The Journal of Nutrition Nutrient Requirements and Optimal Nutrition A Deficiency or Excess of Dietary Threonine Reduces Protein Synthesis in Jejunum and Skeletal Muscle of Young Pigs1,2 Xu Wang,3 Shiyan Qiao,3* Yulong Yin,4 Longyao Yue,3 Zongyi Wang,3 and Guoyao Wu4,5* 3 National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, China 100094; 4Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, China 410125; and 5Faculty of Nutrition and Department of Animal Science Texas A&M University, College Station, TX 77843 Abstract Dietary threonine imbalance is known to reduce the growth of the small intestine, liver, and skeletal muscle in young animals, but the underlying mechanism is largely unknown. Using the pig model, this study was conducted to test the hypothesis that either a deficiency or an excess of dietary threonine impairs protein synthesis in these tissues. Young pigs (25 d of age) were fed diets containing 0.37, 0.74 (current NRC requirement) or 1.11% true ileal digestible threonine (TIDT) (n ¼ 6/diet). Pigs receiving the 0.74 and 1.11% TIDT diets were pair-fed with the same amount of feed as pigs receiving the 0.37% TIDT diet. After a 14-d dietary treatment, the fractional synthesis rate (FSR) of protein in tissues was measured using a flooding dose of L-phenylalanine plus L-[ring-2H5]phenylalanine. The results indicated that the FSR of protein in liver was reduced (P , 0.05) in pigs fed the 0.37% TIDT diet compared with pigs fed the 0.74 or 1.11% TIDT diet, and did not differ between pigs fed the 0.74 and 1.11% TIDT diets. The FSR of protein in longissimus muscle, jejunal mucosa, and mucins was reduced (P , 0.05) in pigs fed the 0.37 or 1.11% TIDT diet compared with pigs fed the 0.74% TIDT diet. The absolute synthesis rate of protein in the jejunal mucosa and muscle was also reduced (P , 0.01) in pigs fed the 0.37 and 1.11% TIDT diets compared with the controls. The absolute synthesis rate of hepatic protein was lower (P , 0.01) in pigs fed the 0.37% TIDT diets when compared with pigs fed the 0.74% TIDT diet. Protein synthesis in skeletal muscle as well as jejunal mucosa and mucins was reduced to a greater extent than that in liver in response to an imbalance of dietary threonine. Collectively, these results indicate that either an excess or a deficiency of dietary threonine decreases protein synthesis in rapidly growing tissues of young pigs. The findings provide a mechanism for the low growth performance of animals fed a threonine-imbalanced diet. J. Nutr. 137: 1442–1446, 2007. Introduction The gastrointestinal tissues have an important impact on the basal metabolism of animals due to their relatively high protein turnover rate and high oxygen consumption (1). In neonatal pigs, the portal-drained viscera (the intestine, pancreas, spleen, and stomach) accounts for only 4–6% of the total body mass, but is responsible for 20–50% of the total protein turnover (1– 3). Remarkably, the gastrointestinal tissues of growing pigs utilize ;50% of amino acids in the diet (4), including 30–50% of lysine, leucine, and phenylalanine, as well as 60% threonine (5–7). Results of a recent study indicate that dietary, rather than systemic, threonine was preferentially utilized for protein 1 Supported in part by grants from the National Natural Science Foundation of China (30525029 and 30528006), the National Basic Research Program of China (2004CB117503), Outstanding Overseas Chinese Scholar Fund of The Chinese Academy of Sciences (2005–1–4), and Texas Agricultural Experiment Station. 2 Author disclosures: X. Wang, S. Qiao, Y. Yin, L. Yue, Z. Wang, and G. Wu, no conflicts of interest. * To whom correspondence should be addressed. E-mail: firstname.lastname@example.org or email@example.com. 1442 synthesis in the small intestinal mucosa of piglets consuming a normal protein diet (8). Interestingly, the portal-drained viscera has a high obligatory requirement for threonine due to its abundance in mucosal proteins (8). Structurally, the intestinal mucosa is protected by a complex network of glycoproteins (mucus), of which mucin is an important component (9). The protein cores of mucins contain large amounts of threonine (10). In rats, dietary threonine restriction has been shown to specifically reduce fractional synthesis rate (FSR)6 of intestinal mucins but had no effect on total mucosal protein (11). However, although a dietary deficiency of threonine is known to impair the growth of young animals, little information is available concerning its effects on protein synthesis in extraintestinal tissues. Likewise, to our knowledge, there are no reports in the literature regarding effects of an excess intake of dietary threonine on protein synthesis in animal tissues. We hypothesized that either a deficiency or an excess of dietary threonine 6 Abbreviations used: ASR, absolute synthesis rate of protein; FSR, fractional synthesis rate of protein; TIDT, true ileal digestible threonine. 