Dietary Supplementation with l-Arginine Partially Counteracts Serum

Sep 27, 2011 - 'INTRODUCTION. Early weaning (weaning at age 15А21 d) often causes psychological and nutritional stress in piglets in commercial...
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Dietary Supplementation with L-Arginine Partially Counteracts Serum Metabonome Induced by Weaning Stress in Piglets Qinghua He,† Huiru Tang,† Pingping Ren,† Xiangfeng Kong,‡ Guoyao Wu,‡,§ Yulong Yin,*,‡ and Yulan Wang*,† †

Wuhan Center for Magnetic Resonance, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, China ‡ Hunan Engineering and Research Center of Animal and Poultry Science and Key Laboratory for Agro-ecological Processes in Subtropical Regions, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China § Department of Animal Science, Texas A&M University, College Station, Texas 77843, United States

bS Supporting Information ABSTRACT: Arginine plays an important role in preventing intestinal dysfunction and metabolic disorders caused by early weaning stress. However, little is known about how arginine mitigates early weaning stress. This study was conducted to evaluate the effects of weaning stress and dietary arginine supplementation on the metabonome in the serum of piglets using 1H NMR spectroscopy in conjunction with multivariate data analysis. Thirty castrated male piglets aged 21 d were evenly divided into three groups and fed in three different regimes: sow-fed (SF), weaned with L-alanine supplementation (ALA), and weaned with arginine supplementation (ARG). We found that early weaning stress led to a significantly reduced bodyweight gain (15.6%) and that supplementation with arginine can improve growth rates in piglets by 5.6% (P < 0.05). The early weaning stress was associated with marked alterations in lipid and amino acid metabolisms and perturbations in population and/or activities of gut microorganisms, which were manifested in increased levels of organic acids, amino acids, and acetyl-glycoproteins and reduced levels of choline metabolism and lipoproteins. Dietary supplementation with arginine could partially counteract the changes of metabolites induced by weaning stress, such as lipid and amino acid metabolisms. However, arginine was not able to restore disturbed gut microbiota. These results demonstrate the central role of arginine supplementation in regulating the metabolisms of weaned piglets. KEYWORDS: weaning stress, arginine, metabonomics, serum, piglets

’ INTRODUCTION Early weaning (weaning at age 15 21 d) often causes psychological and nutritional stress in piglets in commercial swine-production systems because piglets have to adapt to dramatic changes, including maternal and littermate separations, abrupt changes in food source, commingling with unfamiliar piglets, and establishment of a new social hierarchy.1,2 These combined stresses, associated with early weaning, lead to a decrease in food intake and impaired immune function, both of which contribute to an increased susceptibility to disease.3 Early weaning stress also results in increased dyspepsia and diarrhea, intestinal dysfunction and atrophy, impaired mucosal barrier function,4 and an imbalance of intestinal microbiota,5 which are responsible for the impaired growth of piglets.6 Previous studies have shown that problems associated with early weaning stress in piglets can be alleviated by an increase in the endogenous synthesis of arginine through dietary supplementation with glutamine.7,8 Therefore, there is growing interest in arginine nutrition,9 physiology,10 and toxicology11 beyond protein synthesis. Previous studies have also shown that arginine is an essential and functional amino acid for young mammals and exhibits remarkable metabolic regulatory functions.9 In addition to its role as a substrate in protein synthesis, arginine also helps to r 2011 American Chemical Society

regulate key metabolic pathways associated with nutritional metabolism, growth, and immunity in neonatal piglets10,12 and thereby enhances the efficiency of food utilization, promotes growth13,14 and gut development,15 and improves health16 in neonatal piglets. A growing body of evidence demonstrates that arginine effectively prevents intestinal dysfunction and metabolic disorders caused by early weaning.17 However, the metabolic changes associated with weaning stress and dietary intervention with arginine are largely unknown. Metabonomics is a powerful top-down systems biological tool in nutritional research that can provide the end point of consequences of nutritional intervention, which will aid the understanding of how metabolic balances may be disturbed by interventions.18 21 For example, Fardet et al. found that a diet containing whole-grain wheat flour improved redox status and lipid metabolism compared with refined wheat flours.22 A NMRbased metabonomic approach was applied to evaluate the metabolic consequences of rats in response to psychological and/or physiological stresses, as well as the effects of intervention with an enriched long chain polyunsaturated fatty acid diet.23 In these Received: July 21, 2011 Published: September 27, 2011 5214

