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Long-chain n-3 fatty acids enhance neonatal insulin-regulated protein metabolism in piglets by differentially altering muscle lipid composition

2007; Elsevier BV; Volume: 48; Issue: 11 Linguagem: Inglês

10.1194/jlr.m700166-jlr200

ISSN

1539-7262

Autores

Karen Bergeron, Pierre Julien, Teresa A. Davis, Alexandre Myre, M. Carole Thivierge,

Tópico(s)

Muscle metabolism and nutrition

Resumo

This study investigated the role of long-chain n-3 polyunsaturated fatty acids (LCn-3PUFAs) of muscle phospholipids in the regulation of neonatal metabolism. Twenty-eight piglets were weaned at 2 days of age and raised on one of two milk formulas that consisted of either a control formula supplying 0% or a formula containing 3.5% LCn-3PUFAs until 10 or 28 days of age. There was a developmental decline in the insulin sensitivity of amino acid disposal in control pigs during the first month of life, with a slope of −2.24 μmol·kg−1·h−1 (P = 0.01) per unit of insulin increment, as assessed using hyperinsulinemic-euglycemic-euaminoacidemic clamps. LCn-3PUFA feeding blunted this developmental decline, resulting in differing insulin sensitivities (P < 0.001). When protein metabolism was assessed under parenteral feeding-induced hyperinsulinemia, LCn-3PUFAs reduced by 16% whole body oxidative losses of amino acids (from 238 to 231 μmol·kg−1·h−1; P = 0.06), allowing 41% more amino acids to accrete into body proteins (from 90 to 127 μmol·kg−1·h−1; P = 0.06). The fractional synthetic rate of muscle mixed proteins remained unaltered by the LCn-3PUFA feeding. However, LCn-3PUFAs retarded a developmental increase in the essential-to-nonessential amino acid ratio of the muscle intracellular free pool (P = 0.05). Overall, alterations in metabolism were concomitant with a preferential incorporation of LCn-3PUFAs into muscle total membrane phospholipids (P < 0.001), in contrast to intramuscular triglycerides. These results underscore the potential role of LCn-3PUFAs as regulators of different aspects of protein metabolism in the neonate. This study investigated the role of long-chain n-3 polyunsaturated fatty acids (LCn-3PUFAs) of muscle phospholipids in the regulation of neonatal metabolism. Twenty-eight piglets were weaned at 2 days of age and raised on one of two milk formulas that consisted of either a control formula supplying 0% or a formula containing 3.5% LCn-3PUFAs until 10 or 28 days of age. There was a developmental decline in the insulin sensitivity of amino acid disposal in control pigs during the first month of life, with a slope of −2.24 μmol·kg−1·h−1 (P = 0.01) per unit of insulin increment, as assessed using hyperinsulinemic-euglycemic-euaminoacidemic clamps. LCn-3PUFA feeding blunted this developmental decline, resulting in differing insulin sensitivities (P < 0.001). When protein metabolism was assessed under parenteral feeding-induced hyperinsulinemia, LCn-3PUFAs reduced by 16% whole body oxidative losses of amino acids (from 238 to 231 μmol·kg−1·h−1; P = 0.06), allowing 41% more amino acids to accrete into body proteins (from 90 to 127 μmol·kg−1·h−1; P = 0.06). The fractional synthetic rate of muscle mixed proteins remained unaltered by the LCn-3PUFA feeding. However, LCn-3PUFAs retarded a developmental increase in the essential-to-nonessential amino acid ratio of the muscle intracellular free pool (P = 0.05). Overall, alterations in metabolism were concomitant with a preferential incorporation of LCn-3PUFAs into muscle total membrane phospholipids (P < 0.001), in contrast to intramuscular triglycerides. These results underscore the potential role of LCn-3PUFAs as regulators of different aspects of protein metabolism in the neonate. isotopic enrichments long-chain n-3 