0022-3166/07 $8.00 ª 2007 American Society for Nutrition. Manuscript received 14 February 2007. Initial review completed 13 March 2007. Revision accepted 4 April 2007. Downloaded from https://academic.oup.com/jn/article-abstract/137/6/1442/4664791 by guest on 28 March 2018 may impair tissue protein synthesis. We tested this hypothesis using the rapidly growing pig (12). Materials and Methods The China Agricultural University Animal Care Committee approved the protocol used in this experiment. Animals and diets. Three isonitrogenous and isocaloric diets were formulated to meet the NRC (13) recommended requirements of true ileal digestible amino acids for pigs weighing 5–10 kg, except for threonine (13). The ingredients of the diets and the addition of appropriate amounts of synthetic amino acids are summarized in Table 1. The 3 experimental diets were supplemented with different amounts of L-threonine (98.5% purity; Dachen Biochemicals) to provide true ileal digestible threonine (TIDT) levels of 0.37, 0.74, and 1.11%. These levels corresponded to 50, 100 (control), and 150% of the NRC (13) recommended dietary requirement of TIDT for pigs weighing 5–10 kg. Alanine, which is rapidly metabolized by neonatal pigs and does not have any adverse effects (14), was added to the 0.37 and 0.74% TIDT diets to obtain the same levels of total nitrogen as the 1.11% TIDT diet. The TIDT values in corn, peanut meal, and whey were determined using T-cannula fitted 12–15 cm anterior to the ileocecal valve, as described (15). TABLE 1 The composition of diets TIDT levels, % Ingredient composition, % as fed Corn Peanut meal Whey Cornstarch Sucrose Soybean oil Limestone Dicalcium phosphate Salt Vitamin-mineral premix1 Synthetic amino acids2 3 L-Threonine, 98.5% 4 L-Alanine, 98.5% Chemical analysis, % as fed Crude protein Lysine Methionine Cystine Tryptophan Isoleucine Valine Leucine Histidine Threonine Digestible energy, kJ/kg as fed 0.37 0.74 1.11 54.27 20.00 5.00 5.35 5.00 3.00 0.33 2.85 0.20 1.00 2.35 0.00 0.65 54.27 20.00 5.00 5.30 5.00 3.00 0.33 2.85 0.20 1.00 2.35 0.37 0.33 54.27 20.00 5.00 5.25 5.00 3.00 0.33 2.85 0.20 1.00 2.35 0.75 0.00 16.19 1.33 0.53 0.19 0.25 0.73 0.89 1.30 0.40 0.42 14351 16.21 1.35 0.55 0.21 0.28 0.76 0.92 1.29 0.42 0.84 14343 16.17 1.34 0.51 0.20 0.24 0.72 0.90 1.33 0.38 1.24 14339 1 Supplying the following (mg/kg diet): Mn (as MnO), 20; Fe (as FeSO4H2O), 75; Zn (as ZnO), 100; Cu (as CuSO45H2O), 50; I (as CaI2), 0.48; Se (as Na2SeO3), 0.40; retinyl acetate, 1.9; cholecalciferol, 0.055; all-rac-a-tocopheryl acetate, 64; menadione sodium bisulfite (62.5% menadione), 2.2; vitamin B-12, 0.028; riboflavin, 5.5; calcium pantothenate, 13.8; nicotinic acid, 30.3; and choline chloride, 350. 2 Contained L-lysine (78%), 1.0%; DL-methionine (99%), 0.30%; L-tryptophan (99%), 0.11%; L-Isoleucine(99.9%), 0.29%; L-Valine (99.5%), 0.27%; L-Leucine (98.5%), 0.29%; L-Histidine (99%), 0.09%. 3 Feed grade obtained from Dachen Biochemicals. 4 Food grade obtained from Sigma. Eighteen crossbred (Large White 3 Landrace) barrows, weaned at 21 d of age were obtained from the China-Holland Pig Breeding Farm. During a 4-d adaptation period, the pigs were fed a mixture of the 0.74% TIDT diet and a commercial diet (50:50, w:w). The composition of nutrients in the commercial diet was: 14.226 MJ digestible energy/kg; crude protein, 19.5%; lactose, 5.6%; ether extract, 5.2%; crude fiber, 2.8%; ash, 2.8%; L-lysine, 1.3%; L-methionine 1 L-cystine, 0.85%; L-threonine, 0.89%; Ca, 0.82; and P, 0.5% (Provimi). After the adaptation period, all pigs were weighed (7.34 6 0.55 kg) and assigned randomly into 1 of 3 groups (n ¼ 6/group) on the basis of the origin of litters and body weights. The experimental pigs were housed individually in 1.20 3 0.45 m2 pens over plastic-coated, expanded-metal floors, in an environmentally controlled nursery (25–27C) with