬氮減排常用的營(yíng)養(yǎng)調(diào)控技術(shù)，然后分別就肝臟尿素合成的直接前體物(氨)與間接前體物(如甘氨酸和丙氨酸)以及氨基酸代謝燃料功能替代機(jī)制進(jìn)行論述，在此基礎(chǔ)上提出豬氨基酸代謝節(jié)儉機(jī)制新假說(shuō)，即促進(jìn)丙酮酸/葡萄糖等物質(zhì)的供能效率，以降低谷氨酸等氨基酸的代謝速率，從而達(dá)到減少門(mén)靜脈尿素前體物凈流量、肝臟尿素合成以及尿氮排放量的目的。

豬；氮排放；氨基酸；代謝節(jié)儉；丙酮酸脫氫酶

近年來(lái)，氮排放引發(fā)的環(huán)境污染隨畜禽養(yǎng)殖規(guī)模和集約化程度的不斷擴(kuò)大而日趨嚴(yán)重。目前，全球畜禽氮排放量的估計(jì)值高達(dá)8 900萬(wàn)～16 400萬(wàn)t；我國(guó)畜禽氮排放量約為3 000萬(wàn)t，其中單胃動(dòng)物(主要是豬)的氮排放量約占總氮排放量的60%。與此同時(shí)，蛋白質(zhì)資源緊缺是全世界共同面臨的問(wèn)題；2014年，中國(guó)蛋白質(zhì)飼料原料的進(jìn)口量約為4 000萬(wàn)t，魚(yú)粉和大豆的進(jìn)口依存度達(dá)到70%。因此，如何提高蛋白質(zhì)的利用效率、減少氮排放量已成為我國(guó)畜禽養(yǎng)殖業(yè)尤其是養(yǎng)豬業(yè)迫切需要解決的科學(xué)問(wèn)題。

1 豬氮減排常用的營(yíng)養(yǎng)調(diào)控技術(shù)

目前圍繞生豬氮排放已經(jīng)開(kāi)展了大量研究，包括以理想氨基酸模式為基礎(chǔ)配制飼糧[1]、降低飼糧蛋白質(zhì)含量并補(bǔ)充限制性氨基酸[2-7]、增加飼糧中可發(fā)酵性碳水化合物的比例[2,8-9]以及添加酶制劑、益生素和有機(jī)酸等添加劑[10-11]。盡管大量研究已經(jīng)證實(shí)低蛋白質(zhì)飼料可顯著降低豬的氮排放量[2,6,12-13]，但這一營(yíng)養(yǎng)調(diào)控措施尚未成為養(yǎng)豬生產(chǎn)業(yè)的通用技術(shù)，尤其是在以獲取快速生長(zhǎng)為目標(biāo)的集約化生產(chǎn)體系中；其他營(yíng)養(yǎng)調(diào)控技術(shù)也只能在一定程度上減少豬的氮排放量。

鑒此，有必要深入研究豬的氮排放機(jī)制以明確關(guān)鍵調(diào)控靶點(diǎn)。豬尿氮排放量占總氮排放量的比例為60%～70%[9,14-15]，而尿素是尿液中的主要含氮物，其合成速率在很大程度上決定了尿氮以及總氮的排放量。因此，降低豬肝臟尿素合成速率是減少氮排放量的重要策略，而明確尿素前體物的種類(lèi)與來(lái)源則是開(kāi)展氮減排研究的首要前提。

2 尿素前體物

2.1 氨——尿素的直接前體物

氨為尿素的直接前體物，主要來(lái)源于氨基酸的分解代謝。門(mén)靜脈回流組織(portal-drained viscera，PDV)是氨基酸代謝的重要場(chǎng)所，如飼糧中97%的谷氨酸和天門(mén)冬氨酸、70%的谷氨酰胺、40%～50%的絲氨酸和甘氨酸、40%的精氨酸和脯氨酸、20%～40%的支鏈氨基酸以及30%～60%的其他必需氨基酸均在PDV中發(fā)生分解代謝[4,16-20]。氨基酸脫氨后轉(zhuǎn)化為乙酰輔酶A、丙酮酸、草酰乙酸、琥珀酰輔酶A、延胡索酸和α-酮戊二酸等物質(zhì)進(jìn)入三羧酸(tricarboxylic acid，TCA)循環(huán)以氧化供能[21](圖1)。氨基酸在PDV中的廣泛代謝導(dǎo)致門(mén)靜脈血氨濃度遠(yuǎn)高于其他部位，進(jìn)入肝臟后大部分血氨用于尿素的合成[22]。

Glucose：葡萄糖；pyruvate：丙酮酸；threonine：蘇氨酸；cysteine：半胱氨酸；serine：絲氨酸；glycine：甘氨酸；alanine：丙氨酸；tryptophan：色氨酸；lysine：賴(lài)氨酸；leucine：亮氨酸；isoleucine：異亮氨酸；tyrosine：酪氨酸；phenylalanine：苯丙氨酸；acetyl-CoA：乙酰輔酶A；citrate：檸檬酸；isocitrate：異檸檬酸；glutamine：谷氨酰胺；histidine：組氨酸；arginine：精氨酸；proline：脯氨酸；glutamate：谷氨酸；α-ketoglutarate：α-酮戊二酸；succinyl-CoA：琥珀酰輔酶A；methionine：蛋氨酸；valine：纈氨酸；succinate：琥珀酸；fumarate：富馬酸；malate：蘋(píng)果酸；oxaloacetate：草酰乙酸；aspartate：天門(mén)冬氨酸；NAD+：煙酰胺腺嘌呤二核苷酸nicotinamide adenine dinucleotide；NADH：還原型煙酰胺腺嘌呤二核苷酸reduced nicotinamide adenine dinucleotide；GTP：三磷酸鳥(niǎo)苷guanosine triphosphate；GDP：二磷酸鳥(niǎo)苷guanosine diphosphate；FAD：黃素腺嘌呤二核苷酸flavin adenine dinucleotide；FADH2：還原型黃素腺嘌呤二核苷酸reduced flavin adenine dinucleotide。

圖1 氨基酸氧化代謝途徑

Fig.1 The oxidative metabolism pathways of amino acids[21]

2.2 甘氨酸和丙氨酸——尿素的間接前體物

前期研究發(fā)現(xiàn)，采食粗蛋白質(zhì)水平為20%、17%和14%飼糧的仔豬門(mén)靜脈谷氨酸凈吸收速率分別為-4.43、-5.65和-6.64 mg/min；門(mén)靜脈氨的凈吸收速率則分別為2.86、2.68和2.38 mg/min[23]。該結(jié)果與其他報(bào)道一致，即豬PDV中廣泛代謝谷氨酸等氨基酸，同時(shí)也產(chǎn)生大量的氨[4,17,20]。此外，采食上述3個(gè)蛋白質(zhì)水平飼糧的仔豬門(mén)靜脈甘氨酸與丙氨酸的凈吸收量占總氨基酸凈吸收量的比例分別為38.2%、37.3%和37.0%；甘氨酸和丙氨酸在肝臟中的消耗量占總氨基酸代謝量的比例分別為52.0%、49.5%和43.8%。這一氨基酸代謝規(guī)律的發(fā)現(xiàn)引起人們對(duì)甘氨酸和丙氨酸的來(lái)源及代謝去路的深入思考。

傳統(tǒng)觀點(diǎn)認(rèn)為絲氨酸是甘氨酸的主要前體物，而Wu[21]則提出不同的觀點(diǎn)，認(rèn)為僅有10%左右的甘氨酸來(lái)源于絲氨酸；丙氨酸的前體物包括丙酮酸、絲氨酸和天門(mén)冬氨酸[24]。根據(jù)氨基酸的代謝轉(zhuǎn)化途徑[21,24](圖2)，推測(cè)PDV中廣泛代謝的氨基酸(如谷氨酸、谷氨酰胺和天門(mén)冬氨酸等)極有可能是甘氨酸和丙氨酸的重要前體物。為證實(shí)這一推測(cè)，利用血插管與15N穩(wěn)定性同位素示蹤技術(shù)發(fā)現(xiàn)，PDV中轉(zhuǎn)化為甘氨酸和丙氨酸的谷氨酸占谷氨酸代謝總量的比例約為30%。這一氨基酸代謝規(guī)律實(shí)質(zhì)上反映了機(jī)體的一項(xiàng)重要自我保護(hù)機(jī)制：PDV中氨基酸代謝所產(chǎn)生的氨如果全部直接進(jìn)入肝臟會(huì)造成氨的濃度過(guò)高，有可能引起肝損傷，而將其中一部分氨轉(zhuǎn)化為分子質(zhì)量相對(duì)較小的甘氨酸和丙氨酸(分子質(zhì)量分別為75和89 u，遠(yuǎn)低于氨基酸的平均分子質(zhì)量)，不僅能有效降低氨的濃度、減輕肝臟的氨負(fù)擔(dān)，同時(shí)又能發(fā)揮谷氨酸等氨基酸在PDV中的代謝燃料功能。

