Darrell A. Knabe

Arginine Nutrition in Neonatal Pigs

The concentration of arginine (an essential amino acid for neonates) in sows milk is remarkably low, and thus endogenous synthesis of arginine plays a crucial role in maintaining arginine homeostasis in milk-fed piglets. Paradoxically, intestinal synthesis of citrulline from glutamine/glutamate and proline (the endogenous source of arginine) declines markedly in 7- to 21-d-old suckling pigs, compared with 1- to 3-d-old pigs. Therefore, plasma concentrations of arginine and its immediate precursors (ornithine and citrulline) decrease progressively by 20-41%, whereas plasma ammonia levels increase progressively by 18-46%, between d 3 and 14 of life. Dietary supplementation of 0.2 and 0.4% arginine to 7- to 21-d-old pigs (artificially reared on a milk feeding system) dose dependently enhances the plasma arginine concentration (30 and 61%), reduces the plasma ammonia level (20 and 35%), and increases weight gain (28 and 66%). These compelling metabolic and growth data demonstrate unequivocally that arginine is insufficient for supporting the maximal growth in milk-fed young pigs and that this arginine deficiency represents a major obstacle to realizing the growth potential in piglets. A low concentration of mitochondrial N-acetylglutamate (an activator of both pyrroline-5-carboxylate synthase and carbamoylphosphate synthase-I) is responsible for the striking decline in the intestinal synthesis of citrulline and arginine during the suckling period. Accordingly, oral administration of N-carbamoylglutamate [a metabolically stable analogue of N-acetylglutamate; 2 x 50 mg/(kg body wt . d)] enhances plasma arginine level (68%) and weight gain (61%) of 4- to 14-d-old sow-reared pigs. Thus, the metabolic activation of intestinal citrulline and arginine synthesis provides a novel, effective means to increase endogenous arginine provision and therefore piglet growth (a major goal of animal agriculture). Our findings not only generate new fundamental knowledge about amino acid utilization by neonatal pigs, but they also have important practical implications for improving the efficiency of pork production.

Arginine nutrition in development, health and disease.

Abstract As a precursor of nitric oxide, polyamines and other molecules with enormous biologic importance, L‐arginine plays versatile key roles in nutrition and metabolism. Arginine is an essential amino acid in the fetus and neonate, and is conditionally an essential nutrient for adults, particularly in certain disease conditions. L‐Arginine administration is beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, and in facilitating wound healing. The effect of L‐arginine in treating many common health problems is unique among amino acids, and offers great promise for improved health and well‐being in the future.

Triennial Growth Symposium: important roles for L-glutamine in swine nutrition and production.

L-Glutamine (Gln) has traditionally not been considered a nutrient needed in diets for livestock species or even mentioned in classic animal nutrition textbooks. This is due to previous technical difficulties in Gln analysis and the unsubstantiated assumption that animals can synthesize sufficient amounts of Gln to meet their needs. Consequently, the current (1998) version of NRC does not recommend dietary Gln requirements for swine. This lack of knowledge about Gln nutrition has contributed to suboptimal efficiency of global pig production. Because of recent advances in research, Gln is now known to be an abundant AA in physiological fluids and proteins and a key regulator of gene expression. Additionally, Gln can regulate cell signaling via the mammalian target of rapamycin pathway, adenosine monophosphate-activated protein kinase, extracellular signal-related kinase, Jun kinase, mitogen-activated protein kinase, and nitric oxide. The exquisite integration of Gln-dependent regulatory networks has profound effects on cell proliferation, differentiation, migration, metabolism, homeostasis, survival, and function. As a result of translating basic research into practice, dietary supplementation with 1% Gln maintains gut health and prevents intestinal dysfunction in low-birth-weight or early-weaned piglets while increasing their growth performance and survival. In addition, supplementing 1% Gln to a corn- and soybean-meal-based diet between d 90 and 114 of gestation ameliorates fetal growth retardation in gilts and reduces preweaning mortality of piglets. Furthermore, dietary supplementation with 1% Gln enhances milk production by lactating sows. Thus, adequate amounts of dietary Gln, a major nutrient, are necessary to support the maximum growth, development, and production performance of swine.

