RESEARCH Open Access

Effects of adding sodium dichloroacetate tolow-protein diets on nitrogen balance andamino acid metabolism in the portal-drained viscera and liver of pigsWeizhong Sun1†, Yunxia Li2†, Zhiru Tang1, Huiyuan Chen1, Ke Wan1, Rui An1, Liuting Wu1 and Zhihong Sun1*

Abstract

Background: Identifying regulatory measures to promote glucose oxidative metabolism while simultaneouslyreducing amino acid oxidative metabolism is one of the foremost challenges in formulating low-protein (LP) dietsdesigned to reduce the excretion of nitrogen-containing substances known to be potential pollutants. In this study,we investigated the effects of adding sodium dichloroacetate (DCA) to a LP diet on nitrogen balance and aminoacid metabolism in the portal-drained viscera (PDV) and liver of pigs.To measure nitrogen balance, 18 barrows (40 ± 1.0 kg) were fed one of three diets (n = 6 per group): 18% crudeprotein (CP, control), 13.5% CP (LP), and 13.5% CP + 100 mg DCA/kg dry matter (LP-DCA). To measure amino acidmetabolism in the PDV and liver, 15 barrows (40 ± 1.0 kg) were randomly assigned to one of the three diets (n = 5per group). Four essential amino acids (Lys, Met, Thr, and Trp) were added to the LP diets such that these hadamino acid levels comparable to those of the control diet.

Results: The LP-DCA diet reduced nitrogen excretion in pigs relative to that of pigs fed the control diet (P < 0.05),without any negative effects on nitrogen retention (P > 0.05). There were no differences between the control andLP-DCA groups with respect to amino acid supply to the liver and extra-hepatic tissues in pigs (P > 0.05). The netrelease of ammonia into the portal vein and production rate of urea in the liver of pigs fed the LP-DCA diet wasreduced relative to that of pigs fed the control and LP diets (P < 0.05).

Conclusion: The results indicated that addition of DCA to a LP diet can efficiently reduce nitrogen excretion in pigsand maximize the supply of amino acids to the liver and extra-hepatic tissues.

Keywords: Amino acid metabolism, Low-protein diet, Nitrogen excretion, Pig, Sodium dichloroacetate

BackgroundThe pork sector benefits the livelihoods of millions ofproducers and consumers but is also a source of seriousnitrogen pollution. It is estimated that, worldwide, the

pork sector releases 10 Tg of nitrogen into the environ-ment annually, approximately 70% of which is lost towatercourses in the form of organic nitrogen and ni-trates; the remainder is emitted into the atmosphere asnitrogenous gases (e.g., NH3, NOX, and N2O) [1]. In thepast few decades, low-protein (LP) diets supplementedwith indispensable amino acids have shown the potentialto reduce nitrogen excretion [2–4], and numerous stud-ies have shown no adverse effects on the growth

© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: sunzh2002cn@aliyun.com†Weizhong Sun and Yunxia Li contributed equally to this work.1Laboratory for Bio-feed and Molecular Nutrition, College of Animal Scienceand Technology, Southwest University, Chongqing 400715, People’s Republicof ChinaFull list of author information is available at the end of the article

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 https://doi.org/10.1186/s40104-020-00437-2

http://crossmark.crossref.org/dialog/?doi=10.1186/s40104-020-00437-2&domain=pdfhttp://orcid.org/0000-0002-3839-037Xhttp://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/mailto:sunzh2002cn@aliyun.com

performance of pigs when the dietary crude protein (CP)content was reduced by less than 2–3% [2, 5–7]. How-ever, when the dietary protein content is reduced by≥3%, it becomes necessary to add large amounts ofamino acids to LP diets to meet animal requirements;otherwise pigs tend to show poor growth [8–13]. Never-theless, it is deemed inadvisable to reduce the dietary CPcontent while simultaneously supplementing the dietwith amino acids, as this increases both nitrogen intakeand feed cost and reduces economic performance.Urinary nitrogen typically accounts for 60–70% of total

nitrogen excretion [14, 15], indicating that more atten-tion should be paid to porcine urinary nitrogen. Theor-etically, decreasing the entry of the main nitrogenprecursors of urea into the liver is an important strategyfor reducing urinary nitrogen. Ammonia (NH3), which isa direct nitrogen donor for urea, is mainly derived fromthe catabolism of amino acids, particularly those thatrepresent a source of metabolic fuel and undergo exten-sive oxidative metabolism in the portal-drained viscera(PDV; including stomach, small and large intestines,pancreas, spleen, and omental fat) [16, 17]. Althoughglucose is a primary source of metabolic fuel that can beuniversally metabolized, large amounts of amino acidsare metabolized in the PDV, for example, a large part ofall non-essential amino acids (NEAAs) are oxidized bythe absorptive epithelial cells (enterocytes) of mamma-lian small intestines [18–20]; 30–50% of essential aminoacids (EAAs) in the diet may be catabolized by the smallintestines in first-pass metabolism [21]. While glucosehas little effect on the oxidative metabolism of aminoacids, most amino acids, including glutamate and glu-tamine, can suppress glucose oxidation [22, 23]. Al-though intestinal mucosal amino acid metabolism isessential for maintaining intestinal integrity and function[24, 25], it should be noted that most amino acids me-tabolized in the intestines, particularly Gln, Glu, andAsp, are used as metabolic fuel [26] and produce nitro-gen precursors for urea synthesis. Therefore, identifyingthe regulatory measures that promote glucose oxidationin the presence of amino acids is an important step forreducing nitrogen excretion and modifying LP diets.With the aim of achieving this goal, we previously con-

ducted a series of studies in which we made a number ofkey observations [17, 27, 28]. First, we found that NEAAinsufficiency is a major disadvantage of the existing low-protein diets [17]. Second, we established that pyruvate,which is critical to energy metabolism [29, 30], could po-tentially replace the role of amino acids as a metabolicfuel and consequently reduce nitrogen excretion in pigs[27]. Third, we revealed that the pyruvate dehydrogenasekinase (PDK)/pyruvate dehydrogenase (PDH) axis is akey target for shifting amino acid oxidation to glucose/pyruvate metabolism [28]. In addition, we also found

that culturing cells in the presence of sodium dichloroa-cetate (DCA) increased the phosphorylation of PDHA1/pyruvate dehydrogenase phosphatase 1 (PDP1), inhibitedthe phosphorylation of PDK1, and promoted increasesin the mRNA and protein expression of glucose trans-porter 1 (GLUT1) and GLUT4 in cells, thereby increas-ing glucose consumption and reducing amino acidmetabolism [28]. DCA, which is an inhibitor of PDK[31], binds to the N-terminal domain of PDK and pro-motes conformational changes, leading to the inactiva-tion of kinase activity and reactivation of glucoseoxidation [32]. DCA has been shown to reverse theWarburg effect of colorectal cancer [33–35], thereby re-instating oxidative phosphorylation. Oral administrationhas been shown to significantly increase in the activity ofthe PDH complex and tricarboxylic acid (TCA) cycle,not only in the platelets but also in various human tis-sues [36]. Previous studies have also indicated that DCAmay be used as a metabolic modulator to enhancecarbohydrate oxidative metabolism more precisely glu-cose oxidative metabolism, while reducing amino acidoxidative metabolism, thereby achieving the goal of re-ducing porcine nitrogen excretion. In addition, DCA isinexpensive, generally recognized as being safe [37–39],and has no detectable bone marrow, hepatic, renal, pul-monary, or cardiac toxicity [39]. Moreover, DCA-loadedmats had minimal effects on physical function after sub-cutaneous implantation or even attachment to the liverof mice [40].We therefore hypothesized that DCA would be a suit-

able dietary additive to reduce nitrogen excretion in pigsby increasing amino acid utilization efficiency. Accord-ingly, the objective of the present study was to determinethe effects of DCA supplementation of a LP diet (in whichdietary CP was reduced by 4.5%) on nitrogen excretionand amino acid metabolism in the PDV and liver of pigs.