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Table 1. Composition and Nutrient Levels of the Basal Diet composition

calculated nutrient

composition

(g/kg)

valuesa

(g/kg)

corn

582

digestible energy, kJ/g

soybean meal

200

crude protein

ingredients

14.4 212

fish meal

90

total phosphorus

whey powder

50

total calcium

glucose

10

arginine

13.1

sucrose

10

lysine

15.0

soybean oil L-lysine

20 2

threonine tryptophan

8.6 2.6

calcium hydrogen

15

methionine

3.8

cystine

3.2

9.1 12.7

Bodyweight and Food Intake

The bodyweight of each piglet was recorded at the beginning and the end of the trial. The daily food intake of the weaned piglets was also recorded.

phosphate calcium

8

carbonate salt vitamin mineral

3

histidine

6.1

10

isoleucine

8.5

Sample Collection

Approximately 2 mL of blood was collected by venipuncture from the jugular vein from each piglet at the ages of day 21 and 28 following a 12 h fast. Sera were obtained by centrifugation at 1000g and 4 °C for 10 min and stored at 80 °C until NMR analysis.

premixb leucine phenylalanine tyrosine valine a

16.9 9.8 6.9

1

10.2 25

Calculated values according to the National Research Council. b Vitamin mineral premix consists of the following components (mg/ kg diet): Cu (as CuSO4), 15; Zn (as ZnSO4), 104; Fe (as FeSO4), 100; Mn (as MnSO4), 19; vitamin A, 3; vitamin D, 0.025; vitamin E, 27; vitamin K, 2.5; choline, 570; pantothenic acid, 16; riboflavin, 5; folic acid, 2; niacin, 25; thiamine, 1.6; vitamin B6, 1.8; biotin, 0.2; vitamin B12, 0.25; choline chloride (50%), 1000; calcium propionate, 1000; ethoxyquine, 10; and carrier, 6590.

studies, metabonomics was shown to be very useful for exploring the complex relationship between nutritional intervention and metabolism in order to clarify the role of dietary components in maintaining health and the development of disease. Previously, we have reported the metabolic effects of arginine supplementation to growing pigs and found that increased levels of glucose and decreased levels of lipoproteins were associated with arginine supplementation.24 In the present study, we employed NMR spectroscopy coupled with appropriate multivariate data analysis techniques to evaluate the systemic metabolic consequences of piglets in response to early weaning stress and the metabolic effects of dietary intervention with arginine on early weaned piglets. The ultimate aim of this investigation is to provide information on metabolic alterations associated with weaning stress and dietary supplementation with arginine.

’ EXPERIMENTAL METHODS Experimental Design

The basal diet consisted of corn- and soybean-based diets (Tianke Company, Guangzhou, China) that were formulated to meet the National Research Council’s recommended nutrients for weanling piglets.25 L-Alanine and L-arginine were obtained from Ajinomoto Inc. (Tokyo, Japan) and added to the basal diet. All weaned piglets were housed individually in an environmentally controlled pen with free access to their respective diets and drinking water. SF piglets were suckled by sows. The experiment was carried out in accordance with the Chinese guidelines for animal welfare and experimental protocol26 and approved by the Animal Care and Use Committee of the Chinese Academy of Sciences.

Thirty castrated male piglets (Duroc  Landrace  Large Yorkshire strain) aged 21 d with a mean bodyweight of 5436 ( 316 g were obtained from a local commercial swine-breeding farm. They were randomly assigned to one of three groups (n = 10 piglets per group): (1) sow-fed (SF) piglets continued to be nursed by sows, (2) weaned piglets received dietary supplementation with 2.05% L-alanine (wt:wt, isonitrogenous control) (ALA) to the basal diet, and (3) weaned piglets received dietary supplementation with 1.0% L-arginine (wt:wt) (ARG) to the basal diet (Table 1).

H NMR Spectroscopic Measurement

Serum samples were prepared by mixing 200 μL of serum with 400 μL of saline containing 50% D2O (for field frequence lock purposes). The proton NMR spectra of serum were recorded at 298 K on a Bruker Avance DRX-600 spectrometer (Bruker Biospin, Rheinstetten, Germany) operating at a 1H frequency of 600.11 MHz with a cryogenic probe. Standard water-suppressed one-dimensional (1D) NOESY NMR spectra was recorded. A total of 64 transients were collected into 32 k data points for each spectrum. Analysis of NMR Data 1

