Acute and persistent effects of oral glutamine supplementation on growth, cellular proliferation, and tight junction protein transcript abundance in jejunal tissue of low and normal birthweight pre-weaning piglets.

Breeding for higher fertility has resulted in a higher number of low birthweight (LBW) piglets. It has been shown that LBW piglets grow slower than normal birthweight (NBW) littermates. Differences in growth performance have been associated with impaired small intestinal development. In suckling and weaning piglets, glutamine (Gln) supplementation has been associated with improved growth and intestinal development. This study was designed to examine the effects of oral Gln supplementation on growth and small intestinal parameters in LBW and NBW suckling piglets. At birth (day 0), a total of 72 LBW (1.10 ± 0.06 kg) and 72 NBW (1.51 ± 0.06) male piglets were selected. At day 1, litters were standardized to 12 piglets, and experimental piglets supplemented daily with either Gln (1 g/kg BW) or isonitrogenous amounts of Alanine (Ala) as control (1.22 g/kg BW) until day 12. Creep feed was offered from day 14 onward. Subgroups of piglets were euthanized at days 5, 12, and 26 for the analyses of jejunal morphometry, cellular proliferation, glutathione concentration and transcript abundance of tight junction proteins. From age day 11 to 21, Gln supplemented LBW (LBW-Gln) piglets were heavier than Ala supplemented LBW (LBW-Ala) littermates (P = 0.034), while NBW piglets were heavier until age day 26 compared to LBW littermates. Villus height was higher in LBW-Gln compared to LBW-Ala on age day 12 (P = 0.031). Sporadic differences among supplementation and birthweight groups were detected for jejunal cellular proliferation, cellular population and glutathione concentration, whereas age was the most dominant factor. These results show that Gln supplementation improved the growth of LBW piglets compared to LBW-Ala beyond the termination of Gln supplementation, but this was not associated with consistent effects on selected parameters of jejunal development.

Preclinical insights into the gut-skeletal muscle axis in chronic gastrointestinal diseases.

Muscle wasting represents a constant pathological feature of common chronic gastrointestinal diseases, including liver cirrhosis (LC), inflammatory bowel diseases (IBD), chronic pancreatitis (CP) and pancreatic cancer (PC), and is associated with increased morbidity and mortality. Recent clinical and experimental studies point to the existence of a gut-skeletal muscle axis that is constituted by specific gut-derived mediators which activate pro- and anti-sarcopenic signalling pathways in skeletal muscle cells. A pathophysiological link between both organs is also provided by low-grade systemic inflammation. Animal models of LC, IBD, CP and PC represent an important resource for mechanistic and preclinical studies on disease-associated muscle wasting. They are also required to test and validate specific anti-sarcopenic therapies prior to clinical application. In this article, we review frequently used rodent models of muscle wasting in the context of chronic gastrointestinal diseases, survey their specific advantages and limitations and discuss possibilities for further research activities in the field. We conclude that animal models of LC-, IBD- and PC-associated sarcopenia are an essential supplement to clinical studies because they may provide additional mechanistic insights and help to identify molecular targets for therapeutic interventions in humans.

Oral exposure of pigs to the mycotoxin deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction.

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Substitution of Dietary Sulfur Amino Acids by dl-2-Hydroxy-4-Methylthiobutyric Acid Reduces Fractional Glutathione Synthesis in Weaned Piglets.