a 12-h light and12-h dark cycle. Equally sized meals (15 g feed/kg body wt) were provided to pigs 3 times daily at 0800, 1600, and 2400 h. Pigs assigned to the 0.37% TIDT treatment group had free access to feed, whereas pigs receiving the 0.74 and 1.11% TIDT diets were individually pair-fed with the same feed intake as pigs receiving the 0.37% TIDT diet. Pair-feeding was necessary to ensure similar intakes of all dietary nutrients except for threonine. Between meals, pigs had free access to drinking water. The pigs were weighed on d 0 and 14. These values were then used to calculate daily weight gain, daily feed consumption, and feed conversion. Infusion protocol and sample collection. At ;0800 h on the morning of d 14, the pigs received their normal morning allotment of feed. Blood samples (;7 mL) were collected 1 h after feeding by jugular vein puncture using vacutainer tubes coated with EDTA (Greiner Bio-one). Plasma was separated from whole blood by centrifugation at 2,000 3 g for 20 min at 4C and then stored at 220C until needed for plasma amino acid analysis. Immediately after blood sampling, the pigs received i.p. administration of a flooding dose of L-phenylalanine (1.5 mmol/kg body wt) plus 2 L-[ring- H5]phenylalanine (Cambridge Isotopes Laboratories; 0.6 mmol per kg body wt), as previously described (16). The injection was completed in 5–10 s. Thirty minutes after the isotope administration, pigs were killed with an intracardial injection of sodium pentobarbital (50 mg/kg body wt) and jugular exsanguination. After the abdomen was exposed, liver and longissimus muscle were quickly isolated. The whole small intestine was removed and flushed with ice-cold saline to remove the digesta. The jejunum was obtained, and mucosal samples were then collected by scrapping. All the samples were immediately frozen in liquid nitrogen and stored at 270C until analysis. Protein fractional synthesis rates in tissues and mucins. Sample preparation in duplicate and the isotopic enrichment of L-[2H5]phenylalanine in the free and protein-bound pools were measured as described by Bregendahl et al. (16). The preparation and purification of jejunal mucins in duplicate was carried out according to the procedures of Faure et al. (17). The isotopic enrichment of L-[2H5] phenylalanine in the mucosal free pool, mucins, and mucosal protein-bound pool were measured according to the procedures of Faure et al. (17), except that the n-propyl hepta-fluorobutyrate derivative of phenylalanine was measured using a model 6890 GC linked to a 5973N quadruple MS set on Electron Ionization mode (Agilent Technologies) (18,19). Ions with mass-tocharge ratios of 91 and 96 were monitored and converted to percentage of molar enrichment (mol %) using calibration curves. FSR in tissues and mucins was calculated following the procedures of Frank et al. (20). FSR in tissues and mucins was calculated as: FSR (%/d) ¼ (EBound 3 1440 3 100%) / (EFT 3 t). FSR is the fractional protein synthesis rate; EBound is the isotopic enrichment (%) of the tracer phenylalanine in the protein pool; 1440 is the number of min/d; EFT is the enrichment of the tracer phenylalanine in the free pool at time t; and t is the exact time (min) of protein labeling between the end of i.p. tracer injection and the time the tissue sample was placed in liquid nitrogen (16). Absolute protein synthesis rates (ASR) were calculated by fractional synthesis rates (FSR) time tissue protein mass (TPM) as previously described (20,21): ASR (g/d) ¼ FSR (%/d) 3 TPM (g). Tissue protein mass was obtained as dissected tissue mass (DTM) 3 total protein concentration (TPC) of the tissue: TPM (g) ¼ DTM (g) 3 TPC (%). Threonine and tissue protein synthesis Downloaded from https://academic.oup.com/jn/article-abstract/137/6/1442/4664791 by guest on 28 March 2018 1443 Concentrations of free amino acids in plasma were determined simultaneously with the measurement of isotopic enrichment using L-norleucine (200 mL of 2.5 mmol/L L-norleucine solution; Sigma) as an internal standard (18). TABLE 2 Growth performance and relative tissue weights of weaned pigs fed diets containing 0.37, 0.74, and 1.11% TIDT between 25 and 39 d of age TIDT levels, % Statistical analysis. All data are presented as means 6 SEM. The effects of dietary TIDT on the measured variables were analyzed by 1-way ANOVA using the General Linear Model procedures of SAS statistical