Surplus dietary tryptophan inhibits stress hormone kinetics and induces insulin resistance in pigs

Physiology & Behavior 98 (2009) 402–410

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Physiology & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p h b

Surplus dietary tryptophan inhibits stress hormone kinetics and induces insulin resistance in pigs Sietse Jan Koopmans a,⁎, Marko Ruis b, Ruud Dekker a, Mechiel Korte c a b c

BioMedical Research of Wageningen University and Research Centre, 8219 PH Lelystad, The Netherlands Animal Sciences Group of Wageningen University and Research Centre, 8219 PH Lelystad, The Netherlands Rudolf Magnus Institute, Department of Neuroscience and Pharmacology, Utrecht University, 3584 CG Utrecht, The Netherlands

a r t i c l e

i n f o

Article history: Received 30 January 2009 Received in revised form 18 June 2009 Accepted 8 July 2009 Keywords: Pigs Tryptophan Large neutral amino acids Cortisol Adrenaline Noradrenaline Insulin sensitivity Glucose metabolism Protein metabolism

a b s t r a c t Recently we have shown that surplus dietary tryptophan (TRP) reduced the plasma concentrations of cortisol and noradrenaline in pigs. Stress hormones are known to affect insulin sensitivity and metabolism. We now investigated the long-term effects of surplus dietary TRP on 1) plasma and urinary stress hormone kinetics, 2) insulin sensitivity for glucose and amino acid clearance, and 3) whole body nitrogen balance. Pigs were fed for 3 weeks a high (13.2%) vs normal (3.4%) TRP to large neutral amino acids (LNAA) diet, leading to reduced fasting (14 h) plasma cortisol (17.1 ± 3.0 vs 28.9 ± 4.3 ng/mL, p b 0.05) and noradrenaline (138 ± 14 vs 225 ± 21 pg/mL, p b 0.005) concentrations, lower daily urinary noradrenaline (313 ± 32 vs 674 ± 102 ng/kg day, p b 0.001) and adrenaline (124 ± 13 vs 297 ± 42 ng/kg day, p b 0.001) but higher dopamine (5.8 ± 0.5 vs 1.5 ± 0.2 µg/kg day, p b 0.001) excretions, respectively. Insulin sensitivities for both glucose and amino acid clearance, (as measured by the intraportal hyperinsulinaemic (1 mU/kg min) euglycaemic euaminoacidaemic clamp technique), were lower by 22% in pigs on the high vs normal TRP/LNAA diet (14.8 ± 1.4 vs 18.9 ± 0.9, p b 0.05 and 69.7 ± 4.3 vs 89.7 ± 6.8 mL/kg min, p b 0.05, respectively) without affecting urinary nitrogen excretion (35.5 ± 1.0 vs 36.6 ± 1.0% of dietary nitrogen intake, p = ns). In conclusion, long-term feeding of surplus dietary TRP inhibits both baseline adrenocortical and sympathetic nervous system activity, it induces insulin resistance for both glucose and amino acid clearance but it does not affect whole body protein catabolism. This indicates that the bioactive amino acid TRP contributes to homeostasis in neuroendocrinology and insulin action and that low baseline adrenocortical and sympatho-adrenal axis activity are associated with insulin resistance. © 2009 Elsevier Inc. All rights reserved.

1. Introduction The amino acid tryptophan (TRP) is an essential component of food and animal feed. In growing pigs, the TRP requirement for maximum growth and development is approximately 2 g/kg feed [1,2]. Apart from its effect on growth and protein accretion, supplemental TRP (+5 g/kg feed) has been shown to affect brain and nervous system functioning through interference with serotonergic neurotransmission [3–5]. Brain serotonin is known to modulate plasma glucose homeostasis and its regulating hormones [6]. TRP serves as the immediate precursor for serotonin synthesis, and as such, surplus dietary TRP has been shown to increase plasma TRP concentrations and to boost serotonergic activity by mass action [5]. TRP-modulated serotonergic activity, ranging from deficient to surplus TRP, has been implicated in the regulation of many behavioural and physiological processes such as food intake, mood, aggression, stress susceptibility, cortisol, noradre⁎ Corresponding author. Edelhertweg 15, P.O. Box 65, 8200 AB Lelystad, The Netherlands. Tel.: +31 320 237327; fax: +31 320 237320. E-mail address: [email protected] (S.J. Koopmans). 0031-9384/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2009.07.001

naline and insulin secretion [2,7–11]. In more detail, we have recently shown that surplus dietary TRP reduces the long-term plasma cortisol, noradrenaline and adrenaline responses after acute stress in pigs [8]. Stress hormones are considered to be insulin antagonistic and, in general, catabolic by nature with respect to their ability to stimulate glycogenolysis, lipolysis and, specific for cortisol, to stimulate proteolysis [12–16]. If surplus dietary TRP reduces stress hormone concentrations, it may therefore be expected that pigs on a TRP enriched diet show increased insulin sensitivity and reduced protein and amino acid catabolism. As such, a TRP-induced chronic depression of plasma stress hormone concentrations could favour insulin action and nitrogen retention in growing pigs. However, it is unknown whether long-term feeding of surplus dietary TRP affects insulin sensitivity and protein metabolism in vivo. Delivery of TRP to the brain is not only determined by the concentration of TRP itself but also by the concentration of other large neutral amino acids (LNAA) in blood. LNAA (isoleucine, leucine, valine, phenylalanine, and tyrosine) compete with TRP for passage through the blood– brain barrier and may therefore interfere with TRP for serotonin synthesis [17]. To maximize TRP delivery to the brain on a long-term

S.J. Koopmans et al. / Physiology & Behavior 98 (2009) 402–410

basis, it is therefore advantageous to simultaneously increase the TRP and to decrease the LNAA concentration in food [8]. In the present study, we compared the long-term (three weeks) effects of two diets containing 13.2 vs 3.4% (high vs normal) of TRP to LNAA ratios on insulin-antagonistic hormones (cortisol, noradrenaline and adrenaline), on insulin sensitivities for glucose and amino acid utilization and on whole body protein metabolism in pigs. The objective of the study is to test if surplus dietary TRP inhibits stress hormone kinetics, increases insulin sensitivity and reduces whole body protein catabolism. 2. Materials and methods

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Table 2 Analyzed dry matter, crude protein and other calculated compositions of the diets (g/kg). TRP/LNAA ratio Dry matter Crude protein EE (crude fat) Net energy (MJ/kg) Ileal digestible protein Ileal digestible lysine Ileal digestible methionine Ileal digestible cysteine Ileal digestible tryptophan Crude protein digestibility (%)

Normal (3.4%)

High (13.2%)

860.6 205.2 59.7 9.4 158.0 8.7 3.0 2.6 1.4 85.1

855.0 196.0 61.4 9.4 161.7 8.7 2.9 2.6 7.0 86.3

Experimental protocols describing the management, surgical procedures, and animal care were reviewed and approved by the ASGLelystad Animal Care and Use Committee (Lelystad, The Netherlands).

the blood sampling sessions, the catheters were flushed and filled with physiological saline containing 5 IU heparin per mL.