Berthiaume等[25]和Doepel等[26]先后報(bào)道肝臟會(huì)代謝大量的甘氨酸和丙氨酸，且甘氨酸是重要的生氨氨基酸[27]；丙氨酸會(huì)增加饑餓大鼠肝細(xì)胞尿素的合成[28]，丙氨酸也是甘氨酸代謝過(guò)程的重要參與者[29]。以上研究表明，甘氨酸和丙氨酸與肝臟尿素合成密切相關(guān)[27-29]，但尚未有報(bào)道證實(shí)甘氨酸和丙氨酸是尿素合成的重要氮來(lái)源。結(jié)合前人的研究報(bào)道，推測(cè)在肝臟中多余的甘氨酸和丙氨酸用來(lái)合成尿素。為證實(shí)這一推測(cè)，利用血插管與15N穩(wěn)定性同位素示蹤技術(shù)開(kāi)展了甘氨酸和丙氨酸在肝臟中代謝去路的研究，研究表明甘氨酸和丙氨酸是尿素的重要間接前體物[30]。

Ile：異亮氨酸isoleucine；Leu：亮氨酸leucine；Lys：賴(lài)氨酸lysine；Phe：苯丙氨酸phenylalanine；Tyr：酪氨酸t(yī)yrosine；Trp：色氨酸t(yī)ryptophan；Acetyl-CoA：乙酰輔酶A；CO2：二氧化碳carbon dioxide；NH3：氨ammonia；Choline：膽堿；Threonine：蘇氨酸；Glycine：甘氨酸；Serine：絲氨酸；Alanine：丙氨酸；Pyruvate：丙酮酸；Gluc：葡萄糖glucose；Val：纈氨酸valine；Met：蛋氨酸methionine；Oxaloacetate：草酰乙酸；Aspartate：天門(mén)冬氨酸；Asparagine：天門(mén)冬酰胺；α-Ketoglutarate：α-酮戊二酸；BCAA：支鏈氨基酸branched-chain amino acids；Glutamate：谷氨酸；His：組氨酸histidine；Glutamine：谷氨酰胺；Proline：脯氨酸；Ornithine：鳥(niǎo)氨酸；Arginine：精氨酸；Cys：半胱氨酸cysteine；D3PG：D-3-磷酸甘油酸D-3-phosphoglycerate；HYP：羥(基)脯氨酸 hydroxyproline；TF：四氫葉酸 tetrahydrofolic acid。

圖2 氨基酸的代謝轉(zhuǎn)化途徑

Fig.2 The pathways of metabolic transformation between amino acids[21,24]

3 氨基酸代謝燃料功能替代機(jī)制

綜上所述，減少PDV中尿素前體物(主要包括氨、甘氨酸和丙氨酸)的生成是降低尿素合成以及尿氮排放量的關(guān)鍵，而提供氨基酸代謝燃料替代物以降低氨基酸的氧化代謝速率是實(shí)現(xiàn)這一目標(biāo)的重要途徑。有關(guān)氨基酸代謝燃料替代物的探索開(kāi)始于20世紀(jì)90年代，但由于研究甚少，迄今為止尚未取得突破性進(jìn)展。除谷氨酸/谷氨酰胺外，葡萄糖也是各類(lèi)組織細(xì)胞的重要燃料物質(zhì)，但通常情況下葡萄糖難以抑制谷氨酸/谷氨酰胺的氧化分解[17]；不僅如此，谷氨酸/谷氨酰胺還會(huì)顯著降低葡萄糖的氧化代謝速率[31-33]。因此，如何提高葡萄糖在PDV中的氧化供能效率是豬氮減排研究亟待解決的科學(xué)問(wèn)題。

氨基酸、脂肪、葡萄糖的氧化路徑雖不同，但最后都匯聚于同一點(diǎn)，即TCA循環(huán)[34](圖3)。乙酰輔酶A、丙酮酸、草酰乙酸、琥珀酰輔酶A、延胡索酸和α-酮戊二酸是氨基酸進(jìn)入TCA循環(huán)的中間產(chǎn)物[21]，其中丙酮酸在三大物質(zhì)的代謝聯(lián)系中起重要的樞紐作用，若丙酮酸代謝發(fā)生異常將會(huì)導(dǎo)致眾多疾病的發(fā)生，包括糖尿病、肥胖[35]、線粒體功能紊亂[36]、心臟衰竭[37]、神經(jīng)退行性疾病[38]和癌癥[39]。研究表明，丙酮酸是氨基酸氧化代謝的重要調(diào)控因子[40-42]。鑒于丙酮酸在三大物質(zhì)代謝過(guò)程中所發(fā)揮的重要作用，推測(cè)丙酮酸有可能是氨基酸和葡萄糖代謝的共同調(diào)控靶點(diǎn)，促進(jìn)丙酮酸在PDV中的氧化分解有望增加葡萄糖的氧化代謝速率、抑制氨基酸的代謝燃料功能，從而降低尿素前體物(氨、甘氨酸和丙氨酸)的生成以及尿素的合成。