BACKGROUND: Cys is limiting for reduced glutathione (GSH) synthesis and can be synthesized from Met. We hypothesized that the dietary Met hydroxyl analogue dl-2-hydroxy-4-methylthiobutyric acid (dl-HMTBA) affects Cys and GSH metabolism and oxidative stress defense differently than Met. OBJECTIVE: The objective was to elucidate whether dl-HMTBA supplementation of a Met-deficient diet affects Cys flux, GSH fractional synthetic rate (FSR), and the basal oxidative stress level relative to Met supplementation in pigs. METHODS: Twenty-nine male German Landrace piglets aged 28 d were allocated to 3 dietary groups: a basal diet limiting in Met (69% of Met plus Cys requirement) supplemented with either 0.15% l-Met (LMET; n = 9), 0.15% dl-Met (DLMET; n = 11), or 0.17% dl-HMTBA (DLHMTBA; n = 9) on an equimolar basis. At age 54 d the pigs received a continuous infusion of [1-13C]-Cys to calculate Cys flux and Cys oxidation. After 3 d, GSH FSR was determined by [2,2-2H2]-glycine infusion, and RBC GSH and oxidized GSH concentrations were measured. At age 62 d the animals were killed to determine hepatic mRNA abundances of enzymes involved in GSH metabolism, GSH concentrations, and plasma oxidative stress defense markers. RESULTS: The Cys oxidation was 21-39% and Cys flux 5-15% higher in the fed relative to the feed-deprived state (P < 0.001). On average, GSH FSR was 49% lower (P < 0.01), and RBC GSH and total GSH concentrations were 12% and 9% lower, respectively, in DLHMTBA and DLMET relative to LMET pigs (P < 0.05). In the feed-deprived state, Gly flux, the GSH:oxidized glutathione (GSSG) ratio, RBC GSSG concentrations, plasma oxidative stress markers, and the hepatic GSH content did not differ between groups. CONCLUSIONS: Although GSH FSR was higher in LMET compared with DLMET or DLHMTBA feed-deprived pigs, these differences were not reflected by lower oxidative stress markers and antioxidant defense enzymes in LMET pigs.

Substitution of Dietary Sulfur Amino Acids by DL-2-hydroxy-4-Methylthiobutyric Acid Increases Remethylation and Decreases Transsulfuration in Weaned Piglets.

BACKGROUND: DL-2-hydroxy-4-methylthiobutyric acid (DL-HMTBA), an L-methionine (L-Met) hydroxyl analogue, has been suggested to be a dietary L-Met source. How dietary DL-HMTBA compared with L-Met affects whole-body L-Met kinetics in growing individuals is unknown. OBJECTIVES: We determined to what extent DL-HMTBA supplementation of an L-Met-deficient diet affects whole-body L-Met and L-cysteine (L-Cys) kinetics, protein synthesis (PS), and the L-Met incorporation rate in liver protein (L-MetInc) compared with L-Met and DL-Met supplementation in a piglet model. METHODS: Forty-five, 28-d-old weaned piglets (male, German Landrace) were allocated to 4 dietary groups: L-Met-deficient diet [Control: 69% of recommended L-Met plus L-Cys supply; 0.22% standardized ileal digestible (SID) L-Met; 0.27% SID L-Cys; n = 12] and Control diet supplemented equimolarly to 100% of recommended intake with either L-Met (n = 12; LMET), DL-Met (n = 11; DLMET), or DL-HMTBA (n = 10; DLHMTBA). At 47 d of age, the piglets were infused with L-[1-13C; methyl-2H3]-Met and [3,3-2H2]-Cys to determine the kinetics and PS rates. Plasma amino acid (AA) concentrations, hepatic mRNA abundances of L-Met cycle and transsulfuration (TS) enzymes, and L-MetInc were measured. RESULTS: During feed deprivation, L-Met kinetics did not differ between groups, and were ≤3 times higher in the fed state (P < 0.01). Remethylation (RM) was 31% and 45% higher in DLHMTBA than in DLMET and Control pigs, respectively, and the RM:transmethylation (TM) ratio was 50% higher in DLHMTBA than in LMET (P < 0.05). Furthermore, TS and the TS:TM ratio were 32% lower in DLHMTBA than in LMET (P < 0.05). L-MetInc was 42% lower in DLMET and DLHMTBA than in L-Met-deficient Control pigs, whereas plasma AA and hepatic mRNA abundances were similar among DL-HMTBA-, L-Met-, and DL-Met-supplemented pigs. CONCLUSIONS: In piglets, DL-HMTBA compared with L-Met and DL-Met supplementation increases RM and reduces the TS rate to conserve L-Met, but all 3 Met isomers support growth at a comparable rate.