Materials and methodsAnimal use and careAll animal procedures complied with the relevant ethicaland animal welfare standards and were approved by theethics committee of Southwest University.Crossbred pigs (Yorkshire-Landrace sow × Duroc

boar) were obtained from a local commercial swine farmand were reared individually in stainless-steel metaboliccages (1.8 m × 1.2 m × 0.75 m). All pigs had ad libitumaccess to fresh water. The room temperature was main-tained at approximately 26 °C (range, 25.4–26.6 °C) usingthermostatically controlled heaters and exhaust fans.

Assessment of nitrogen balanceTo measure nitrogen excretion, 18 barrows (40 ± 1.0 kg)were randomly assigned to receive one of three dietarytreatments: 18% CP (control), 13.5% CP (LP), and 13.5%

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 Page 2 of 12

CP + 100mg DCA/kg dry matter (LP-DCA). The controldiet was formulated according to the recommendationsof the National Research Council [41] and Lys, Met,Thr, and Trp were added to the LP diets (Table 1), mak-ing the actual amino acid intake (with the exception ofLys, Met, Thr, and Trp) of pigs in the LP and LP-DCAgroups lower than that of pigs in the control group(Table 2). The dose of DCA selected for use in this studywas based on that reported in the literature [38, 42]. Theexperimental period lasted 14 d, including habituationfor the first 7 d and sample collection for the remainderof the period. The daily feed (45 g/kg body weight) wasprovided as two equal meals at 07:00 and 17:00 h. Fromd 8 to d 14, the feed was supplemented with 0.3% TiO2(as a digesta marker). Feces and urine were collected at08:00, 15:00, and 23:00 h from d 11 to d 14. The sampleswere collected according to previously describedmethods [17]. Briefly, the collected feces (approximately0.2 kg per pig per collection) were combined with 10%sulfuric acid (10 mL/100 g feces) and frozen at − 20 °C.All urine samples were collected in plastic bottles,weighed, recorded, and stored at − 20 °C. Five millilitersof 6 mol/L HCl was added daily to each urine collectionbottle, thereby preventing evaporation and nitrogen loss.Feed intake was recorded daily during the sample collec-tion period.At the end of the sample collection period, the

urine and feces samples obtained from each pig werepooled per pig, mixed, and divided into subsamples.The fecal subsamples were dried, ground, and passedthrough a 1-mm screen for nutrient analysis, with allsamples being analyzed in duplicate. The dry matterand organic matter content in the diet and feces; theCP content in the diet, urine, and feces; the amountsof phosphorus and calcium in the diet; and theamounts of titanium in the feces and diet were ana-lyzed according to previously described methods [43,44]. The energy in the feed, feces, and urine was de-termined using an adiabatic bomb calorimeter(C5000; IKA Werke GmbH, Staufen im Breisgau,Germany). The energy content of urine was deter-mined after freeze-drying approximately 50 mL ofurine in polyethylene bags. After hydrolysis with 6mol/L at 110 °C, the amounts of amino acids (withthe exception of Trp, Cys, and Met) in the feed weremeasured using an automatic amino acid analyzer (L-8900; Hitachi, Tokyo, Japan). The analytical proce-dures used for Trp, Cys, and Met followed previouslydescribed methods [45].

Assessment of nutrient fluxesAfter fasting for 24 h, 15 barrows (40 ± 1.0 kg) were sur-gically catheterized in the carotid artery, hepatic vein,portal vein, and mesenteric vein using polyurethane

Table 1 Ingredients and composition of diets differing in crudeprotein content (dry matter basis, %)Items Diets

Control LP

Ingredients

Corn 61.20 74.55

Soybean meal 25.90 12.07

Wheat bran 7.80 7.39

Soybean oil 2.00 2.00

Lys 0.18 0.55

Met 0.05 0.12

Thr 0.01 0.24

Trp 0.00 0.08

Dicalcium phosphate 0.69 0.90

Calcium carbonate 0.87 0.80

Salt 0.30 0.30

1% Premixa 1.00 1.00

Total 100.00 100.00

Composition

Metabolic energy, MJ/kgb 13.9 13.9

Crude proteinc 18.0 13.5

Lysc 1.11 1.12

Metc 0.35 0.35

Thrc 0.73 0.73

Trpc 0.22 0.22

Argc 1.01 0.76

Hisc 0.44 0.33

Ilec 0.71 0.55

Leuc 1.35 1.02

Phec 0.79 0.61

Valc 0.68 0.52

Proc 0.73 0.56

Asx (Asp+Asn)c 1.76 1.34

Serc 0.74 0.57

Glx (Glu + Gln)c 2.95 2.23

Glyc 0.58 0.44

Alac 0.79 0.60

Cysc 0.23 0.18

Tyrc 0.37 0.28

Calciumd 0.63 0.63

Phosphorusd 0.54 0.54

Abbreviations: Control 18.0% CP, LP 13.5% CPaProviding the following per kg diet: Cu (as copper sulfate), 100mg;Fe (as ferrous sulfate), 100mg; Zn (as zinc oxide), 120mg; Mn (asmanganese sulfate), 20mg; I (as calcium iodate), 0.3 mg; and Se (assodium selenite), 0.3 mg; vitamin A, 3, 800 IU; vitamin D3, 800 IU;vitamin E, 10 IU; vitamin K, 1 mg; choline, 200mg; pantothenic, 5 mg;vitamin B2, 2 mg; folic acid, 0.8 mg; niacin, 10mg; vitamin B1, 1 mg;vitamin B6, 1 mg; biotin, 0.08mg; vitamin B12, 0.01mgbCalculated valuescValues for standardized ileal digestible concentrations of aminoacids in diets were calculated using standardized ileal digestiblecoefficients for the various ingredients provided by NRC (2012) [41]dAnalyzed values

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 Page 3 of 12

(2.41 mm OD) under isoflurane anesthesia and sterileconditions according to previously described methods[46]. After surgery, pigs were maintained in metabolismcages for recovery and received an intramuscular injec-tion of penicillin twice daily (1.6 × 107 units) for 7 d.Catheters were flushed daily aseptically with 200 IU ofheparinized normal saline to maintain their patency andwere protected in pockets attached to a jacket made ofsize 7 Surgilast (Glenwood Laboratories Canada, Oak-ville, ON, Canada). Wound Clear Spray (Guangan Ani-mal Health Co., Changsha, Hunan, China) was appliedto the sutures and to the exit site of catheters.One week after surgery (or at the point at which pigs

achieved > 80% of their pre-surgery feed intake for than2 d), the pigs were randomly assigned to one of the threedietary treatments: 18% CP (control), 13.5% CP (LP), or13.5% CP + 100mg DCA/kg dry matter (LP-DCA). The

experiment continued for 7 d, with blood samples col-lected on the final day. During the experimental period,the feed (45 g/kg body weight) was divided into threeequal portions, provided at 08:00, 14:00, and 20:00 h. Onthe day of blood sampling, the animals received a prim-ing dose of p-aminohippuric acid (PAH) solution (0.75mL/kg; 0.14 μmol/mL) via the mesenteric vein at 07:30h, followed by a constant infusion of PAH solution at arate of 3.2 mL/kg/h. At 08:30, 10:00, 12:00, 14:30, and17:30 h, 5 mL blood samples were collected from the ca-rotid artery, portal vein, and hepatic vein, to which so-dium heparin solution (100 IU/mL) was added as ananticoagulant. The blood samples were immediatelyplaced on ice and centrifuged at 3,000×g for 10 min at4 °C, and the plasma was stored at − 20 °C. The plasmaobtained from each site at the five time points waspooled for each pig, and the concentration of PAH inthe plasma was measured following previously describedmethods [47] with some modification [48]. The hepaticvein plasma flow (HVPF; mL/kg/h) and the portal veinplasma flow (PVPF; mL/kg/h) were calculated using thefollowing equations [17, 49]:

HVPF ¼ Ci � IR= PAHhv � PAHcað Þ

PVPF ¼ Ci � IR= PAHpv � PAHca� �

where Ci is the concentration of infused PAH solution(mg/mL); IR is the infusion rate (mL/kg/h) of PAH; andPAHhv, PAHca, and PAHpv are the PAH concentrations(mg/mL) in the hepatic vein, carotid artery, and portalvein, respectively. The hepatic artery plasma flow(HAPF; mL/kg/h) was then calculated as follows:

HAPF ¼ HVPF� PVPF

The concentrations of plasma urea and NH3 weremeasured following previously described procedures[50]. The analysis of fresh plasma NH3 was performedwithin 2 h of blood collection. The frozen plasmasamples were thawed at 4 °C and precipitation of theproteins therein was carried out as follows. Briefly, 1mL of the sample and 2.5 mL of 7.5% (w/v)trichloroacetic acid solution were mixed thoroughlyand centrifuged at 12,000×g and 4 °C for 15 min. Thesupernatant was collected and analyzed for aminoacids using an automatic amino acid analyzer (L-8900; Hitachi, Tokyo, Japan). The fluxes of plasmaamino acids (mg/kg/h), NH3 (μmol/kg/h), and urea(mmol/kg/h) across the carotid artery, portal vein, orhepatic vein were calculated as the concentration of aplasma nitrogen-containing compound in a veinmultiplied by the plasma flow across the vein. ThePDV balance of a nitrogen-containing compound wascalculated as follows:

Table 2 The effects of adding sodium dichloroacetate (DCA) tolow-protein diets on the dry matter and amino acid intakes of40-kg pigs in a nitrogen balance experiment

Items Treatments SEM P

Control LP LP-DCA

Intake of feed, kg 2.12 2.20 2.18 0.04 0.332

Intakes of amino acids, g/d

Lys 24.9 24.0 23.4 0.18 0.417

Met 7.40 7.12 6.97 0.05 0.514

Thr 16.7 16.2 15.8 0.12 0.889

Trp 4.43 4.29 4.19 0.04 0.356

Arg 21.9a 15.8b 15.3b 0.13

A positive value means net uptake and a negative valuesignifies net release. The hepatic balance of nitrogen-containing compound was calculated as follows:the concentration of nitrogen-containing compound in

the hepatic artery × HAPF + the AA concentration in theportal vein × PVPF – the AA concentration in the hepaticvein × HVPF. A positive value indicates that the amount ofnitrogen-containing compound leaving the liver is smallerthan that entering the liver, whereas a negative value indi-cates that the amount of nitrogen-containing compoundleaving the liver is greater than that entering the liver.

Statistical analysisData were analyzed based on a randomized completeblock design considering the pig as the experimental

unit. The MIXED procedure of SAS (SAS Institute, Cary,NC, USA) was used to analyze the variables. The fixedeffect consisted of dietary treatment (control, LP, andLP-DCA), with the pig as the random component. Themodel used was as follows:

Yijk ¼ μþ Ti þ Li þ Aj þ Eijk

where Yijk is the dependent variable, μ is the overallmean, Ti is the dietary treatment, Li is the period effect,Ai is the pig effect, and Eijk is the residual error. Differ-ences between treatment means were determined byTukey multiple comparison tests. Results are reported asmeans ± SEM, and P < 0.05 was considered statisticallysignificant.

PVPF� �the concentration of plasma nitrogen

�containing compound in the portal vein

�the concentration of the corresponding compound in the carotid arteryÞ:

Table 3 The effects of adding sodium dichloroacetate (DCA) to low-protein diets on the nitrogen balance and apparent digestibilityof other nutrients in 40-kg pigs

Items Treatments SEM P

Control LP LP-DCA

Nitrogen utilization

Average daily feed intake 1.60 1.66 1.66 0.07 0.543

Intake of nitrogen, g/d 46.0a 35.8b 35.9b 0.54

ResultsNitrogen balanceAs shown in Table 3, LP diets (LP and LP-DCA) re-duced urinary nitrogen excretion, fecal nitrogen excre-tion, and total nitrogen excretion in pigs (P < 0.05).Compared with the control and LP-DCA diets, the LPdiet reduced the retention of nitrogen in pigs (P < 0.05).Furthermore, we found that the digestive energy digest-ibility and metabolic energy/digestive energy ratio of pigsin the LP-DCA group were both higher than those ofpigs in the control group (P < 0.05).

AA metabolism in the PDV and liverAs shown in Table 4, with the exception of Lys, Trp,and urea, the concentrations of plasma nitrogenouscompounds in the portal vein of pigs fed the LP dietwere lower than those of pigs fed the control diet (P <0.05). The concentrations of plasma Met, Arg, His, Ile,

Phe, Val, Pro, Ser, Gly, Ala, and EAAs in the hepaticvein of pigs fed the LP were lower than those of pigs fedthe control diet (P < 0.05). Similarly, the concentrationsof plasma Gly, Ala, and NH3 in the portal vein and thoseof plasma Gly and Ala in the hepatic vein of pigs fed theLP-DCA diet were lower than those of pigs fed the con-trol diet (P < 0.05). Furthermore, the concentrations ofplasma Thr, Pro, Ser, and Gln in the portal vein andthose of plasma Met, Arg, His, Ile, Val, Pro, and EAAsin the hepatic vein of pigs fed the LP diet were lowerthan those of pigs fed the LP-DCA diet (P < 0.05).As shown in Table 5, plasma flow across the hepatic

artery of pigs fed the LP diet was lower than that of pigsin the control group (P < 0.05). However, there were nosignificant differences in plasma flow across the portaland hepatic veins in pigs among the three groups (P >0.05). The fluxes of plasma nitrogenous compoundsacross the portal vein (with the exception of Lys, Asn,and urea) and the fluxes of plasma nitrogen-compounds

Table 4 The effects of adding sodium dichloroacetate (DCA) to low-protein diets on the concentrations of amino acids (mg/dL),NH3 (μmol/L), and urea (mmol/L) in the portal vein, hepatic artery, and hepatic vein of 40-kg pigsItems Portal vein SEM P Hepatic artery SEM P Hepatic vein SEM P

Control LP LP-DCA Control LP LP-DCA Control LP LP-DCA

Lys 3.30 2.93 3.15 0.07 0.076 2.42 2.14 2.32 0.06 0.071 2.88 2.57 2.87 0.08 0.058

Met 1.39a 1.18b 1.32ab 0.03 0.023 1.00a 0.86b 0.96ab 0.03 0.049 1.22a 1.03b 1.20a 0.03 0.012

Thr 3.97a 3.55b 3.86a 0.09 0.028 3.22 2.91 3.15 0.09 0.100 3.65 3.25 3.63 0.11 0.054

Trp 0.64 0.56 0.59 0.01 0.068 0.45 0.40 0.44 0.01 0.077 0.50 0.46 0.51 0.01 0.053

Arg 2.95a 2.56b 2.80ab 0.06 0.024 2.21 1.98 2.11 0.06 0.130 2.55a 2.21b 2.53a 0.06 0.026

His 2.08a 1.74b 1.94ab 0.04 0.014 1.63 1.43 1.51 0.04 0.087 1.83a 1.53b 1.78a 0.04 0.009

Ile 2.32a 1.97b 2.16ab 0.08 0.026 1.51a 1.30b 1.40ab 0.04 0.038 1.98a 1.69b 1.93a 0.05 0.013