polyunsaturated fatty acid protein breakdown protein synthesis Considerable attention has been devoted to the refinement of the nutritional composition of human infant formula. Recent improvements in formulation include the incorporation of long-chain n-3 polyunsaturated fatty acids (LCn-3PUFAs) in conjunction with arachidonic acid, because these play a prominent role in visual and neural development and in the biogenesis of prostaglandins and leukotrienes (1.Makrides M. Gibson R.A. Udell T. Ried K. Supplementation of infant formula with long-chain polyunsaturated fatty acids does not influence the growth of term infants.Am. J. Clin. Nutr. 2005; 81: 1094-1101Crossref PubMed Scopus (83) Google Scholar, 2.Lauritzen L. Hansen H.S. Jorgensen M.H. Michaelsen K.F. The essentiality of long chain n-3 fatty acids in relation to development and function of the brain and retina.Prog. Lipid Res. 2001; 40: 1-94Crossref PubMed Scopus (831) Google Scholar). Neonatal nutrition, which typically involves feeding numerous meals of small size to the newborn, also affects certain regulatory aspects of postprandial metabolism that are of paramount importance for development. Insulin is a primary regulator of postprandial metabolism, and the high insulin sensitivity of the neonate enables a highly efficient anabolism that critically sustains neonatal growth. As development progresses, the skeletal musculature becomes less sensitive to insulin (3.Wray-Cahen D. Beckett P.R. Nguyen H.V. Davis T.A. Insulin-stimulated amino acid utilization during glucose and amino acids clamps decreases with development.Am. J. Physiol. Endocrinol. Metab. 1997; 273: E305-E314Crossref PubMed Google Scholar, 4.Davis T.A. Burrin D.G. Fiorotto M.L. Reeds P.J. Jahoor F. Roles of insulin and amino acids in the regulation of protein synthesis in the neonate.J. Nutr. 1998; 128: 347-350Crossref Google Scholar), and this is parallel to a simultaneous reduction in muscle anabolism. The mechanisms behind this developmental regulation include a reduction in both the responsiveness and the sensitivity of muscle to insulin (3.Wray-Cahen D. Beckett P.R. Nguyen H.V. Davis T.A. Insulin-stimulated amino acid utilization during glucose and amino acids clamps decreases with development.Am. J. Physiol. Endocrinol. Metab. 1997; 273: E305-E314Crossref PubMed Google Scholar, 4.Davis T.A. Burrin D.G. Fiorotto M.L. Reeds P.J. Jahoor F. Roles of insulin and amino acids in the regulation of protein synthesis in the neonate.J. Nutr. 1998; 128: 347-350Crossref Google Scholar). Refinements of knowledge regarding insulin action on body tissues are critically relevant to the development of new approaches targeting the prevention and treatment of growth retardation, preterm birth nutrition, and early life programming. Insulin resistance leading to defects in glucose metabolism in certain pathological states, such as obesity, type II diabetes, and insulin resistance attributable to high-fat feeding, can be improved through dietary LCn-3PUFAs from fish oil (5.Liu S. Baracos V.E. Quinney H.A. Clandinin M.T. Dietary omega-3 and polyunsaturated fatty acids modify fatty acyl composition and insulin binding in skeletal-muscle sarcolemma.Biochem. J. 1994; 299: 831-837Crossref PubMed Scopus (94) Google Scholar, 6.Borkman M. Storlien L.H. Pan D.A. Jenkins A.B. Chishlom D.J. Campbell L.V. The relation between insulin sensitivity and the fatty-acid composition of skeletal-muscle phospholipids.N. Engl. J. Med. 1993; 328: 238-244Crossref PubMed Scopus (781) Google Scholar, 7.Storlien L.H. Pan D.A. Kriketos A.D. O'Connor J. Caterson I.D. Cooney G.J. Jenkins A.B. Baur L.A. Skeletal muscle membrane lipids and insulin resistance.Lipids. 