Stable production of cyanophycinase in Nicotiana benthamiana and its functionality to hydrolyse cyanophycin in the murine intestine.

Food supplementation with the conditionally essential amino acid arginine (Arg) has been shown to have nutritional benefits. Degradation of cyanophycin (CGP), a peptide polymer used for nitrogen storage by cyanobacteria, requires cyanophycinase (CGPase) and results in the release of ß-aspartic acid (Asp)-Arg dipeptides. The simultaneous production of CGP and CGPase in plants could be a convenient source of Arg dipeptides. Different variants of the cphB coding region from Thermosynechococcus elongatus BP-1 were transiently expressed in Nicotiana benthamiana plants. Translation and enzyme stability were optimized to produce high amounts of active CGPase. Protein stability was increased by the translational fusion of CGPase to the green fluorescent protein (GFP) or to the transit peptide of the small subunit of RuBisCO for peptide production in the chloroplasts. Studies in mice showed that plant-expressed CGP fed in combination with plant-made CGPase was hydrolysed in the intestine, and high levels of ß-Asp-Arg dipeptides were found in plasma, demonstrating dipeptide absorption. However, the lack of an increase in Asp and Arg or its metabolite ornithine in plasma suggests that Arg from CGP was not bioavailable in this mouse group. Intestinal degradation of CGP by CGPase led to low intestinal CGP content 4 h after consumption, but after ingestion of CGP alone, high CGP concentrations remained in the large intestine; this indicated that intact CGP was transported from the small to the large intestine and that CGP was resistant to colonic microbes.

The Effects of Oral Quercetin Supplementation on Splanchnic Glucose Metabolism in 1-Week-Old Calves Depend on Diet after Birth.

BACKGROUND: Inadequate colostrum supply results in insufficient intake of macronutrients and bioactive factors, thereby impairing gastrointestinal development and the maturation of glucose metabolism in neonatal calves. The flavonoid quercetin has been shown to have health-promoting properties, including effects in diabetic animals. However, quercetin interacts with intestinal glucose absorption and might therefore exert negative effects in neonates. OBJECTIVE: We evaluated the interaction between neonatal diet and quercetin feeding on splanchnic glucose metabolism in neonatal calves. METHODS: Calves (n = 28) were assigned to 4 groups and fed either colostrum or a milk-based formula on days 1 and 2 and supplemented daily with 148 µmol quercetin aglycone/kg body weight [colostrum with quercetin (CQ+)/formula with quercetin (FQ+)] or without this substance [colostrum without quercetin (CQ-)/formula with quercetin (FQ-)] from days 2-8. From day 3 onward, all calves received milk replacer. A xylose absorption test was performed on day 3, and on day 7, blood samples were collected to study glucose first-pass uptake after [(13)C6]-glucose feeding and intravenous [6,6-(2)H2]-glucose bolus injection. Plasma concentrations of metabolites and hormones were measured by taking additional blood samples. A biopsy specimen of the liver was harvested on day 8 to measure the mRNA expression of gluconeogenic enzymes. RESULTS: Higher postprandial plasma concentrations of glucose, lactate, urea, adrenaline, noradrenaline, insulin, and glucagon on day 7 in colostrum-fed calves indicate that metabolic processes were stimulated. Postabsorptive xylose and glucose plasma concentrations each increased by an additional 26%, and splanchnic glucose turnover decreased by 35% in colostrum-fed calves, suggesting improved glucose absorption and lower splanchnic glucose utilization in colostrum-fed calves. Quercetin supplementation resulted in higher noradrenaline concentrations and enhanced peak absorption and oxidation of [(13)C6]-glucose by 10%. Liver mitochondrial phosphoenolpyruvate carboxykinase mRNA abundance was reduced by 34% in colostrum-deprived calves. CONCLUSIONS: Feeding colostrum during the first 2 d of life is crucial for maturation of splanchnic glucose metabolism in calves. Supplementing quercetin improves gastrointestinal absorption capacity, particularly in colostrum-deprived calves.