Leu 3.48a 3.07b 3.29ab 0.07 0.024 2.50 2.26 2.41 0.07 0.157 3.12 2.79 3.05 0.08 0.057

Phe 2.77a 2.37b 2.60ab 0.06 0.018 1.99 1.78 1.89 0.05 0.136 2.45a 2.11b 2.38ab 0.06 0.030

Val 2.22a 1.93b 2.12ab 0.06 0.024 1.86 1.64 1.79 0.05 0.092 2.04a 1.78b 2.00a 0.05 0.040

Pro 1.86a 1.54b 1.78a 0.04 0.002 1.44 1.28 1.41 0.04 0.143 1.60a 1.37b 1.59a 0.04 0.017

Asn 3.30a 2.94b 3.16ab 0.07 0.046 2.81 2.55 2.72 0.07 0.167 3.06 2.78 2.97 0.07 0.116

Ser 2.15a 1.66b 2.01a 0.04

across the hepatic vein (with the exception of Asn, Gln,Tyr, NH3, and urea) were lower in pigs fed the LP dietthan those of pigs fed the control diet (P < 0.05). Thefluxes of plasma Gly, Ala, and NH3 across the portalvein and the fluxes of plasma Gly, Ala, and urea acrossthe hepatic vein of pigs fed the LP-DCA diet were lowerthan those of pigs fed the control diet (P < 0.05). Fur-thermore, the fluxes of plasma Met, Thr, Arg, His, Pro,Ser, Gln, and Tyr across the portal vein and the fluxes ofplasma nitrogenous compounds across the hepatic veinof pigs fed the LP diet (with the exception of Leu, Asn,Gln, Gly, Ala, Cys, NEAAs, NH3, and urea) were lowerthan those of pigs fed the LP-DCA diet (P < 0.05).Compared with the control diet, the LP diet reduced the

net release of all nitrogenous compounds (with exceptionof Lys and Cys) into the portal vein (P < 0.05); the LP-DCA diet reduced the net release of Trp, Gly, Ala,

NEAAs, total amino acids (TAAs), and NH3 into the por-tal vein (P < 0.05) (Table 6). Moreover, we observed thatthe net release of Met, Thr, Arg, His, Ile, Leu, Phe, Val,Pro, Asn, Ser, Gln, Tyr, EAAs, TAAs, and NH3 to the por-tal vein of pigs fed the LP-DCA diet was higher than thatof pigs fed the LP (P < 0.05). The net release of Gly, Ala,and NH3 to the portal vein of pigs fed the LP-DCA dietwas lower than that of pigs fed the LP diet (P < 0.05).Compared with pigs fed the control diet, those fed the

LP diet showed reduced metabolism of Met, Trp, Ile, Pro,Asn, Se, Gln, Gly, Ala, Cys, Tyr, EAAs, NEAAs, TAAs,and NH3, as well as a reduction in the production of ureain the liver (P < 0.05) (Table 7). Similarly, relative to thepigs in the control group, pigs in the LP-DCA groupshowed reduced metabolism of Lys, Met, Thr, Trp, Arg,His, Ile, Leu, Phe, Val, Ser, Gly, Ala, Cys, Tyr, EAAs,NEAAs, TAAs, and NH3 and lower production of urea in

Table 5 The effects of adding sodium dichloroacetate (DCA) to low-protein diets on the fluxes of plasma (mL/kg/h), amino acids(mg/kg/h), NH3 (μmol/kg/h), and urea (mmol/kg/h) across the portal vein, hepatic artery, and hepatic vein of 40-kg pigsItems Portal vein SEM P Hepatic artery SEM P Hepatic vein SEM P

Control LP LP-DCA Control LP LP-DCA Control LP LP-DCA

Plasma 1614 1627 1659 35.6 0.878 409a 346b 404a 13.9 0.004 2023 1973 2063 51.3 0.330

Lys 53.4 47.5 52.1 1.25 0.060 9.88a 7.59b 9.36a 0.23

the liver (P < 0.05). Pigs fed the LP-DCA diet showed re-duced metabolism of Lys, Met, Thr, Arg, His, Ile, Leu,Phe, Val, Gly, Ala, EAAs, TAAs, and NH3 and reducedproduction of urea in the liver relative to those fed the LPdiet (P < 0.05). The metabolism of Ser, Glu, Cys, Tyr,NEAAs in the liver of pigs fed the LP-DCA diet washigher than that of pigs fed the LP diet (P < 0.05).

DiscussionNitrogen balanceIn this study, we found that the provision of a LP diet, re-gardless of whether it was supplemented with DCA, re-sulted in reduced nitrogen excretion by pigs. Specifically,compared with pigs in the control group, those in the LP

and LP-DCA groups showed reductions in urinary nitro-gen excretion by 23.5% and 44.7%, respectively, and reduc-tions in fecal nitrogen of 35.0% and 38.6%, respectively.Although the reductions in the fecal nitrogen excretion ofthe two groups were comparable, the reduction in urinarynitrogen excretion in pigs fed a DCA-supplemented LPdiet was considerably higher than that of pigs fed theunsupplemented LP diet. In addition, compared with pigsfed the control diet, we found that the pigs fed a LP dietwithout DCA supplementation showed reduced nitrogenretention. These observations are similar to the findings ofour earlier study [17] and other studies [8, 51], which havereported that nitrogen retention was reduced when theamount of dietary CP was reduced by ≥3%. However,

Table 6 The effects of adding sodium dichloroacetate (DCA) tolow-protein diets on the portal-drained viscera balance ofamino acids (mg/kg/h) and NH3 (μmol/kg/h) in pigs*

Items Treatments SEM P

Control LP LP-DCA

Lys 14.3 12.9 14.2 0.29 0.087

Met 6.36a 5.16b 6.16a 0.12 0.005

Thr 12.2a 10.4b 12.3a 0.32 0.013

Trp 3.12a 2.56b 2.70b 0.05 0.031

Arg 11.9a 9.44b 11.8a 0.25

DCA supplementation compensated for the adverse ef-fects of the LP diets on nitrogen retention in pigs, which isin accordance with our hypothesis that DCA may be asuitable dietary additive for reducing nitrogen excretion inpigs via an enhancement of nitrogen utilization efficiency.In this regard, we suspect that the mechanisms underlyingthe enhanced nitrogen utilization are related to thechanges in nutrient metabolism in response to dietaryDCA supplementation.

Nutrient metabolismIn a previous study, we demonstrated that the provisionof a LP diet supplemented with Lys, Met, Thr, and Trpreduced the supply of NEAAs to the portal vein and in-creased the rate of EAA metabolism in the liver of pigs[17]. NEAAs play important roles in the synthesis of nu-merous bioactive compounds and the normal growthand maintenance of animals [52, 53]. The daily bodyweight gain and feed efficiency of growing pigs are re-duced when the dietary NEAA supply is insufficient [9].Therefore, we assume that an insufficient supply ofNEAAs is a major disadvantage of LP diets. In thepresent study, although the net amount of NEAAs andTAAs released into the portal vein of pigs fed a DCA-supplemented LP diet were still lower than those of pigsfed the control diet, addition of DCA was observed toreduce the metabolism of EAAs and TAAs in the liver.Eventually the differences in the fluxes of NEAAs, EAAs,and TAAs across the hepatic vein between the controlgroup and the pigs that received a LP diet were abol-ished. In addition, compared with pigs in the controland LP groups, pigs in the LP-DCA group showed a re-duced release of NH3 into the portal vein and reducedurea production in the liver. These results indicate thatsupplementing a LP diet with DCA can enhance nitro-gen utilization efficiency in pigs by increasing and opti-mizing the supply of amino acids to the liver and extra-hepatic tissues, thereby reducing urea production in theliver and urinary nitrogen excretion. Notably, dietaryDCA supplementation promoted a reduction in the ca-tabolism of amino acids such as Glx (Glu + Gln) in thePDV, which are known to be major energy substrates inenterocytes and colonocytes and are beneficial for intes-tinal health [16, 54, 55]. Therefore, theoretically, dietaryDCA supplementation may have some adverse effects onpigs, primarily on the intestines. We suggest that in fu-ture studies, more attention should be devoted to asses-sing the effects of DCA on intestinal health.Recent evidence has indicated that gut microbiota in

