Winfried Otten

Tryptophan metabolism, from nutrition to potential therapeutic applications

Tryptophan is an indispensable amino acid that should to be supplied by dietary protein. Apart from its incorporation into body proteins, tryptophan is the precursor for serotonin, an important neuromediator, and for kynurenine, an intermediary metabolite of a complex metabolic pathway ending with niacin, CO2, and kynurenic and xanthurenic acids. Tryptophan metabolism within different tissues is associated with numerous physiological functions. The liver regulates tryptophan homeostasis through degrading tryptophan in excess. Tryptophan degradation into kynurenine by immune cells plays a crucial role in the regulation of immune response during infections, inflammations and pregnancy. Serotonin is synthesized from tryptophan in the gut and also in the brain, where tryptophan availability is known to influence the sensitivity to mood disorders. In the present review, we discuss the major functions of tryptophan and its role in the regulation of growth, mood, behavior and immune responses with regard to the low availability of this amino acid and the competition between tissues and metabolic pathways for tryptophan utilization.

Variation in fat mobilization during early lactation differently affects feed intake, body condition, and lipid and glucose metabolism in high-yielding dairy cows

Fat mobilization to meet energy requirements during early lactation is inevitable because of insufficient feed intake, but differs greatly among high-yielding dairy cows. Therefore, we studied milk production, feed intake, and body condition as well as metabolic and endocrine changes in high-yielding dairy cows to identify variable strategies in metabolic and endocrine adaptation to overcome postpartum metabolic load attributable to milk production. Cows used in this study varied in fat mobilization around calving, as classified by mean total liver fat concentrations (LFC) postpartum. German Holstein cows (n=27) were studied from dry off until d 63 postpartum in their third lactation. All cows were fed the same total mixed rations ad libitum during the dry period and lactation. Plasma concentrations of metabolites and hormones were measured in blood samples taken at d 56, 28, 15, and 5 before expected calving and at d 1 and once weekly up to d 63 postpartum. Liver biopsies were taken on d 56 and 15 before calving, and on d 1, 14, 28, and 49 postpartum to measure LFC and glycogen concentrations. Cows were grouped accordingly to mean total LFC on d 1, 14, and 28 in high, medium, and low fat-mobilizing cows. Mean LFC (±SEM) differed among groups and were 351±14, 250±10, and 159±9 mg/g of dry matter for high, medium, and low fat-mobilizing cows, respectively, whereas hepatic glycogen concentrations postpartum were the highest in low fat-mobilizing cows. Cows in the low group showed the highest dry matter intake and the least negative energy balance postpartum, but energy-corrected milk yield was similar among groups. The decrease in body weight postpartum was greatest in high fat-mobilizing cows, but the decrease in backfat thickness was greatest in medium fat-mobilizing cows. Plasma concentrations of nonesterified fatty acids and β-hydroxybutyrate were highest around calving in high fat-mobilizing cows. Plasma triglycerides were highest in the medium group and plasma cholesterol concentrations were lowest in the high group at calving. During early lactation, the decrease in plasma glucose concentrations was greatest in the high group, and plasma insulin concentrations postpartum were highest in the low group. The revised quantitative insulin sensitivity check index values decreased during the transition period and postpartum, and were highest in the medium group. Plasma cortisol concentrations during the transition period and postpartum period and plasma leptin concentrations were highest in the medium group. In conclusion, cows adapted differently to the metabolic load and used variable strategies for homeorhetic regulation of milk production. Differences in fat mobilization were part of these strategies and contributed to the individual adaptation of energy metabolism to milk production.

Dissecting the effects of mtDNA variations on complex traits using mouse conplastic strains

Previous reports have demonstrated that the mtDNA of mouse common inbred strains (CIS) originated from a single female ancestor and that mtDNA mutations occurred during CIS establishment. This situation provides a unique opportunity to investigate the impact of individual mtDNA variations on complex traits in mammals. In this study, we compiled the complete mtDNA sequences of 52 mouse CIS. Phylogenetic analysis demonstrated that 50 of the 52 CIS descended from a single female Mus musculus domesticus mouse, and mtDNA mutations have accumulated in 26 of the CIS. We then generated conplastic strains on the C57BL/6J background for 12 mtDNA variants with one to three functional mtDNA mutations. We also generated conplastic strains for mtDNA variants of the four M. musculus subspecies, each of which contains hundreds of mtDNA variations. In total, a panel of conplastic strains was generated for 16 mtDNA variants. Phenotypic analysis of the conplastic strains demonstrated that mtDNA variations affect susceptibility to experimental autoimmune encephalomyelitis and anxiety-related behavior, which confirms that mtDNA variations affect complex traits. Thus, we have developed a unique genetic resource that will facilitate exploration of the biochemical and physiological roles of mitochondria in complex traits.