Effects of a 6-wk intraduodenal supplementation with quercetin on energy metabolism and indicators of liver damage in periparturient dairy cows.

Periparturient dairy cows experience metabolic challenges that result in a negative energy balance (EB) and a range of postpartum health problems. To compensate for the negative EB, cows mobilize fatty acids from adipose tissues, which can lead to fatty liver disease, a periparturient metabolic disorder. Flavonoids, such as quercetin (Q), are polyphenolic substances found in all higher plants and have hepatoprotective potential and the ability to prevent or reduce lipid accumulation in the liver. In ruminants, few studies on the metabolic effects of Q are available, and thus this study was conducted to determine whether Q has beneficial effects on EB, lipid metabolism, and hepatoprotective effects in periparturient dairy cows. Quercetin was supplemented intraduodenally to circumvent Q degradation in the rumen. Cows (n=10) with duodenal fistulas were monitored for 7wk. Beginning 3wk before expected calving, 5 cows were treated with 100mg of quercetin dihydrate per kilogram of body weight daily in a 0.9% sodium chloride solution for a total period of 6wk, whereas the control cows received only the sodium chloride solution. The plasma flavonoid levels were higher in the Q-treated cows than in the control cows. A tendency for higher postpartum (pp) than antepartum (ap) plasma flavonoid levels was observed in the Q-treated cows than in the controls, which was potentially caused by a reduced capacity to metabolize Q. However, the metabolic status of the Q-treated cows did not differ from that of the control cows. The pp increases in plasma aspartate aminotransferase and glutamate dehydrogenase activities were less in the Q-treated cows than in the control cows. The Q had no effect on energy expenditures, but from ap to pp the cows had a slight decline in respiratory quotients. Irrespective of the treatment group, the oxidation of fat peaked after calving, suggesting that the increase occurred because of an increased supply of fatty acids from lipomobilization. In conclusion, supplementation with Q resulted in lower pp plasma aminotransferase and glutamate dehydrogenase, which indicated reduced liver damage. However, the direct effects of Q on the liver and the implications for animal performance remain to be investigated.

Feeding of selenium alone or in combination with glucoraphanin differentially affects intestinal and hepatic antioxidant and phase II enzymes in growing rats.

The anti-carcinogenic effects of sulforaphane (SFN) are based on the up-regulation of antioxidant enzymes (AE) and phase II enzymes (PIIE) through the transcription factor Nrf2. Current knowledge on the roles of the SFN precursor glucoraphanin (GRA) on these processes is limited. Anti-carcinogenic effects of Se depending on glutathione peroxidase (GPx) activity have also been reported. We studied effects and possible synergisms of Se and GRA on the expression and activity of a broad spectrum of AE and PIIE in jejunum, colon and the liver of rats fed diets differing in Se and GRA concentration. In all organs, GPx1 mRNA expression was 70 % to 90 % lower in Se deficiency than in Se sufficiency. GPx2 expression increased in jejunum and liver under Se deficiency and decreased in the colon. Se deficiency increased most colonic AE and PIIE compared to Se adequacy. Adequate and in particular supranutritive Se combined with GRA increased colonic AE and PIIE expression up to 3.72-fold. In the liver Se deficiency raised the expression of AE and PIIE up to 4.49-fold. GRA attenuated liver AE and PIIE response in Se deficiency. Expression- and correlation analyses revealed that Keap1 mRNA better reflects AE and PIIE gene expression than Nrf2 mRNA. We conclude that: (1) GPx1 sensitively indicates Se deficiency; (2) the influence of Se and Nrf2/Keap1 on GPx2 expression depends on the organ; (3) GRA combined with supranutritive Se may effectively protect against inflammation and colon cancer; (4) future investigations on AE and PIIE expression should consider the role of Keap1 to a higher extent.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Maternal dietary protein restriction and excess affects offspring gene expression and methylation of non-SMC subunits of condensin I in liver and skeletal muscle.