H NMR spectra were processed according to the parameters previously published.24,27 NMR spectra (δ 0.5 8.5) were integrated into regions of 0.002 ppm wide using the AMIX package (v 3.8.3, Bruker Biospin, Germany). The water region (δ 4.60 5.16) was removed to avoid the effects of imperfect water suppression. In addition, the ethanol peaks (δ 1.15 1.20 and δ 3.62 3.68) originating from the process of blood collection were removed. Subsequently, each integral region was normalized to the total sum of all integral regions for each spectrum prior to pattern recognition analyses. An overview of the data distribution and intersample variations was first investigated by principal component analysis with Simca-P 11.0 software (Umetrics, Sweden).27 NMR spectral data scaled to unit variance were further analyzed using the orthogonal projection to latent structure with discriminant analysis (OPLS-DA) method,28 and the loadings were backtransformed in Excel (Microsoft) and plotted with color-coded absolute coefficient values (|r|) of the variables in MATLAB.29 The coefficient plot showed the significance of variables (resonances) that contributed to the differentiation of classes of interest, with red color being more significant than blue. In the present study, a correlation coefficient of |r| > 0.60 was regarded as being significant, on the basis of the significance of discrimination at the level of P < 0.05, as determined according to the test for the significance of the Pearson’s product-moment correlation coefficient. The OPLS-DA models were cross-validated using a 7-fold cross-validation method29 and a permutation test.30 5215

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Table 2. Effects of Continuous Suckling and Arginine Supplementation on Bodyweight Gain and Food Intake of Piglets from 21 to 28 d of Agea treatment

ALA

SF

ARG

bodyweight at d 21 (g)

5488 ( 323

5434 ( 307

5387 ( 344

bodyweight at d 28 (g)

6344 ( 432

7338 ( 332b

6697 ( 376b

(15.6%)

(5.6%)

122 ( 25

272 ( 50b

187 ( 32b

mean daily gain (g) mean daily food intake (g/d) food intake/bodyweight

203 ( 33 1.66 ( 0.21

218 ( 24 1.18 ( 0.11b

gain ratio

Values are presented as means ( SD compared to the ALA piglets using Student’s t-tests. b P < 0.05.

a

Statistical Analysis

Data from bodyweights, food intake, and the relative integrals for the metabolites that contributed significantly to the classification are expressed as mean ( SD and were subjected to Student’s t-test (SAS Institute, NC). P < 0.05 was used for the level of significance.

’ RESULTS Food Intake and Bodyweight

SF and ARG piglets gained more bodyweight compared to ALA piglets measured at 28 d (15.6 and 5.6%). In addition, the food intake to bodyweight gain ratio was lower for ARG piglets than for ALA piglets (P < 0.05, Table 2). 1

H NMR Spectra of Serum Samples

Typical 1D 1H NMR spectra of serum taken from SF piglet at 21 d of age and SF, ALA, and ARG piglets at 28 d of age are shown in Figure 1. A total of 34 metabolites were unambiguously assigned and listed in Table S1 (Supporting Information). Assignment of metabolites was made on the basis of a comparison with published literature24,30 32 and confirmed by 2D 1 H 1H COSY and TOCSY spectra (data not shown). The spectra for all of the serum samples contained resonances from several amino acids, glucose, organic acids, and urea, as well as choline and lipoproteins. Tricarboxylic acid-cycle metabolites, such as succinate, citrate, and fumarate, were also detected in serum. Multivariate Data Analysis of NMR Data

The serum metabolic changes from 21 to 28 d of age in the SF piglets were analyzed by OPLS-DA strategy. A plot of OPLS-DA scores showed a clear separation between the SF piglets at 21 and 28 d of age (Figure 2). The corresponding coefficient plot showed that serum levels of pyruvate, succinate, citrate, fumarate, acetate, valine, arginine, creatinine, and threonine were higher in SF piglets at 28 than 21 d of age (P < 0.05), whereas serum levels of lipoproteins, formate, and proline were lower in SF piglets at 28 d of age (P < 0.05) (Figure 2 and Table 3). Projection to latent structure with discriminant analysis (PLSDA) of serum spectra of SF, ALA, and ARG piglets at 28 d of age was performed, and the scores plot (Figure 3) clearly highlighted three clusters corresponding to the three groups of different dietary regimes. To further identify the important serum metabolic changes induced by weaning in piglets, the metabolic profiles of SF piglets were compared with those of ALA piglets using the OPLS-DA strategy. Multivariate data analysis showed