software (22). Duncan’s multiple range test was performed to identify differences among groups. Significance was set at P , 0.05. Results Feed intake, growth performance, and tissue weights. Feed intake [g/(d kg body wt)] by pigs fed the 0.37% TIDT diet during the experimental period was reduced by 6.0% compared with the value for the basal period. Intakes by pigs fed the 0.37, 0.74, and 1.11% TIDI diets during the experimental period were 470, 477, and 474 g/d, respectively. Dietary intakes of digestible energy by the corresponding 3 groups of pigs during the experimental period were 6745, 6841, and 6795 kJ/d, respectively. Pigs fed the 0.37% TIDT diet had lower daily weight gain and feed conversion efficiency (P , 0.05) than pigs fed the 0.74% (control) and 1.11% TIDT diets (Table 2). However, there were no differences in growth performance between pigs fed the 0.74 and 1.11% TIDT diets. Feed intakes did not differ among the 3 groups, because pigs fed the 0.74 and 1.11% TIDT diets were pair-fed with the same amount of feed consumed by pigs fed the 0.37% TIDT diet (Table 2). The relative tissue weights (g/kg body wt) for liver, longissimus muscle, and jejunum did not differ among the 3 groups of pigs. Plasma amino acid concentrations. Increasing dietary levels of threonine dose-dependently increased (P , 0.001) its concentration in plasma (Table 3). Concentrations of glycine in plasma also increased (P , 0.010) with increasing dietary levels of TIDT from 0.37 to 0.74% and leveled off thereafter. Dietary levels of threonine did not affect concentrations of other amino acids, including alanine, arginine, glutamine, and branched-chain amino acids (data not shown). Growth performance Initial body weight, kg Final body weight, kg Body weight gain, g/d Daily feed intake, g/(d kg body wt) Feed conversion, g weight gain/g feed Tissue weights, g/kg body wt Liver Longissimus muscle Jejunum 1444 Wang et al. Downloaded from https://academic.oup.com/jn/article-abstract/137/6/1442/4664791 by guest on 28 March 2018 0.74 1.11 SEM1 P-Value 7.36 10.92 254b 43.0 7.32 11.77 318a 40.5 7.34 11.85 322a 40.0 0.55 0.58 17 1.2 0.987 0.180 0.021 0.920 0.03 0.001 1.42 0.62 1.61 0.734 0.476 0.990 0.540b 26.1 20.0 28.0 0.667a 0.679a 24.5 19.0 28.0 25.4 19.8 28.2 1 Values are means and pooled SEM, n ¼ 6/group. Means in a row with superscripts without a common letter differ, P , 0.05. Discussion The results of growth performance indicated that pigs fed the 0.37% TIDT diet had a 20% lower weight gain and 19% lower feed efficiency compared with pigs fed the control (0.74% TIDT) diet. Similar reductions in the efficiency of feed utilization have been observed for pigs fed threonine-deficient diets (23– 25). Interestingly, in contrast to rats (11), dietary levels of threonine did not affect the relative weights of any tissues in young pigs (Table 2). This discrepancy can be explained likely by a more severe restriction of dietary threonine in the rodent study (11). Nonetheless, the finding that pig growth performance did not differ between the 0.74 and 1.11% TIDT diets (Table 2) suggests that feeding 150% of NRC (13) TIDT is not beneficial for pigs weighing 5–10 kg. TABLE 3 Fractional and absolute protein synthesis rates in the liver, muscle, and jejunum. The FSR of protein was reduced (P , 0.05) in the liver of pigs fed the 0.37% TIDT diet, compared with pigs fed the 0.74 and 1.11% TIDT diets, and did not differ between pigs fed the 0.74 and 1.11% TIDT diets (Table 3). However, in both longissimus muscle and jejunal mucins, the FSR of protein was reduced (P , 0.05) in pigs fed the 0.37 or 1.11% TIDT diets compared with pigs fed the 0.74% TIDT diet, and did not differ between pigs fed the 0.37% and 1.11% TIDT diets. Notably, the FSR of protein in the jejunal mucosa of pigs fed the 1.11% TIDT diet was lower (P , 0.05) than that in pigs fed the 0.74% TIDT diet but was greater (P , 0.05) than the value for pigs fed the 0.37% TIDT diet. The ASR of hepatic protein was lower (P , 0.05) in pigs fed the 0.37% TIDT diet than in pigs fed the 0.74 and 1.11% TIDT diets (Table 3). In contrast, the ASR of protein in longissimus muscle was lower (P , 0.05) in pigs fed the 0.37 and 1.11% TIDT diets than in pigs fed the 0.74% TIDT diet, and did not differ between pigs fed the 0.37 and 1.11% TIDT diets. Interestingly, the ASR of protein synthesis was highest and lowest (P , 0.05) in pigs fed the 0.74 and 0.37% TIDT diets, respectively, with the intermediate value for pigs fed the 1.11% TIDT diet (P , 0.05). 