Recent evidence indicates that maternal nutrition during pregnancy influences gene expression in offspring through epigenetic alterations. In the present study we evaluated the effect of protein excess and deficiency during porcine pregnancy on offspring hepatic and skeletal muscular expression patterns of key genes of methionine metabolism (DNMT1, DNMT3a, DNMT3b, BHMT, MAT2B and AHCYL1), condensin I subunit genes (NCAPD2, NCAPG and NCAPH), important for chromosome condensation and segregation, global DNA methylation and gene-specific DNA methylation. German Landrace sows were randomly assigned to control (CO), high protein (HP) and low protein (LP) diet groups. Tissue samples of offspring were collected from fetal (dpc95), newborn (dpn1), weanling (dpn28) and finisher pigs (dpn188). Gene expression of DNMT1, DNMT3a and DNMT3b was influenced by both HP and LP diets, indicating an involvement of DNA methylation in fetal programming by maternal protein supply. Moreover, hepatic global methylation was significantly affected by protein restriction at dpc95 (p = 0.004) and by protein excess at dpn188 (p = 0.034). Gene expression in fetal liver was significantly different between CO and LP for NCAPD2 (p = 0.0005), NCAPG (p = 0.0009) and NCAPH (p < 0.0001). In skeletal muscle, LP fetuses had significantly altered gene expression of NCAPD2 (p = 0.020) and NCAPH (p = 0.001), compared with CO. Furthermore, NCAPG was differentially methylated among LP, HP and CO; indeed, a significant positive correlation was detected with transcript amount in fetal pigs (r = 0.47, p = 0.002). These data demonstrate that both restriction and excess dietary protein during pregnancy alters the offspring's epigenetic marks and influences gene expression.

High and low protein∶ carbohydrate dietary ratios during gestation alter maternal-fetal cortisol regulation in pigs.

Imbalanced maternal nutrition during gestation can cause alterations of the hypothalamic-pituitary-adrenal (HPA) system in offspring. The present study investigated the effects of maternal low- and high-protein diets during gestation in pigs on the maternal-fetal HPA regulation and expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-HSD1 and 11ß-HSD2) and c-fos mRNAs in the placenta and fetal brain. Twenty-seven German Landrace sows were fed diets with high (HP, 30%), low (LP, 6.5%) or adequate (AP, 12.1%) protein levels made isoenergetic by varying the carbohydrate levels. On gestational day 94, fetuses were recovered under general anesthesia for the collection of blood, brain and placenta samples. The LP diet in sows increased salivary cortisol levels during gestation compared to the HP and AP sows and caused an increase of placental GR and c-fos mRNA expression. However, the diurnal rhythm of plasma cortisol was disturbed in both LP and HP sows. Total plasma cortisol concentrations in the umbilical cord vessels were elevated in fetuses from HP sows, whereas corticosteroid-binding globulin levels were decreased in LP fetuses. In the hypothalamus, LP fetuses displayed an enhanced mRNA expression of 11ß-HSD1 and a reduced expression of c-fos. Additionally, the 11ß-HSD2 mRNA expression was decreased in both LP and HP fetuses. The present results suggest that both low and high protein∶carbohydrate dietary ratios during gestation may alter the expression of genes encoding key determinants of glucocorticoid hormone action in the fetus with potential long-lasting consequences for stress adaptation and health.

Practical recommendations for the reduction of memory effects in compound-specific 15N/14N-ratio analysis of enriched amino acids by gas chromatography/combustion/isotope ratio mass spectrometry.

Gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS) is a highly sensitive approach which allows the analysis of the (13)C/(12)C and (15)N/(14)N isotope composition of amino acids in the range of natural abundance or in slightly (13)C- and (15)N-enriched samples. However, the accuracy of measurements remains a permanent challenge. Here we show the effect of the presence of slightly (15)N-enriched compounds in physiological samples on the accuracy and reproducibility of (15)N-abundances of amino acids within or between analytical runs. We spiked several individual amino acids with the respective (15)N-labelled isotopomer and measured the (15)N/(14)N ratios of other amino acids in the same sample or in the following analytical runs. Intra- and inter-run memory effects can be observed in (15)N/(14)N ratios of amino acids. Sample throughput is reduced when cleaning runs using standard mixtures are required to restore initial conditions after runs of samples with (15)N-enriched analytes. Possible reasons for the observed phenomenon and its implications for work in the lower (15)N-enrichment range (<0.5 APE) are discussed and include different aspects of gas chromatography, derivatisation, and hot catalytic metal surface effects. Results need to be interpreted with caution if complex physiological samples contain (15)N-enriched amino acids beyond 500‰ δ(15)N (~0.18 APE).

Effect of a high-protein diet on food intake and liver metabolism during pregnancy, lactation and after weaning in mice.

Major hepatic metabolic pathways are involved in the control of food intake but how dietary proteins affect global metabolism to adjust food intake is incompletely understood, particularly under physiological challenging conditions such as lactation. In order to identify these molecular events, mice were fed a high-protein (HP) diet from pregnancy, during lactation until after weaning and compared with control fed counterparts. Liver specimens were analyzed for regulated proteins using 2-DE and MALDI-TOF-MS and plasma samples for metabolites. Based on the 26 differentially expressed proteins associated with depleted liver glycogen content, elevated urea and citrulline plasma concentrations, we conclude that HP feeding during lactation leads to an activated amino acid, carbohydrate and fatty acid catabolism while it activates gluconeogenesis. From pregnancy to lactation, plasma arginine, tryptophan, serine, glutamine and cysteine decreased, whereas urea concentrations increased in both groups. Concomitantly, hepatic glycogen content decreased while total fat content remained unaltered in both groups. Consideration of 59 proteins differentially expressed between pregnancy and lactation highlights different strategies of HP and control fed mice to meet energy requirements for lactation by adjusting amino acid degradation, carbohydrate and fat metabolism, citrate cycle, but also ATP-turnover, protein folding, secretion of proteins and (de)activation of transcription factors.

Role of beta-hydroxybutyric acid in the central regulation of energy balance.

Although the phenomenon of beta-hydroxybutyric acid (BHBA) impact on satiety and thermogenesis has been described in the past decades, the underlying molecular mechanisms involved remain unresolved. Other metabolites such as glucose, fatty or branched chain amino acids are known to activate the AMP kinase pathway leading to an increase of anorexic and a decrease of orexigenic neuropeptides in the hypothalamus, one of the central regulators of energy homeostasis. Since BHBA is utilized as an energy source by the brain particularly in suckling newborns and under starving conditions, it is supposed to be a further central signal and energy providing substrate involved in the regulation of food intake. Moreover, BHBA might present a therapeutic approach for treating neuronal diseases because of its neuroprotective properties. Therefore, the purpose of this review is to summarize the known central effects of BHBA and to point out the importance of the identification of cellular pathways triggered in response to BHBA.

Effects of dietary energy intake during gestation and lactation on milk yield and composition of first, second and fourth parity sows.

In order to determine the effects of a varied level of dietary energy intake during pregnancy and lactation on milk yield and composition, first, second and fourth parity sows (Large White x German Landrace) were provided with energy at a level of either: (i) 100% of ME requirement (MEreq) during pregnancy and lactation, (ii) 120% MEreq during pregnancy and 80% during lactation, and (iii) 80% MEreq during pregnancy and 120% during lactation. In spite of equal target levels feed analysis revealed that gestating first parity sows with 120/80 treatment combination and lactating sows of 80/120 treatment combination received 25, and 11-17% more digestible N than in the respective 100/100 treatment combination. Irrespective of this 120/80 sows responded with the highest milk DM, fat, and energy contents, and the lowest lactose concentrations whereas protein levels where not affected, irrespective of parity (p < 0.05). Milk yield of sows in 1st and 4th lactation was 85 and 106% of that in 2nd lactation, respectively. Average milk composition was 18.1% DM, 4.9% protein, 6.8% fat, 5.6% lactose, and 0.8% ash. Milk composition changes ceased at day 7 of lactation with a reduction of milk GE and protein, and an increase of lactose content. Concentrations of threonine, arginine, valine, leucine, tyrosine, phenylalanine, cystine, and tryptophan, as well as stearic, oleic, and linoleic acid were higher in colostrum than in milk at later lactation stages. In contrast, laurine, myristic, palmitic, and palmitoleic acids were lower concentrated in colostrum. In conclusion, these results illustrate the importance of body reserve mobilization for milk production in sows and indicate that low energy supply during gestation cannot be compensated by higher energy supply during lactation.