2.1. Animals, housing, feed intake and surgery

2.2. Diets and experimental design

Sixteen crossbred barrows (Dutch Landrace × Yorkshire × Finnish Landrace) with an initial body weight of 30–35 kg were used in this study. Two weeks before surgery the pigs were kept in specially designed metabolic pens (1.15 × 1.35 m) and adapted to the light/dark cycle (lights on at 05:00 h and off at 19:00 h) and the feeding schedule. During the 2-week adaptation period, a commercial pig diet (containing 2 g/kg TRP, 10 g/kg lysine and 18% crude protein) was fed in a liquid form in a feed:water ratio of 1:4 (w/v). The pigs were fed twice daily (at 06:00 and 16:00 h) at a feeding level of 2.8 times maintenance requirements (MR) for metabolisable energy (ME) (MR = 293 kJ/kg BW0.75) throughout the study. This feeding level is practically close to ad libitum access to feed [1]. Pigs were provided with 3 permanent blood vessel catheters in the jugular vein, carotid artery and portal vein, as previously described [18,19]. During the period between surgery and the first series of experiments (at 9 days post-surgery), the pigs were habituated to the blood and saliva sampling procedures. The carotid artery was used for blood sampling and the jugular vein catheter was used as a back-up in case of a malfunctioning arterial catheter. The latter did not occur. The portal vein was used for the infusion of fluids during the hyperinsulinaemic euglycaemic euaminoacidaemic clamp experiments. During

On day 2 post-surgery the pigs were switched to the two experimental diets (normal and high ratio of TRP to LNAA i.e. VAL, ILE, LEU, TYR, and PHE). The diets used in the present study were part of a larger batch which was stored at −40 °C for a period of 1 year. Characteristics of the diets are shown in Tables 1 (raw material), 2 (composition) and 3 (amino acid content). In short, to obtain a normal TRP/ LNAA diet, dietary protein was selected with a normal endogenous TRP/LNAA ratio. To obtain a high TRP to LNAA ratio in the diet, 0.5% of L-TRP was added to dietary protein characterized by a high endogenous TRP/LNAA ratio. The normal TRP to LNAA ratio was 3.4% (or 1:29.4). The high TRP to LNAA ratio was 13.2% (or 1:7.6). The two diets were fed to the pigs (n = 8 per diet group) for 3 weeks comprising the following phases: 1) the first week adaptation to the diets followed by blood sampling after (14 h) fasting and an intraportal hyperinsulinaemic euglycaemic euaminoacidaemic clamp study; 2) the second week quantitative collection of urine and faeces (N-balance study and urinary catecholamine excretion); 3) the third week blood sampling after (14 h) fasting and followed by an intraportal hyperinsulinaemic euglycaemic euaminoacidaemic clamp study. 2.3. Hyperinsulinaemic euglycaemic euaminoacidaemic clamp technique The intraportal hyperinsulinaemic euglycaemic euaminoacidaemic clamp technique was used to quantitate insulin-stimulated net

Table 1 Dietary raw materials (g/kg). TRP/LNAA ratio Normal (3.4%) Maize Wheat Peas (CP N220) Soya beans extracted (CF b50) Tapioca starch (625–675) Palm kernel expeller (CF N220) Maize gluten feed (CP N200) Maize gluten meal Wheat middlings Linseed Cane molasses (sugar b475) Animal fat Trace mineral vitamin premix Calcium carbonate Salt Choline chloride MCP-aliphos Fumaric acid L-Lysine HCl DL-Methionine L-Tryptophan Total

High (13.2%)

290.6 347.9 75.0 183.6 61.5 35.0 180.0 53.0

60.0 32.5 5.0 11.5 0.5 0.3 7.8 2.0 1.7

1000.0

280.0 80.0

150.0 5.0 60.0 44.6 5.0 11.2 1.3 0.3 7.3 2.0 0.4 5.0 1000.0

Table 3 Analyzed composition of amino acids in the diets (g/kg). Amino acids ASP THR SER GLU GLY ALA VAL ILE LEU TYR PHE HIS LYS ARG PRO CYS MET TRP

g

TRP/LNAA ratio

LNAA

Normal (3.4%)

High (13.2%)

20.4 8.1 11.4 38.5 8.9 12.5 10.2 8.7 20.8 8.6 10.6 6.2 11.2 12.7 14.2 3.8 3.3 2.0

21.5 8.1 12.3 42.4 9.9 9.6 10.0 9.0 15.8 7.9 10.2 6.5 11.2 14.5 12.7 3.6 3.2 7.0

g

58.9

g

52.9

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utilization of plasma glucose and of individual amino acids in pigs [18– 20]. Nutrient utilization was expressed as nutrient clearance (infusion rate of nutrients (in mol/kg min) divided by the plasma nutrient concentration (in mol/mL). When comparing blood nutrient fluxes at different nutrient concentrations in blood (in particular for some of the 20 amino acids), the calculation of nutrient clearance normalizes the nutrient fluxes for differences in nutrient concentrations, thereby eliminating the mass action effect of concentration on flux and thus exposing whole body nutrient utilization at identical blood nutrient concentrations. Insulin (Actrapid MC, porcine monocomponent, Novo, Copenhagen, Denmark), D-glucose (Merck, Darmstadt, Germany) and a tailor-made mixture of 20 amino acids (Sigma, Zwijndrecht, The Netherlands) (Table 4) were prepared as sterile solutions and passed through a 0.22 µm Millipore filter into sterile containers before use. Insulin was diluted in a saline solution containing 3% pig plasma in order to avoid sticking of insulin to the plastic containers and tubings. D-glucose and the amino acids were dissolved in water. At 06:00 and 06:15, approximately 14 h after the previous meal, two heparinized blood (10 mL) and saliva (1 mL) samples were collected. At 06:30, hyperinsulinaemic euglycaemic euaminoacidaemic clamp experiments were started by a prime (17 mU/kg)-continuous (1 mU/kg min) intraportal infusion (pump Ad Fusor E, SP280, Adquipment medical BV, The Netherlands) of insulin for 3 h. Simultaneously, a variable intraportal infusion of a D-glucose solution (330 g/L) and an amino acid solution (65 g/L) (Table 4) was started and the infusion rate was adjusted every 10 (glucose solution) and 20 min (amino acid solution) to maintain the plasma glucose and phenylalanine concentrations at preclamp levels. The infused, tailor made, amino acid mixture contains 20 amino acids in concentrations which are in proportion to the utilization rates per individual amino acid in the pig. By experience, this amino acid mixture was defined in previous experiments [18,19] which allows us to study insulin sensitivity in the absence of hypo- or hyperaminoacidaemia. Steady state conditions for plasma glucose and phenylalanine concentrations and the infusion rates of glucose and amino acids were achieved within 120 min after initiation of the hyperinsulinaemic clamp and steady state calculations for whole body glucose and amino acid utilization were carried out during the last 40 min of the clamp (t = 140, 160 and 180 min). At 140, 160 and 180 min, blood (10 mL) samples were collected in heparinized tubes for measurement of plasma insulin, glucose, 20 amino acids, and urea concentrations. 2.4. Justification to infuse insulin, glucose and amino acids in portal blood Insulin, glucose and amino acids were infused in the portal blood because it reflects the physiological route of appearance in the blood stream after food intake. After a meal, carbohydrates and proteins are enzymatically digested in the gastrointestinal tract and, subsequently, the fluxes of glucose and amino acids increase in the portal vein. The postprandial rise in blood glucose and amino acid concentrations triggers insulin secretion from the pancreatic beta-cells into the portal vein. Insulin stimulates glucose and amino acid utilization and whole body anabolism by net synthesis of glycogen, lipids and protein. Starting in the portal vein, the mixture of insulin, glucose and amino acids Table 4 Tailor-made mixture of 20 amino acids (mmol/L) for intraportal infusion in pigs. Alanine, 51.5 Arginine, 20.3 Asparagine, 14.4 Aspartate, 1.5 Cysteine, 5.8