Psychological Stress-Induced, IDO1-Dependent Tryptophan Catabolism: Implications on Immunosuppression in Mice and Humans

It is increasingly recognized that psychological stress influences inflammatory responses and mood. Here, we investigated whether psychological stress (combined acoustic and restraint stress) activates the tryptophan (Trp) catabolizing enzyme indoleamine 2,3-dioxygenase 1(IDO1) and thereby alters the immune homeostasis and behavior in mice. We measured IDO1 mRNA expression and plasma levels of Trp catabolites after a single 2-h stress session and in repeatedly stressed (4.5-days stress, 2-h twice a day) naïve BALB/c mice. A role of cytokines in acute stress-induced IDO1 activation was studied after IFNγ and TNFα blockade and in IDO1−/− mice. RU486 and 1-Methyl-L-tryptophan (1-MT) were used to study role of glucocorticoids and IDO1 on Trp depletion in altering the immune and behavioral response in repeatedly stressed animals. Clinical relevance was addressed by analyzing IDO1 activity in patients expecting abdominal surgery. Acute stress increased the IDO1 mRNA expression in brain, lung, spleen and Peyers patches (max. 14.1±4.9-fold in brain 6-h after stress) and resulted in a transient depletion of Trp (−25.2±6.6%) and serotonin (−27.3±4.6%) from the plasma measured 6-h after stress while kynurenine levels increased 6-h later (11.2±9.3%). IDO1 mRNA up-regulation was blocked by anti-TNFα and anti-IFNγ treatment. Continuous IDO1 blockade by 1-MT but not RU486 treatment normalized the anti-bacterial defense and attenuated increased IL-10 inducibility in splenocytes after repeated stress as it reduced the loss of body weight and behavioral alterations. Moreover, kynurenic acid which remained increased in 1-MT treated repeatedly stressed mice was identified to reduce the TNFα inducibility of splenocytes in vitro and in vivo. Thus, psychological stress stimulates cytokine-driven IDO1 activation and Trp depletion which seems to have a central role for developing stress-induced immunosuppression and behavioral alteration. Since patients showed Trp catabolism already prior to surgery, IDO is also a possible target enzyme for humans modulating immune homeostasis and mood.

Limited and excess dietary protein during gestation affects growth and compositional traits in gilts and impairs offspring fetal growth

The aim of this study was to investigate whether dietary protein intake during gestation less than or greater than recommendations affects gilts growth and body composition, gestation outcome, and colostrum composition. German Landrace gilts were fed gestation diets (13.7 MJ of ME/kg) containing a low (n = 18; LP, 6.5% CP), an adequate (n = 20; AP, 12.1%), or a high (n = 16; HP, 30%) protein content corresponding to a protein:carbohydrate ratio of 1:10.4, 1:5, and 1:1.3, respectively, from mating until farrowing. Gilts were inseminated by semen of pure German Landrace boars and induced to farrow at 114 d postcoitum (dpc; Exp. 1). Energy and protein intake during gestation were 33.3, 34.4, and 35.8 MJ of ME/d (P < 0.001) and 160, 328, and 768 g/d, respectively, in LP, AP, and HP gilts (P < 0.001). From insemination to 109 dpc, BW gain was least in LP (42.1 kg), intermediate in HP (63.1 kg), and greatest in AP gilts (68.3 kg), whereas increase of backfat thickness was least in gilts fed the HP diet compared with LP and AP diets (3.8, 5.1, 5.0 mm; P = 0.01). Litter size, % stillborn piglets, and mummies were unaffected (P > 0.28) by the gestation diet. Total litter weight tended to be less in the offspring of LP and HP gilts (14.67, 13.77 vs. 15.96 kg; P = 0.07), and the percentage of male piglets was greater in litters of HP gilts (59.4%; P < 0.01). In piglets originating from LP and HP gilts, individual birth weight was less (1.20, 1.21 vs. 1.40 kg; P = 0.001) and birth weight/crown-rump length ratio was reduced (45.3, 46.4 vs. 50.7 g/cm; P = 0.003). Colostrum fat (7.8, 7.4 vs. 8.1%) and lactose concentrations (2.2, 2.1 vs. 2.6%) tended to be reduced in LP and HP gilts (P = 0.10). In Exp. 2, 28 gilts (LP, 10; AP, 9; HP, 9) were treated as in Exp. 1 but slaughtered at 64 dpc. At 64 dpc, LP gilts were 7% lighter than AP gilts (P = 0.03), whereas HP gilts were similar to AP gilts. Body composition was markedly altered in response to LP and HP feeding with less lean (P < 0.01) and greater fat content (P = 0.02 to 0.04) in LP and less fat content (P = 0.02 to 0.04) in HP gilts. Fetal litter weight and number, and embryonic survival at 64 dpc were not affected by the diets. These results indicated that gestation diets containing protein at 50 and 250% of recommendations and differing in protein:carbohydrate ratio led to marked changes in protein and fat metabolism in gilts resulting in fetal growth retardation of 15%, which mainly occurred during the second half of gestation.