Lactating Porcine Mammary Tissue Catabolizes Branched-Chain Amino Acids for Glutamine and Aspartate Synthesis

The uptake of branched-chain amino acids (BCAA) from plasma by lactating porcine mammary gland substantially exceeds their output in milk, whereas glutamine output is 125% greater than its uptake from plasma. In this study, we tested the hypothesis that BCAA are catabolized for glutamine synthesis in mammary tissue. Mammary tissue slices from sows on d 28 of lactation were incubated at 37 degrees C for 1 h in Krebs buffer containing 0.5 or 2 mmol/L l-[1-(14)C]- or l-[U-(14)C]-labeled leucine, isoleucine, or valine. Rates of BCAA transport and degradation in mammary tissue were high, with approximately 60% of transaminated BCAA undergoing oxidative decarboxylation and the remainder being released as branched-chain alpha-ketoacids (BCKA). Most ( approximately 70%) of the decarboxylated BCAA were oxidized to CO(2). Rates of net BCAA transamination were similar to rates of glutamate, glutamine, aspartate, asparagine, and alanine synthesis. Consistent with the metabolic data, mammary tissue expressed BCAA aminotransferase (BCAT), BCKA decarboxylase, glutamine synthetase (GS), glutamate-oxaloacetate aminotransferase, glutamate-pyruvate aminotransferase, and asparagine synthetase, but no phosphate-activated glutaminase, activity. Western blot analysis indicated relatively high levels of mitochondrial and cytosolic isoforms of BCAT, as well as BCKA dehydrogenase and GS proteins in mammary tissue. Our results demonstrate that glutamine and aspartate (abundant amino acids in milk protein) were the major nitrogenous products of BCAA catabolism in lactating porcine mammary tissue and provide a biochemical basis to explain an enrichment of glutamine and aspartate in sow milk.

Effect of partitioning pens on aggressive behavior of pigs regrouped at weaning

Two trials were conducted to evaluate the effect on agonistic behavior of a temporary partition to create a more complex pen for newly weaned, crossbred pigs. The partition, which contained two 20 × 30 cm ports, was placed diagonally across alternate pens within the same room. Pigs were assigned to pens by body weight. Short (< 10 s) and long (⩾ 10 s) fights and the number of lying pigs were quantified by direct observation for the first 3 h and again for 1 h at 24 and 48 h post-grouping. The number of scratches on each pig were counted 25 h post-grouping. The treatments did not influence aggressive behavior of the piglets. There was a significant difference (P = 0.003) in agonistic behavior between the two trials. Long fights were significantly correlated with ear (P = 0.05), shoulder (P = 0.03) and total (P = 0.03) scratches. The heavier pigs were associated with more shoulder (P = 0.006) and total (P = 0.04) scratches. When a fight occurred, the non-combatants frequently moved to the other side of the partition, but this was variable. No pigs were seen using corners to avoid aggression. Using a partition to make weaning pens into a more complex environment did not reduce agonistic behavior or injuries caused by agonistic behavior.

Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes

Hypocitrullinemia and hypoargininemia but hyperprolinemia are associated with elevated plasma concentration of lactate in infants. Because the small intestine may be a major organ for initiating proline catabolism via proline oxidase in the body and is the major source of circulating citrulline and arginine in neonates, we hypothesized that lactate is an inhibitor of intestinal synthesis of citrulline and arginine from proline. To test this hypothesis, jejunum was obtained from 14-day-old suckling pigs for preparation of enterocyte mitochondria and metabolic studies. Mitochondria were used for measuring proline oxidase activity in the presence of 0-10 mM L-lactate. For metabolic studies, enterocytes were incubated at 37 degrees C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose, 2 mM L-glutamine, 2 mM L-[U-14C]proline, and 0, 1, 5, or 10 mM L-lactate. Kinetics analysis revealed noncompetitive inhibition of intestinal proline oxidase by lactate (decreased maximal velocity and unaltered Michaelis constant). Lactate had no effect on either activities of other enzymes for arginine synthesis from proline or proline uptake by enterocytes but decreased the synthesis of ornithine, citrulline, and arginine from proline in a concentration-dependent manner. These results demonstrate that lactate decreased intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase and provide a biochemical basis for explaining hyperprolinemia, hypocitrullinemia, and hypoargininemia in infants with hyperlactacidemia.Hypocitrullinemia and hypoargininemia but hyperprolinemia are associated with elevated plasma concentration of lactate in infants. Because the small intestine may be a major organ for initiating proline catabolism via proline oxidase in the body and is the major source of circulating citrulline and arginine in neonates, we hypothesized that lactate is an inhibitor of intestinal synthesis of citrulline and arginine from proline. To test this hypothesis, jejunum was obtained from 14-day-old suckling pigs for preparation of enterocyte mitochondria and metabolic studies. Mitochondria were used for measuring proline oxidase activity in the presence of 0-10 mMl-lactate. For metabolic studies, enterocytes were incubated at 37°C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mMd-glucose, 2 mMl-glutamine, 2 mMl-[U-14C]proline, and 0, 1, 5, or 10 mM l-lactate. Kinetics analysis revealed noncompetitive inhibition of intestinal proline oxidase by lactate (decreased maximal velocity and unaltered Michaelis constant). Lactate had no effect on either activities of other enzymes for arginine synthesis from proline or proline uptake by enterocytes but decreased the synthesis of ornithine, citrulline, and arginine from proline in a concentration-dependent manner. These results demonstrate that lactate decreased intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase and provide a biochemical basis for explaining hyperprolinemia, hypocitrullinemia, and hypoargininemia in infants with hyperlactacidemia.

Salivary and plasma cortisol response to adrenocorticotropin administration in pigs

Two experiments were conducted to determine the responsiveness of salivary and plasma cortisol to acute (i.v.), depot (i.m.) and chronic (repeated i.m.) adrenocorticotropin (ACTH) administration in swine. In Experiment 1, barrows (castrated pigs) were assigned to one of three injection treatments: (1) saline i.m. (SHAM1 n = 2); (2) 0.75 IU/kg BW ACTH in saline i.v. (ACUTE, n = 2); (3) 2.25 IU/kg BW ACTH in gel i.m. (DEPOT, n = 3). Total cortisol concentrations were determined for concurrent saliva and blood samples. Correlations between salivary and plasma cortisol within treatments were: SHAM1, r = 0.60; ACUTE, r = 0.58; DEPOT, r = 0.79. In Experiment 2, barrows were assigned to one of two injection treatments: (1) gel i.m. (SHAM2, n = 3); (2) 2.25 IU/kg BW ACTH in gel i.m. (CHRONIC, n = 4). The injections occurred every 6 h for a total of eight injections. Concurrent saliva and blood samples were obtained every 3 h for 72 h followed by an increasing sampling interval until day 6. Overall correlations between salivary and plasma cortisol were: SHAM2, r = 0.30 and CHRONIC, r = 0.61. Experiment 1 found that the relationship between salivary and plasma cortisol was stronger during longer (DEPOT) than shorter (ACUTE) ACTH stimulation. Experiment 2 found a strong relationship between the two measurements during chronic ACTH stimulation, but that relationship weakened after ACTH stimulation ceased.