0.37 Plasma concentrations of threonine and glycine as well as fractional and absolute synthesis rates of protein in jejunum, longissimus muscle, and liver of weaned pigs fed diets containing 0.37, 0.74, and 1.11% TIDT TIDT levels, % Tissue or variable Plasma amino acid, mmol/L Threonine Glycine FSR,2 %/d Jejunal mucosa Jejunal mucins Longissimus muscle Liver ASR,3 g/d Jejunual mucosa Longissimus muscle Liver 1 0.37 0.74 1.11 SEM1 P-Value 34c 485b 294b 862a 1230a 877a 67 79 0.001 0.010 75.5c 52.8b 5.0b 99.9b 115.8a 96.7a 9.8a 136.8a 88.8b 52.6b 5.2b 144.0a 3.12 3.06 1.18 5.20 0.001 0.001 0.017 0.001 0.87 0.04 1.39 0.001 0.001 0.001 17.8c 0.60b 46.3b 29.6a 1.95a 69.4a 22.8b 0.68b 70.3a Values are means and pooled SEM, n ¼ 6/group. Means in a row with superscripts without a common letter differ, P , 0.05. 2 FSR, fractional synthesis rate. 3 ASR, absolute synthesis rate. Plasma threonine concentrations increased markedly as the dietary TIDT level increased (Table 3), as previously reported (26,27). An exceedingly low concentration of threonine in plasma of pigs fed the 0.37% TIDT diet indicates its gross inadequacy for supporting tissue protein synthesis. A high concentration of threonine in plasma of pigs fed the 1.11% TIDT diet may result from an increase in exogenous provision and a reduced rate of tissue protein synthesis. Because glycine is a product of threonine catabolism in mammals, including pigs (28), increasing dietary levels of TIDT from 0.37 to 0.74% increased plasma concentrations of glycine (Table 3). This result aids in explaining previous observations that glycine is the most abundant amino acid in pigs fed a diet that met NRC nutrient requirement (29,30). However, a further increase in dietary threonine provision beyond 0.74% TIDT did not result in an additional increase in plasma concentrations of glycine, suggesting a limit in threonine degradation via the hepatic threonine dehydrogenase pathway. The mucus layer forms a gel adherent to the mucosal surface to resist exogenous and endogenous luminal irritants and, therefore, plays an important protective function in the intestine (31–33). The mucin is an important component of the mucus (34) and contributes to the lubrication of the gut epithelium, the protection of the intestinal lumen from an acidic environment and bacterial protease, colonization resistance, and the repair of the epithelium (35). Malnutrition decreases the absolute amount of intestinal mucin, resulting in impaired resistance to enteric infection (36). In turn, this affects nutrient uptake by the intestine (37). Although one study with rats reports that a dietary deficiency of threonine reduced intestinal mucin synthesis (11), it is not known whether an excess dietary threonine can affect this biochemical event. Also, little information is available regarding the effects of dietary threonine levels on protein synthesis in tissues of young pigs. A novel and important finding from the present study is that the FSR of small intestinal mucosal protein and mucins was reduced by an imbalanced intake of dietary threonine (both a threonine deficiency and an excess). It appears that, in young pigs, the small intestine is more sensitive to dietary threonine levels than the liver with regard to tissue protein synthesis (both fractional and absolute protein synthesis rates), particularly in response to an excess of dietary threonine. A reduced availability of threonine in the intestinal lumen is expected to impair intestinal protein synthesis (1). However, it is not clear how an excess of threonine in the intestinal lumen also results in the same outcome. A possible explanation may be a reduction in the uptake of neutral amino acids (including branched-chain amino acids) by the intestinal mucosa due to their sharing the same transport systems (4), thereby limiting the local synthesis of both global and specific proteins. Additionally, an imbalance of threonine intake may affect the secretion of local and systemic hormones that regulate intestinal protein metabolism. Further studies are required to test this hypothesis. Another new and important finding from the present study is that a dietary deficiency of threonine also reduces protein synthesis in skeletal muscle and liver of young pigs (Table 3). Particularly, the