Glutamine, 47.2 Glutamate, 32.6 Glycine, 102 Histidine–HCl, 9.6 Isoleucine, 24.7

Leucine, 42.0 Lysine–HCl 19.6 Methionine, 10.5 Phenylalanine, 14.0 Proline, 42.9

Serine, 31.5 Threonine, 25.0 Tryptophan, 4.8 Tyrosine, 12.2 Valine, 34.7

The TRP/LNAA ratio of the amino acid mixture is 3.8%, closely reflecting the TRP/LNAA ratio of the normal diet (3.4%).

enters firstly metabolic pathways in the liver and afterwards via the hepatic vein it is delivered to the whole body. To mimic the physiological action of insulin on food derived glucose and amino acids, we chose to infuse insulin, glucose and amino acids via the portal vein during the hyperinsulinaemic euglycaemic euaminoacidaemic clamp procedure. In a comparable study where insulin action on whole body glucose and amino acid metabolism was studied in pigs [20], insulin, glucose and amino acids were infused via the jugular vein thereby ignoring the physiological portal-peripheral gradient in insulin concentrations and, with respect to food derived glucose and amino acids, the portal-peripheral gradient in glucose and amino acid concentrations as well. In the latter situation the central role of the liver in orchestrating insulin action and the partitioning of nutrients at the whole body level is not included in the clamp procedure. 2.5. Plasma, saliva and urine analyses Samples were immediately chilled at 0 °C on ice, and centrifuged at 4 °C for 10 min at 3000 rpm. Plasma was stored at −80 °C in 4 aliquots of 1.5 mL for later analyses. Saliva was collected by allowing the pigs to chew on two large cotton-buds, until they were thoroughly moistened. Cotton-buds were spun down at 3000 rpm for 10 min and saliva fluid was stored at −20 °C until analysis for cortisol concentration. Urine was stored at −80 °C in 2 aliquots of 10 mL for later analyses. Plasma and saliva cortisol concentrations and plasma catecholamines (noradrenaline and adrenaline) were measured as described before [5,8]. Plasma insulin concentration was measured using a Delfia assay (test kit No B080-1101 by Perkin Elmer Life Sciences Trust by Wallac Oy, Turku, Finland). This specific pig insulin assay was validated using pig insulin standards, as indicated before [21]. Plasma glucose was analyzed with a blood autoanalyzer of Radiometer (ABL and AML, Copenhagen, Denmark). Plasma urea was analyzed by the method described by Gutmann and Bergmeyer [22]. Plasma tryptophan and phenylalanine concentrations were measured by reversed-phase liquid chromatography (HPLC System Gold, Beckman, Fullerton, CA, USA) using a C18 (Hypersil) column (Alltech, Deerfield, IL, USA), and detected with a fluorescence detector at 217 nm [23]. For rapid plasma phenylalanine determination during the clamp studies, blood samples (0.5 mL) were immediately centrifuged in a microcentrifuge for 0.5 min, 0.1 mL of a 8% salicylic acid (SSA) solution was added to 0.1 mL plasma, mixed thoroughly, centrifuged in a microcentrifuge for 0.5 min and 0.02 mL of supernatant was injected in the reversed-phase HPLC system. Retention time for phenylalanine and tryptophan analyses was 4 and 6 min respectively. The concentrations of amino acids in plasma (except for tryptophan) were analyzed as described previously [23]. Urinary catecholamines (noradrenaline, adrenaline and dopamine) were assayed using a high performance liquid chromatography procedure (Perkin Elmer 410, Perkin Elmer, Norwalk, CT, USA using a C18 (Nucleosil) column (Macherey Nagel, Düren, Germany) with electrochemical detection [24]. 2.6. Nitrogen balance study Retention and excretion of nitrogen were assessed by the balance method (daily intake–output in faeces–output in urine). Faeces were collected quantitatively over 7 days by a temporary fixation of polyethylene bags (15 × 30 cm) around the anus of the pig [25]. These bags were replaced twice daily, and after weighing, the contents were stored at − 20 °C. Before subjecting to chemical analyses, the 7-day collection of faeces for each pig was pooled, homogenized, and representative subsamples were freeze-dried and milled to pass through a 1-mm sieve. Urine was collected quantitatively over periods of 24 h (06:00–06:00) for 7 days. Urine was collected via a tray under the metabolic pen floor via a filter into a plastic barrel containing concentrated H2SO4 (density = 1.3) to obtain pH in the range of 4.0 to

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3.5 for the analysis of N. Volume of urine was registered and a well mixed aliquot of 10 mL was frozen at −80 °C and stored for further analyses (nitrogen and catecholamines). 2.7. Diet analyses and N analysis in faeces and urine Diets and freeze-dried faeces were analyzed for the content of dry matter (DM). Nitrogen content in diets, faeces and urine was assayed by the Dumas method [26].

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3.2. Plasma TRP and LNAA concentrations After overnight (14 h) fasting, basal plasma TRP and LNAA concentrations were similar among pigs on the surplus and normal TRP diets with factual plasma ratios of TRP/LNAA between 6.1 and 6.7% (Tables 5 and 6). 3.3. Plasma and salivary stress hormone concentrations

Each pig was an experimental unit. Results are expressed as means ± SEM and the criterion of statistical significance was set at p b 0.05. The data were subjected to the analysis of variance procedure (ANOVA) for repeated measurements followed by the unpaired or paired student's t-test of Genstat 5 [27] for determination of differences between or within the two dietary groups, respectively.

One week after introduction of the surplus and normal TRP diets, fasting (14 h) plasma cortisol, noradrenaline and adrenaline concentrations were not different between the pigs (Fig. 1). By contrast, three weeks after introduction of the surplus and normal TRP diets, fasting (14 h) plasma cortisol and noradrenaline concentrations were lower in pigs on the surplus TRP diet (Fig.1). Fasting (14 h) saliva cortisol concentrations did not differ significantly between pigs after one and three weeks on the surplus vs normal TRP diets (1.24 ± 0.24 vs 1.41 ± 0.14 and 0.78 ± 0.07 vs 1.43 ± 0.46 ng/mL, respectively).

3. Results

3.4. Urinary catecholamine excretion

3.1. Dietary composition

Two weeks after introduction of the surplus and normal TRP diets, daily urinary noradrenaline and adrenaline excretions were reduced ~2-fold in pigs fed the surplus TRP diet (Fig. 2). By contrast, daily urinary dopamine excretion was increased ~ 4-fold in pigs fed the surplus TRP diet (Fig. 2).

2.8. Statistical analyses

Raw material, calculated composition, the analyzed chemical characteristics and the amino acid profile of the experimental diets are shown in Tables 1–3. In short, the surplus and normal contents of TRP in the diets were 7 and 2 g/kg, respectively. The respective amounts of LNAA were 52.9 and 58.9 g/kg feed. Consequently, the factual ratios of TRP/LNAA were 13.2 and 3.4% for the surplus and normal TRP diets, respectively. This means the levels were differing 3.9-fold (13.2/3.4). Regardless of a 3.9-fold difference in TRP to LNAA ratio between the two diets, the content of crude protein (Table 2) and total amino acids (Table 3) were similar between the two diets.