Effects of limited and excess protein intakes of pregnant gilts on carcass quality and cellular properties of skeletal muscle and subcutaneous adipose tissue in fattening pigs

The aim of this study was to investigate whether dietary protein intake of gilts during gestation below (50%) or above (250%) recommendations affects body composition, carcass and meat quality, and properties of skeletal muscle and subcutaneous adipose tissue (SCAT) in offspring at d 83 and 188 of age. German Landrace gilts were fed isoenergetic gestation diets (~13.7 MJ of ME/kg) containing a low (LP, 6.5%; n = 18), an adequate (AP, 12.1%; n = 20), or a high (HP, 30%; n = 16) protein content from mating until farrowing. Within 48 h of birth, offspring were cross-fostered to sows fed a standard diet. On d 83 of age, no effects of the LP diet on BW and body composition were detected, whereas HP pigs showed a slight growth delay (P = 0.06) associated with increased relative weights of small intestine (P < 0.01) and brain (P = 0.08), and reduced relative thymus weight (P < 0.01). On d 188 of age, BW was not different among the dietary groups. However, the carcass of LP pigs contained less (P = 0.01) lean and more (P = 0.07) fat compared with AP and HP pigs, which was only pronounced in pigs originating from large litters (P < 0.05). Like skeletal muscles (P = 0.06), the heart muscle weighed less (P = 0.02) in LP than AP pigs. Compared with AP pigs, LP pigs exhibited a fewer (P = 0.09) total number of myofibers in semitendinosus muscle plus LM both at d 83 and 188 of age, whereas total muscular DNA was less (P = 0.02) at d 188 only. The mRNA abundance of IGF2 measured on d 188 was reduced in SCAT (P = 0.03) and LM (P = 0.07) of LP compared with AP pigs. No changes in muscular fiber type frequency, capillary density, or creatine kinase activity, as well as SCAT adipocyte size and number, were observed at either stages of age. Meat quality characteristics remained unchanged at d 83, whereas Warner-Bratzler shear force value in LM was decreased (P = 0.03) in LP compared with AP pigs on d 188 of age. The results suggest that the maternal LP diet impairs prenatal myofiber formation, reduces the potential of postnatal lean growth related to reduced IGF2 mRNA expression and myonuclear accumulation, and consequently changes carcass quality toward reduced lean proportion and improved tenderness at market weight. In contrast, except for a slight transient growth delay, excess dietary protein during gestation seems to have little effect on the fetal programming of postnatal muscle and adipose tissue phenotype of the progeny.

Effects of weaning and restraint stress on glucocorticoid receptor binding capacity in limbic areas of domestic pigs

Changes in glucocorticoid receptor (GR) binding in different brain areas were investigated in neonatal and adult pigs exposed to psychological stress (weaning) and a physical stressor (repeated snaring). The GR binding was significantly decreased 4 days after weaning in both the hippocampus and the amygdala, but there were no changes in the hypothalamus. Repeated snaring of adult pigs resulted in a significant diminished GR binding only in the hippocampus.