Dietary protein modifies hepatic gene expression associated with oxidative stress responsiveness in growing pigs.

Understanding the basis for differences in nutrient requirements and for nutrient effects on health and performance requires an appreciation of the links between nutrition and gene expression. We developed and applied molecular probes to characterize diet-associated postabsorptive hepatic gene expression in growing pigs chronically fed protein-restricted diets based on either casein (CAS) or soy protein isolate (SPI). Eighty-eight expressed sequence tags (ESTs) were identified on the basis of diet-related changes in expression, by using an mRNA differential display method. Expression profiling based on transcription analysis by real-time reverse transcriptase-polymerase chain reaction showed that the SPI diet significantly changed the pattern of gene expression as compared with the CAS diet and allowed identification of coregulated genes. The expression of six genes involved in the metabolism of stress response (glutathione S-transferase, peptide methionine sulfoxide reductase, apolipoprotein A-I, organic anion transport polypeptide 2, calnexin, heat shock transcription factor 1) exhibited significant changes in the transcription level and indicated an increased oxidative stress response in pigs fed the SPI diet. Hierarchical clustering of gene expression data of all 33 ESTs analyzed across 14 pigs fed the two different diets resulted in clustering of genes related to the oxidative stress response with genes related to the regulation of gene expression and neuronal signaling.

Low-abundance plasma and urinary [(15)N]urea enrichments analyzed by gas chromatography/combustion/isotope ratio mass spectrometry.

We report a method for determining plasma und urinary [(15)N]urea enrichments in an abundance range between 0.37 and 0.52 (15)N atom% (0-0.15 atom% excess (APE) (15)N) using a dimethylaminomethylene derivative. Compared with conventional off-line preparation and (15)N analysis of urea, this method requires only small sample volumes (0.5 ml of plasma and 25 microl of urine). The (15)N/(14)N ratio of urea derivatives was measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Two peaks were separated; one was identified by gas chromatography/mass spectrometry (GC/MS) as the complete derivatized urea. Calibration of the complete urea derivative was performed by linear regression of enrichment values of known standard mixtures. Replicate standard (6-465 per thousand delta(15)N) derivatizations showed a relative standard deviation ranging from 0.1 to 7%. In order to test the feasibility of the method, human subjects and rats ingested a single meal containing either 200 mg of [(15)N]glycine (95 AP (15)N) or 0.4 mg of [(15)N]-alpha-lysine (95 AP (15)N), respectively. Urine and plasma were collected at hourly intervals over 7 h after the meal intake. After (15)N glycine intake, maximum urinary urea (15)N enrichments were 330 and 430 per thousand delta(15)N (0.12 and 0.16 APE (15)N) measured by GC/C/IRMS, whereas plasma [(15)N]glycine enrichments were 2.5 and 3.3 APE (15)N in the two human subjects 2 h after the meal. (15)N enrichments of total urine and urine samples devoid of ammonia were higher enriched than urinary [(15)N]urea measured by GC/C/IRMS, reflecting the presence of other urinary N-containing substances (e.g. creatinine). In rats plasma urea (15)N enrichments were 15-20 times higher than those in urinary urea (10-20 per thousand delta(15)N). The different [(15)N]urea enrichments observed after ingestion of [(15)N]-labeled glycine and lysine confirm known differences in the metabolism of these amino acids.