ASR of protein in longissimus muscle and liver of threonine-deficient piglets decreased by 70 and 33%, respectively, compared with the control group. This result is consistent with the previous report that rates of protein synthesis in skeletal muscle of young pigs are more responsive to changes in dietary intake of nutrients (including amino acids) than those in the liver (38–40). Furthermore, it is noteworthy that FSR of protein in longissimus muscle (;7%/d) was much lower than that in intestinal mucosal protein (;80%/d) and mucin protein (;65%/d). On the basis of a percentage of decline in FSR of protein, longissimus muscle or the intestinal mucosa responded similarly to both a deficiency and an excess of dietary threonine. Our results are in contrast to the previous report that a dietary deficiency of threonine did not reduce muscle protein synthesis in rats (11). This discrepancy may be explained by a difference in either species or the relative age of the animals used in that the previous study involved rats at several weeks postweaning (11). Additionally, a deficiency of dietary threonine (30% of the control intake) in rats reduced plasma levels of threonine by only 48%, whereas a more severe reduction (by 88%) was observed in pigs fed the 0.37% TIDT diet (Table 3). Although the Faure et al. method (11) may not be the best for purifying mucins from the large intestine that contains a large amount of proteoglycans, it was the only technique that was published for isolating mucins from the small intestine at the time of our study. Thus, the values for jejunal mucin synthesis in the present study represent all mucins combined. Also, our determination of muscle FRS deserves comment. There were no differences in plasma concentrations of adrenocorticotropic hormone or cortisol in pigs between the time of the morning blood sampling and 30 min after isotope administration (data not shown), suggesting a lack of stress from the procedures. In the previous study that measured protein synthesis in tissues of pigs using the i.p. administration of [2H]phenylalanine (16), its isotopic enrichment in the free pool of longissimus doris muscle and visceral tissues did not differ between 15 and 75 min post tracer injection. The isotopic enrichment of the protein pool increased at 30 min post i.p. administration of the tracer in skeletal muscle as in visceral tissues when compared with the values at 15 min, but did not further increase in the muscle at 45–75 min (16). Thus, muscle FSR values were substantially lower at 45–75 min than at 15 min, but differed by ;20% between 15 and 30 min (16). Therefore, although FRS values of longissimus doris muscle measured at 30 min post i.p. administration of [2H]phenylalanine may be modestly underestimated in the present study, it is valid to assess their relative changes in pigs fed the 0.37 and 1.11% TIDT diets, compared with pigs fed the 0.74% TIDT diet (control, 100% NRC requirement for threonine). In support of this view, we found that a marked decline in plasma concentrations of threonine in pigs fed the 0.37% TIDT diet, compared with pigs fed the 0.74% TIDT diet was associated with a substantial reduction in muscle protein synthesis (Table 3). Feed intake by the 3 groups of pigs (Table 2) was 96% of that recommended by NRC for pigs weighing 5–10 kg (13). Note that dietary intakes of energy and essential amino acids (EAA) were ;96% of the values recommended by NRC (13). On the basis of a previous study with young pigs (41), such a small reduction in energy and EAA intake may not result in a substantial decrease in tissue protein synthesis. Because all groups of pigs had a similar intake of nutrients except for threonine, we believe that the changes in tissue FSR values in pigs fed the 0.37 and 1.11% TIDT diets, in comparison with pigs fed the 0.74% TIDT diet (100% NRC requirement), could be interpreted to be brought about by a deficiency or excess of dietary threonine. In conclusion, a dietary deficiency or excess of threonine reduced the synthesis of intestinal mucosal protein and mucins as well as muscle protein in weaned pigs. An inadequate intake of threonine also impairs hepatic protein synthesis. The syntheses of global proteins and mucins in the small intestine and of skeletal-muscle protein were reduced to a greater extent than those in the liver. 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