Glutamine and glucose metabolism in intraepithelial lymphocytes from pre- and post-weaning pigs

The metabolism of glutamine (Gln) and glucose was studied in intraepithelial lymphocytes (IEL) from 21-, 29- and 56-day-old pigs. Pigs were weaned at 21 days of age. Cells were incubated at 37 degrees C in the presence of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 mM [U-14C]glutamine plus 5 mM glucose, or 5 mM [U-14C]glucose plus 1 mM glutamine. Glucose was converted to lactate, pyruvate and CO2, which accounted for 81, 11 and 8% of measured glucose carbon, respectively. Glutamine was metabolized mainly to glutamate (92% of Gln C) and ammonia, and to a lesser extent, to aspartate (4% of Gln C) and CO2 (4% of Gln C). In the presence of both glucose and glutamine, glucose provided 2-3-fold more ATP to IELs than glutamine in 21-56-day-old pigs, on the basis of their measured end products. The rates of ammonia and glutamate production from glutamine in IELs from 29-day-old pigs were 112 and 90% greater than those in cells from 56-day-old pigs, respectively. The rates of glucose oxidation to CO2 in IELs from 29-day-old pigs were elevated 56 and 64% respectively, compared with 21- and 56-day-old pigs. Elevated rates of substrate metabolism in IELs from 29-day-old post-weaning pigs indicated a metabolic alteration of these cells possibly due to changes in diet and intestinal bacterial population.

A diet containing myristoleic plus palmitoleic acids elevates plasma cholesterol in young growing swine.

The objective of this study was to test the effect of a novel fatty acid mixture, enriched with myristoleic and palmitoleic acids, on plasma lipoprotein cholesterol concentrations. Weanling pigs were assigned to one of six groups and each group received a diet differing in fatty acid composition. Diets were fed for 35 days and contained 10 g added cornstarch/100 g (to provide baseline data) or 10 g added fatty acids/100 g. For those diets containing added fatty acids, extracted lipids contained 36% myristoleic plus palmitoleic acid combined (14∶1/16∶1 diet), 52% palmitic acid (16∶0 diet), 51% stearic acid (18∶0 diet), 47% oleic acid (18∶1 diet), or 38% linoleic acid (18∶2 diet). Witht the exception of the cornstarch diet, all diets contained approximately 30% myristic acid. There were no significant differences in weight gain across treatment groups (P=0.22). All diets caused a significant increase in triglycerides and in total, low density lipoprotein, high density lipoprotein, and very low density lipoprotein cholesterol. The increase in total plasma cholesterol from pretreatment values was greatest in pigs fed the 14∶1/16∶1 and 18∶1 diets. However, the increase in low density lipoprotein cholesterol from the pretreatment concentration was greatest in the 14∶1/16∶1-fed pigs. Increases in very low density lipoprotein cholesterol above pretreatment concentrations were lowest in 16∶0-fed pigs and greatest in 18∶1-fed pigs. Dietary fatty acids elicited changes in plasma fatty acids which generally were reflective of the diets, although the 18∶0 diet did not alter plasma fatty acid concentrations and the 16∶0 diet increased plasma 16∶0 only at the end of the study. These results demonstrated that the combination of myristoleic plus palmitoleic acids increased plasma cholesterol in young pigs, suggesting that fatty acid chain length, rather than degree of unsaturation, is primarily responsible for the effects of fatty acids on circulating lipoprotein cholesterol concentrations.

Chapter 5 Amino acid metabolism in the small intestine: biochemical bases and nutritional significance

Publisher Summary This chapter reviews recent work on intestinal amino acid metabolism, with an emphasis on its biochemical bases and nutritional significance. The small intestine is a highly differentiated and complex organ, responsible for the terminal digestion and absorption of nutrients, and also plays an important role in amino acid metabolism. Most of glutamine and almost all of glutamate and aspartate in the diet are catabolized by the small intestinal mucosa in the first pass. The small intestinal mucosa also degrades enteral arginine, ornithine, proline, branched-chain amino acids, and lysine. In the postabsorptive state, the small intestine takes up arterial glutamine and releases ammonia, alanine, citrulline, and proline as the major nitrogenous products. The intestine-derived citrulline is effectively utilized for arginine synthesis by extrahepatic cells and organs. The extensive catabolism of enteral amino acids by the small intestine substantially reduces their availability to extraintestinal tissues and alters the patterns of amino acids that enter the systemic circulation. It has important practical implications for the utilization efficiency and recommended requirements of dietary protein and amino acids by animals, including humans.