1996; 31: 261-265Crossref PubMed Google Scholar). In such instances, enhanced glucose metabolism in response to insulin stimulation relies on the complex biophysical and biochemical effects of highly unsaturated fatty acid content in membranes. The latter includes enhanced membrane fluidity that affects the functions of membranes and transmembrane proteins and membrane-associated enzymes, which also can lead to alterations in the activation of insulin signaling intermediates (8.Daveloose D. Linard A. Asfi T. Viret J. Christon R. Simultaneous changes in lipid composition, fluidity and enzyme activity in piglet intestinal brush border membrane as affected by dietary polyunsaturated fatty acid deficiency.Biochim. Biophys. Acta. 1993; 1166: 229-237Crossref PubMed Scopus (38) Google Scholar, 9.Taouis M. Dagou C. Ster C. Durand G. Pinault M. Delarue J. N-3 polyunsaturated fatty acids prevent the defect of insulin receptor signaling in muscle.Am. J. Physiol. Endocrinol. Metab. 2002; 282: E664-E671Crossref PubMed Scopus (144) Google Scholar, 10.Kamada T. Yamashita T. Baba Y. Kai M. Setoyama S. Chuman Y. Otsuji S. Dietary sardine oil increases erythrocyte membrane fluidity in diabetic patients.Diabetes. 1986; 35: 604-611Crossref PubMed Scopus (92) Google Scholar). To date, most of the focus has been devoted to the improvement of glucose metabolism for different dysregulations of insulin action. LCn-3PUFAs were recently shown to be effective at upregulating protein anabolism through enhanced insulin sensitivity in a healthy growing animal model that was otherwise more insulin-resistant as a result of maturity (11.Gingras A.A. White P.J. Chouinard P.Y. Julien P. Davis T.A. Dombroski L. Couture Y. Dubreuil P. Myre A. Bergeron K. et al.Long-chain omega-3 fatty acids regulate whole-body protein metabolism by promoting muscle insulin signaling to the Akt-mTOR-S6K1 pathway and insulin sensitivity.J. Physiol. (Lond.). 2007; 579: 269-284Crossref Scopus (142) Google Scholar). Increased insulin-mediated amino acid disposal and activation of Akt-mTOR-S6K1 insulin signaling machinery, combined with reduced whole body oxidative metabolism, were among the protein-anabolic benefits observed (11.Gingras A.A. White P.J. Chouinard P.Y. Julien P. Davis T.A. Dombroski L. Couture Y. Dubreuil P. Myre A. Bergeron K. et al.Long-chain omega-3 fatty acids regulate whole-body protein metabolism by promoting muscle insulin signaling to the Akt-mTOR-S6K1 pathway and insulin sensitivity.J. Physiol. (Lond.). 2007; 579: 269-284Crossref Scopus (142) Google Scholar). The current study was undertaken to establish whether LCn-3PUFAs would similarly affect protein metabolism in the neonatal pig to explore new avenues for intervening at different stages of development. Twenty-eight cross-bred male piglets [Duroc×(Yorshire×Landrace)] were purchased at 2 days of age (1.97 ± 0.3 kg; Alfred Couture Ltée, St. Anselme, Québec, Canada). Piglets were weighed and randomly assigned to a 2 × 2 factorial arrangement of treatments according to a completely randomized design. The latter consisted of two age groups: 10 or 28 days of age, and of two semipurified milk formulas: control (0% LCn-3PUFAs) or enriched to 3.5% LCn-3PUFAs on formula total dry matter. Control oil was substituted on an isoenergetic basis with menhaden oil (Table 1). Five hours after their arrival, piglets were introduced to their respective milk formula and raised on these. Piglets were housed individually and had free access to water. Twice a week, they were weighed on two consecutive days throughout the study.TABLE 1.Ingredients and chemical composition of milk formulas fed to neonatal pigletsIngredients and CompositionControlLCn-3PUFAIngredient (% dry matter basis) LactoseaGrober Nutrition, Inc. (Cambridge, Ontario, Canada).31.031.0 Sodium caseinateaGrober