Lipids：脂類(lèi)；fatty acids：脂肪酸；acyl-CoA：?；o酶A；acetyl-CoA：乙酰輔酶A；carbohydrates：碳水化合物；glucose：葡萄糖；ADP：二磷酸腺苷adenosine diphosphate；ATP：三磷酸腺苷adenosine triphosphate；glycolysis：醣酵解；pyruvate：丙酮酸；proteins：蛋白質(zhì)；amino acids：氨基酸；transamination：轉(zhuǎn)氨基；deamination：脫氨；CO2：二氧化碳carbon dioxide；TCA cycle：三羧酸循環(huán)；β-oxidation：β-氧化；NADH：還原型煙酰胺腺嘌呤二核苷酸reduced nicotinamide adenine dinucleotide；FADH2：還原型黃素腺嘌呤二核苷酸reduced flavin adenine dinucleotide；Pi：磷酸基；ETC：電子傳遞鏈electron transfer chain。

圖3 三大營(yíng)養(yǎng)物質(zhì)氧化代謝途徑

Fig.3 The oxidative metabolism pathways of three major nutrients[34]

哺乳動(dòng)物細(xì)胞中，丙酮酸脫氫酶復(fù)合體(pyruvate dehydrogenase complex，PDC)負(fù)責(zé)催化丙酮酸轉(zhuǎn)化為乙酰輔酶A。PDC由3種酶[丙酮酸脫氫酶(pyruvate dehydrogenase，PDH)、二氫硫辛酰轉(zhuǎn)乙?；浮⒍淞蛐了崦摎涿竇和6種輔助因子[焦磷酸硫胺素、硫辛酸、黃素腺嘌呤二核苷酸(flavin adenine dinucleotide，F(xiàn)AD)、煙酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide，NAD)、輔酶A(coenzyme A，CoA)和Mg2+]組成。PDH上游調(diào)控因子主要包括丙酮酸脫氫酶激酶(pyruvate dehydrogenase kinase，PDK)和丙酮酸脫氫酶磷酸酶(pyruvate dehydrogenase phosphatase，PDP)，調(diào)控機(jī)制如圖4所示[43]。PDK1通過(guò)磷酸化PDH分子上的絲氨酸殘基(包括Ser-293、Ser-300、Ser-232)抑制其活性，而PDP則通過(guò)去磷酸化恢復(fù)PDH以及PDC的活性[44]。酪氨酸磷酸化將分別激活PDK活性和抑制PDP活性[45]。綜上所述，PDK/PDP/PDH軸極有可能是葡萄糖/氨基酸的調(diào)控靶點(diǎn)。

Gluconeogenesis：糖異生；cytosol：細(xì)胞溶質(zhì)；glucose：葡萄糖；PEP：磷酸烯醇式丙酮酸phosphoenolpyruvate；pyruvate：丙酮酸；glycolysis：糖酵解；lipid biosynthesis：脂類(lèi)生物合成；acetyl-CoA：乙酰輔酶A；CO2：二氧化碳carbon dioxide；H+：氫離子；NAD+：煙酰胺腺嘌呤二核苷酸nicotinamide adenine dinucleotide；NADH：還原型煙酰胺腺嘌呤二核苷酸reduced nicotinamide adenine dinucleotide；mitochondrial matrix：線粒體基質(zhì)；PDC active：有活性的丙酮酸脫氫酶復(fù)合體active pyruvate dehydrogenase complex；PDC inactive：無(wú)活性的丙酮酸脫氫酶復(fù)合體inactive pyruvate dehydrogenase complex；PDK：丙酮酸脫氫酶激酶pyruvate dehydrogenase kinase；isoenzymes：同功異構(gòu)酶；PDP：丙酮酸脫氫酶磷酸酶pyruvate dehydrogenase phosphatase；insulin：胰島素：Ca2+：鈣離子；ATP：三磷酸腺苷adenosine triphosphate；P1-3：磷酸基1-3；TCA cycle：三羧酸循環(huán)。

圖4 丙酮酸脫氫酶復(fù)合體調(diào)節(jié)機(jī)制

Fig.4 The regulatory mechanisms of pyruvate dehydrogenase complex[43]

