Tissue Amino Acid Concentrations Research Articles

Alterations in one‑carbon metabolism and protein synthesis signals due to methionine supplementation and lipopolysaccharide challenge in Holstein fetal liver explants

One‑carbon metabolism (OCM) fueled by methionine (Met), choline, and folic acid is key for embryo development and fetal growth. We investigated effects of lipopolysaccharide (LPS) to induce inflammation in fetal liver tissue with or without Met on components of OCM and protein synthesis activity. Fetal liver harvested at slaughter from six multiparous pregnant Holstein dairy cows (37 ± 6 kg milk/d, 100 ± 3 d gestation) were incubated (0.2 ± 0.02 g) for 4 h at 37 °C with each of the following: ideal profile of amino acids (control; Lysine:Met 2.9:1), control plus LPS (1 μg/mL), increased Met supply (Met, Lys:Met 2.5:1), and Met+LPS. Data were analyzed as a 2 × 2 factorial (PROC MIXED, SAS 9.4). Ratios of mechanistic target of rapamycin (p-mTOR:mTOR) and eukaryotic elongation factor 2 (p-eEF2:eEF2) protein were lowest (P < 0.0 5) with LPS and highest with Met. Tissue amino acid concentrations were lowest (P < 0.0 5) with Met regardless of LPS suggesting enhanced use via mTOR. The marked increase (P = 0.02) in phosphorylation of S6 ribosomal protein (p-RPS6) with LPS suggested a pro-inflammatory response that was partly alleviated with Met+LPS. No effect (P = 0.4 5) on methionine adenosyl transferase 1 A (MAT1A) protein abundance was detected. Activity of betaine-homocysteine S-methyltransferase (BHMT) was greatest with Met, but Met+LPS dampened this effect (P = 0.0 5). Overall, fetal liver responds to inflammatory challenges and Met supply. The latter can stimulate protein synthesis via mTOR and alter some OCM reactions while having a modest anti-inflammatory effect.

Low feed intake at weaning reduces intestinal glutathione levels and promotes cysteine oxidation to taurine in pigs.

Weaning stress in pigs is associated with low feed intake and poor nutrient utilization. Cysteine is a sulfur amino acid with key roles in pig production, but how cysteine metabolism and requirements are affected by weaning stress should be better defined. The objective of this study was to determine the collective impact of weaning and feed restriction on tissue cysteine metabolism. Pigs were weaned at 21-d age without access to feed (W; 6.90 ± 0.81kg; n = 9; reflecting acute nutritional stress) or were not weaned and remained with the sow (nonweaned [NW]; 6.81 ± 0.65kg; n = 8). At euthanasia (23-d age), blood, bile, liver, jejunum, and ileum tissues were collected. Plasma, bile, and tissue amino acid and amino thiol concentrations were analyzed by HPLC. The activity of glutamate cysteine ligase (GCL) and glutathione synthetase (GSS), enzymes needed for glutathione (GSH) production, and cysteine dioxygenase 1 (CDO1) were determined with activity assays followed by HPLC analysis of reaction products. Plasma (271 vs. 192 ± 19 µmol/L; P < 0.001) and liver (417 vs. 298 ± 33 nmol/g; P < 0.05) Cys concentrations were increased in W compared to NW pigs. Despite greater plasma Cys, jejunum and ileum Cys content were not affected by weaning (P > 0.10), whereas γ-glutamylcysteine (γ-GlyCys), the immediate precursor of GSH, declined in both jejunum (14.3 vs. 9.7 ± 1.4 nmol/g; P < 0.01) and ileum (11.2 vs. 6.4 ± 0.8 nmol/g; P < 0.001) in W pigs. Glutathione content was lower in the jejunum (1,379 vs. 1,720 ± 70 nmol/g; P < 0.05) and ileum (1,497 vs. 1,740 ± 74 nmol/g; P < 0.05) in W pigs. In the jejunum, GCL activity tended to be greater (0.56 vs. 0.39 ± 0.07 nmol γ-GluCys • mg-1 • min-1; P < 0.10), whereas GSS activity tended to be lower (1.11 vs. 1.38 ± 0.10 nmol GSH • mg-1 • min-1; P < 0.10) in W compared to NW pigs. In the ileum, the activities of GCL and GSS were not affected by weaning (P > 0.10). Although liver CDO1 activity was not different between groups (P < 0.10), liver taurine was greater in W compared to NW pigs (5,115 vs. 2,336 ± 912 nmol/g; P = 0.001). Bile concentrations of Cys (1,203 vs. 279 ± 103 µmol/L; P < 0.001) and cysteinylglycine (203 vs. 117 ± 33 µmol/L; P < 0.10), the direct product of GSH degradation, were greater in W compared to NW pigs. Collectively, these results suggest that systemic Cys is not effectively utilized for gut GSH production in newly weaned pigs; instead, it is oxidized to taurine and eliminated in bile.

Tissue and plasma free amino acid detection by LC-MS/MS method in high grade glioma patients.

The changes in serum amino acid profiles are evaluated in different types of cancers and screening tests were developed for estimating the risk of cancer by rapid analysis of plasma free amino acid (PFAA) levels. There is scarce evidence about the metabolomics analysis of PFAA in malignant gliomas. The aim of the present study was to identify the most promising diagnostic amino acid biomarkers that could be objectively measured for high-grade glioma and to compare their level with the tissue counterpart. In this prospective study, we collected serum samples from 22 patients with the pathological diagnosis of high-grade diffuse glioma according to WHO 2016 classification and 22 healthy subjects, and brain tissue from 22 controls. Plasma and tissue amino acid concentrations were analyzed applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Serum alanine, alpha-aminobutyric acid (AABA), lysine (Lys) and cysteine concentrations were significantly higher in high-grade glioma patients despite low levels of alanine and Lys in the tumor tissue. Aspartic acid, histidine and taurine were significantly decreased in both serum and tumors of glioma patients. A positive correlation was detected between tumor volumes and serum levels of latter three amino acids. This study demonstrated potential amino acids which may have diagnostic value for high-grade glioma patients by utilizing LC-MS/MS method. Our results are preliminary to compare serum and tissue levels of amino acids in patients with malignant gliomas. The data presented here may provide feature ideas about the metabolic pathways in the pathogenesis of gliomas.

Root exudation of carbon and nitrogen compounds varies over the day-night cycle in pea: The role of diurnal changes in internal pools.

Rhizodeposition is the export of organic compounds from plant roots to the soil. Carbon allocation towards rhizodeposition has to be balanced with allocation for other physiological functions, which depend on both newly assimilated and stored nonstructural carbohydrate (NSC). To test whether the exudation of primary metabolites scales with plant NSC status, we studied diurnal dynamics of NSC and amino acid (AA) pools and fluxes within the plant and the rhizosphere. These diurnal dynamics were measured in the field and under hydroponic-controlled conditions. Further, C-limiting treatments offered further insight into the regulation of rhizodeposition. The exudation of primary metabolites fluctuated diurnally. The diurnal dynamics of soluble sugars (SS) and AA concentrations in tissues coincided with exudate pool fluctuations in the rhizosphere. SS and AA pools in the rhizosphere increased with NSC and AA pools in the roots. C starvation treatments offset the balance of exudates: AA exudate content in the rhizosphere significantly decreased while SS exudate content remained stable. Our results suggest that rhizodeposition is to some extent controlled by plant C:N status. We propose that SS exudation is less controlled than AA exudation because N assimilation depends on controlled C supply while SS exudation relies to a greater extent on passive diffusion mechanisms.

An acute increase in water temperature can increase free amino acid concentrations in the blood, brain, liver, and muscle in goldfish (Carassius auratus).

Water temperature directly affects the body temperature in fish, so increasing water temperatures in oceans and rivers will lead to increases in fish body temperatures. Whilst a range of responses of fish to increases in water temperature have been measured, amino acid metabolism in a fish under high water temperature (HT) conditions has not been investigated. The aim of this study was to determine the effects of an acute increase in water temperature on oxygen consumption, plasma cortisol concentrations, and free amino acid concentrations in plasma and several tissues in goldfish (Carassius auratus). Oxygen consumption and plasma cortisol concentrations were increased in goldfish exposed to HT (30 ± 1°C) for 200min compared with goldfish at a control water temperature (CT 17 ± 1°C). Oxygen consumption and plasma cortisol concentrations in both groups of fish combined were positively correlated. When goldfish were exposed to HT for 300min oxygen consumption and plasma concentrations of 15 free amino acids were increased compared with goldish at CT. Concentrations of several free amino acids were increased to varying extents in the brain, liver, and muscle tissues. In conclusion, an acute increase in water temperature affected amino acid metabolism differently in the brain, liver, and muscle tissues. Goldfish will be a useful species for further studies of the possible roles of various amino acids in the brain, muscle, and liver during acute increases in water temperature in fish.

L-DOPA Reverses the Increased Free Amino Acids Tissue Levels Induced by Dopamine Depletion and Rises GABA and Tyrosine in the Striatum.

Perturbations in the cerebral levels of various amino acids are associated with neurological disorders, and previous studies have suggested that such alterations have a role in the motor and non-motor symptoms of Parkinson's disease. However, the direct effects of chronic L-DOPA treatment, that produces dyskinesia, on neural tissue amino acid concentrations have not been explored in detail. To evaluate whether striatal amino acid concentrations are altered in peak dose dyskinesia, 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian mice were treated chronically with L-DOPA and tissue amino acid concentrations were assessed by HPLC analysis. These experiments revealed that neither 6-OHDA nor L-DOPA treatment are able to alter glutamate in the striatum. However, glutamine increases after 6-OHDA and returns back to normal levels with L-DOPA treatment, suggesting increased striatal glutamatergic transmission with lack of dopamine. In addition, glycine and taurine levels are increased following dopamine denervation and restored to normal levels by L-DOPA. Interestingly, dyskinetic animals showed increased levels of GABA and tyrosine, while aspartate striatal tissue levels are not altered. Overall, our results indicate that chronic L-DOPA treatment, besides normalizing the altered levels of some amino acids after 6-OHDA, robustly increases striatal GABA and tyrosine levels which may in turn contribute to the development of L-DOPA-induced dyskinesia.

Characterization of the putative amino acid transporter genes AtCAT2, 3 &4: The tonoplast localized AtCAT2 regulates soluble leaf amino acids

The plant vacuole constitutes a large transient storage compartment for nutrients, proteins and metabolites, and is a major cellular sink for toxic waste compounds. Amino acids can cross the vacuolar membrane via specific transport proteins, which are molecularly not well characterized. Two members of a small subfamily of the cationic amino acid transporters, AtCAT2 and AtCAT4, were primarily localized at the tonoplast when tagged with GFP. The closely related AtCAT3, by contrast, was detected in the endoplasmic reticulum membrane. The exchange of a di-acidic motif at the carboxy-tail affected their sub-cellular localization, with larger effects visible in transiently transformed protoplasts compared to stably expressing plant lines. The genes have broad, partially overlapping tissue expression, with CAT2 dominating in most tissues. Loss-of-function mutants of individual CATs showed no visible phenotype under various conditions, but the overall tissue concentration of amino acids was increased in soil-grown cat2 mutants. The data suggest that CAT2 is a critical target of leaf amino acid concentrations and manipulation of this tonoplast transporter can significantly alter total tissue amino acid concentrations.

Stimulation of the Po-shen and Shen-hun scalp-acupuncture bands modifies levels of inhibitory and excitatory amino acids in the immature rat brain

ObjectiveIn the present study, the effect of stimulation of the Po-shen and Shen-hun scalp-acupuncture bands on tissue amino acid concentrations in several brain regions in awake and pentobarbital-sedated immature rats was evaluated. Materials and methodsSprague–Dawley rats (aged 15days) were organized in four groups of at least eight animals: control groups received saline solution 0.9% or sodium pentobarbital at 30mg/kg dosage via intraperitoneal. Experimental groups received saline solution or sodium pentobarbital plus stimulation in Po-shen and Shen-hun scalp-acupuncture bands for one continuous hour during 10 sessions by using scalp-acupuncture. ResultsAs compared to rats receiving saline solution, scalp-acupuncture produced significant changes in amino acid concentrations, depending on the analyzed region, as follows: in inhibitory amino acids, a GABA increase was observed in amygdala and hippocampus (491 and 184%, respectively), but a decrease in the substantia nigra (80%); glycine showed decrease in all the analyzed regions, except for an increase in brainstem(78%); glutamine presented an increase in hippocampus and cortex (42 and 149%, respectively). In the case of excitatory amino acids, glutamate decreased in all the analyzed regions; whereas aspartate decreased in substantia nigra and brainstem (77.08 and 35%, correspondingly) but increased in hippocampus and cortex (32 and 54%, respectively). The combined treatment of scalp-acupuncture and a GABAergic depressant drug like pentobarbital resulted in almost all changes induced in amino acids for scalp-acupuncture alone being significantly reverted. ConclusionStimulation of the Po-shen and Shen-hun scalp-acupuncture bands by using scalp-acupuncture alone might produce depressant activity by changes in amino acids, but the combination with a GABAergic tranquilizer like sodium pentobarbital can interfere with this response.

Citrulline a More Suitable Substrate than Arginine to Restore NO Production and the Microcirculation during Endotoxemia

BackgroundImpaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia.Methodology/Principal FindingsTo study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg•g bodyweight−1•h−1), combined with either L-Citrulline (6.25 mg•h-1), L-Arginine (6.25 mg•h−1), or L-Alanine (isonitrogenous control; 12.5 mg•h−1) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot.Conclusion/SignificanceL-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

Adverse effects of chronic intake of glutamine-supplemented diet on amino acid concentrations and protein metabolism in rat: Effect of short-term starvation

Summary Background & aims Glutamine supplementation is recommended in severe illness, preterm infants, and athletes. However, the information about side effects of chronic intake of glutamine-supplemented diet is limited. Methods Male Wistar rats were fed with glutamine-supplemented diet (GlnD). The controls were fed by standard or high-protein diets. After three months, the effect of GlnD on amino acid concentration in body fluids and protein metabolism was examined in fed and in 24 h-fasted animals. Protein synthesis was examined by the “flooding method” using phenylalanine; proteolysis on the basis of chymotrypsin-like activity (CHTLA) of proteasome and cathepsin B and L activities; amino acid concentrations were estimated using HPLC. Student’s t -tests with Bonferroni correction were used for statistical analysis. Results In the blood of GlnD fed animals sacrificed in fed state we found higher concentrations of glutamine, urea, and histidine, and lower concentration of creatinine and of a number of amino acids than in controls. The main alterations in intracellular free amino acid concentration were increased concentration of glutamine, increased concentration of histidine in skeletal muscle, and decreased concentrations of glycine, serine, and threonine in several tissues. In GlnD fed animals we found enhanced amount and concentration of protein in the kidneys, decreased protein content in tibialis muscle, and enhanced CHTLA in soleus muscle. We did not find significant effect on protein synthesis in any tissue. Starvation of animals previously fed by GlnD led to a marked decrease in glutamine concentration and an increase in glycine, serine, and threonine in plasma and in most of examined tissues, and in more significant decrease in protein synthesis in the liver, jejunum, colon, and spleen than in animals fed before starvation by standard diet. Conclusions It is concluded that chronic intake of GlnD has negative effect on protein balance in skeletal muscle, significantly alters amino acid concentrations in plasma and tissues, and results in impaired response to starvation, particularly to the rapid development of glutamine deficiency in body fluids and impaired protein synthesis in visceral tissues.

Effect of feeding a high-protein diet following an 18-hour period of feed withholding on mammalian target of rapamycin-dependent signaling in skeletal muscle of mature horses

To determine the effect of refeeding following an 18-hour period of feed withholding on the phosphorylation of translation initiation factors in the skeletal muscle of mature horses. 8 adult horses. Following an 18-hour period of feed withholding, horses either continued to have feed withheld (postabsorptive state) or were fed 2 g/kg of a high-protein feed (33% crude protein) at time 0 and 30 minutes (postprandial state). Blood samples were taken throughout the experimental period. At 90 minutes, a biopsy specimen was taken from the middle gluteal muscle to measure the phosphorylation of translation initiation factors and tissue amino acid concentrations. Plasma glucose, insulin, and amino acid concentrations were also measured. Horses in the postprandial state had significantly higher plasma insulin, glucose, and amino acid concentrations than did those in the postabsorptive state at the time of biopsy. Refeeding significantly increased the phosphorylation state of riboprotein S6 and eukaryotic initiation factor 4E binding protein 1. In mature horses, feeding resulted in increased mammalian target of rapamycin signaling and the mechanism appeared to be independent of an increase in Akt phosphorylation at Ser⁴⁷³. Results indicate that adult horses may be able to increase rates of muscle protein synthesis in response to feeding and that dietary amino acids appear to be the main mediators of this effect.

Adaptative response of nitrogen metabolism in early endotoxemia: role of ornithine aminotransferase

Arginine (Arg) and glutamine (Gln) utilization is greatly increased during catabolic stress. While the supply of both amino acids has been advocated in this situation, arginine administration is possibly associated with deleterious effects. From a metabolic point of view, these two amino acids are reciprocal precursors via ornithine aminotransferase (OAT). We hypothesized that OAT plays a key role in the interconversion between Arg and Gln. To test this hypothesis, we evaluated the influence of OAT activity in a model of septic shock induced by intraperitoneal injection of lipopolysaccharide (LPS) in wild-type (WT) and transgenic mice overexpressing OAT (OAT) in the liver, kidney and intestine, i.e. the three main organs of OAT expression. Plasma and tissue amino acid concentrations and tissue OAT expression and activity were measured. Five hours after LPS injection, WT and OAT mice showed a similar response to LPS in terms of inflammatory cytokine production and protein catabolism, suggesting that the interconversion between Arg and Gln through this pathway remains limited. Endotoxemia led to a significant decrease in plasma Orn levels and an increase in liver Orn levels. Of note, Orn levels were always lower in OAT mice. While only plasma Arg and Gln remained unaffected by LPS treatment, hepatic Gln was significantly increased without any difference between the two genotypes. In this model of early endotoxemia, arginine and glutamine maintained their metabolic homeostasis. Our results show an inhibition of OAT activity and expression in the liver following LPS treatment. These data highlight the importance of OAT in ornithine metabolism, especially in the liver, and suggest a post-transcriptional regulation of OAT by LPS in the liver.

Overexpression of ornithine aminotransferase: consequences on amino acid homeostasis

Ornithine aminotransferase (OAT) is a reversible enzyme expressed mainly in the liver, kidney and intestine. OAT controls the interconversion of ornithine into glutamate semi-aldehyde, and is therefore involved in the metabolism of arginine and glutamine which play a major role in N homeostasis. We hypothesised that OAT could be a limiting step in glutamine-arginine interconversion. To study the contribution of the OAT enzyme in amino acid metabolism, transgenic mice that specifically overexpress human OAT in the liver, kidneys and intestine were generated. The transgene expression was analysed by in situ hybridisation and real-time PCR. Tissue (liver, jejunum and kidney) OAT activity, and plasma and tissue (liver and jejunum) amino acid concentrations were measured. Transgenic male mice exhibited higher OAT activity in the liver (25 (sem 4) v. 11 (sem 1) nmol/min per microg protein for wild-type (WT) mice; P < 0.05) but there were no differences in kinetic parameters (i.e. Km and maximum rate of reaction (Vmax)) between WT and transgenic animals. OAT overexpression decreased plasma and liver ornithine concentrations but did not affect glutamine or arginine homeostasis. There was an inverse relationship between ornithine levels and OAT activity. We conclude that OAT overexpression has only limited metabolic effects, probably due to the reversible nature of the enzyme. Moreover, these metabolic modifications had no effect on phenotype.

Amino acid composition of Arctic charr, Salvelinus alpinus (L.) and the prediction of dietary requirements for essential amino acids

Embryo somatic tissues, non-somatic yolk-sac materials, and whole, individual fingerlings (age 0+) of Arctic charr, Salvelinus alpinus (L.), as well as a commercial trout diet, were analysed for a wide spectrum of amino acids. Analytical material consisted of prefeeding swim-up fry that were separated into discrete yolk sac and somatic embryo tissue samples. Amino acid concentrations in fry somatic tissue and whole fingerlings were generally very similar to each other, but were lower than those measured in yolk materials. Higher correlations were observed between the majority of specific amino acid concentrations in the trout diet when compared with fingerling data (r2 = 0.91) and fry somatic tissue data (r2 = 0.89), than when correlated with fry yolk sac material (r2 = 0.76). These results indicate that the essential amino acid profiles of fry somatic tissue and whole fingerlings are closer to that of a commercial feed than they are to the endogenous profiles found in the embryonic yolk sac material itself. The dietary ratios of individual essential amino acids were also compared with the total essential amino acid concentrations (A/E ratios) in whole fingerling tissues, and these ratios could be used to accurately estimate the apparent essential amino acid requirements of Arctic charr. The rationale for using carcass amino acid composition data to estimate the dietary essential amino acid requirements of Arctic charr is discussed.

Staphylococcus aureus Subvert Autophagy for Induction of Caspase-independent Host Cell Death

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Role of Glutamine Depletion in Directing Tissue-specific Nutrient Stress Responses to L-Asparaginase

Ornithine aminotransferase (OAT) and the response to endotoxemia in mice.

OAT catalyzes the only reversible step in the of Arginine (Arg) to Glutamine (Gln) metabolism. Since both these amino acids are strongly involved in the metabolic response to stress, we hypothesized that OAT could act as a limiting enzyme in Gln-Arg interconversion during hypercatabolic stress. The aim of this study was to evaluate the role of OAT using a model of endotoxemia in transgenic mice overexpressing human OAT (liver, kidney, and intestine). Methods Thirty wild-type mice (WT) and 30 transgenic mice (OAT) received an i.p. injection of either lipopolysaccharide (LPS; 10 mg/kg) or NaCl. The animals were sacrificed 5 hours after injection: plasma and tissue (muscle, liver, jejunum) amino acid concentrations and OAT tissue (liver, jejunum) expression and activity. OAT expression and activity in the jejunum and plasma, and tissue Gln and Arg concentrations were similar in all groups. Conclusion Endotoxemia led to a decrease in OAT expression and activity, regardless of whether OAT was overexpressed. OAT overexpression did not affect Gln and Arg homeostasis in endotoxemic mice. Hence, OAT does not appear to be the limiting step in Gln-Arg homeostasis during endotoxemic stress.

Optimization of dipeptide–protein mixtures in experimental diet formulations for rainbow trout ( Oncorhynchus mykiss) alevins

It has previously been shown that diets in which the amino acid portion is based on synthetic dipeptides result in positive growth of rainbow trout alevins. However, the protein-based diets used as a control performed significantly better. One objective of the present study was to identify a protein–dipeptide mixture ratio that results in comparable growth to a protein-based control, in alevins over the first 6 weeks of feeding. Seven diets were tested, including the casein–gelatin control, three diets with differing proportions of the casein–gelatin replaced with synthetic dipeptides (50P, 75P and 100P), one diet based on free amino acids and one reference commercial diet. In the seventh diet, arginine-containing dipeptides were withdrawn from the 100P dipeptide-based diet. To evaluate the diets, effects on growth, survival, feed intake and free amino acid (FAA) concentrations in muscle tissues were studied. The results on rainbow trout alevin performance using synthetic dipeptide (100P)-based diets resulted in a relative growth of 34% of the growth observed when alevins were fed the casein–gelatin control diet. The free amino acid (FAA)-based diet, however, resulted in a negative growth of the alevins. In contrast, a 1:1 ratio of synthetic dipeptides to casein–gelatin (50P diet) resulted in similar growth as the casein–gelatin control diet fed fish. Furthermore, the 50P diet resulted in a survival that was not significantly different from that found when using a commercial diet, which showed the highest survival. The concentrations of indispensable FAA (IAA) in muscle were with few exceptions similar in the control and 50P group, while the 100P dipeptide- or free amino acid-based diet fed fish showed lower levels of free IAA in muscle. Alevins fed dipeptide-based diets without arginine (100P w/o Arg) showed high mortality within 2 weeks and negative or minimal growth. This dietary group was restarted at 2 and 4 weeks, and similar results were obtained. In conclusion, the present study indicates that a 50% replacement of the protein portion with synthetic dipeptides is an acceptable diet formulation for rainbow trout alevins in terms of growth, survival and muscle levels of indispensable FAA. The effects of arginine-containing dipeptide withdrawal on fish growth and survival indicates that arginine is an indispensable amino acid in first-feeding alevins, despite their expression of the ornithine–urea cycle and related enzymes that may function in net de novo synthesis of arginine. The synthetic dipeptide diets used here were able to identify such limiting amino acids and may be further used to re-evaluate indispensable amino acid requirements in fish early life stages characterized with high growth.

Contrasting effects of ammonium enrichment on fen bryophytes

In the second half of the 20th century, frequency and diversity of brown mosses strongly declined in Dutch fens while species of Sphagnum and Polytrichum increased markedly. We hypothesized that high ammonium (NH4+) concentrations in Dutch precipitation have promoted these species changes. To test this hypothesis, we examined the effects of varying concentrations (1–1000 μM) of NH4+ on a brown moss (Calliergonella cuspidata), two species of Sphagnum (S. contortum and S. squarrosum) and Polytrichum commune in a hydroponic experiment. A second goal was to identify possible interspecific differences in the mechanisms responsible for NH4+ detoxification. Dry matter production and tissue cation concentrations indicated decreasing sensitivity to NH4+ in the order C. cuspidata>S. contortum>S. squarrosum and P. commune. In S. squarrosum and P. commune, the highest NH4+ treatments induced strong increases in tissue nitrogen (N) and amino acid concentrations. Sphagnum contortum showed a similar, but less marked response. In contrast, C. cuspidata showed a negative relationship between external NH4+ concentrations and both tissue N and amino acid concentrations (except arginine). Our findings support the hypothesis that the present rates of NH4+ deposition in The Netherlands are detrimental to brown mosses, but not to fast-growing Sphagnum and Polytrichum species. The efficiency of the NH4+ assimilation apparatus, producing N-rich amino acids, probably plays a key role in determining sensitivity to increased NH4+ deposition.

The role of autophagy during the early neonatal starvation period

At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3-12 h before returning to basal levels within 1-2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (approximately 12 h) is much shorter than that of wild-type mice (approximately 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of 'self' proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.