Figure 1. Typical 600 MHz 1H NMR standard 1D spectra of serum taken from a SF piglet at 21 d of age (A) and SF (B), ALA (C), and ARG piglets (D) at 28 d of age. The spectra in the aromatic region (δ 5.7 8.5) were magnified 4 times compared to the aliphatic region (δ 0.6 5.4). A total of 34 metabolites were unambiguously assigned. Key: 1, isoleucine; 2, leucine; 3, valine; 4, lysine; 5, alanine; 6, arginine; 7, methionine; 8, glutamate; 9, glutamine; 10, proline; 11, threonine; 12, glycine; 13, tyrosine; 14, 1-methylhistidine; 15, phenylalanine; 16, creatine; 17, creatinine; 18, lactate; 19, citrate; 20, pyruvate; 21, succinate; 22, fumarate; 23, β-glucose; 24, α-glucose; 25, acetate; 26, formate; 27, trimethylamine; 28, TMAO; 29, urea; 30, choline; 31, GPC; 32, Nacetyl-glycoproteins; 33, albumin; 34, lipoproteins. Their chemical shifts and peak multiplicities are given in Table S1 (Supporting Information).

that serum levels of lactate, succinate, fumarate, creatine, creatinine, alanine, glycine, threonine, urea, and N-acetyl-glycoproteins were higher in ALA piglets than in SF piglets (P < 0.05), whereas serum levels of lipoproteins, trimethylamine, trimethylamine-N-oxide (TMAO), choline, proline, and valine were lower in ALA piglets (P < 0.05) (Figure 4A and Table 3). In order to investigate the effect of arginine supplementation, we compared the metabolic profiles of ARG piglets with those of ALA piglets using the same strategy. We found that most of the altered metabolites in ARG piglets were similar to those of suckling piglets with exceptional changes in the levels of a few amino acids, N-acetyl-glycoproteins, trimethylamine, TMAO, creatinine, creatine, urea, and fumarate. In addition, levels of arginine, glutamine, and glutamate were different in the serum of ARG piglets (Figure 4B and Table 3). In order to verify the results obtained from multivariate data analysis, integrals of the altered metabolites were taken from NMR spectra and were compared using Student’s t-test (Table 3).

’ DISCUSSION Early weaning stress is associated with gastrointestinal dysfunction and an overall metabolic disorder.4,33 Arginine can alleviate intestinal dysfunction and promote immune function in piglets.12 To explore the molecular mechanisms of early weaning stress and the ameliorative role of arginine, the variations of 5216

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Figure 2. OPLS-DA scores (left) and coefficient plots (right) obtained from standard 1D spectra of SF piglets at 28 (9) and 21 d (0) of age (R2X = 0.65, Q2 = 0.72). The color scale in the coefficient plots shows the significance of metabolite variations between SF piglets at 28 and 21 d of age.

Table 3. OPLS-DA Coefficients and Relative Integrals of Significantly Changed Metabolites in Serum OPLS-DA coefficient (r)a metabolites

28 d (vs 21 d)

SF (vs ALA)

relative integrals (%)b

ARG (vs ALA)

21 d

28 d

ALA

ARG

0.902 ( 0.021

0.947 ( 0.030c,d

valine (1.04)

0.81

0.75

0.921 ( 0.018

0.922 ( 0.026

lipoproteins (1.29) lactate (1.33)

0.87

0.91 0.79

0.82 0.76

6.45 ( 0.34 1.29 ( 0.18

5.73 ( 0.42c,d 1.27 ( 0.12d

4.49 ( 0.37 1.53 ( 0.14

5.35 ( 0.43d 1.26 ( 0.23d

0.71

0.80

1.18 ( 0.03

1.19 ( 0.04d

1.25 ( 0.07

1.08 ( 0.04d

0.88

1.84 ( 0.04

1.92 ( 0.06

1.93 ( 0.08

2.00 ( 0.04d

0.297 ( 0.007

0.307 ( 0.005

0.304 ( 0.004

0.301 ( 0.009

1.174 ( 0.017

1.123 ( 0.020c,d

1.082 ( 0.026

1.142 ( 0.030d

alanine (1.48) arginine (1.73)

0.78

acetate (1.92)

0.83

proline (2.00)

0.84

0.77

0.81

c c

1.29 ( 0.03

1.27 ( 0.03

1.41 ( 0.04

1.46 ( 0.07

glutamate (2.35)

0.89

0.834 ( 0.019

0.851 ( 0.023

0.865 ( 0.021

0.823 ( 0.020d

glutamine (2.45) pyruvate (2.37)