丙酮酸氧化代謝速率隨PDC活性的升高而提高[46]。小分子物質(zhì)二氯乙酸(dichloroacetate，DCA)具有誘導(dǎo)細(xì)胞自噬、降低細(xì)胞增殖的重要功能。此外，研究表明DCA通過(guò)抑制PDK活性來(lái)激活PDH活性，從而降低糖酵解比例、提高葡萄糖的氧化代謝速率[47-48]。谷氨酰胺氧化代謝速率隨葡萄糖氧化代謝速率的升高而降低[49]。研究表明，促進(jìn)丙酮酸的氧化代謝將導(dǎo)致谷氨酸脫氫酶的活性降低，從而降低來(lái)源于谷氨酰胺的乙酰輔酶A的生成[49]。由此可見(jiàn)，通過(guò)調(diào)控丙酮酸/葡萄糖氧化代謝速率來(lái)抑制氨基酸代謝燃料功能是可行的。

4 小 結(jié)

綜上所述，在PDV中異常增加的甘氨酸和丙氨酸歸因于谷氨酸等氨基酸的過(guò)度代謝，甘氨酸和丙氨酸是肝臟尿素合成的重要前體物。降低氨基酸的氧化代謝速率是減少尿素合成前體物和肝臟尿素合成的關(guān)鍵。促進(jìn)丙酮酸/葡萄糖在豬PDV中的供能效率有望增加葡萄糖的氧化代謝速率、抑制氨基酸的代謝燃料功能，從而減少尿素前體物的生成以及尿氮排放量，而PDK/PDP/PDH軸可能是丙酮酸氧化代謝的調(diào)控靶點(diǎn)。雖然在體外試驗(yàn)、老鼠試驗(yàn)以及人類(lèi)臨床試驗(yàn)上已經(jīng)證實(shí)通過(guò)促進(jìn)丙酮酸/葡萄糖的氧化代謝速率來(lái)降低氨基酸的供能效率是可行的，但豬體代謝與細(xì)胞、老鼠和人類(lèi)相比差異極大，且研究目的不同，因此這一假說(shuō)需要開(kāi)展大量的體內(nèi)和體外試驗(yàn)進(jìn)行驗(yàn)證。

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Author, SUN Zhihong, professor, E-mail: sunzh2002cn@aliyun.com

(責(zé)任編輯 李慧英)

A New Hypothesis for the Mechanism of Metabolic Saving of Amino Acids of Pigs

SUN Zhihong1LI Mao1XU Qingqing1YIN Yulong2ZHU Weiyun3JIANG Qingyan4HUANG Feiruo5

(1. Laboratory for Bio-Feed and Animal Nutrition, Southwest University, Chongqing 400715, China; 2. Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha 410125, China; 3. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China; 4. College of Animal Science and Technology, Huanan Agricultural University, Guangzhou 510642, China; 5. College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, China)

Urinary nitrogen excretion accounts for 60% to 70% of the total nitrogen excretion of pigs. The production rate of urea, which is the main nitrogen-containing substance in the urine, to a large extent determines the urinary nitrogen and total nitrogen excretion. Therefore, declining the production rate of urea in liver of pigs is a fundamental approach for reducing total nitrogen excretion. This review summarized the existing nutrition regulatory measures for reducing nitrogen excretion in pigs, characterized the nitrogen direct precursors (ammonia) and indirect precursors (glycine and alanine) of urea synthesis in liver, and the mechanism of metabolic fuel function substitution of amino acid (AA). On this basis, a new hypothesis for the regulatory mechanism of metabolic saving of AA was proposed, the essence of which is to promote the efficiency of substances like as pyruvate/glucose being as metabolic fuel, decline metabolic rate of AA especially of glutamate, decrease the net flow of nitrogen precursors for urea synthesis in portal vein, urea synthesis in liver and urinary nitrogen excretion.[ChineseJournalofAnimalNutrition, 2016, 28(11)：3369-3376]

nitrogen excretion; amino acids; metabolic saving; pyruvate dehydrogenase

2016-04-11

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃(2013CB127300)；農(nóng)業(yè)部"948"項(xiàng)目(2015Z74)；國(guó)家科技支撐計(jì)劃課題(2012BAD14B18)；重慶市自然科學(xué)基金(cstc2012jjA80001)

孫志洪(1975—)，男，四川成都人，教授，碩士生導(dǎo)師，博士，從事動(dòng)物營(yíng)養(yǎng)研究。E-mail: sunzh2002cn@aliyun.com

10.3969/j.issn.1006-267x.2016.11.001

S828

A

1006-267X(2016)11-3369-08