3.5. Insulin-mediated glucose and amino acid metabolism One and three weeks after introduction of the surplus and normal TRP diets, fasting (14 h) plasma insulin, glucose, urea and amino acid concentrations were similar among pigs (Tables 5 and 6). Upon insulin stimulation (+9–10 mU/L increment over fasting plasma insulin

Table 5 Plasma data after one week of feeding diets with a normal or high TRP/LNAA ratio in pigs. Diet

Normal TRP/LNAA ratio (3.4%) Preclamp

Insulin (mU/L)

Steady state

High TRP/LNAA ratio (13.2%) Stimulation

Preclamp

5±2

14 ± 2

9

5.1 ± 0.1 3.6 ± 0.2

4.7 ± 0.3 3.3 ± 0.2

CV (%) 11 4

Dev. % −9 −9

Plasma amino acid (µmol/L) Tryptophan 43 ± 3 Phenylalanine 63 ± 3 Leucine 200 ± 9 Isoleucine 90 ± 4 Valine 237 ± 11 Tyrosine 69 ± 5 Total LNAA 658 ± 28 TRP/LNAA (%) 6.4 ± 0.3 Threonine 132 ± 9 Serine 126 ± 2 Asparagine 48 ± 3 Aspartate 15 ± 1 Glutamine 474 ± 17 Glutamate 127 ± 9 Glycine 768 ± 35 Alanine 295 ± 15 Cysteine 31 ± 1 Methionine 33 ± 3 Lysine 84 ± 9 Histidine 83 ± 5 Arginine 101 ± 8 Proline 205 ± 8 Total amino acids 3222 ± 87

49 ± 3 56 ± 2 173 ± 4 95 ± 2 257 ± 9 60 ± 4 641 ± 15 7.6 ± 0.5 140 ± 12 121 ± 6 46 ± 3 14 ± 1 471 ± 29 115 ± 9 889 ± 61 291 ± 18 37 ± 2 31 ± 3 79 ± 6 72 ± 4 103 ± 8 230 ± 9 3331 ± 132

7 6 8 7 5 8 6 4 6 9 10 15 4 8 4 7 8 8 12 5 9 8 5

15 − 10# − 13⁎⁎ 6 8 − 13 −3 19⁎ 7 −4 −4 −4 0 − 10 16# −2 20⁎⁎

Glucose (mmol/L) Urea (mmol/L)

−7 −5 − 13 2 12⁎ 3

Steady state

Stimulation

2±1

12 ± 3

10

4.9 ± 0.2 3.4 ± 0.3

5.0 ± 0.2 3.3 ± 0.4

CV (%) 12 3

Dev. % 2 −5

42 ± 1 60 ± 3 165 ± 7 122 ± 7 279 ± 11 62 ± 4 687 ± 20 6.1 ± 0.1 133 ± 13 143 ± 10 50 ± 3 20 ± 3 460 ± 31 143 ± 16 783 ± 64 406 ± 25 35 ± 2 34 ± 2 112 ± 18 75 ± 3 98 ± 5 197 ± 12 3417 ± 101

49 ± 3 61 ± 4 155 ± 13 103 ± 12 287 ± 17 56 ± 4 663 ± 39 7.5 ± 0.6 138 ± 14 145 ± 7 53 ± 3 20 ± 2 525 ± 22 166 ± 20 905 ± 72 403 ± 34 41 ± 3 37 ± 3 87 ± 4 49 ± 3 108 ± 7 247 ± 13 3636 ± 115

6 7 8 10 5 9 6 3 9 13 9 15 3 5 4 6 8 8 8 6 9 7 4

16⁎ 2 −6 − 16 3 −9 − 4⁎ 22 3 2 6 −1 14# 16 16 −1 19⁎ 10 − 23 − 34⁎⁎⁎ 10 26⁎⁎ 6

Means ± SEM. CV% = coefficient of variation within pigs for steady state concentration. Dev% = deviation of steady state concentration from preclamp concentration. ***p b 0.001; **p b 0.01; *p b 0.05; #p b 0.1.

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Table 6 Plasma data after three weeks of feeding diets with a normal or high TRP/LNAA ratio in pigs. Diet

Normal TRP/LNAA ratio (3.4%) Preclamp

Insulin (mU/L)

Steady state

7±2

17 ± 3

4.9 ± 0.1 3.2 ± 0.2

4.5 ± 0.2 2.7 ± 0.2

Plasma amino acid (µmol/L) Tryptophan 45 ± 3 Phenylalanine 62 ± 2 Leucine 194 ± 9 Isoleucine 93 ± 5 Valine 230 ± 11 Tyrosine 71 ± 7 Total LNAA 651 ± 27 TRP/LNAA (%) 6.7 ± 0.3 Threonine 135 ± 11 Serine 120 ± 5 Asparagine 47 ± 3 Aspartate 12 ± 1 Glutamine 464 ± 26 Glutamate 101 ± 10 Glycine 822 ± 61 Alanine 291 ± 17 Cysteine 34 ± 1 Methionine 37 ± 3 Lysine 90 ± 7 Histidine 73 ± 5 Arginine 95 ± 7 Proline 207 ± 10 Total amino acids 3224 ± 106

51 ± 4 58 ± 2 154 ± 5 79 ± 10 239 ± 9 59 ± 5 590 ± 14 8.5 ± 0.6 134 ± 10 124 ± 5 45 ± 2 13 ± 1 464 ± 25 99 ± 8 912 ± 47 305 ± 12 38 ± 1 34 ± 2 84 ± 6 67 ± 2 93 ± 6 227 ± 8 3280 ± 97

Glucose (mmol/L) Urea (mmol/L)

High TRP/LNAA ratio (13.2%) Stimulation

Preclamp

10 CV (%) 9 3

Dev. % − 8# − 14

7 7 7 11 7 7 7 4 7 5 8 8 4 5 4 4 6 8 10 6 9 6 4

14 −7 − 21 − 15⁎⁎⁎ 4 − 17 − 9# 27⁎ 1 3 −4 7 0 −2 11 5 12# −8 −6 −9 −2 10 2

Steady state

Stimulation

4±1

13 ± 2

9

4.9 ± 0.1 3.5 ± 0.2

4.6 ± 0.3 3.1 ± 0.2

CV (%) 13 4

Dev. % −6 − 11

5 8 7 13 3 7 5 5 5 8 8 12 5 10 4 5 6 9 4 5 7 6 4

12⁎ − 5# −7 − 24 3 −6 −6 24⁎⁎ 5 10# −5 24 4 17⁎ 11⁎

45 ± 2 57 ± 2 169 ± 6 120 ± 4 292 ± 10 65 ± 5 703 ± 20 6.2 ± 0.2 151 ± 11 128 ± 3 52 ± 4 14 ± 1 463 ± 27 110 ± 6 838 ± 24 341 ± 28 35 ± 2 32 ± 4 82 ± 6 69 ± 3 112 ± 8 194 ± 7 3369 ± 100