0.73 0.78

0.870 ( 0.027 0.230 ( 0.012

0.863 ( 0.033 0.241 ( 0.010c

0.878 ( 0.017 0.236 ( 0.025

0.857 ( 0.020d 0.241 ( 0.006

succinate (2.40)

0.88

0.84

0.179 ( 0.004

0.188 ( 0.004c,d

0.192 ( 0.003

0.186 ( 0.004d

0.453 ( 0.013

0.467 ( 0.016

c

0.475 ( 0.010

0.467 ( 0.014

0.214 ( 0.006

0.219 ( 0.005d

0.213 ( 0.003

0.211 ( 0.007

0.87

0.236 ( 0.017

0.219 ( 0.024d

0.200 ( 0.015

0.187 ( 0.011d

0.87

0.720 ( 0.036

0.654 ( 0.092

d

0.399 ( 0.046

0.492 ( 0.034d

d

N-acetyl-glycoproteins (2.05)

citrate (2.52)

0.87

0.69

0.84

trimethylamine (2.92)

0.74

TMAO (3.26)

0.87

choline (3.21)

0.93

d

glycine (3.56)

0.73

0.327 ( 0.013

0.331 ( 0.045

0.372 ( 0.035

0.383 ( 0.043

creatine (3.93) creatinine (4.05)

0.83

0.93 0.91

0.317 ( 0.027 0.173 ( 0.006

0.304 ( 0.012d 0.181 ( 0.005c,d

0.360 ( 0.032 0.198 ( 0.003

0.362 ( 0.037 0.196 ( 0.005

threonine (4.25)

0.70

0.84

0.437 ( 0.012

0.453 ( 0.014c,d

0.480 ( 0.006

0.485 ( 0.010

0.77

0.162 ( 0.008

0.159 ( 0.018d

0.206 ( 0.024

0.223 ( 0.026

0.77

0.0367 ( 0.0014

0.0404 ( 0.0017c,d

0.0428 ( 0.0010

0.0419 ( 0.0016

0.0547 ( 0.0019

0.0571 ( 0.0020c

0.0581 ( 0.0019

0.0593 ( 0.0025

urea (5.78) fumarate (6.52)

0.87

formate (8.45)

0.68

a

The coefficients from the OPLS-DA results; positive and negative signs indicate positive and negative correlations in the concentrations of serum metabolites in piglets relative to groups in brackets. A coefficient of 0.60 was used as the cutoff value for significance (P < 0.05). b Data are means ( SD. Normalized integral of metabolites in the spectrum (normalized to 100, chemical shift region over the ranges of δ 0.5 1.15, 1.20 3.62, 3.68 4.60, and 5.16 8.50). c Different from SF piglets at 21 d of age, P < 0.05. d Different from ALA piglets at 28 d of age, P < 0.05.

serum metabonome were investigated using a metabonomic strategy. Dosage Justification

In our current investigation, we have chosen L-alanine as isonitrogenous feeding control because L-alanine is simple and has no antagonism with arginine and hence is widely accepted as control treatment in the assessment of the effects of arginine supplementation.13,15,34 36 This is in contrast to lysine. Lysine has similar structure to arginine and hence may compete with arginine for the same amino acid transporters and may inhibit the absorption of arginine in the small intestine of animals; lysine is therefore not a

suitable compound to keep isonitrogen consistent with arginine. In our investigation, 1.0% arginine was deemed to be the suitable level for supplementation, which was on the basis of previous investigations. It was shown that dosage of arginine at the high level of 4000 mg/kg body weight caused damage to the pancreas.11 Researchers evaluated the effects of dietary arginine levels on microvascular development and suggested that the optimum level of arginine was 0.7%.15 A recent investigation demonstrated that the arginine supplementation at the level of 1.0% can enhance intestinal development of weaned piglets without noticeable adverse effects.36 Hence, we adopted this value in the current investigation. 5217

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Journal of Proteome Research Growth Performance

The mean daily gains were significantly lower in both ALA and ARG piglets than in SF piglets (Table 2), which clearly demonstrated negative effects of weaning on the growth performance of piglets. We also noted that ARG piglets had better growth performance than ALA piglets, which is in contrast to our previous results showing that arginine supplementation to growing pigs was unable to achieve such growth performance.24 These results suggest that arginine supplementation could be more effective in improving the growth of early weaned piglets than in growing pigs. Metabolic Variations Associated with Growth