Nutrition, Inc. (Cambridge, Ontario, Canada).16.516.5 Whey proteinaGrober Nutrition, Inc. (Cambridge, Ontario, Canada).9.89.8 Plasma proteinbNutrapro; JEFO Nutrition (St. Hyacinthe, Quebec, Canada).3.03.0 Mineral and vitamin mixaGrober Nutrition, Inc. (Cambridge, Ontario, Canada).5.55.5 Palm oilcBedessee (Toronto, Ontario, Canada).20.021.6 Cottonseed oildCedar Vale Natural Health (Cedar Vale, KS).6.20.0 Coconut oileBunge Canada (Oakville, Ontario, Canada).4.00.0 Menhaden oilfOmega Protein, Inc. (Reedville, VA).0.09.3 Soybean lecithineBunge Canada (Oakville, Ontario, Canada).2.72.7 Soybean emulsifieraGrober Nutrition, Inc. (Cambridge, Ontario, Canada).0.70.7Chemical composition (% dry matter basis) CarbohydratesgEstimated by difference: carbohydrates = total dry matter − [crude protein + fat + ashes].26.426.1 Crude protein29.829.9 Fat38.238.4 Ashes5.65.6LCn-3PUFA, long-chain n-3 polyunsaturated fatty acids. Milk replacers had a chemical composition similar to that of the sow's milk (13.Pond W.G. Mersmann H.J. Biology of the Domestic Pig. Cornell University Press, Ithaca, NY2001Google Scholar).a Grober Nutrition, Inc. (Cambridge, Ontario, Canada).b Nutrapro; JEFO Nutrition (St. Hyacinthe, Quebec, Canada).c Bedessee (Toronto, Ontario, Canada).d Cedar Vale Natural Health (Cedar Vale, KS).e Bunge Canada (Oakville, Ontario, Canada).f Omega Protein, Inc. (Reedville, VA).g Estimated by difference: carbohydrates = total dry matter − [crude protein + fat + ashes]. Open table in a new tab LCn-3PUFA, long-chain n-3 polyunsaturated fatty acids. Milk replacers had a chemical composition similar to that of the sow's milk (13.Pond W.G. Mersmann H.J. Biology of the Domestic Pig. Cornell University Press, Ithaca, NY2001Google Scholar). Two semipurified milk formulas were formulated to meet piglet nutritional requirements (Table 1) (12.National Research CouncilNutrient Requirements of Swine. 10th revised edition. National Academy of Sciences, Washington, DC1998Google Scholar). These were isoenergetic and isonitrogenous, and they differed only in their fatty acid composition (Table 2). The composition of the formula's fatty acids and macronutrients was similar to that of the sow's milk (13.Pond W.G. Mersmann H.J. Biology of the Domestic Pig. Cornell University Press, Ithaca, NY2001Google Scholar). Oils in the milk formulas were emulsified by heating at 50°C for 15 min, with soybean lecithin and a soybean emulsifier, before mixing with other constituents. Milk replacers were offered at a minimum of 6.25% body weight on a dry matter basis to ensure ad libitum consumption (12.National Research CouncilNutrient Requirements of Swine. 10th revised edition. National Academy of Sciences, Washington, DC1998Google Scholar). The dry matter concentration of the formulas was initially 10% and was increased gradually to 15% until 28 days of age, taking care to avoid diarrhea. Feeding frequency of the newborn consisted of five diurnal meals with a maximum of a 12 h overnight fast; this was reduced to four diurnal meals with a maximum of a 12 h overnight fast at 14 days old onward.TABLE 2.Fatty acid composition of milk fat formulas fed to neonatal pigletsMilk FormulaFatty AcidControlLCn-3PUFAC8:00.26NDC10:00.72NDC12:06.490.74C14:03.993.45C16:033.3034.81C16:1 n-70.322.95C18:04.524.78C18:130.9731.54C18:2 n-618.269.54C18:3 n-30.570.88C18:4 n-30.251.22C20:00.340.38C20:1ND0.55C20:4 n-6ND0.27C20:4 n-3ND0.56C20:5 n-3ND3.29C22:5 n-3ND0.69C22:6 n-3ND4.35SAT49.644.2PUFA19.120.8Total n-618.39.8Total n-30.811.0n-3/n-60.041.12LCn-n-3PUFAND8.3aEquivalent to 3.5% LCn-3PUFAs on a dry matter basis of total formula constituents.PUFA/SAT0.380.47Values shown are percentage of total fatty acids. SAT, sum of saturated fatty acids; PUFA, sum of polyunsaturated fatty acids; Total n-6, sum