50 ± 2 54 ± 2 156 ± 10 91 ± 15 302 ± 11 60 ± 4 664 ± 30 7.7 ± 0.5 159 ± 9 141 ± 7 50 ± 3 18 ± 2 481 ± 34 128 ± 6 933 ± 30 306 ± 14 43 ± 2 33 ± 3 79 ± 4 50 ± 2 111 ± 7 212 ± 8 3450 ± 107

− 10 22# 3 −5 27⁎⁎⁎ −1 10 2

Plasma insulin, glucose, urea and amino acid concentrations are shown at preclamp (basal) and at steady state during the hyperinsulinaemic euglycaemic euaminoacidaemic clamp. Means ± SEM. CV% = coefficient of variation within pigs for steady state concentration. Dev% = deviation of steady state concentration from preclamp concentration. ***p b 0.001; **p b 0.01; *p b 0.05; #p b 0.1.

concentrations), steady state euglycaemia and euaminoacidaemia were maintained within 9% and 6% deviation from their preclamp concentrations, respectively. For some individual amino acids, a significant positive or negative deviation of the steady state concentration from their preclamp concentration was observed. In the surplus TRP diet group, plasma histidine showed the most prominent deviation (−34 and −27%) at steady state concentrations indicating that the infused amino acid mixture should have contained more histidine. Steady state plasma urea concentrations did not differ among pigs and diets. One week after introduction of the experimental diets, insulinstimulated glucose clearance tended (p = 0.07) to be lower in the surplus TRP diet group but amino acid clearance rates were identical between diet groups (Fig. 3A + B). Three weeks after introduction of the experimental diets, both insulin-stimulated glucose and amino acid clearance were lower by 22% (p b 0.05) in pigs on the surplus TRP diet (Fig. 3A + B). Not all amino acids were affected equally: significant reductions in insulin-stimulated clearance rates were observed for valine (35%), threonine (34%), serine (28%), asparagine (24%), aspartate (37%), glutamate (39%), alanine (17%) and cysteine (27%) in pigs after 3 weeks on the surplus TRP diet (Table 7). All other amino acids showed a numerical but not a significant reduction in clearance rates. 3.6. Nitrogen retention and excretion Two weeks after introduction of the diets, whole body nitrogen retention, as percentage of dietary nitrogen intake, was greater in the surplus TRP diet compared to the normal TRP diet. The increase in nitrogen retention occurred at decreased nitrogen excretion via faeces and similar nitrogen excretion via urine (Fig. 4). Dietary nitrogen intake was similar in the normal and surplus TRP groups (0.97 ± 0.02 and 0.93 ± 0.04 grams of nitrogen per kg body weight per day).

4. Discussion 4.1. Insulin-stimulated glucose and amino acid metabolism After 3 weeks, but not after one week of feeding the surplus TRP diet, insulin sensitivity was reduced by 22% for both glucose and amino acid clearance (Fig. 3). However, both fasting plasma glucose and total amino acid concentrations were similar between dietary groups indicating that the baseline setpoint for glucose and amino acid metabolism was unaffected by the surplus TRP diet. Taken together, this suggests that TRP-induced insulin resistance was caused by a general reduction in insulin signalling with respect to carbohydrate and protein metabolism and that insulin resistance could not be pinpointed to either whole body glucose and amino acid production or glucose and amino acid uptake. At identical feed intake, baseline and insulinmediated plasma urea concentrations were similar between the dietary groups, suggesting unchanged metabolic nitrogen-efficiency and thus an unaffected balance in whole body protein anabolism and catabolism. The latter was confirmed by the similar urinary nitrogen excretion during the nitrogen balance study. Surplus dietary TRP chronically reduced adrenocortical and sympathoadrenal activity which was paralleled by insulin resistance for both glucose and amino acid clearance. Cortisol and catecholamines are considered to be insulin antagonistic and as such a reduction in the levels of these insulin-counterregulatory hormones should increase insulin sensitivity [12–16]. Surprisingly, in the present study, the opposite was observed showing insulin resistance for both glucose and amino acid metabolism. This phenomenon may be explained by the fact that it has been reported that an acute (hours) increase in the levels of noradrenaline and adrenaline induced insulin resistance whereas a chronic (several days) elevation in plasma catecholamine

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Fig. 1. Fasting (14 h) plasma cortsiol, noradrenaline and adrenaline concentrations are depicted, one and three weeks after introduction of the diets to the pigs. Pigs were fed two meals per day at 06:00 and 16:00 h. Open bars = 8 pigs on the normal (3.4%) TRP/LNAA diet, black bars = 8 pigs on the high (13.2%) TRP/LNAA diet. Means ± SEM. **p b 0.005; *p b 0.05.

concentrations increased insulin sensitivity [28–31]. If this is true, then the chronic reduction in plasma noradrenaline concentrations, as present in our study, is expected to decrease insulin sensitivity. The chronic effect of noradrenaline may over-rule the chronic effect of cortisol on insulin action and the net result appears to be the induction of insulin resistance. Another factor which could be responsible for the induction of insulin resistance is the reduction in physical activity when pigs are fed a diet enriched with TRP [5,8,17]. Pig be-

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Fig. 3. (A + B) Insulin-stimulated whole body glucose clearance (A) and insulinstimulated whole body amino acid clearance (B) during the steady state phase of the intraportal hyperinsulinaemic euglycaemic euaminoacidaemic clamp technique, one and three weeks after introduction of the diets to the pigs. Open bars = 8 pigs on the normal (3.4%) TRP/LNAA diet, black bars = 8 pigs on the high (13.2%) TRP/LNAA diet. Means ± SEM. #p = 0.07 and *p b 0.05.

haviour was not monitored in the present study but from our previous studies [5,8], using comparable diets, we observed more lying and less standing in pigs. This indicates reduced physical activity. It has been shown [32] that physical activity closely correlates with insulin sensitivity. This fits nicely with the observation that low physical activity reduces noradrenaline outflow [24,33] and, as such, reduces insulin sensitivity. Finally, dopamine has been shown to induce hyperglycaemia [34] which was not related to changes in insulin or glucagon secretion [35]. This suggests that the high levels of dopamine excretion in the urine, as observed in our study, may be related to the induction of insulin resistance.

Fig. 2. Daily urinary noradrenaline, adrenaline and dopamine excretions are depicted, two weeks after introduction of the diets to the pigs. Urine was collected daily over a 7-day period. Open bars = 8 pigs on the normal (3.4%) TRP/LNAA diet, black bars = 8 pigs on the high (13.2%) TRP/LNAA diet. Means ± SEM. ***p b 0.001.

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Table 7 Insulin-stimulated amino acid clearance rates during the steady state phase of the hyperinsulinaemic euglycaemic euaminoacidaemic clamp in pigs fed diets with a normal or high TRP/LNAA ratio. Time

Week 1

Dietary TRP/ Normal LNAA ratio (%) (3.4)

Week 3 High (13.2)

Normal (3.4)

Plasma amino acid clearance (mL/kg min) Tryptophan 2.2 ± 0.1 2.4 ± 0.2 2.0 ± 0.2 Phenylalanine 5.5 ± 0.3 5.7 ± 0.5 5.1 ± 0.5 Leucine 5.4 ± 0.2 6.6 ± 0.8 5.7 ± 0.4 Isoleucine 5.7 ± 0.3 6.6 ± 1.6 8.5 ± 2.6 Valine 3.0 ± 0.1 2.8 ± 0.2 3.1 ± 0.3 Tyrosine 4.6 ± 0.2 5.2 ± 0.6 4.6 ± 0.5 Total LNAA 24.2 ± 1.0 26.9 ± 3.4 27.0 ± 3.1 TRP/LNAA 8.4 ± 0.5 8.3 ± 0.4 7.4 ± 0.7 ratio (%) Threonine 4.1 ± 0.3 4.4 ± 0.6 4.1 ± 0.5 Serine 5.8 ± 0.3 5.0 ± 0.5 5.4 ± 0.4 Asparagine 7.0 ± 0.5 6.4 ± 0.4 6.7 ± 0.4 Aspartate 2.4 ± 0.3 1.9 ± 0.3 2.4 ± 0.2 Glutamine 2.2 ± 0.1 2.1 ± 0.2 2.2 ± 0.2 Glutamate 6.5 ± 0.7 5.2 ± 0.5 7.3 ± 0.9 Glycine 2.6 ± 0.2 2.7 ± 0.3 2.4 ± 0.2 Alanine 4.0 ± 0.2 3.0 ± 0.3a 3.5 ± 0.2 Cysteine 3.5 ± 0.1 3.5 ± 0.3 3.3 ± 0.3 Methionine 7.8 ± 0.6 7.3 ± 0.7 6.5 ± 0.5 Lysine 5.2 ± 0.4 4.3 ± 0.4 4.8 ± 0.6 Histidine 2.7 ± 0.2 2.7 ± 0.2 2.9 ± 0.2 Arginine 4.5 ± 0.5 4.5 ± 0.2 4.7 ± 0.4 Proline 4.1 ± 0.1 4.2 ± 0.2 4.0 ± 0.3

p-value High (13.2) 1.6 ± 0.1 4.5 ± 0.4 4.8 ± 0.3 6.7 ± 1.9 2.0 ± 0.1c 3.5 ± 0.1 21.5 ± 2.3 7.6 ± 0.6 2.7 ± 0.1b 3.9 ± 0.3b 5.1 ± 0.3c 1.5 ± 0.2c 1.7 ± 0.1b 4.5 ± 0.4c 1.9 ± 0.1b 2.9 ± 0.2b 2.4 ± 0.1b 5.6 ± 0.3 4.0 ± 0.5 2.2 ± 0.2b 3.3 ± 0.3b 3.5 ± 0.2

Diet Week d × w

⁎ # # ⁎⁎

# #

⁎ #

⁎

⁎ ⁎ ⁎

# ⁎ #

⁎

# ⁎⁎ ⁎ ⁎⁎ ⁎

⁎ ⁎⁎ # #

# ⁎

Clamps were performed 1 and 3 weeks after introduction of the diets. Plasma insulin, glucose, urea and amino acid concentrations are shown at preclamp (basal) and at steady state during the hyperinsulinaemic euglycaemic euaminoacidaemic clamp. Means ± SEM. a p b 0.05 compared to Normal diet, Week 1. b p b 0.05 compared to Normal diet, Week 3. c p b 0.01 compared to Normal diet, Week 3. **p b 0.01; *p b 0.05; #p b 0.1 for an overall diet effect or a week (time) effect or an interaction between diet and week.

With regard to insulin resistance for amino acid clearance, not all amino acids were affected equally: percentage-wise, the reduction in insulin-stimulated clearance ranged from 12% (phenylalanine and proline) to 39% (glutamic acid), suggesting that insulin resistance towards protein metabolism was amino acid specific. The reason for this amino acid specificity is unknown but could be linked to the disbalance in amino acid feeding which leads to an imbalance in the systemic clearance rates of specific amino acids. However, no clear correlation could be observed between the dietary intake levels of specific amino acids and their respective blood clearance rates. Probably, the time lag between feeding and the blood clearance study (16– 17 h) leads to a new steady state in amino acid metabolism in which excess dietary amino acids have been disposed of by oxidation and limiting amino acids have been spared. Moreover, the clearance rate of TRP to LNAA ratio from blood remained constant (1.03-fold) in pigs fed the high (13.2%) compared to the low (3.2%) TRP/LNAA diet, although the dietary TRP/LNAA ratio differed ~ 4-fold. The induction of insulin resistance by surplus dietary TRP is reflected in a previous study [36] in which low, normal and high dietary TRP were compared in pigs. Both low and high dietary TRP-induced higher plasma insulin and glucose concentrations compared to normal dietary TRP supply. This suggests that an optimal (intermediate) concentration of dietary TRP exists which promotes maximum insulin sensitivity. 4.2. Whole body nitrogen retention Pigs on the surplus vs normal TRP diet showed greater whole body N-retention at decreased fecal N-excretion but comparable urinary

N-excretion. This suggests that the increase in N-retention was mainly caused by an increase in N-digestion and/or N-absorption at similar metabolic N-efficiency. Dietary TRP may locally boost the serotonergic system in the gastrointestinal tract (GIT) and as such enhance peristaltic activity by smooth muscle and stimulate intestinal secretion by enterocytes [37]. Also, intestinal perfusion with L-TRP has been found to induce cholecystokinin production and subsequent pancreatic enzyme production in dogs and humans [38]. L-TRP was found to be the most potent amino acid for stimulating pancreatic synthesis in dogs [39]. Of the total body serotonin, 80% is located in the gut. The TRP driven effect on GIT serotonin may therefore be responsible for the observed increase in N-digestion and/or N-absorption in the present study. In addition to this local TRP-serotonergic effect on the GIT, TRP may increase GIT functionality indirectly through its inhibitory effect on the peripheral sympathetic nervous system. It is well known that stress and a general stimulation of the sympathetic nervous system lead to a reduction in both GIT blood flow [40–42] and digestion and in an increase of intestinal fluid secretion and defecation [13,43–45]. The increase in N-digestion and/or N-absorption by surplus dietary TRP may therefore be caused by both serotonergic driven mechanisms and by inhibition of stress hormone kinetics. 4.3. Plasma catecholamine concentration and urinary catecholamine excretion The sympathetic nervous system is activated due to a general state of arousal and physical activity in domestic pigs [24], which results in the release of catecholamines in blood. Blood adrenaline originates from the adrenal medulla and blood noradrenaline originates mainly from peripheral nerves endings in muscle [33] and subsequently both catecholamines appear in urine [46,47]. Daily urinary catecholamine excretion is thought to be a good indicator of chronic sympathetic nervous system activity [46,47]. Three weeks, but not one week of feeding the surplus TRP diet reduced overnight fasting (14 h) plasma noradrenaline concentrations. Plasma adrenaline concentrations remained unaffected throughout the study. A previous study by us using the same diet for 1 week showed reduced plasma noradrenaline and normal adrenaline concentrations when blood samples were collected after 6 h of fasting [8]. This indicates that the effect of surplus dietary TRP on noradrenaline fades between 6 and 14 h after the meal when pigs are on the diets for a time period of 1 week. Taken together, these results suggest that long-lasting effects (14 h after the meal) of surplus dietary TRP on plasma noradrenaline concentrations take 3 weeks to develop whereas no effect on plasma adrenaline concentrations can be detected.