We noted alterations in lipoproteins and energy metabolism during the growth of SF piglets. For example, elevated concentrations of tricarboxylic acid-cycle metabolites and decreased concentrations of lipoproteins were noted in the serum of SF piglets at 28 d compared to that at 21 d of age. In practice, growth leads to an increase in bodyweight and size, requiring the synthesis and deposition of fat and muscle. Therefore, the observed increase in the levels of tricarboxylic acid-cycle intermediates is associated with the increased energy requirement and consumption in the growing process of piglets. In addition,

Figure 3. PLS-DA scores plot (R2X = 0.53, Q2 = 0.53) for standard 1D spectra of SF (9), ALA (2), and ARG piglets (b) at 28 d of age. The scores plots show ARG piglets in between SF and ALA piglets.

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researchers showed that lipoprotein lipases and growth factor-1, both of which are the determinants of subcutaneous fat accumulation, increased markedly during the neonatal periods.37 Hence, the decreased levels of lipoproteins observed here are likely contributed to by the requirement for fat deposition during growth. We also noted the association of elevated levels of creatinine with growth. Creatinine is an index of muscle mass,38 which supported our finding. In addition, a previous study also observed an increased excretion of urinary creatinine with age in dogs.39 Metabolic changes during growth were also exhibited in the elevated levels of serum valine, arginine, and threonine, which could indicate further the increased energy expenditure during growth. Valine is a precursor for arginine synthesis, as well as one of the most efficient energy-generating amino acids in mammals.40 Moreover, arginine has an important regulatory function in improving muscle synthesis.10 Therefore, the concurrent increase in the serum levels of arginine and valine are also associated with increased levels of creatinine during growth. Metabolic Variations Associated with Weaning and Arginine Supplementation

Weaned piglets mainly experienced two stresses: psychological stress and stress related to the change of diet. Previous research has established that psychological stress caused the reduction in the levels of lipoproteins and increased levels of amino acids, creatine, citrate, and lactate in plasma.23 These changes are mainly related to increases in the secretion of stress hormones23 because stress hormones such as glucocorticoids and catecholamines are able to stimulate gluconeogenesis and lipolysis and release amino acids from protein. Here, we observed an increase in serum levels of lactate, succinate, fumarate, creatine, creatinine, and some amino acids and decreased serum levels of lipoproteins, which suggested that the weaned piglets were in a condition of psychological stress and could experience slow growth.

Figure 4. OPLS-DA scores (left) and coefficient plots (right) obtained from standard 1D spectra in SF (9), ALA (2), and ARG piglets (b) at 28 d of age (A: R2X = 0.67, Q2 = 0.86; B: R2X = 0.45, Q2 = 0.59). The color scale in the coefficient plot shows the significance of metabolite variation between SF, ALA, and ARG piglets. 5218

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Journal of Proteome Research The implication of reduced levels of lipoproteins may lie in the impairment of immune systems in early weaned piglets because lipoproteins play an important role in host defense.41 One of their key defensive functions is the nonspecific immune ability of high density lipoprotein to bind to endotoxin and other bacterial products to neutralize their toxic effects.42 In support of this notion, suppressed innate immunity and defensive functions in piglets were found in early weaned piglets.3 Certainly, the functions of lipoproteins in the immune system of early weaned piglets need to be tested by further studies. A novel and unexpected finding was the decreased levels of serum choline, trimethylamine, and TMAO in weaned piglets (Table 3). Choline is mainly ingested via diet, and there are three major metabolic pathways of choline degradation. One of the pathways is via the gut microbiota, producing dimethylamine, trimethylamine, and TMAO.43,44 The other two pathways produce phosphorylcholine and creatinine.45 The decrease in trimethylamine and TMAO in ALA piglets could suggest that disturbance of intestinal microorganisms is associated with weaned piglets. Previous supporting evidence comes from the significantly altered yeast levels between weaning and weaned piglets.46 The reduced levels of choline found in weaned piglets could imply that the levels of phosphorylcholine was limited, which could in turn contribute to the reduction in the levels of lipoproteins observed in weaned piglets because phosphorylcholine is a necessary component for the assembly and secretion of very-low-density lipoproteins in the liver. Dietary supplementation with arginine could partially counteract the changes of metabolites induced by weaning stress, including the increased levels of choline and lipoproteins and the decreased levels of succinate, lactate, and a range of amino acids. It is known that arginine plays a vital role in promoting protein synthesis;10,13 consequently, more amino acids are converted into proteins, leading to decreased levels of amino acids present in serum. Our previous investigation of arginine supplementation to growing pigs found a similar trend.24 Here, we observed a reduction in the levels of alanine, glutamine, and glutamate in the serum of ARG piglets, which agreed well with the role of arginine in promoting protein synthesis and explained the enhanced bodyweight gains in the ARG piglets. The reductions in the levels of serum glutamine and glutamate after supplementation with arginine were also presented in rats47 and pigs.14,35 However, the observed increased levels of proline in the ARG piglets appeared to be contrary to the above discussion. This is because proline can be synthesized from arginine via the arginase pathway in mammals;48 hence, an excess amount of proline was noted in ARG piglets. In the current investigation, L-alanine was used for isonitrogenous control; therefore, partial contributions of added alanine in the diet cannot be ruled out for the observed relative reduction in the levels of alanine in the plasma of ARG piglets. Arginine supplementation alleviating the effects induced by weaning stress was also manifested in the restoring levels of lipoproteins in the serum. Because of the role of lipoproteins in immune function, it is clear that arginine can enhance innate immunity functions in piglets, which is consistent with a previous report on the role of arginine in the regulation of immune function.12 Having discussed this, it has to be mentioned that arginine supplementation to growing pigs resulted in decreased levels of lipoproteins,24 which is in contrast to the current investigation. This discrepancy implies again that arginine supplementation was more effective for piglets than for growing pigs.

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The observation of a further reduction in the levels of TMAO in serum from ARG piglets (Table 3) demonstrated that supplementation of arginine was unable to balance the disturbed gut microbiota caused by weaning. In fact, arginine enhanced the generation of nitric oxide by both intestinal epithelial cells and luminal bacteria,49 whereas nitric oxide, in turn, can reduce both the number and metabolic activity of gut bacteria.24 Therefore, supplementation with arginine could disturb gut microbiota even further. The reductive metabolism of choline via gut microbiota associated with ARG piglets could suggest that additional amounts of choline were available for phosphorylcholine synthesis, leading to an enhanced secretion of very-low-density lipoproteins, which explained the restored levels of choline and lipoproteins in ARG piglets. The changes in the metabolism of intestinal microorganisms affected by supplementation with arginine have not been previously investigated, and metabonomics is a sensitive strategy for measuring the fluctuations of cometabolites between microorganisms and hosts. Future microbiological studies are warranted to determine exactly how arginine regulates the population and/or activities of gut microbiota.

’ CONCLUSIONS Metabonomic investigations have been used to characterize metabolic signatures associated with growth, early weaning stress, and dietary supplementation with arginine. Variations in energy metabolism were mainly associated with the growth of piglets. Several altered metabolisms induced by weaning stress were identified, including perturbations in gut microbiota, alterations in the lipid and amino acid metabolisms, and elevation of N-acetyl-glycoproteins, which could contribute to reduced growth in weaned piglets. Dietary supplementation with arginine is more effective in improving the growth performance of weaned piglets than growing pigs and can partially counteract the metabolic consequences associated with early weaning stress, such as lipid and amino acid metabolism; however, arginine supplementation cannot restore the perturbed population/activity of gut microbiota. Our results provide metabolic consequences associated with weaning stress and dietary supplementation with arginine, which has important implications in nutritional research in infants. Our work has demonstrated that the metabonomic technique is a useful tool to probe the effects of nutritional intervention in a mammalian system. ’ ASSOCIATED CONTENT

bS

Supporting Information NMR assignments of serum metabolites in piglets are available. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel.: 86-27-8719-7143 (Y.W.), 86-731-8461-9703 (Y.Y.). Fax: 86-27-8719-9291 (Y.W.), 86-731-8461-2685 (Y.Y.). E-mail: yulan. [email protected] (Y.W.), [email protected] (Y.Y.).

’ ACKNOWLEDGMENT We acknowledge financial support from the Ministry of Science and Technology of China (2009CB118804 and 2009CB118806), 5219

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Journal of Proteome Research the Chinese Academy of Sciences (KJCX2-YW-W11), and the National Science Foundation (20921004, 20825520, and 31101729). Author disclosures: Q.H. He, H.R. Tang, P.P. Ren, X.F. Kong, G.Y. Wu, Y.L. Yin, and Y.L. Wang declare no conflicts of interest.