the intestines play an important role in dietary protein/amino acid metabolism. Fermentation of amino acids bygut bacteria produces metabolites that can affect hostprotein/amino acid uptake (transport) and metabolism,as well as influenc host cell physiology [56]. Moreover,

bacteria can synthesize certain amino acids for hostutilization [57]. Reducing the amount of dietary proteinhas been viewed as an alternative option that can beused to reduce excessive protein fermentation in thelarge intestine of pigs [58]. This reduction can inhibitthe growth of potential pathogens, such as species ofBacteroides and Clostridium, and reduce amino acid de-amination [59]. These studies indicate that the reduc-tions in nitrogen excreted in the feces and urine of pigsfed LP diets with or without DCA supplementation maybe related to a reduction in the amino acid deaminationactivities of intestinal microbiota. However, given thatwe did not investigate the effects of DCA on the intes-tinal microbiota in this study, we were unable to test thishypothesis.We assume that enhanced amino acid utilization effi-

ciency is closely related to changes in energy utilizationefficiency. The LP diets formulated in the present studywere designed to be isocaloric with the control diet,which we achieved by increasing the proportion of car-bohydrates in the diet. Accordingly, the LP diets werecharacterized as being LP high-starch diets. The net en-ergy provided by starch is considerably higher than thatprovided by the protein [60]. Feeding LP diets has beenfound to reduce the amount of energy required for de-amination of excess amino acids, lower body proteinturnover and heat production, and enhance energyutilization efficiency [61]. In the present study, we ob-served that there were no significant differences in thedigestive energy digestibility and metabolic energy/di-gestive energy ratio of pigs provisioned with the controland LP diets, although the values recorded for pigs inthe LP group tended to be higher, whereas those for pigsin the LP-DCA group were significantly higher. Accord-ingly, these results indicate that the energy utilization ef-ficiency of pigs was enhanced by dietary DCAsupplementation.Of note in this regard is the fact that DCA is similar

to acetate, which is known to serve as a source of energyin several cell types, including intestinal cells, and hasprofound effects on gut metabolism and health [62]. Al-though, to the best of our knowledge, there is no directevidence to indicate that DCA can be used as an energysubstrate or energy precursor similar to acetate, the doseof DCA used in the present study was very low. Thus,we could effectively ignore the putative role of DCA asan energy substrate/energy precursor, even though DCAis functionally similar to acetate.

Possible effect mechanisms of DCAWe have previously shown that DCA supplementationcan reduce the phosphorylation of PDK1 in porcine in-testinal epithelial (IPEC-J2) cells and also promotes the

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 Page 9 of 12

phosphorylation of PDP1 and PDHA1 [28], which isconsistent with the findings of other studies [32]. Thephosphorylation of PDK and deactivation of PDH reducecarbohydrate flux [63, 64], whereas dephosphorylationand activation by PDP promote carbohydrate oxidation[65]. In addition, we have found that DCA supplementa-tion increases cellular GDH1 activity and reduces GDH2activity [28], the latter being responsible for catalyzingthe conversion of glutamate to α-ketoglutarate (a sub-strate in the TCA cycle); the former plays the oppositerole. Thus, supplementation of a LP diet would, in the-ory, promote glucose oxidation, while reducing theamount of amino acids entering the TCA cycle.

ConclusionsIn this study, we demonstrated that addition of DCA toLP diets can enhance the nitrogen utilization efficiencyby increasing the amino acid supply from the intestines,reducing amino acid metabolism in the PDV and liver,and reducing the production of urea in the liver. Conse-quently, DCA supplementation increased the amino acidmetabolic thrift and reduced nitrogen excretion withoutnegatively affecting nitrogen retention in pigs fed a dietin which dietary CP was reduced by 4.5%. However, eventhough we have demonstrated the value of DCA as adietary supplement, a considerable amount of additionalresearch is necessary to determine the effects of DCA ongrowth, immunity, and meat quality before DCA can bewidely used in pig diets.

AbbreviationsAAs: Amino acids; CP: Crude protein; DCA: Sodium dichloroacetate;EAAs: Essential amino acids; GDH1: NADPH-dependent glutamatedehydrogenase; GDH2: NAD+-dependent glutamate dehydrogenase;GLUT: Glucose transporter; HAPF: Hepatic artery plasma flow; HVPF: Hepaticvein plasma flow; IR: Infusion rate; NEAAs: Non-essential amino acids;NH3: Ammonia; PAH: P-aminohippuric acid; PAHhv, PAHca, and PAHpv: are thePAH concentrations in the hepatic vein, carotid artery, and portal vein,respectively; PDH: Pyruvate dehydrogenase; PDK: Pyruvate dehydrogenasekinase; PDP: Pyruvate dehydrogenase phosphatase; PVPF: Portal vein plasmaflow; TAAs: Total amino acids

AcknowledgementsWe thank Professor Yulong Yin, Professor Weiyun Zhu, and ProfessorQingyan Jiang for their insightful suggestions on experimental design.

Authors’ contributionsThe authors’ contributions are as follows: ZHS contributed to experimentaldesign; WZS, YXL, ZRT, HYC, KW, RA, LTW, and ZHS conducted the animaland laboratory experiments, acquired and analyzed the data and interpretedthe results; WZS, YXL, and ZHS contributed to manuscript writing andfinalizing. All authors read and approved the final version of the manuscriptand approved publication.

FundingThis study was funded by grants from the National Natural ScienceFoundation of China (31872370; 31670123), the Fundamental Research Fundsfor the Central Universities (XDJK2019B014; XDJK2013C097), and the NaturalScience Foundation Project of CQ CSTC (cstc2018jcyjAX0025).

Availability of data and materialsThe data analyzed during the current study are available from thecorresponding author on reasonable request.

Ethics approvalAll animal procedures such as ethical and animal welfare issues wereapproved by the ethics committee of Southwest University.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Laboratory for Bio-feed and Molecular Nutrition, College of Animal Scienceand Technology, Southwest University, Chongqing 400715, People’s Republicof China. 2Institute of Animal Nutrition, Sichuan Agricultural University,Chengdu 611130, People’s Republic of China.

Received: 8 November 2019 Accepted: 17 February 2020

References1. Uwizeye A, Gerber PJ, Opio CI, Tempio G, Mottet A, Makkar HPS, et al.

Nitrogen flows in global pork supply chains and potential improvementfrom feeding swill to pigs. Resour Conserv Recy. 2019;146:168–79. https://doi.org/10.1016/j.resconrec.2019.03.032.

2. Gallo L, Montà GD, Carraro L, Cecchinato A, Carnier P, Schiavon S. Growthperformance of heavy pigs fed restrictively diets with decreasing crudeprotein and indispensable amino acids content. Livest Sci. 2014;161:130–8.https://doi.org/10.1016/j.livsci.2013.12.027.

3. Zhang S, Chu L, Qiao S, Mao X, Zeng X. Effects of dietary leucinesupplementation in low crude protein diets on performance, nitrogenbalance, whole-body protein turnover, carcass characteristics and meatquality of finishing pigs. Anim Sci J. 2016;87:911–20. https://doi.org/10.1111/asj.12520.

4. Li Y, Li F, Duan Y, Guo Q, Wang W, Wen C, et al. The protein and energymetabolic response of skeletal muscle to the low-protein diets in growingpigs. J Agr Food Chem. 2017;65:8544–51. https://doi.org/10.1021/acs.jafc.7b02461.

5. Hinson RB, Schinckel AP, Radcliffe JS, Allee GL, Sutton AL, Richert BT. Effectof feeding reduced crude protein and phosphorus diets on weaning-finishing pig growth performance, carcass characteristics, and bonecharacteristics. J Anim Sci. 2009;87:1502–17. https://doi.org/10.2527/jas.2008-1325.

6. Galassi G, Colombini S, Malagutti L, Crovetto GM, Rapetti L. Effects of highfibre and low protein diets on performance, digestibility, nitrogen excretionand ammonia emission in the heavy pig. Anim Feed Sci Tech. 2010;16:1140–8. https://doi.org/10.1016/j.anifeedsci.2010.08.009.

7. Hong JS, Lee GI, Jin XH, Kim YY. Effect of dietary energy levels and phasefeeding by protein levels on growth performance, blood profiles andcarcass characteristics in growing-finishing pigs. J Anim Sci Tech. 2016;58:37.https://doi.org/10.1186/s40781-016-0119-z.

8. Otto ER, Yokoyama M, Ku PK, Ames NK, Trottier NL. Nitrogen balance andileal amino acid digestibility in growing pigs fed diets reduced in proteinconcentration. J Anim Sci. 2003;81:1743–53. https://doi.org/10.2527/2003.8171743x.

9. He L, Wu L, Xu Z, Li T, Yao K, Cui Z, et al. Low-protein diets affect ilealamino acid digestibility and gene expression of digestive enzymes ingrowing and finishing pigs. Amino Acids. 2016;48:21–30. https://doi.org/10.1007/s00726-015-2059-1.

10. Guay F, Donovan SM, Trottier NL. Biochemical and morphologicaldevelopments are partially impaired in intestinal mucosa from growing pigsfed reduced-protein diets supplemented with crystalline amino acids. JAnim Sci. 2006;84:1749–60. https://doi.org/10.2527/jas.2005-558.

11. Mansilla WD, Htoo JK, de Lange CFM. Nitrogen from ammonia is as efficientas that from free amino acids or protein for improving growth performanceof pigs fed diets deficient in nonessential amino acid nitrogen. J Anim Sci.2017;95:3093–102. https://doi.org/10.2527/jas.2016.0959.

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 Page 10 of 12

https://doi.org/10.1016/j.resconrec.2019.03.032https://doi.org/10.1016/j.resconrec.2019.03.032https://doi.org/10.1016/j.livsci.2013.12.027https://doi.org/10.1111/asj.12520https://doi.org/10.1111/asj.12520https://doi.org/10.1021/acs.jafc.7b02461https://doi.org/10.1021/acs.jafc.7b02461https://doi.org/10.2527/jas.2008-1325https://doi.org/10.2527/jas.2008-1325https://doi.org/10.1016/j.anifeedsci.2010.08.009https://doi.org/10.1186/s40781-016-0119-zhttps://doi.org/10.2527/2003.8171743xhttps://doi.org/10.2527/2003.8171743xhttps://doi.org/10.1007/s00726-015-2059-1https://doi.org/10.1007/s00726-015-2059-1https://doi.org/10.2527/jas.2005-558https://doi.org/10.2527/jas.2016.0959

12. Roux ML, Donsbough AL, Waguespack AM, Powell S, Bidner TD, Payne RL,et al. Maximizing the use of supplemental amino acids in corn-soybeanmeal diets for 20- to 45-kilogram pigs. J Anim Sci. 2011;89:2415–24. https://doi.org/10.2527/jas.2010-3756.

13. Yue L, Qiao S. Effects of low-protein diets supplemented with crystallineamino acids on performance and intestinal development in piglets over thefirst 2 weeks after weaning. Livest Sci. 2008;115:144–52. https://doi.org/10.1016/j.livsci.2007.06.018.

14. Patráš P, Nitrayová S, Brestenský M, Heger J. Effect of dietary fiber and crudeprotein content in feed on nitrogen retention in pigs. J Anim Sci. 2012;90:158–60. https://doi.org/10.2527/jas.53837.

15. Shirali M, Doeschl-Wilson A, Knap PW, Duthie C, Kanis E, van Arendonk JA,et al. Nitrogen excretion at different stages of growth and its associationwith production traits in growing pigs. J Anim Sci. 2012;90:1756–65. https://doi.org/10.2527/jas.2011-4547.

16. Burrin DG, Stoll B. Metabolic fate and function of dietary glutamate in thegut. Am J Clin Nutr. 2009;90:850S–6S. https://doi.org/10.3945/ajcn.2009.27462Y.

17. Wu LT, Zhang XX, Tang ZR, Li YX, Li TJ, Xu QQ, et al. Low-protein dietsdecrease porcine nitrogen excretion but with restrictive effects on aminoacid utilization. J Agr Food Chem. 2018;66:8262–71. https://doi.org/10.1021/acs.jafc.8b03299.

18. Windmueller HG, Spaeth AE. Metabolism of absorbed aspartate, asparagine,and arginine by rat small intestine in vivo. Arch Biochem Biophys. 1976;175:670–6. https://doi.org/10.1016/0003-9861(76)90558-0.

19. Reeds PJ, Burrin DG, Jahoor F, Wykes L, Henry J, Frazer EM. Enteralglutamate is almost completely metabolized in first pass by thegastrointestinal tract of infant pigs. Am J Phys. 1996;270:E413–8. https://doi.org/10.1016/0167-0115(95)00163-8.

20. Matthews DE, Marano MA, Campbell RG. Splanchnic bed utilization ofglutamine and glutamic acid in humans. Am J Phys. 1993;264:848–54.https://doi.org/10.1152/ajpendo.1993.264.6.E848.

21. Stoll B, Henry J, Reeds PJ, Yu H, Jahoor F, Burrin DG. Catabolism dominatesthe first-pass intestinal metabolism of dietary essential amino acids in milkprotein-fed piglets. J Nutr. 1998;128:606–14. https://doi.org/10.1093/jn/128.3.606.

22. Kight CE, Fleming SE. Oxidation of glucose carbon entering the TCA cycle isreduced by glutamine in small intestine epithelial cells. Am J Phys. 1995;268:G879–88. https://doi.org/10.1152/ajpgi.1995.268.6.G879.

23. Le A, Lane AN, Hamaker M, Bose S, Gouw A, Barbi J, et al. Glucose-independent glutamine metabolism via TCA cycling for proliferation andsurvival in B-cells. Cell Meta. 2012;15:110–21. https://doi.org/10.1016/j.cmet.2011.12.009.

24. Reeds PJ, Burrin DG, Stoll B, Jahoor F, Frazer ME. Enteral glutamate is thepreferential source for mucosal glutathione synthesis in fed piglets. Am JPhys. 1997;273:E408–15. https://doi.org/10.1152/ajpendo.1997.273.2.E408.

25. Alican I, Kubes P. A critical role for nitric oxide in intestinal barrier functionand dysfunction. Am J Physiol-Gastr L. 1996;270:G225–37. https://doi.org/10.1152/ajpgi.1996.270.2.G225.

26. Burrin DG, Reeds PJ. Alternative fuels in the gastrointestinal tract. Curr OpinGastroenterol. 1997;13:165–70. https://doi.org/10.1097/00001574-199703000-00015.

27. Li YX, Tang ZR, Li TJ, Chen C, Huang FR, Yang J, et al. Pyruvate is aneffective substitute for glutamate in regulating porcine nitrogen excretion. JAnim Sci. 2018;96:3804–14. https://doi.org/10.1093/jas/sky237.

28. An R, Tang ZR, Li YX, Li TJ, Xu QQ, Zhen JF, et al. Activation of pyruvatedehydrogenase by sodium dichloroacetate shifts metabolic consumptionfrom amino acids to glucose in IPEC-J2 cells and intestinal bacteria in pigs. JAgr Food Chem. 2018;66:3793–800. https://doi.org/10.1021/acs.jafc.7b05800.

29. Bricker DK, Taylor EB, Schell JC, Orsak T, Boutron A, Chen YC, et al. Amitochondrial pyruvate carrier required for pyruvate uptake in yeast,Drosophila, and humans. Science. 2012;337:96–100. https://doi.org/10.1126/science.1218099.

30. Gray LR, Sultana MR, Rauckhorst AJ, Oonthonpan L, Tompkins SC, Sharma A,et al. Hepatic mitochondrial pyruvate carrier 1 is required for efficientregulation of gluconeogenesis and whole-body glucose homeostasis. CellMeta. 2015;22:669–81. https://doi.org/10.1016/j.cmet.2015.07.027.

31. Michelakis ED, Webster L, Mackey JR. Dichloroacetate (DCA) as a potentialmetabolic-targeting therapy for cancer. Br J Cancer. 2008;99:989–94. https://doi.org/10.1038/sj.bjc.6604554.

32. Kato M, Li J, Chuang JL, Chuang DT. Distinct structural mechanisms forinhibition of pyruvate dehydrogenase kinase isoforms by AZD7545,dichloroacetate, and radicicol. Structure. 2007;15:992–1004. https://doi.org/10.1016/j.str.2007.07.001.

33. Madhok BM, Yeluri S, Perry SL, Hughes TA, Jayne DG. Dichloroacetateinduces apoptosis and cell-cycle arrest in colorectal cancer cells. Br J Cancer.2010;102:1746–52. https://doi.org/10.1038/bjc.2014.281.

34. Babu E, Ramachandran S, Coothankandaswamy V, Elangovan S, Prasad PD,Ganapathy V, et al. Role of SLC5A8, a plasma membrane transporter and atumor suppressor, in the antitumor activity of dichloroacetate. Oncogene.2010;30:4026–37. https://doi.org/10.1038/onc.2011.113.

35. Sanchez-Arago M, Chamorro M, Cuezva JM. Selection of cancer cells withrepressed mitochondria triggers colon cancer progression. Carcinogenesis.2011;31:567–76. https://doi.org/10.1093/carcin/bgq012.

36. Naito E, Kuroda Y, Toshima K, Takeda E, Saijo T, Kobashi H, et al. Effect ofsodium dichloroacetate on human pyruvate metabolism. Brain andDevelopment. 1989;11:195–7. https://doi.org/10.1016/S0387-7604(89)80098-1.

37. Berendzen K, Theriaque DW, Shuster J, Stacpoole PW. Therapeutic potentialof dichloroacetate for pyruvate dehydrogenase complex deficiency.Mitochondrion. 2006;6:126–35. https://doi.org/10.1016/j.mito.2006.04.001.

38. Stacpoole PW, Barnes CL, Hurbanis MD, Cannon SL, Kerr DS. Treatment ofcongenital lactic acidosis with dichloroacetate. Arch Dis Child. 1997;77:535–41. https://doi.org/10.1136/adc.77.6.535.

39. Stacpoole PW, Kerr DS, Barnes C, Bunch ST, Carney PR, Fennell EM, et al.Controlled clinical trial of dichloroacetate for treatment of congenital lacticacidosis in children. Pediatrics. 2006;117:1519–31. https://doi.org/10.1542/peds.2005-1226.

40. Liu D, Wang F, Yue J, Jing X, Huang Y. Metabolism targeting therapy ofdichloroacetate-loaded electrospun mats on colorectal cancer. Drug Deliv.2015;22:136–43. https://doi.org/10.3109/10717544.2013.870258.

41. NRC. Nutrient Requirements of Swine. 11th ed. Washington, DC: NationalAcademy Press; 2012.

42. Stacpoole PW, Neiberger RE, Carney PR, Valenstein E, Theriaque DW, ShusterJJ. Evaluation of long-term treatment of children with congenital lacticacidosis with dichloroacetate. Pediatrics. 2008;121:e1223–8. https://doi.org/10.1542/peds.2007-2062.

43. AOAC. Official methods of analysis. 17th revision. Association of OfficialAnalytical Chemists: Arlington, MA; 2000.

44. Myers WD, Ludden PA, Nayigihugu V, Hess BW. Technical note: a procedurefor the preparation and quantitative analysis of samples for titaniumdioxide. J Anim Sci. 2004;82:179–83. https://doi.org/10.2527/2004.821179x.

45. Pan L, Ma XK, Wang HL, Xu X, Zeng ZK, Tian QY, et al. Enzymatic feathermeal as an alternative animal proteinsource in diets for nursery pigs. AnimFeed Sci Tech. 2015;212:112–21. https://doi.org/10.1016/j.anifeedsci.2015.12.014.

46. Yen JT, Killefer J. A method for chronically quantifying net absorption ofnutrients and gut metabolites into hepatic portal vein in conscious swine. JAnim Sci. 1987;64:923–34. https://doi.org/10.2527/jas1987.643923x.

47. Harvey RB, Brothers AJ. Renal extraction of Para-aminohippurate andcreatinine measured by continuous in vivo sampling of arterial and renal-vein blood. Ann N Y Acad Sci. 1962;102:46–53. https://doi.org/10.1111/j.1749-6632.1962.tb13624.x.

48. Fernández-Fígares I, Lachica M, Rojas-Cano ML, González-Casado A.Influence of Para-aminohippuric acid analysis on net portal-drained visceraflux of nutrients in pigs. Animal. 2018;12:232–8. https://doi.org/10.1017/s1751731117001598.

49. Li L, Zhang P, Zheng P, Bao Z, Wang Y, Huang FR. Hepatic cumulative netappearance of amino acids and related gene expression response todifferent protein diets in pigs. Livest Sci. 2015;182:11–21. https://doi.org/10.1016/j.livsci.2015.10.012.

50. Zhu LH, Armentano LE, Bremmer DR, Grummer RR, Bertics SJ. Plasmaconcentration of urea, ammonia, glutamine around calving, and the relationof hepatic triglyceride, to plasma ammonia removal and blood acid-basebalance. J Dairy Sci. 2000;83:734–40. https://doi.org/10.3168/jds.S0022-0302(00)74935–6.

51. Figueroa JL, Lewis AJ, Miller PS, Fischer RL, Gómez RS, Diedrichsen RM.Nitrogen metabolism and growth performance of gilts fed standard corn-soybean meal diets or low-crude protein, amino acid-supplemented diets. JAnim Sci. 2002;80:2911–9. https://doi.org/10.2527/2002.80112911x.

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 Page 11 of 12

https://doi.org/10.2527/jas.2010-3756https://doi.org/10.2527/jas.2010-3756https://doi.org/10.1016/j.livsci.2007.06.018https://doi.org/10.1016/j.livsci.2007.06.018https://doi.org/10.2527/jas.53837https://doi.org/10.2527/jas.2011-4547https://doi.org/10.2527/jas.2011-4547https://doi.org/10.3945/ajcn.2009.27462Yhttps://doi.org/10.3945/ajcn.2009.27462Yhttps://doi.org/10.1021/acs.jafc.8b03299https://doi.org/10.1021/acs.jafc.8b03299https://doi.org/10.1016/0003-9861(76)90558-0https://doi.org/10.1016/0167-0115(95)00163-8https://doi.org/10.1016/0167-0115(95)00163-8https://doi.org/10.1152/ajpendo.1993.264.6.E848https://doi.org/10.1093/jn/128.3.606https://doi.org/10.1093/jn/128.3.606https://doi.org/10.1152/ajpgi.1995.268.6.G879https://doi.org/10.1016/j.cmet.2011.12.009https://doi.org/10.1016/j.cmet.2011.12.009https://doi.org/10.1152/ajpendo.1997.273.2.E408https://doi.org/10.1152/ajpgi.1996.270.2.G225https://doi.org/10.1152/ajpgi.1996.270.2.G225https://doi.org/10.1097/00001574-199703000-00015https://doi.org/10.1097/00001574-199703000-00015https://doi.org/10.1093/jas/sky237https://doi.org/10.1021/acs.jafc.7b05800https://doi.org/10.1126/science.1218099https://doi.org/10.1126/science.1218099https://doi.org/10.1016/j.cmet.2015.07.027https://doi.org/10.1038/sj.bjc.6604554https://doi.org/10.1038/sj.bjc.6604554https://doi.org/10.1016/j.str.2007.07.001https://doi.org/10.1016/j.str.2007.07.001https://doi.org/10.1038/bjc.2014.281https://doi.org/10.1038/onc.2011.113https://doi.org/10.1093/carcin/bgq012https://doi.org/10.1016/S0387-7604(89)80098-1https://doi.org/10.1016/S0387-7604(89)80098-1https://doi.org/10.1016/j.mito.2006.04.001https://doi.org/10.1136/adc.77.6.535https://doi.org/10.1542/peds.2005-1226https://doi.org/10.1542/peds.2005-1226https://doi.org/10.3109/10717544.2013.870258https://doi.org/10.1542/peds.2007-2062https://doi.org/10.1542/peds.2007-2062https://doi.org/10.2527/2004.821179xhttps://doi.org/10.1016/j.anifeedsci.2015.12.014https://doi.org/10.1016/j.anifeedsci.2015.12.014https://doi.org/10.2527/jas1987.643923xhttps://doi.org/10.1111/j.1749-6632.1962.tb13624.xhttps://doi.org/10.1111/j.1749-6632.1962.tb13624.xhttps://doi.org/10.1017/s1751731117001598https://doi.org/10.1017/s1751731117001598https://doi.org/10.1016/j.livsci.2015.10.012https://doi.org/10.1016/j.livsci.2015.10.012https://doi.org/10.3168/jds.S0022-0302(00)74935�6https://doi.org/10.3168/jds.S0022-0302(00)74935�6https://doi.org/10.2527/2002.80112911x

52. Rhoads JM, Wu G. Glutamine, arginine, and leucine signaling in theintestine. Amino Acids. 2009;37:111–22. https://doi.org/10.1007/s00726-008-0225-4.

53. Wu G, Bazer FW, Dai Z, Li D, Wang J, Wu Z. Amino acid nutrition in animals:protein synthesis and beyond. Annu Rev Anim Biosci. 2014;2:387–417.https://doi.org/10.1146/annurev-animal-022513-114113.

54. Newsholme P, Procopio J, Lima MM, Pithon-Curi TC, Curi R. Glutamine andglutamate-their central role in cell metabolism and function. Cell BiochemFunct. 2003;21:1–9. https://doi.org/10.1002/cbf.1003.

55. Riedijk MA, de Gast-Bakker DA, Wattimena JL, van Goudoever JB. Splanchnicoxidation is the major metabolic fate of dietary glutamate in enterally fedpreterm infants. Pediatr Res. 2007;62:468–73. https://doi.org/10.1203/PDR.0b013e31813cbeba.

56. Davila AM, Blachier F, Gotteland M, Andriamihaja M, Benetti PH, Sanz Y,et al. Intestinal luminal nitrogen metabolism: Role of the gut microbiotaand consequences for the host. Pharmacol Res. 2013;69:114–26. https://doi.org/10.1016/j.phrs.2012.11.005.

57. Metges CC. Contribution of microbial amino acids to amino acidhomeostasis of the host. J Nutr. 2000;130:1857S–64S. https://doi.org/10.1093/jn/130.7.1857S.

58. Heo JM, Kim JC, Hansen CF, Mullan BP, Hampson DJ, Pluske JR. 2008. Effectsof feeding low protein diets to piglets on plasma urea nitrogen, faecalammonia nitrogen, the incidence of diarrhoea and performance afterweaning. Arch Anim Nutri. 2008;62:343–58. https://doi.org/10.1080/17450390802327811.

59. Macfarlane S, Macfarlane GT. Proteolysis and amino acid fermentation. In:Gibson GR, Macfarlane GT, editors. Human colonic bacteria: role in nutrition,physiology and pathology. Boca Raton (Florida): CRC Press; 1995. p. 75–100.

60. Noblet J, Fortune H, Shi XS, Dubois S. Prediction of net energy value offeeds for growing pigs. J Anim Sci. 1994;72:344–54. https://doi.org/10.1016/0168-1591(94)90137-6.

61. Noblet J, Henry Y, Dubois S. Effect of protein and lysine levels in the diet onbody gain composition and energy utilization in growing pigs. J Anim Sci.1987;65:717–26. https://doi.org/10.1051/gse:19870309.

62. Tremaroli V, Bäckhed F. Functional interactions between the gut microbiotaand host metabolism. Nature. 2012;489:242–9. https://doi.org/10.1038/nature11552.

63. Denton RM, Randle PJ, Bridges BJ, Cooper RH, Kerbey AL, Pask HT, et al.Regulation of mammalian pyruvate dehydrogenase. Mol Cell Biochem.1975;9:27–53. https://doi.org/10.1007/BF01731731.

64. Wieland OH. The mammalian pyruvate dehydrogenase complex: structureand regulation. Rev Physiol Biochem Pharmacol. 1983;96:123–70. https://doi.org/10.1007/BFb0031008.

65. Leblanc PJ, Mulligan M, Antolic A, Macpherson L, Inglis JG, Martin D, et al.Skeletal muscle type comparison of pyruvate dehydrogenase phosphataseactivity and isoform expression: effects of obesity and endurance training.Am J Physiol Regul Integr Comp Physiol. 2008;295:R1224–30. https://doi.org/10.1152/ajpregu.90320.2008.

Sun et al. Journal of Animal Science and Biotechnology (2020) 11:36 Page 12 of 12

https://doi.org/10.1007/s00726-008-0225-4https://doi.org/10.1007/s00726-008-0225-4https://doi.org/10.1146/annurev-animal-022513-114113https://doi.org/10.1002/cbf.1003https://doi.org/10.1203/PDR.0b013e31813cbebahttps://doi.org/10.1203/PDR.0b013e31813cbebahttps://doi.org/10.1016/j.phrs.2012.11.005https://doi.org/10.1016/j.phrs.2012.11.005https://doi.org/10.1093/jn/130.7.1857Shttps://doi.org/10.1093/jn/130.7.1857Shttps://doi.org/10.1080/17450390802327811https://doi.org/10.1080/17450390802327811https://doi.org/10.1016/0168-1591(94)90137-6https://doi.org/10.1016/0168-1591(94)90137-6https://doi.org/10.1051/gse:19870309https://doi.org/10.1038/nature11552https://doi.org/10.1038/nature11552https://doi.org/10.1007/BF01731731https://doi.org/10.1007/BFb0031008https://doi.org/10.1007/BFb0031008https://doi.org/10.1152/ajpregu.90320.2008https://doi.org/10.1152/ajpregu.90320.2008

AbstractBackgroundResultsConclusion

BackgroundMaterials and methodsAnimal use and careAssessment of nitrogen balanceAssessment of nutrient fluxesStatistical analysis

ResultsNitrogen balanceAA metabolism in the PDV and liver

DiscussionNitrogen balanceNutrient metabolismPossible effect mechanisms of DCA

ConclusionsAbbreviationsAcknowledgementsAuthors’ contributionsFundingAvailability of data and materialsEthics approvalConsent for publicationCompeting interestsAuthor detailsReferences