of n-6 fatty acids; Total n-3, sum of n-3 fatty acids; n-3/n-6, ratio of total n-3 to total n-6 fatty acids; LCn-3PUFA, 20:5 n-3 + 22:5 n-3 + 22:6 n-3; PUFA/SAT, ratio of polyunsaturated fatty acids to saturated fatty acids; ND, not detected.a Equivalent to 3.5% LCn-3PUFAs on a dry matter basis of total formula constituents. Open table in a new tab Values shown are percentage of total fatty acids. SAT, sum of saturated fatty acids; PUFA, sum of polyunsaturated fatty acids; Total n-6, sum of n-6 fatty acids; Total n-3, sum of n-3 fatty acids; n-3/n-6, ratio of total n-3 to total n-6 fatty acids; LCn-3PUFA, 20:5 n-3 + 22:5 n-3 + 22:6 n-3; PUFA/SAT, ratio of polyunsaturated fatty acids to saturated fatty acids; ND, not detected. Three days before the onset of measurements (at 5 and 23 days old), piglets were anesthetized for surgical procedures after an overnight fast, as described previously (14.Thivierge M.C. Bush J.A. Suryawan A. Nguyen H.V. Orellana R.A. Burrin D.G. Jahoor F. Davis T.A. Whole body and hindlimb protein breakdown are differentially altered by feeding in neonatal piglets.J. Nutr. 2005; 35: 1430-1435Crossref Scopus (13) Google Scholar). A catheter for sampling was placed in a carotid artery, and an infusion catheter was placed in a jugular vein. Catheters were kept patent by filling with heparinized saline (200 IU·ml−1). Piglets were returned to their crate after surgery. The experimental procedures were approved by the Animal Care and Use Committee of the Université Laval and were conducted in accordance with Canadian Council on Animal Care guidelines (15.Canadian Council on Animal CareGuide to Care and Use of Experimental Animals. 2nd edition. Bradda Printing Services, Inc., Ottawa, Ontario, Canada1993Google Scholar). Insulin sensitivity of whole-body amino acid and glucose disposal was measured at either 7.5 ± 0.5 days of age (2.2 ± 0.3 kg body weight) or 26 ± 0.5 days of age (6.9 ± 1 kg body weight) in six piglets per milk replacer per age, for n = 12 per age group as a result of catheter patency, using the hyperinsulinemic-euglycemic-euaminoacidemic clamp procedure (3.Wray-Cahen D. Beckett P.R. Nguyen H.V. Davis T.A. Insulin-stimulated amino acid utilization during glucose and amino acids clamps decreases with development.Am. J. Physiol. Endocrinol. Metab. 1997; 273: E305-E314Crossref PubMed Google Scholar). After an overnight fast with free access to water, piglets were placed in a sling restraining system. Insulin stock solutions (1 mg·ml−1) were prepared daily by dissolving lyophilized porcine insulin (29.2 IU/mg; I-5523; Sigma Chemical, St. Louis, MO) in 0.01 N HCl followed by mixing with sterile physiological saline containing 4% filter-sterilized porcine plasma. Insulin was infused into the jugular vein at 100 ng·(kg−0.66)−1·min−1. This infusion rate was specifically selected to generate fed plasma insulinemia that approximates 30 μU·ml−1 at both ages (16.Wray-Cahen D. Nguyen H.V. Burrin D.G. Beckett P.R. Fiorotto M.L. Reeds P.J. Wester T.J. Davis T.A. Response of skeletal muscle protein synthesis to insulin in suckling pigs decreases with development.Am. J. Physiol. Endocrinol. Metab. 1998; 275: E602-E609Crossref PubMed Google Scholar). A constant insulin infusion rate (12 ml·h−1) was used by individually preparing insulin syringes and by diluting the appropriate amount of stock solution with physiological saline. Fasting baseline concentrations of glucose and branched-chain amino acids, the latter used as an index of essential amino acid concentration, were established by sampling three arterial blood samples at 5 min intervals before the onset of insulin infusion. Glucose was analyzed immediately in fresh whole blood by peroxidase reaction (YSI 2300 STAT Plus analyzer; Yellow Springs Instruments, Yellow Springs, OH). A branched-chain amino acid assay was performed on plasma according to the enzymatic method of Beckett et al. (17.Beckett P.R. Hardin D.S. Davis T.A. Nguyen H.V. Wray-Cahen D. Copeland K.C. Spectrophotometric assay for measuring branched-chain amino acid concentrations: application for measuring the sensitivity of protein metabolism to insulin.Anal. Biochem. 1996; 240: 48-53Crossref PubMed Scopus (61) Google Scholar). Remaining plasma was frozen at −20°C until further analyses of baseline concentrations of insulin and amino acids were conducted. Once glucose and branched-chain amino acid baselines were established, the clamp was initiated with a 10 min priming insulin infusion (18.DeFronzo R.A. Tobin J.D. Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance.Am. J. Physiol. Endocrinol. Metab. 1979; 237: E214-E223Crossref PubMed Google Scholar). During the clamp procedure, blood samples were taken every 5 min and immediately analyzed for glucose and branched-chain amino acids. Glucose and amino acid concentrations were maintained at ±10% of baseline concentrations by adjustments of auxiliary infusions of dextrose [50% (w/v) sterile solution; Baxter Healthcare, Deerfield, IL] and a complete amino acid solution using Plum Lifecare pumps (series 1.6; Abbott Laboratories, Chicago, IL). The amino acid mixture used was adapted from Davis et al. (19.Davis T.A. Fiorotto M.L. Burrin D.G. Reeds P.J. Nguyen H.V. Beckett P.R. Vann R.C. O'Connor P.M.J. Stimulation of protein synthesis by both insulin and amino acids is unique to skeletal muscle in neonatal pigs.Am. J. Physiol. 2002; 282: E880-E890Crossref PubMed Scopus (169) Google Scholar) using the l-form of medical-grade amino acids as follows (mmol·l−1): alanine, 27.3; arginine, 20.1; aspartic acid, 12.0; glutamic acid, 23.9; glycine, 54.3; histidine, 17.2; isoleucine, 28.5; leucine, 34.2; lysine, 27.4; methionine, 15.1; phenylalanine, 15.5; proline, 34.8; serine, 23.8; threonine, 21.1; tryptophan, 4.4; tyrosine, 1.1; and valine, 34.1 (Ajinomoto, Sigma-Aldrich, Oakville, Ontario, Canada). Cysteine and glutamine were not added to the mixture because of solubility and stability problems. The clamp procedures lasted 150 min on average, and a 90 min period was required to reach steady utilization rates; an additional 60 min was allocated for the monitoring of glucose and amino acid disposal rates. During the last steady 45 min of the clamp procedures, four blood samples were collected at 15 min intervals. These were centrifuged and the plasma was frozen at −20°C until further analyses of insulin and individual amino acids were conducted. Whole body protein metabolism was measured at 2 days after the clamp procedure at either 10 ± 0.5 days of age (2.7 ± 0.4 kg body weight) or 28 ± 0.8 days of age (7.5 ± 1.4 kg body weight) on seven piglets per milk replacer per age group for n = 14 piglets per age. Phenylalanine kinetics was assessed at a fed steady state after 12 h of food deprivation. The total parenteral nutrition was selected to elicit fed plasma insulin concentrations (20.Burrin D.G. Stoll B. Jiang R. Cahng X. Hartmann B. Holst H.J. Greeley G.H. Reeds P.J. Minimal enteral nutrient requirements for intestinal growth in neonatal piglets: how much is enough?.Am. J. Clin. Nutr. 2000; 71: 1603-1610Crossref PubMed Scopus (192) Google Scholar, 21.Bertolo R.F.P. Chen C.Z.L. Law G. Pencharz P.B. Ball R.O. Threonine requirement of neonatal piglets receiving total parenteral nutrition is considerably lower than that of piglets receiving an identical diet intragastrically.J. Nutr. 1998; 128: 1752-1759Crossref PubMed Scopus (116) Google Scholar). Tracers used to conduct phenylalanine kinetics studies were as follows: l-[1-13C]phenylalanine (97.9% mole percent excess; Cambridge Isotopes Laboratories, Andover, MA), NaH13CO3 (99% atom percent excess; Cambridge Isotopes Laboratories), and NaH14CO3 (2 mCi·ml−1; MP Biochemicals, Inc., Irvine, CA). The tracer used for the quantification of plasma phenylalanine using the isotopic ratio method (22.Calder A.G. Garden K.E. Anderson S.E. Lobley G.E. Quantification of blood and plasma amino acids using isotope dilution electron impact gas chromatography/mass spectrometry with U-13C amino acids as internal standards.Rapid Commun. Mass Spectrom. 1999; 13: 2080-2083Crossref PubMed Scopus (149) Google Scholar) on blood collected during the phenylalanine kinetics was l-[ring-2H5]phenylalanine (98% mole percent excess; Cambridge Stable Isotopes). Stock solutions of labeled phenylalanine were dissolved in 0.1 N HCl; stock solutions of labeled bicarbonate were dissolved in 0.05 M NaOH. Whole body phenylalanine kinetics were assessed over a 240 min period with piglets restrained in a sling system. The onset of the kinetic study was preceded by three arterial background samples taken every 10 min over a 30 min period to determine the natural abundance of plasma phenylalanine carbon isotope, with basal concentrations of glucose, phenylalanine, and insulin. According to a similar schedule, breath gases were sampled using a facemask equipped with a one-way valve bag for determination of CO2 isotopic ratios. Four separate lines were used for infusions: l-[1-13C]phenylalanine, NaH14CO3, fat emulsion, and the mixture of the remaining constituents of the parenteral nutrition (glucose, amino acids, vitamins, minerals). These were connected to the venous catheter using three-way valves. At time 0, body pools were primed for l-[1-13C]phenylalanine (22 μmol·kg−1), NaH13CO3 (6 μmol·kg−1), and NaH14CO3 (0.75 μCi·kg−1). Continuous infusions of l-[1-13C]phenylalanine (22 μmol·kg−1·h−1), NaH14CO3 (1 μCi·kg−1·h−1), intralipids (2.1 ml·kg−1·h−1), and the remaining parenteral nutrition (7.9 ml·kg−1·h−1) were immediately initiated and maintained throughout the 240 min period. After 195 min of infusion, four blood and breath samples were taken at 15 min intervals. Glucose and blood gas (ABL 77 series; Radiometer Medical, Copenhagen, Denmark) were immediately measured on fresh blood. A subsample of fresh blood was processed for the measurement of blood specific radioactivity (14.Thivierge M.C. Bush J.A. Suryawan A. Nguyen H.V. Orellana R.A. Burrin D.G. Jahoor F. Davis T.A. Whole body and hindlimb protein breakdown are differentially altered by feeding in neonatal piglets.J. Nutr. 2005; 35: 1430-1435Crossref Scopus (13) Google Scholar). Remaining blood was centrifuged and kept frozen (−20°C) until analyses of plasma amino acid and insulin concentrations and of plasma phenylalanine isotopic enrichments were conducted. At 240 min, the infusions were stopped and the piglets were euthanized by an intravenous lethal injection (pentobarbital sodium, 50 mg·kg−1; CDMV, St. Hyacinthe, Quebec). Longissimus dorsi skeletal muscle was rapidly sampled and immediately frozen in liquid N2. Muscle samples were stored at −80°C for subsequent analyses of fatty acid profiling of total membrane phospholipids and intramuscular triglycerides. The concentrations of free amino acids plus the isotopic labeling of free and bound phenylalanine in muscle mixed protein homogenate were also determined. During the 240 min period of the phenylalanine kinetic studies, piglets continuously received total parenteral nutrition adapted from Burrin et al. (20.Burrin D.G. Stoll B. Jiang R. Cahng X. Hartmann B. Holst H.J. Greeley G.H. Reeds P.J. Minimal enteral nutrient requirements for intestinal growth in neonatal piglets: how much is enough?.Am. J. Clin. Nutr. 2000; 71: 1603-1610Crossref PubMed Scopus (192) Google Scholar) and Bertolo et al. (21.Bertolo R.F.P. Chen C.Z.L. Law G. Pencharz P.B. Ball R.O. Threonine requirement of neonatal piglets receiving total parenteral nutrition is considerably lower than that of piglets receiving an identical diet intragastrically.J. Nutr. 1998; 128: 1752-1759Crossref PubMed Scopus (116) Google Scholar) (Table 3). The amino acid composition was modified to approximate that of the sow's milk protein. Vitamins and minerals were added to the parenteral nutrition solution on the morning of their use. Emulsified lipids (20% Intralipid®, composition weight·100 ml−1: 20 g of sterile emulsion purified soybean oil, 1.2 g of purified egg phospholipids, 2.2 g of anhydrous glycerol, and water; Fresenius Kabi, Uppsala, Sweden), were administered using a separate line. The parenteral nutrition, including intralipids, provided 14.5 g amino acids·kg−1·day−1 and 1.1 MJ metabolizable energy·kg−1·day−1, for which glucose and lipid each supplied 50% of nonprotein energy intake (21.Bertolo R.F.P. Chen C.Z.L. Law G. Pencharz P.B. Ball R.O. Threonine requirement of neonatal piglets receiving total parenteral nutrition is considerably lower than that of piglets receiving an identical diet intragastrically.J. Nutr. 1998; 128: 1752-1759Crossref PubMed Scopus (116) Google Scholar).TABLE 3.Total parenteral nutrition continuously infused into the jugular vein of neonatal piglets during steady-fed-state phenylalanine kinetic studiesIngredient, MacronutrientsAmountIngredient, MicronutrientsAmountGlucose, mmol·l−1526.1MineralIntralipids 20%, ml·l−1210.9aThis amount provided 42.2 ml of intralipids in total per liter of elementary diet. NaCl, mmol·l−119.2Amino acids, mmol·l−1 [(mmol/l)(mmol/l)]2470.2 NaOH, mmol·l−130.3 Alanine32.7 K acetate, mmol·l−17.7 Arginine14.6 KPO4, mmol·l−122.3 Aspartic acid15.8 Mg sulfate, mmol·l−13.2 Glutamic acid23.1 Ca gluconate, mmol·l−15.1 Glycine23.0 Zn, mg·l−16.24 Histidine12.7 Mn, mg·l−11.25 Isoleucine29.9 Cu, mg·l−11.56 Leucine53.6 Cr, mg·l−115.6 Lysine32.9 Se, μg·l−1133.3 Methionine11.9Vitamin Phenylalanine30.8 Folic acid, mg·l−10.13 Proline59.2 Thiamin, mg·l−18.0 Serine54.6 Riboflavin, mg·l−10.4 Threonine28.5 Pyridoxine, mg·l−10.4 Tryptophan5.6 Penthotenol, mg·l−10.8 Tyrosine1.1 Niacin, mg·l−110.0 Valine40.3 Vitamin B12, μg·l−18.0 Vitamin A, IU·l−1916.7 Vitamin D, IU·l−191.7 Vitamin E, IU·l−12.75Adapted from Bertolo et al. (21.Bertolo R.F.P. Chen C.Z.L. Law G. Pencharz P.B. Ball R.O. Threonine requirement of neonatal piglets receiving total parenteral nutrition is considerably lower than that of piglets receiving an identical diet intragastrically.J. Nutr. 1998; 128: 1752-1759Crossref PubMed Scopus (116) Google Scholar) and Burrin et al. (20.Burrin D.G. Stoll B. Jiang R. Cahng X. Hartmann B. Holst H.J. Greeley G.H. Reeds P.J. Minimal enteral nutrient requirements for intestinal growth in neonatal piglets: how much is enough?.Am. J. Clin. Nutr. 2000; 71: 1603-1610Crossref PubMed Scopus (192) Google Scholar). The parenteral nutrition composition was 48% glucose, 21% intralipids, and 31% protein on a dry matter basis; vitamin and mineral volume represented 7.6% of the nutritive solution. In total, the parenteral nutrition provided 60 g of medical-grade l-amino acids (Sigma-Aldrich) per solution liter; 75% of gross energy was provided by nonprotein nutrients such as glucose and fat at a ratio of 50% each (based on equivalence of physiological fuel values). Gross energy from nonprotein nutrients represented 2.2 MJ metabolizable energy intake·kg−1·d−1.a This amount provided 42.2 ml of intralipids in total per liter of elementary diet. Open table in a new tab Adapted from Bertolo et al. (21.Bertolo R.F.P. Chen

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