’ ABBREVIATIONS: 1D, one-dimensional; 2D, two-dimensional; ALA, alanine-supplemented weaned piglets; ARG, arginine-supplemented weaned piglets; COSY, correlation spectroscopy; CPMG, Carr Purcell Meiboom Gill; NMR, nuclear magnetic resonance; OPLS-DA, orthogonal projection to latent structure with discriminant analysis; PLS-DA, projection to latent structure discriminant analysis; SF, sow-fed; TMAO, trimethylamine-N-oxide; TOCSY, total correlation spectroscopy ’ REFERENCES (1) Moeser, A. J.; Ryan, K. A.; Nighot, P. K.; Blikslager, A. T. Gastrointestinal dysfunction induced by early weaning is attenuated by delayed weaning and mast cell blockade in pigs. Am. J. Physiol. Gastrointest. Liver Physiol. 2007, 293 (2), G413–421. (2) Moeser, A. J.; Klok, C. V.; Ryan, K. A.; Wooten, J. G.; Little, D.; Cook, V. L.; Blikslager, A. T. Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig. Am. J. Physiol. Gastrointest. Liver Physiol. 2007, 292 (1), G173–181. (3) Kong, X. F.; Yin, Y. L.; He, Q. H.; Yin, F. G.; Liu, H. J.; Li, T. J.; Huang, R. L.; Geng, M. M.; Ruan, Z.; Deng, Z. Y.; Xie, M. Y.; Wu, G. Dietary supplementation with Chinese herbal powder enhances ileal digestibilities and serum concentrations of amino acids in young pigs. Amino Acids 2009, 37 (4), 573–582. (4) Smith, F.; Clark, J. E.; Overman, B. L.; Tozel, C. C.; Huang, J. H.; Rivier, J. E.; Blikslager, A. T.; Moeser, A. J. Early weaning stress impairs development of mucosal barrier function in the porcine intestine. Am. J. Physiol. Gastrointest. Liver Physiol. 2010, 298 (3), G352–363. (5) Lalles, J. P.; Bosi, P.; Smidt, H.; Stokes, C. R. Weaning - A challenge to gut physiologists. Livest. Sci. 2007, 108 (1 3), 82–93. (6) Pluske, J. R.; Hampson, D. J.; Williams, I. H. Factors influencing the structure and function of the small intestine in the weaned pig: A review. Livest. Prod. Sci. 1997, 51 (1 3), 215–236. (7) Wang, J.; Chen, L.; Li, P.; Li, X.; Zhou, H.; Wang, F.; Li, D.; Yin, Y.; Wu, G. Gene expression is altered in piglet small intestine by weaning and dietary glutamine supplementation. J. Nutr. 2008, 138 (6), 1025–1032. (8) Wu, G.; Meier, S. A.; Knabe, D. A. Dietary glutamine supplementation prevents jejunal atrophy in weaned pigs. J. Nutr. 1996, 126 (10), 2578–2584. (9) Wu, G.; Knabe, D. A.; Kim, S. W. Arginine nutrition in neonatal pigs. J. Nutr. 2004, 134 (10 Suppl), S2783–2790. (10) Wu, G.; Bazer, F. W.; Davis, T. A.; Kim, S. W.; Li, P.; Marc Rhoads, J.; Carey Satterfield, M.; Smith, S. B.; Spencer, T. E.; Yin, Y. Arginine metabolism and nutrition in growth, health and disease. Amino Acids 2009, 37 (1), 153–168. (11) Bohus, E.; Coen, M.; Keun, H. C.; Ebbels, T. M.; Beckonert, O.; Lindon, J. C.; Holmes, E.; Noszal, B.; Nicholson, J. K. Temporal metabonomic modeling of L-arginine-induced exocrine pancreatitis. J. Proteome Res. 2008, 7 (10), 4435–4445. (12) Tan, B.; Li, X. G.; Kong, X.; Huang, R.; Ruan, Z.; Yao, K.; Deng, Z.; Xie, M.; Shinzato, I.; Yin, Y.; Wu, G. Dietary L-arginine supplementation enhances the immune status in early-weaned piglets. Amino Acids 2009, 37 (2), 323–331. (13) Yao, K.; Yin, Y. L.; Chu, W.; Liu, Z.; Deng, D.; Li, T.; Huang, R.; Zhang, J.; Tan, B.; Wang, W.; Wu, G. Dietary arginine supplementation increases mTOR signaling activity in skeletal muscle of neonatal pigs. J. Nutr. 2008, 138 (5), 867–872. (14) Kim, S. W.; Wu, G. Dietary arginine supplementation enhances the growth of milk-fed young pigs. J. Nutr. 2004, 134 (3), 625–630.

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