Fig. 4. Balance of nitrogen in pigs fed the normal (3.4%) or high (13.2%) TRP/LNAA diets, two weeks after introduction of the diets (faeces and urine were collected daily over a 7 days period). Whole body N-retention, N-excretion via faeces and N-excretion via urine were expressed as percentage of dietary N-intake. Open bars = pigs on the normal (3.4%) TRP/LNAA diet, black bars = pigs on the high (13.2%) TRP/LNAA diet. Means ± SEM. **p b 0.005.

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By contrast, 2–3 weeks after feeding the surplus TRP diet, daily urinary noradrenaline and adrenaline excretions were both reduced and dopamine excretion was increased. Reduced plasma noradrenaline concentrations parallel reduced daily urinary noradrenaline excretion, but the unchanged plasma adrenaline concentrations do not match the reduction in daily urinary adrenaline excretion in pigs on the high TRP diet. The reason for the discrepancy between the single time point plasma adrenaline concentration and the average 24 h urinary adrenaline excretion could be that 1) average 24 h plasma adrenaline concentrations (but not measured in the present study) were significantly lower compared to the plasma adrenaline concentration measured at 06:00 h, 2) dietary TRP exerts an effect on adrenaline clearance by the kidneys. If TRP reduces the glomerular filtration rate of adrenaline, then a reduction in urinary adrenaline excretion could be paralleled by unchanged plasma adrenaline concentrations. The latter explanation could also be applicable to the increased (~4-fold) daily urinary dopamine excretion at high TRP feeding, since it has been argued [33, 46] that the origin and functional relevance of urinary dopamine are unclear and that urinary dopamine may not reflect average plasma dopamine concentrations. Urinary dopamine may originate from active glomerular filtration, especially due to the active secretion by the renal tubular epithelium [33,46]. Feeding a diet with surplus TRP could therefore stimulate kidney dopamine excretion and inhibit kidney adrenaline secretion. Another speculative explanation for increased urinary dopamine excretion could be a direct inhibitory effect of TRP on the enzyme dopamine-beta-hydroxylase which converts dopamine into noradrenaline [13]. Such an inhibition would cause a build up of dopamine levels and a depletion of noradrenaline and adrenaline levels. 4.4. Diets In a previous study [8] we have shown that the surplus TRP diet induced an elevation (2.9-fold) in the plasma TRP to PHE ratio in pigs, measured 6 h after feeding. In the present study, plasma amino acid concentrations were measured after overnight (14 h) fasting and at this postprandial time, the TRP concentration, the TRP to PHE ratio and the TRP to LNAA ratio in plasma were similar between the two diet groups. It seems therefore that excess TRP in plasma is catabolised to prevent the accumulation of TRP in plasma after feeding surplus dietary TRP. However, it takes between 6 and 14 h after the meal before plasma TRP homeostasis is restored. This indicates that this diet has intermediate-lasting effects on the amino acid pool in the vascular system (fades between 6 and 14 h postprandially) but long-lasting effects on endocrinology, glucose and amino acid metabolism (N14 h postprandially). 5. Conclusion This study shows that surplus dietary TRP inhibits baseline adrenocortical and sympatho-adrenal axis activity and it reduces insulin sensitivity at unchanged whole body protein catabolism in pigs. Although brain serotonin concentration and turnover have been shown to increase 4–6 days after the onset of surplus dietary TRP feeding [3,5], chronic effects on peripheral neuroendocrinology, insulin sensitivity and metabolism take more than one week (i.e. 3 weeks) to develop. The results clearly indicate that adjustment of the TRP/LNAA ratio in diets is a powerful tool to modulate the kinetics of hormones and shows how nutritional signals contribute to homeostasis in neuroendocrinology and insulin action. Furthermore, this study shows that low baseline adrenocortical and sympatho-adrenal tone are associated with insulin resistance. References [1] (CVB (Centraal Veevoeder Bureau)). Dutch norms for livestock feeding and nutritive values for feedstuffs24th Ed. Lelystad, The Netherlands: CVB Press; 2000.

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[2] Le Floc'h N, Sève B. Biological roles of tryptophan and its metabolism: potential implications for pig feeding. Livest Sci 2007;112:23–32. [3] Adeola O, Ball RO. Hypothalamic neurotransmitter concentrations and meat quality in stressed pigs offered excess dietary tryptophan and tyrosine. J Anim Sci 1992;70: 1888–94. [4] Huether G, Kochen W, Simat TJ, Steinhart H eds. Tryptophan, serotonin, and melatonin: Basic aspects and applications. Kluwer academic/Plenum Publishers, New York; 1999. [5] Koopmans SJ, Guzik AC, Van der Meulen J, Dekker R, Kogut J, Kerr BJ, Southern LL. Effects of supplemental L-tryptophan on serotonin, cortisol, intestinal integrity, and behavior in weanling piglets. J Anim Sci 2006;84:963–71. [6] Scheurink AJW, De Boer SF, Van Dijk G, Steffens AB. Central and peripheral mechanisms involved in the regulation of sympathoadrenal outflow. In: McCarty R, Aguilera G, Sabban E, Kvetnansky R, editors. Stress: molecular genetic and neurobiological advances. New York: Gordon and Breach Science Publishers S.A; 1996. p. 227–41. [7] Cortamira NO, Sève B, Lebreton Y, Ganier P. Effect of dietary tryptophan on muscle, liver and whole body protein synthesis in weaned piglets: relationship to plasma insulin. Br J Nutr 1991;66:423–35. [8] Koopmans SJ, Ruis M, Dekker R, van Diepen H, Korte M, Mroz Z. Surplus dietary tryptophan reduces plasma cortisol and noradrenaline concentrations and enhances recovery after social stress in pigs. Physiol Behav 2005;85:469–78. [9] Lepage O, Vilchez IM, Pottinger TG, Winberg S. Time-course of the effect of dietary L-tryptophan on plasma cortisol levels in rainbow trout. J Exp Biol 2003;206: 3589–99. [10] Markus CR, Olivier B, Panhuysen GEM, Van der Gugten J, Alles MS, Tuiten A, et al. The bovine protein alpha-lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino acids, and in vulnerable subjects raises brain serotonin activity, reduces cortisol concentration, and improves mood under stress. Am J Clin Nutr 2000;71:1536–44. [11] Zhang H, Yin J, Li D, Zhou X, Li X. Tryptophan enhances ghrelin expression and secretion associated with increased food intake and weight gain in weanling pigs. Domest Anim Endocrinol 2007;33:47–61. [12] Bratusch-Marrain PR. Insulin-counteracting hormones: their impact on glucose metabolism. Diabetologia 1983;24:74–9. [13] Newsholm EA, Leech AR. Biochemistry for the medical sciences. UK: John Wiley & Sons Ltd; 1983. [14] Ruzzin J, Wagman AS, Jensen J. Glucocorticoid-induced insulin resistance in skeletal muscles: defects in insulin signalling and the effects of a selective glycogen synthase kinase-3 inhibitor. Diabetologia 2005;48:2119–30. [15] Simmons PS, Miles JM, Gerich JE, Haymond MW. Increased proteolysis: an effect of increases in plasma cortisol within the physiologic range. J Clin Invest 1984;73: 412–20. [16] Strack AM, Sebastian RJ, Schwartz MW, Dallman MF. Glucocorticoids and insulin: reciprocal signals for energy balance. Am J Physiol 1995;268:R142–9. [17] Sève B. Physiological roles of tryptophan in pig nutrition. In: Huether, editor. Tryptophan, serotonin, and melatonin: Basic aspects and applications. New York: Kluwer academic/Plenum Publishers; 1999. p. 729–41. [18] Koopmans SJ, Mroz Z, Dekker R, Corbijn H, Wijdenes J, Van der Crabben S, Ackermans M, Sauerwein HP. Insulin-stimulated net utilization of plasma glucose and amino acids in growing pigs. In: Souffrant WB, Metges CC, editors. Progress in research on energy and protein metabolism, vol. 109; 2003. p. 197–200. [19] Koopmans SJ, Van der Meulen J, Dekker R, Corbijn H, Mroz Z. Diurnal variation in insulin-stimulated systemic glucose and amino acid utilization in pigs fed with identical meals at 12-hour intervals. Horm Metab Res 2006;38:607–13. [20] Wray-Cahen D, Beckett PR, Nguyen HV, Davis TA. Insulin-stimulated amino acid utlization during glucose and amino acid clamps decreases with development. Am J Physiol 1997;273:E305–14. [21] Koopmans SJ, Van der Meulen J, Dekker R, Corbijn H, Mroz Z. Diurnal rhythms in plasma cortisol, insulin, glucose, lactate and urea in pigs fed identical meals at 12hourly intervals. Physiol Behav 2005;84:497–503. [22] Gutmann I, Bergmeyer HU. Urea. In: Bergmeyer HU, editor. Methods of enzymatic analyses. 2nd edition. New York and London: Verlag Chemie Weinheim and Academic Press Inc 1974; p. 1791. [23] de LH Jonge, Breuer M. Modification of the analysis of amino acids in pig plasma. J Chromatogr B 1994;652:90–6. [24] Ruis MAW, de Groot J, te Brake JHA, Ekkel ED, van de Burgwal JA, Erkens JHF, Engel B, Buist WG, Blokhuis HJ, Koolhaas JM. Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment. Appl Anim Behav Sci 2001;70:201–25. [25] Mroz Z, Jongbloed AW, Partanen KH, Vreman K, Kemme PA, Kogut J. The effects of calcium benzoate in diets with or without organic acids on dietary buffering capacity, apparent digestibility, retention of nutrients, and manure characteristics in swine. J Anim Sci 2000;78:2622–32. [26] AOAC. Official methods of analysis14th Ed. Arlington (VA: Association of Official Analytical Chemists; 1984. [27] Payne RW, Lane PW, Ainsley AE, Bicknell KE, Digby PGN, Harding SA, et al. Genstat 5. Reference manual. Oxford (UK: Oxford University Press; 1987. [28] Budohoski L, Challiss RAJ, Dubaniewicz A, Kaciuba-Uscilko H, Leighton B, Lozeman FJ, et al. Effects of prolonged elevation of plasma adrenaline concentration in vivo on insulin-sensitivity in soleus muscle of the rat. Biochem J 1987;244:655–60. [29] Jensen J, Ruzzin J, Jebens E, Brennesvik EO, Knardahl S. Improved insulin-stimulated glucose uptake and glycogen synthase activation in rat skeletal muscles after adrenaline infusion: role of glycogen content and PKB phosphorylation. Acta Physiol Scand 2005;184:121–30. [30] Lupien JR, Hirschman MF, Horton ES. Effects of norepinephrine infusion on in vivo insulin sensitivity and responsiveness. Am J Physiol 1990;259:E210–5.