Physiological and behavioral effects of different success during social confrontation in pigs with prior dominance experience

The impact of a 10-h social confrontation on behavior, plasma adrenaline, noradrenaline and cortisol was studied in 14 domestic pigs (eight castrated males and six females) with prior dominance experience. Prior to the experiments, animal groups, each consisting of nine growing pigs (12 weeks old) from different litters, were composed randomly. After 5 days, the pig with the highest rank from each group was removed, provided with a jugular vein catheter and kept in single housing for 2-3 weeks. After this period, each test animal was returned into its familiar group for a 10-h social confrontation. The reintroduction of the test animals into the familiar groups caused frequent agonistic interactions during the first 30 min. Seven animals succeeded to win most of their encounters during the confrontation test and were classified as High Success (HS) animals, whereas seven other animals lost most of their encounters and were classified as Low Success (LS) animals. The reintroduction of the test animals into the groups provoked also marked changes in behavioral and physiological measures. The frequent fighting behavior during the first 30 min was accompanied by a rapid increase of plasma catecholamines and a delayed increase of cortisol. Immediately after introduction, LS pigs tended to show higher plasma adrenaline and noradrenaline concentrations than HS pigs. There was also a tendency for a sustained increase of noradrenaline/adrenaline ratios in HS pigs, whereas the ratios of LS pigs remained nearly unchanged. No significant differences between both groups were found in cortisol levels and in the frequency of agonistic interactions. However, LS animals showed less locomotion, more lying and spent less time exploring the pen or other animals. These results show that during a social confrontation the experience of defeats for formerly high-ranking pigs is accompanied by increased submissive or passive behavior and a higher sympathoneural and adrenomedullary reaction, which may indicate more emotional distress and fear compared to successful animals.

Low and High Dietary Protein:Carbohydrate Ratios during Pregnancy Affect Materno-Fetal Glucose Metabolism in Pigs

Inadequate dietary protein during pregnancy causes intrauterine growth retardation. Whether this is related to altered maternal and fetal glucose metabolism was examined in pregnant sows comparing a high-protein:low-carbohydrate diet (HP-LC; 30% protein, 39% carbohydrates) with a moderately low-protein:high-carbohydrate diet (LP-HC; 6.5% protein, 68% carbohydrates) and the isoenergetic standard diet (ST; 12.1% protein, 60% carbohydrates). During late pregnancy, maternal and umbilical glucose metabolism and fetal hepatic mRNA expression of gluconeogenic enzymes were examined. During an i.v. glucose tolerance test (IVGTT), the LP-HC-fed sows had lower insulin concentrations and area under the curve (AUC), and higher glucose:insulin ratios than the ST- and the HP-LC-fed sows (P < 0.05). Insulin sensitivity and glucose clearance were higher in the LP-HC sows compared with ST sows (P < 0.05). Glucagon concentrations during postabsorptive conditions and IVGTT, and glucose AUC during IVGTT, were higher in the HP-LC group compared with the other groups (P < 0.001). (13)C glucose oxidation was lower in the HP-LC sows than in the ST and LP-HC sows (P < 0.05). The HP-LC fetuses were lighter and had a higher brain:liver ratio than the ST group (P < 0.05). The umbilical arterial inositol concentration was greater in the HP-LC group (P < 0.05) and overall small fetuses (230-572 g) had higher values than medium and heavy fetuses (≥573 g) (P < 0.05). Placental lactate release was lower in the LP-HC group than in the ST group (P < 0.05). Fetal glucose extraction tended to be lower in the LP-HC group than in the ST group (P = 0.07). In the HP-LC and LP-HC fetuses, hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) was higher than in the ST fetuses (P < 0.05). In conclusion, the HP-LC and LP-HC sows adapted by reducing glucose turnover and oxidation and having higher glucose utilization, respectively. The HP-LC and LP-HC fetuses adapted via prematurely expressed hepatic gluconeogenic enzymes.

Intrauterine growth retarded progeny of pregnant sows fed high protein:low carbohydrate diet is related to metabolic energy deficit.

High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR). To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW) at birth, sow body weight and back fat thickness. Sows were fed diets with low (6.5%, LP), adequate (12.1%, AP), and high (30%, HP) protein levels, made isoenergetic by adjusted carbohydrate content. At −5, 24, 66, and 108 days post coitum (dpc) fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC), %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA) concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein∶low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein∶high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal.