Release Of Alanine Research Articles

The Uptake of Glutamine and Release of Arginine, Citruline and Proline by the Small Intestine of Developing Pigs ,

Arteriovenous (A-V) differences in the plasma concentrations of amino acids across the jejunum were studied in preweaning (14- to 21-d-old) and post-weaning (29- to 58-d-old) pigs in the postabsorptive state. Glutamine was the only amino acid that was extracted by the small intestine in both pre- and post-weaning pigs. The production of citrulline by the jejunum was low in preweaning pigs, but was threefold greater in the post-weaning pigs than in the preweaning pigs. The output of proline by the intestine was observed in the post-weaning pigs but not in the preweaning pigs. Arginine and alanine were the predominant amino acids released by the jejunum of the pre- and post-weaning pigs, respectively. Whereas glutamate was the major amino acid formed from glutamine in pig enterocytes in vitro, the jejunum of the post-weaning pigs released comparable amounts of citrulline, proline and glutamate in vivo. There were no significant A-V differences in the concentrations of urea, ornithine, taurine, tyrosine, serine, glycine and nutritionally essential amino acids in either the preweaning or the post-weaning pigs. Thus, we demonstrated for the first time the uptake in vivo of glutamine and the release of arginine, alanine, citrulline, glutamate and proline by the small intestine of developing pigs. Our findings on the release in vivo of citrulline (the precursor of arginine) and proline by the small intestine of the pre- and post-weaning pigs provide a basis for explaining why arginine and proline are nutritionally essential amino acids for young suckling piglets but not for adult pigs.

Resveratrol protects against chronic alcohol-induced liver disease in a rat model

Hepatic ethanol metabolism participates in the pathogenesis of alcoholic liver disease (ALD). We aimed to evaluate the protecting effects and underlying mechanisms of Resveratrol against ALD. Adult male rats were fed with liquid ethanol diet, in the presence or absence of Resveratrol for 23 weeks. It was demonstrated that Resveratrol attenuated ethanol-induced release of serum alanine and aspartate aminotransferase. Ethanol insult caused malondialdehyde elevation and the impairment of antioxidant defense system, leading to reactive oxygen species generation, which could be greatly reversed by Resveratrol treatment. Moreover, Resveratrol protected against chronic ethanol-induced upregulation in hepatic cytochrome P-4502E1 expression, whereas hepatic alcohol dehydrogenase expression was unaffected by Resveratrol. Unexpectedly, hepatic aldehyde dehydrogenase2 expression was markedly diminished in ethanol-fed rats, which was gradually improved by Resveratrol treatment. Our data demonstrate that the protecting effects of Resveratrol on ALD might be associated with changes in the regulation of alcohol-metabolizing enzymes. Our findings could provide new perspective into the pharmacological targets of Resveratrol in the treatment of ALD.

OPTIMIZATION OF AMINO ACID PRODUCTION FROM CHICKEN FEATHER BY BACILLUS AMYLOLIQUEFACIENS USING RESPONSE SURFACE METHODOLOGY AS A NEW TOOL FOR POWDERY MILDEW DISEASE BIOCONTROL

The present work aimed to optimize amino acids production by Bacillus amyloliquefaciens 35s from chicken feather using response surface methodology (RSM) and applying the produced amino acids in biological control of powdery mildew disease. Variables affecting amino acids production from B. amyloliquefaciens 35s were screened using Plackett Burman design. Factors showed to be significant to produce amino acids were optimized using central composite design (CCD) of RSM at three coded levels (-1, 0, +1). All obtained data were analyzed by ANOVA with post hoc multiple comparison analysis performed using Tukey’s HSD. TGY (tryptone, glucose, yeast extract) was the basal medium. Impacts of medium components were studied using Plackett-Burman design. “Design Expert 12” StateEase was used to analyze the experimental Plackett- Burman design. Feather, yeast extract, CaCO3, KH2PO4 and agitation rate (using shake flask) were optimized statistically by the CCD design of the RSM. Validation of statistical model of the medium components was done by carrying out the experiment at optimum conditions of the process parameters as determined by the model. Amino acids types were identified by amino acid analyzer. Among the significant medium components, feather, yeast extract, CaCO3, KH2PO4 and agitation rate showed to have significant effect on amino acids production. Predicted maximum amino acids production was observed near the mid-point concentrations of the mentioned above factors and the experimental value (6.55 g/l) was very close to the predicted value of (6.60 g/l) predicted by the model. Amino acid analyzer analysis for the produced amino acids shows the release of asparagine, threonine, serine, glutamine, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenyl alanine, histidine, lysine and arginine, respectively. Effect of chicken feather hydrolysate on cucumber powdery mildew disease was tested by applying foliar application with different concentrations (0, 5, 10, 15, and 20% v/v), then degree of infection and sporulation on cucumber plants were assessed. The lowest concentration of chicken feather hydrolysate showed the lowest effect on disease index. Conversely, cucumber resistance increased by the increasing of feather’s hydrolysate concentrations associated with a clear reduction in the disease index and sporulation. Aside from, extra foliar application showed a decrease in the disease index for all concentrations compared with untreated plants. Foliar application with 20% concentration of chicken feather hydrolysate had the highest stimulation of the studied oxidative enzymes i.e., peroxidase (POX), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and superoxide dismutase (SOD).

Optimization of amino acid production from chicken feather by Bacillus amyloliquefaciens using Response surface methodology as a new tool for powdery mildew disease biocontrol.

The present work aimed to optimize amino acids production by Bacillus amyloliquefaciens 35s from chicken feather using response surface methodology (RSM) and applying the produced amino acids in biological control of powdery mildew disease. Variables affecting amino acids production from B. amyloliquefaciens 35s were screened using Plackett Burman design. Factors showed to be significant to produce amino acids were optimized using central composite design (CCD) of RSM at three coded levels (-1, 0, +1). All obtained data were analyzed by ANOVA with post hoc multiple comparison analysis performed using Tukey’s HSD. TGY (tryptone, glucose, yeast extract) was the basal medium. Impacts of medium components were studied using Plackett-Burman design. “Design Expert 12” StateEase was used to analyze the experimental Plackett- Burman design. Feather, yeast extract, CaCO3, KH2PO4 and agitation rate (using shake flask) were optimized statistically by the CCD design of the RSM. Validation of statistical model of the medium components was done by carrying out the experiment at optimum conditions of the process parameters as determined by the model. Amino acids types were identified by amino acid analyzer. Among the significant medium components, feather, yeast extract, CaCO3, KH2PO4 and agitation rate showed to have significant effect on amino acids production. Predicted maximum amino acids production was observed near the mid-point concentrations of the mentioned above factors and the experimental value (6.55 g/l) was very close to the predicted value of (6.60 g/l) predicted by the model. Amino acid analyzer analysis for the produced amino acids shows the release of asparagine, threonine, serine, glutamine, proline, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenyl alanine, histidine, lysine and arginine, respectively. Effect of chicken feather hydrolysate on cucumber powdery mildew disease was tested by applying foliar application with different concentrations (0, 5, 10, 15, and 20% v/v), then degree of infection and sporulation on cucumber plants were assessed. The lowest concentration of chicken feather hydrolysate showed the lowest effect on disease index. Conversely, cucumber resistance increased by the increasing of feather’s hydrolysate concentrations associated with a clear reduction in the disease index and sporulation. Aside from, extra foliar application showed a decrease in the disease index for all concentrations compared with untreated plants. Foliar application with 20% concentration of chicken feather hydrolysate had the highest stimulation of the studied oxidative enzymes i.e., peroxidase (POX), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and superoxide dismutase (SOD).

Causes and Consequences of A Glutamine Induced Normoxic HIF1 Activity for the Tumor Metabolism.

The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1α was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1α only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, the Warburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells.

Supporting Survival of Transplanted Stem-Cell-Derived Insulin-Producing Cells in an Encapsulation Device Augmented with Controlled Release of Amino Acids.

Pancreatic islet transplantation is a promising treatment for type I diabetes, which is a chronic autoimmune disease in which the host immune cells attack insulin-producing beta cells. The impact of this therapy is limited due to tissue availability and dependence on immunosuppressive drugs that prevent immune rejection of the transplanted cells. These issues can be solved by encapsulating stem cell-derived insulin-producing cells in an immunoprotective device. However, encapsulation exacerbates ischemia, and the lack of vasculature at the implantation site post-transplantation worsens graft survival. Here, an encapsulation device that supplements nutrients to the cells is developed to improve the survival of encapsulated stem cell-derived insulin-producing cells in the poorly vascularized subcutaneous space. An internal compartment in the device is fabricated to provide zero-order release of alanine and glutamine for several weeks. The amino acid reservoir sustains viability of insulin-producing cells in nutrient limiting conditions in vitro. Moreover, the reservoir also increases cell survival by 30% after transplanting the graft in the subcutaneous space.

Erratum for Mansilla et al. Ammonia-nitrogen added to low-crude-protein diets deficient in dispensable amino acid–nitrogen increases the net release of alanine, citrulline, and glutamate post–splanchnic organ metabolism in growing pigs

Erratum for Mansilla et al. Ammonia-nitrogen added to low-crude-protein diets deficient in dispensable amino acid–nitrogen increases the net release of alanine, citrulline, and glutamate post–splanchnic organ metabolism in growing pigs

Lactate production without hypoxia in skeletal muscle during electrical cycling: Crossover study of femoral venous-arterial differences in healthy volunteers.

BackgroundAim of the study was to compare metabolic response of leg skeletal muscle during functional electrical stimulation-driven unloaded cycling (FES) to that seen during volitional supine cycling.MethodsFourteen healthy volunteers were exposed in random order to supine cycling, either volitional (10-25-50 W, 10 min) or FES assisted (unloaded, 10 min) in a crossover design. Whole body and leg muscle metabolism were assessed by indirect calorimetry with concomitant repeated measurements of femoral venous-arterial differences of blood gases, glucose, lactate and amino acids.ResultsUnloaded FES cycling, but not volitional exercise, led to a significant increase in across-leg lactate production (from -1.1±2.1 to 5.5±7.4 mmol/min, p<0.001) and mild elevation of arterial lactate (from 1.8±0.7 to 2.5±0.8 mM). This occurred without widening of across-leg veno-arterial (VA) O2 and CO2 gaps. Femoral SvO2 difference was directly proportional to VA difference of lactate (R2 = 0.60, p = 0.002). Across-leg glucose uptake did not change with either type of exercise. Systemic oxygen consumption increased with FES cycling to similarly to 25W volitional exercise (138±29% resp. 124±23% of baseline). There was a net uptake of branched-chain amino acids and net release of Alanine from skeletal muscle, which were unaltered by either type of exercise.ConclusionsUnloaded FES cycling, but not volitional exercise causes significant lactate production without hypoxia in skeletal muscle. This phenomenon can be significant in vulnerable patients’ groups.

Ammonia-Nitrogen Added to Low-Crude-Protein Diets Deficient in Dispensable Amino Acid–Nitrogen Increases the Net Release of Alanine, Citrulline, and Glutamate Post–Splanchnic Organ Metabolism in Growing Pigs

Dietary ammonia is rapidly absorbed but poorly used for urea synthesis in pigs fed low-crude-protein (low-CP) diets deficient in dispensable amino acid (DAA)-nitrogen. We explored the effect of dietary ammonia on net amino acid (AA) balances in portal-drained viscera (PDV) and livers of pigs fed a diet deficient in DAA-nitrogen. Eight barrows with an initial body weight (BW) of 26.5±1.4 kg (mean + SD) were surgically fitted with 4 catheters each (portal, hepatic, and mesenteric veins and carotid artery). The pigs were restricted-fed (2.8×191 kcal/kg BW0.60) for 7 d, and every 8 h a diet deficient in DAA-nitrogen supplemented with increasing amounts of ammonia-nitrogen (CP=7.76%, 9.27%, and 10.77% for the control and low- and high-ammonia diets, respectively). The treatment sequence was based on a 3×3 Latin-square design with 3 consecutive periods. On the last day of each period, blood flows in portal and hepatic veins were determined with a continuous infusion of ρ-amino hippuric acid into the mesenteric vein. Consecutive blood samples were taken for AA concentration in blood plasma, and AA balances were calculated for PDV and the liver. Cumulative release of citrulline (Cit) and proline (Pro) increased with ammonia supplementation in PDV but decreased for glutamine (Gln) and glycine (Gly) (Gln: -19.32±3.56, -32.50±3.73, and -42.11±3.55 mmol/meal for the control and low- and high-ammonia groups, respectively; P≤0.05). Cumulative release of alanine (Ala), glutamic acid (Glu), and Gln increased with ammonia supplementation across the liver (P≤0.05). When combined, PDV+liver, the cumulative release of Ala, Cit, and Glu increased with ammonia-nitrogen supplementation (P≤0.05). Dietary ammonia could be used as a nitrogen supplement to increase the synthesis of Ala, Cit, and Glu across splanchnic organs in pigs fed a diet deficient in DAA-nitrogen.

Metabolic response of glioblastoma cells associated with glucose withdrawal and pyruvate substitution as revealed by GC-MS.

BackgroundTumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate.MethodsGlioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed.ResultsThe abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate.ConclusionsOur experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0131-9) contains supplementary material, which is available to authorized users.

Human splanchnic amino-acid metabolism

Plasma levels of several amino acids are correlated with metabolic dysregulation in obesity and type 2 diabetes. To increase our understanding of human amino-acid metabolism, we aimed to determine splanchnic interorgan amino-acid handling. Twenty patients planned to undergo a pylorus preserving pancreatico-duodenectomy were included in this study. Blood was sampled from the portal vein, hepatic vein, superior mesenteric vein, inferior mesenteric vein, splenic vein, renal vein, and the radial artery during surgery. The difference between arterial and venous concentrations of 21 amino acids was determined using liquid chromatography as a measure of amino-acid metabolism across a given organ. Whereas glutamine was significantly taken up by the small intestine (121.0 ± 23.8 µmol/L; P < 0.0001), citrulline was released (−36.1 ± 4.6 µmol/L; P < 0.0001). This, however, was not seen for the colon. Interestingly, the liver showed a small, but a significant uptake of citrulline from the circulation (4.8 ± 1.6 µmol/L; P = 0.0138) next to many other amino acids. The kidneys showed a marked release of serine and alanine into the circulation (−58.0 ± 4.4 µmol/L and −61.8 ± 5.2 µmol/L, P < 0.0001), and a smaller, but statistically significant release of tyrosine (−12.0 ± 1.3 µmol/L, P < 0.0001). The spleen only released taurine (−9.6 ± 3.3 µmol/L; P = 0.0078). Simultaneous blood sampling in different veins provides unique qualitative and quantitative information on integrative amino-acid physiology, and reveals that the well-known intestinal glutamine–citrulline pathway appears to be functional in the small intestine but not in the colon.

From population ecology to metabolism: growth of Trypanosoma evansi, and implications of glucose depletion, in a live host

Little attention has been paid to the potential top-down effects of population ecology on metabolism. Because it is capable of a fast growth that dramatically modifies its blood environment, the parasitic protozoan, Trypanosoma evansi, is a promising model for the study of density-dependent metabolic processes. We assess the in vivo growth rate, doubling time, biomass yield, glycolytic flux, and enzyme expression of T. evansi. Then, we explore the metabolic changes likely occurring during its growth. At low T. evansi densities, most host glucose is used to produce energy, which results in the release of pyruvate. Part of glucose is used for NADPH production through the pentose phosphate pathway. At high T. evansi densities, fructose, mannose, and glycerol become additional energy sources. Part of host glucose is used for biosynthesis and for NADPH production through alternative metabolic pathways, which results in the release of succinate, alanine, and acetate. The ability of organisms to adjust to resource changes is crucial to their survival. Irrespective if the triggering mechanism is direct (nutrient limitation) or indirect (a pheromone-like factor), nutrient availability is necessarily the main evolutionary factor responsible for the density-dependent metabolic properties of trypanosome populations.

Properties of an Inward-Facing State of LeuT: Conformational Stability and Substrate Release

The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters, which are important pharmaceutical targets. There are no high-resolution structures of the human transporters available; however, LeuT has been crystallized in several different conformational states. Recently, an inward-facing conformation of LeuT was solved revealing an unexpectedly large movement of transmembrane helix 1a (TM1a). We have performed molecular dynamics simulations of the mutated and wild-type transporter, with and without the cocrystallized Fab antibody fragment, to investigate the properties of this inward-facing conformation in relation to transport by LeuT within the membrane environment. In all of the simulations, local conformational changes with respect to the crystal structure are consistently observed, especially in TM1a. Umbrella sampling revealed a soft potential for TM1a tilting. Furthermore, simulations of inward-facing LeuT with Na+ ions and substrate bound suggest that one of the Na+ ion binding sites is fully disrupted. Release of alanine and the second Na+ ion is also observed, giving insight into the final stage of the translocation process in atomistic detail.

Ischemia Induces Release of Endogenous Amino Acids from the Cerebral Cortex and Cerebellum of Developing and Adult Mice

Ischemia enhanced release of endogenous neuroactive amino acids from cerebellar and cerebral cortical slices. More glutamate was released in adult than developing mice. Taurine release enhanced by K+ stimulation and ischemia was more than one magnitude greater than that of GABA or glutamate in the developing cerebral cortex and cerebellum, while in adults the releases were almost comparable. Aspartate release was prominently enhanced by both ischemia and K+ stimulation in the adult cerebral cortex. In the cerebellum K+ stimulation and ischemia evoked almost 10-fold greater GABA release in 3-month olds than in 7-day olds. The release of taurine increased severalfold in the cerebellum of 7-day-old mice in high-K+ media, whereas the K+-evoked effect was rather small in adults. In 3-month-old mice no effects of K+ stimulation or ischemia were seen in the release of aspartate, glycine, glutamine, alanine, serine, or threonine. The releases from the cerebral cortex and cerebellum were markedly different and also differed between developing and adult mice. In developing mice only the release of inhibitory taurine may be large enough to counteract the harmful effects of excitatory amino acids in ischemia in both cerebral cortex and cerebellum, in particular since at that age the release of glutamate and aspartate cannot be described as massive.

Conformational Stability and Substrate Translocation - A Computational Study of the Leucine Transporter

The human monoamine transporters are involved in a variety of disorders and thus important medicinal targets.1 There are no available high-resolution structures of the transporters. The leucine transporter (LeuT) is a bacterial homolog of the human monoamine transporters with a sequence similarity of 20-24 %.2 LeuT has been crystallized with a number of different ligands and in several different conformations. Most recently, an inward facing conformation was solved.3 The crystallization process required the use of four point mutations as well as the association of an antibody. We have applied computational methods to investigate whether the conformation of this manipulated protein is relevant for the wild-type transporter in a membrane environment. We have performed several molecular dynamics simulations of both the mutated and wild-type transporter, and analyzed the stability through measurements of helix tilt angles. The simulations reveal that the mutant and wild-type systems types behave similarly and both maintain a relatively stable inward facing conformation. The crystal structure of LeuT in an inward facing conformation does not contain any ligands or ions. To investigate the release of substrate and ions from the transporter, we have also performed simulations of wild-type LeuT in the inward facing conformation with sodium and either leucine or alanine bound. From this, we can conclude that one of the two sodium binding sites is fully disrupted in this conformation leading to the release of this ion in almost all of the simulations. Furthermore, in one of the simulations we observe release of alanine along with the second sodium ion, and are thus able to describe the translocation process in atomic detail.1. Br J Pharmacol (2012) 167, 256-278.2. Mol Pharmacol (2006) 70, 1630-1642.3. Nature (2012) 481, 469-474

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K(+)-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K(+) stimulation (50mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K(+) stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.

Detoxification of Ammonia in Mouse Cortical GABAergic Cell Cultures Increases Neuronal Oxidative Metabolism and Reveals an Emerging Role for Release of Glucose-Derived Alanine

Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus

The human alanine–serine–cysteine transporter 1 (Asc-1) belongs to the slc7a family of solute carrier transporters. Asc-1 mediates the uptake of d-serine in an exchanger-type fashion, coupling the process to the release of alanine and cysteine. Among the bacterial Asc-1 homologues, one transporter shows a significantly higher sequence identity (35%) than other bacterial homologues. Therefore, this homologue from Gloeobacter violaceus might represent the best bacterial target for structural studies probing the molecular mechanism of Asc-1. We have over-expressed the G. violaceus transporter by auto-induction, and performed purification and biophysical characterization. In addition, growth studies indicate a preference for alanine as nitrogen source in cells expressing the G. violaceus transporter. It was observed that use of the auto-induction method and subsequent optimization of the length of auto-induction was crucial for obtaining high yields and purity of the transporter. The transporter was purified with yields in the range of 0.2–0.4 mg per L culture and eluted in a single peak from a size-exclusion column. The circular dichroism spectrum revealed a folded and apparently all-helical protein.

Net flux of amino acids across the portal-drained viscera and liver of the ewe during abomasal infusion of protein and glucose12

The objective of the study was to measure net AA flux rates across the portal-drained viscera (PDV) and liver in the presence and absence of abomasal glucose infusion. Decreasing the fraction of AA metabolized by the mucosal cells may increase the fraction of AA being released into the blood. A potential mechanism to reduce AA catabolism by mucosal cells is to provide an alternative source of energy. We hypothesized that increasing glucose flow to the small intestine would increase net appearance of AA across the PDV. Eighteen mature ewes with sampling catheters were placed on study. The experimental design was a split-plot with a complete randomized design on the whole-plot and a Latin-square subplot with 5 periods and incremental levels of protein infusion. One-half of the ewes received abomasal glucose infusions (3.84 g/h), and all ewes received each of 5 protein abomasal infusion levels over 5 periods (0, 2.6, 5.2, 7.8, and 10.5 g/h). Net PDV release of isoleucine, leucine, methionine, phenylalanine, aspartate, glutamate, glutamine, proline, serine, and tyrosine increased linearly with increased protein infusion, and net PDV release of histidine, lysine, threonine, valine, alanine, and glycine did not differ with protein infusion. Net hepatic glucose release decreased with glucose infusion. With the exception of histidine, phenylalanine, and valine, net hepatic AA uptake increased linearly with increased delivery of AA to the liver. Glucose infusion increased the hepatic lysine and valine uptake and decreased phenylalanine uptake. Based on the observations in the current study, we reject our hypothesis that glucose can spare AA metabolism by PDV tissue. Our findings suggest that hepatic gluconeogenesis can be increased in the presence of increased AA delivery to the liver and that hepatic gluconeogenesis can be decreased with increased absorption of dietary glucose. Our findings support the concept that for most AA, hepatic transport of AA can be described by mass action kinetics; however, the rates of hepatic uptake of specific AA are upregulated directly or indirectly by elevated glucose.

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

BackgroundThe first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.Methods and FindingsWe found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.ConclusionsWe found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.