410

S.J. Koopmans et al. / Physiology & Behavior 98 (2009) 402–410

[31] Sleeman MW, Christopher MJ, Martin IK, Ward GM, Alford FP, Best JD. Effects of acute and chronic counterregulatory hormone infusions on glucose tolerance and insulin sensitivity in diabetic dogs. Diabetes 1992;41:1446–52. [32] Schmitz KH, Jacobs Jr DR, Hong CP, Steinberger J, Moran A, Sinaiko AR. Association of physical activity with insulin sensitivity in children. Int J Obes 2002;26:1310–6. [33] Goldstein DS. Clinical assessment of catecholaminergic function. In: Goldstein DS, editor. Stress, Catecholamines, and Cardiovascular Disease. New York (USA: Oxford University Press; 1995. p. 234–86. [34] Håkanson R, Lundquist I, Rerup C. On the hyperglycaemic effect of dopa and dopamine. Euro J Pharmacol 1967;1:114–9. [35] Arnerić SP, Chow SA, Bhatnagar RK, Webb RL, Fischer LJ, Long JP. Evidence that central dopamine receptors modulate sympathetic neuronal activity to the adrenal medulla to alter glucoregulatory mechanisms. Neuropharmacology 1984;23:137–47. [36] Ponter AA, Cortamira NO, Sève B, Salter DN, Morgan LM. The effects of energy source and tryptophan on the rate of protein synthesis and hormones of the enteroinsular axis in the piglet. Br J Nutr 1994;71:661–74. [37] Spiller RC. Effects of serotonin on intestinal secretion and motility. Curr Opin Gastroenterol 2001;17:99–103. [38] Massey KA, Blakeslee CH, Pitkow HS. A review of physiological and metabolic effects of essential amino acids. Amino Acids 1998;14:271–300. [39] Singer M, Solomon T, Grossman M. Pancreatic response to intestinal perfusion with tryptophan and phenylalanine. Gastroenterol 1976;76:124. [40] Hayashi N, Someya N, Endo MY, Miura A, Fukuba Y. Vasoconstriction and blood flow responses in visceral arteries to mental task in humans. Exp Physiol 2006;91: 215–20.

[41] Knardahl S, Sanders BJ, Johnson AK. Haemodynamic responses to conflict stress in borderline hypertensive rats. J Hypertens 1989;7:585–93. [42] Vatner SF. Effects of exercise and excitement on mesenteric and renal dynamics in conscious, unrestrained baboons. Am J Physiol 1978;234:H210–4. [43] Gruys E, Toussaint MJM, Landman WJM, Tivapasi M, Chamanza R, Van Veen L. Infection, inflammation and stress inhibit growth. Mechanisms and non-specific assessment of the processes by acute phase proteins. In: Wensing Th, editor. Production diseases in farm animals. 10th International conference. The Netherlands: Wageningen press; 1998. p. 72–87. [44] Kraft R, Ruchti Ch, Burkhardt A, Cottier H. Pathogenic principles in the development of gut-derived infectious-toxic shock (GITS) and multiple organ failure. Curr Stud Haematol Blood Transfus 1992;59:204–40. [45] Sanger GJ, Yoshida M, Yahyah M, Kitazumi K. Increased defecation during stress or after 5-hydroxytryptophan: selective inhibition by the 5-HT4 receptor antagonist, SB-207266. Br J Pharmacol 2000;130:706–12. [46] Hay M, Mormède P. Urinary excretion of catecholamines, cortisol and their metabolites in Meishan and Large White sows: validation as a non-invasive and integrative assessment of adrenocortical and sympathoadrenal axis activity. Veter Res 1998;29:119–28. [47] Hay M, Meunier-Salaün MC, Brulaud F, Monnier M, Mormède P. Assessment of hypothalamic-pituitary-adrenal axis and sympathetic nervous system activity in pregnant sows through the measurement of glucocorticoids and catecholamines in urine. J Anim Sci 2000;78:420–8.