Concentrations Of Intermediary Metabolites Research Articles

The utilization of N-acetylgalactosamine and its effect on the metabolism of amino acids in Erysipelotrichaceae strain

BackgroundThe metabolism of gut microbiota produces bioactive metabolites that modulate host physiology and promote self-growth. Erysipelotrichaceae is one of the most common anaerobic microorganism families in the gut, which has been discovered to play a vital role in host metabolic disorders and inflammatory diseases. Our previous study found that N-acetylgalactosamine (GalNAc) in caecal content of pigs significantly affected the abundance of Erysipelotrichaceae strains. However, it remains unknown how GalNAc feeding in vitro culture affects the expression levels of genes in the GalNAc metabolic pathway and the concentrations of intermediate metabolites in the Erysipelotrichaceae strain. Whether GalNAc feeding should influence the metabolism of other nutrients, such as amino acids, remains unrevealed.ResultsIn this study, whole-genome sequence, transcriptome, and metabolome data were analyzed to assess the utilization of a Erysipelotrichaceae strain on GalNAc. The results showed the presence of a complete GalNAc catabolism pathway in the genome of this Erysipelotrichaceae strain. GalNAc feeding to this Erysipelotrichaceae strain significantly changed the expression levels of genes involved in glycolysis and tricarboxylic acid (TCA) cycle. Meanwhile, the concentrations of lactate, pyruvate, citrate, succinate and malate from the glycolysis and TCA cycle were significantly increased. In addition, transcriptome analysis indicated that the genes involved in the metabolism of amino acids were affected by GalNAc, including lysA (a gene involved in lysine biosynthesis) that was significantly down-regulated. The intracellular concentrations of 14 amino acids in the Erysipelotrichaceae strain were significantly increased after feeding GalNAc.ConclusionsOur findings comfirmed and extended our previous works that demonstrated the utilization of GalNAc by Erysipelotrichaceae strain, and explained the possible mechanism of GalNAc affecting the abundance of Erysipelotrichaceae strain in vitro.

SpHsfA4c from Sedum plumbizincicola Enhances Cd Tolerance by the AsA–GSH Pathway in Transgenic Populus × canescens

The ascorbate (AsA)–glutathione (GSH) metabolism pathway is an important antioxidant system in cadmium (Cd) detoxification; the AsA–GSHpathway is generally regulated by a specific set of functional genes. However, transcription factors involved in AsA–GSH pathway have yet to be identified. Herein, we transformed a heat shock transcription factor SpHsfA4c from Sedum plumbizincicola into Populus. × canescens. Under 100 μM CdCl2 stress for 30 d, the leaf chlorosis of wild-type poplars (WT) is more serious than that in transgenic poplars. The root biomass, shoot biomass and tolerance index (TIs) of transgenic poplars were higher than those in WT. In addition, transgenic poplars have higher Cd2+ uptake and Cd content. Compared with WT, the contents of hydrogen peroxide (H2O2) and superoxide anion (O2•−) in transgenic poplars were significantly reduced in leaves under Cd treatment. The expression levels of five enzymes (ascorbate peroxidase (APX), catalases (CAT), superoxide dismutase (SOD), peroxidase (POD) and glutathione S-transferase (GST)) were higher in transgenic poplars than those in WT. Transgenic poplars contained higher concentrations of intermediate metabolites, including GSH, AsA and phytochelatins (PCs), and a higher GSH/GSSG ratio in the AsA–GSH metabolism pathway. In Fourier transform infrared (FTIR) spectra, the characteristic peaks indicated that the contents of cysteine, GSH and AsA in transgenic poplars were exceeded compared to those in WT. These results suggested that SpHsfA4c can activate the AsA–GSH metabolism pathway to reduce Cd-associated oxidative stress. Therefore, overexpressing SpHsfA4c in P. × canescens can give rise to a superior Cd tolerance. Our results provide a theoretical significance for breeding potential new germplasm resources with high biomass and high Cd tolerance for remediation of soil heavy metal pollution.

Adaptive exhaustion during prolonged intermittent hypoxia causes dysregulated skeletal muscle protein homeostasis.

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8h hypoxia followed by 16h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients.

Identification of metabolomic profile related to adult Fontan pathophysiology.

BackgroundMetabolic disorders are important pathophysiologies that can cause multiple organ dysfunction and worsen prognosis in Fontan patients. This study aimed to comprehensively evaluate the metabolomic profile of adult Fontan patients and characterize its pathophysiology in relation to 2 control groups.Methods and ResultsWe performed metabolomic analysis of 31 plasma samples using capillary electrophoresis time-of-flight mass spectrometry. This observational cross-sectional study compared plasma metabolites of 14 heterogeneous adult Fontan patients with those of control groups, including 9 patients with congenital heart disease after biventricular repair and 8 normal healthy controls. Fontan patients exhibited significant differences in intermediate metabolite concentrations related to glycolysis, the tricarboxylic acid (TCA) cycle, and the urea cycle. The plasma concentrations of lactic acid, 2-oxoglutarate, isocitric acid, malic acid, cis-aconitic acid, arginine, citrulline, and the ratio of ornithine/citrulline showed significantly differences among the groups. Multiple logistic regression analysis with a stepwise selection-elimination method identified 2-oxoglutaric acid (odds ratio [OR] 1.98, 95% confidence interval [CI] 1.05–3.76) and cis-aconitic acid (OR 2.69, 95% CI 1.04–6.99) as independently associated with Fontan patients. After adjustment for the covariates of age and gender, 2-oxoglutaric acid (OR 1.97, 95% CI 0.98–3.93) and cis-aconitic acid (OR 3.88, 95% CI 0.99–15.2) showed remarkable relationships with Fontan patients.ConclusionsThe present findings suggest that abnormalities in the TCA cycle and amino acid metabolism are distinguishing features in the pathophysiology of Fontan patients. Future metabolomic studies will assist in developing biomarkers for the early prediction of “silent” Fontan pathophysiologies.

Resource Uptake and the Evolution of Moderately Efficient Enzymes.

Enzymes speed up reactions that would otherwise be too slow to sustain the metabolism of selfreplicators. Yet, most enzymes seem only moderately efficient, exhibiting kinetic parameters orders of magnitude lower than their expected physically achievable maxima and spanning over surprisingly large ranges of values. Here, we question how these parameters evolve using a mechanistic model where enzyme efficiency is a key component of individual competition for resources. We show that kinetic parameters are under strong directional selection only up to a point, above which enzymes appear to evolve under near-neutrality, thereby confirming the qualitative observation of other modeling approaches. While the existence of a large fitness plateau could potentially explain the extensive variation in enzyme features reported, we show using a population genetics model that such a widespread distribution is an unlikely outcome of evolution on a common landscape, as mutation–selection–drift balance occupy a narrow area even when very moderate biases towards lower efficiency are considered. Instead, differences in the evolutionary context encountered by each enzyme should be involved, such that each evolves on an individual, unique landscape. Our results point to drift and effective population size playing an important role, along with the kinetics of nutrient transporters, the tolerance to high concentrations of intermediate metabolites, and the reversibility of reactions. Enzyme concentration also shapes selection on kinetic parameters, but we show that the joint evolution of concentration and efficiency does not yield extensive variance in evolutionary outcomes when documented costs to protein expression are applied.

Multiomics-Identified Intervention to Restore Ethanol-Induced Dysregulated Proteostasis and Secondary Sarcopenia in Alcoholic Liver Disease.

Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment. An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

Light-based control of metabolic flux through assembly of synthetic organelles.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here, we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically-active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation by six-fold and product specificity by 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

Pareto Optimality Explanation of the Glycolytic Alternatives in Nature

The Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) glycolytic pathways are largely conserved across glycolytic species in nature. Is this a coincidence, convergent evolution or there exists a driving force towards either of the two pathway designs? We addressed this question by first employing a variant of the optStoic algorithm to exhaustively identify over 11,916 possible routes between glucose and pyruvate at different pre-determined stoichiometric yields of ATP. Subsequently, we analyzed the thermodynamic feasibility of all the pathways at physiological metabolite concentrations and quantified the protein cost of the feasible solutions. Pareto optimality analysis between energy efficiency and protein cost reveals that the naturally evolved ED and EMP pathways are indeed among the most protein cost-efficient pathways in their respective ATP yield categories and remain thermodynamically feasible across a wide range of ATP/ADP ratios and pathway intermediate metabolite concentration ranges. In contrast, pathways with higher ATP yield (>2) while feasible, are bound within stringent and often extreme operability ranges of cofactor and intermediate metabolite concentrations. The preponderance of EMP and ED is thus consistent with not only optimally balancing energy yield vs. enzyme cost but also with ensuring operability for wide metabolite concentration ranges and ATP/ADP ratios.

Maternal nutrition and stage of early pregnancy in beef heifers: impacts on hexose and AA concentrations in maternal and fetal fluids1.

We examined the hypothesis that maternal nutrition and day of gestation would affect the concentrations of AAs and hexoses in bovine utero-placental fluids and maternal serum from days 16 to 50 of gestation. Forty-nine cross-bred Angus heifers were bred via artificial insemination and fed a control diet (CON = 100% of requirements for growth) or a restricted diet (RES = 60% of CON) and ovariohysterectomized on days 16, 34, or 50 of gestation; nonpregnant controls were not bred and ovariohysterectomized on day 16 of the synchronized estrous cycle. The resulting design was a completely randomized design with a 2 × 3 factorial + 1 arrangement of treatments. Maternal serum, histotroph, allantoic fluid, and amniotic fluid were collected at time of ovariohysterectomy. Samples were then analyzed for concentrations of AAs and intermediary metabolites: alanine (Ala), arginine, asparagine (Asn), aspartate (Asp), citrulline, cysteine, glutamine, glutamate (Glu), glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine (Met), ornithine, phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, tyrosine (Tyr), and valine (Val). The concentrations of Gly, Ser, and Thr in maternal serum were greater (P ≤ 0.05) in CON compared with RES. Furthermore, day of gestation affected (P ≤ 0.05) concentrations of Asn, Glu, Phe, Thr, and Tyr in maternal serum. Status of maternal nutrition affected the Asp concentration of histotroph where RES was greater (P = 0.02) than CON. In histotroph, Ala, Leu, Met, and Val concentrations were greater (P ≤ 0.05) on day 50 compared with day 16. Additionally, Glu and Pro concentrations in histotroph were greater (P < 0.01) on days 34 and 50 compared with day 16. A day × treatment interaction was observed for the concentration of Val in allantoic fluid where day 34 CON was greater (P = 0.05) than all other days and nutritional treatments. In addition, the concentration of Gln in amniotic fluid experienced a day × treatment interaction where day 34 RES was greater (P ≤ 0.05) than day 34 CON, which was greater (P ≤ 0.05) than day 50 CON and RES. These data support our hypothesis that day of gestation and maternal nutrition affect the concentrations of various neutral and acidic AA in beef heifer utero-placental fluids and maternal serum from days 16 to 50 of gestation.

Index markers of chronic fatigue syndrome with dysfunction of TCA and urea cycles.

Chronic fatigue syndrome (CFS) is a persistent and unexplained pathological state characterized by exertional and severely debilitating fatigue, with/without infectious or neuropsychiatric symptoms, lasting at least 6 consecutive months. Its pathogenesis remains incompletely understood. Here, we performed comprehensive metabolomic analyses of 133 plasma samples obtained from CFS patients and healthy controls to establish an objective diagnosis of CFS. CFS patients exhibited significant differences in intermediate metabolite concentrations in the tricarboxylic acid (TCA) and urea cycles. The combination of ornithine/citrulline and pyruvate/isocitrate ratios discriminated CFS patients from healthy controls, yielding area under the receiver operating characteristic curve values of 0.801 (95% confidential interval [CI]: 0.711–0.890, P < 0.0001) and 0.750 (95% CI: 0.584–0.916, P = 0.0069) for training (n = 93) and validation (n = 40) datasets, respectively. These findings provide compelling evidence that a clinical diagnostic tool could be developed for CFS based on the ratios of metabolites in plasma.

Skeletal muscle mitochondria of NDUFS4−/− mice display normal maximal pyruvate oxidation and ATP production

Mitochondrial ATP production is mediated by the oxidative phosphorylation (OXPHOS) system, which consists of four multi-subunit complexes (CI–CIV) and the FoF1-ATP synthase (CV). Mitochondrial disorders including Leigh Syndrome often involve CI dysfunction, the pathophysiological consequences of which still remain incompletely understood. Here we combined experimental and computational strategies to gain mechanistic insight into the energy metabolism of isolated skeletal muscle mitochondria from 5-week-old wild-type (WT) and CI-deficient NDUFS4−/− (KO) mice. Enzyme activity measurements in KO mitochondria revealed a reduction of 79% in maximal CI activity (Vmax), which was paralleled by 45–72% increase in Vmax of CII, CIII, CIV and citrate synthase. Mathematical modeling of mitochondrial metabolism predicted that these Vmax changes do not affect the maximal rates of pyruvate (PYR) oxidation and ATP production in KO mitochondria. This prediction was empirically confirmed by flux measurements. In silico analysis further predicted that CI deficiency altered the concentration of intermediate metabolites, modestly increased mitochondrial NADH/NAD+ ratio and stimulated the lower half of the TCA cycle, including CII. Several of the predicted changes were previously observed in experimental models of CI-deficiency. Interestingly, model predictions further suggested that CI deficiency only has major metabolic consequences when its activity decreases below 90% of normal levels, compatible with a biochemical threshold effect. Taken together, our results suggest that mouse skeletal muscle mitochondria possess a substantial CI overcapacity, which minimizes the effects of CI dysfunction on mitochondrial metabolism in this otherwise early fatal mouse model.

Model of Tryptophan Metabolism, Readily Scalable Using Tissue-specific Gene Expression Data

Tryptophan is utilized in various metabolic routes including protein synthesis, serotonin, and melatonin synthesis and the kynurenine pathway. Perturbations in these pathways have been associated with neurodegenerative diseases and cancer. Here we present a comprehensive kinetic model of the complex network of human tryptophan metabolism based upon existing kinetic data for all enzymatic conversions and transporters. By integrating tissue-specific expression data, modeling tryptophan metabolism in liver and brain returned intermediate metabolite concentrations in the physiological range. Sensitivity and metabolic control analyses identified expected key enzymes to govern fluxes in the branches of the network. Combining tissue-specific models revealed a considerable impact of the kynurenine pathway in liver on the concentrations of neuroactive derivatives in the brain. Moreover, using expression data from a cancer study predicted metabolite changes that resembled the experimental observations. We conclude that the combination of the kinetic model with expression data represents a powerful diagnostic tool to predict alterations in tryptophan metabolism. The model is readily scalable to include more tissues, thereby enabling assessment of organismal tryptophan metabolism in health and disease.

Metabolome Analysis of Erythrocytes from Patients with Chronic Hepatitis C Reveals the Etiology of Ribavirin-Induced Hemolysis

Ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis C, but is often associated with hemolytic anemia as a serious adverse event. Employing metabolome analysis, we demonstrated that the concentrations of intermediate metabolites produced by glycolysis and the pentose phosphate cycle in patients' erythrocytes were significantly decreased after administration of ribavirin. Our findings suggest that hemolysis associated with ribavirin is triggered by an energy crisis and consequent oxidative stress, thus having implications for the prevention of such hemolysis.

The metabolic activity of gut microbiota in obese children is increased compared with normal-weight children and exhibits more exhaustive substrate utilization

Objective:The gut microbiota contribute otherwise impossible metabolic functions to the human host. Shifts in the relative proportions of gut microbial communities in adults have been correlated with intestinal disease and have been associated with obesity. The aim of this study was to elucidate differences in gut microbial compositions and metabolite concentrations of obese versus normal-weight children.Materials and methods:Fecal samples were obtained from obese (n=15; mean body mass index (BMI) s.d. score=1.95) and normal-weight (n=15; BMI s.d. score=−0.14) Swiss children aged 8–14 years. Composition and diversity of gut microbiota were analyzed by qPCR and temperature gradient gel electrophoresis (TGGE).Results:No significant quantitative differences in gut microbiota communities of obese and normal-weight children were identified. Microbial community profiling by TGGE revealed a high degree of both intra- and intergroup variation. Intergroup comparison of TGGE profiles failed to identify any distinct populations exclusive to either obese or normal-weight children. High-pressure liquid chromatography analysis identified significantly higher (P<0.05) concentrations of short-chain fatty acids (SCFA) butyrate and propionate in obese versus normal-weight children. Significantly lower concentrations of intermediate metabolites were detected in obese children, suggesting exhaustive substrate utilization by obese gut microbiota.Conclusions:Our results indicate that a dysbiosis may be involved in the etiology of childhood obesity. In turn, aberrant and overactive metabolic activity within the intestine could dictate survival or loss of individual microbial communities, leading to the altered population ratios previously identified in adult obesity.

Carbon metabolism and the sign of control coefficients in metabolic adaptations underlying K- ras transformation

Metabolic adaptations are associated with changes in enzyme activities. These adaptations are characterized by patterns of positive and negative changes in metabolic fluxes and concentrations of intermediate metabolites. Knowledge of the mechanism and parameters governing enzyme kinetics is rarely available. However, the signs—increases or decreases—of many of these changes can be predicted using the signs of metabolic control coefficients. These signs require the only knowledge of the structure of the metabolic network and a limited qualitative knowledge of the regulatory dependences, which is widely available for carbon metabolism. Here, as a case study, we identified control coefficients with fixed signs in order to predict the pattern of changes in key enzyme activities which can explain the observed changes in fluxes and concentrations underlying the metabolic adaptations in oncogenic K- ras transformation in NIH-3T3 cells. The fixed signs of control coefficients indicate that metabolic changes following the oncogenic transformation—increased glycolysis and oxidative branch of the pentose-phosphate pathway, and decreased concentration in sugar-phosphates—could be associated with increases in activity for glucose-6-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, and decrease for transketolase. These predictions were validated experimentally by measuring specific activities. We conclude that predictions based on fixed signs of control coefficients are a very robust tool for the identification of changes in enzyme activities that can explain observed metabolic adaptations in carbon metabolism. This article is part of a Special Issue entitled: Bioenergetics of Cancer.

Feeding preferences and host associations of specialist marine herbivores align with quantitative variation in seaweed secondary metabolites

Consequences of congeneric quantitative variation in secondary metabolites of seaweeds on diet specificity and host association in specialist marine herbivores have received little attention. We investigated quantitative variation in caulerpenyne and oxytoxin 1 in 7 species of green seaweeds from the genus Caulerpa, along with the feeding preferences and host associations of 4 co-occurring sacoglossan molluscs. C. taxifolia and C. sertularioides contained high concentrations of metabolites and were preferred least by all herbivores. Algae with intermediate metabolite concentrations (C. racemosa, C. serrulata, and C. cupressoides) were preferred by Elysia tomentosa and Lobiger viridis. Oxynoe viridis and Stiliger smaragdinus had strong preferences for different low concentration Caulerpa species (C. racemosa var. laetevirens and C. lentillifera), suggesting not all feeding preferences are based exclusively on the major metabolites. In situ host associations of L. viridis and S. smaragdinus mirrored their feeding preferences, but this was not the case for E. tomentosa. Furthermore, those algal species with the highest and lowest metabolite concentrations had the lowest overall densities of sacoglossans. The results imply that the direct influence of quantitative variation in Caulerpa chemistry may only be limited to host associations in some sacoglossans. However, feeding pressure from multiple herbivore species with unique preferences could still contribute to variation in chemical defence amongst congeneric algae.

Effect of feeding allowance level on embryonic survival, IGF‐1, insulin, GH, leptin and progesterone secretion in early pregnancy gilts

The present experiment was conducted to evaluate the effects of feeding allowance level on embryonic survival, uterine development and reproductive hormone secretion in early gestation gilts. A total of 54 F1 crosses of Landrace x Large white gilts were randomly allocated to three treatment groups of high (H, 2 x maintenance), medium (M, 1.2 x maintenance) and low (L, 0.6 x maintenance) feeding level after mating. Blood samples and uterine flushings were collected on day 12, 25 and 35 of pregnancy, and embryonic survival rate was estimated. Concentrations of insulin-like growth factor I (IGF)-1, insulin, growth harmone (GH), leptin and progesterone in serum and uterine flushings were determined by enzyme-linked immunosorbent assay. Embryonic survival was affected by dietary treatment; total number of viable embryos and embryo survival of group M were higher than other groups in the early pregnancy (p < 0.05). Greater foetal weight in M gilts was achieved when gestation advanced to day 35 (p < 0.05), though there was no difference on day 25 of pregnancy among treatments. No appreciable differences were observed in the crown-rump length on day 25 and 35 of pregnancy among groups. Greater weight of uterus and products of conception were identified in M gilts compared with group H and L (p = 0.024 and p = 0.005, respectively) on day 25 of pregnancy. The hormone level was greatly affected by feeding allowance level. In serum, concentrations of IGF-1, leptin and insulin tended to be greater in H than in M and L during the early gestation, while concentrations of GH were greater in M and progesterone were the lowest in H. At the same time, feed allowance level affected the concentration of IGF-1, insulin, GH, leptin and progesterone in uterine flushings. These data demonstrated that feed allowance level after mating has important consequence on embryo survival, embryo development and uterine development, possibly mediated by nutrition level inducing changes in concentrations of reproductive hormones and/or intermediary metabolites in early pregnancy.

A computer model of gluconeogenesis and lipid metabolism in the perfused liver

A mathematical model of the perfused rat liver was developed to predict intermediate metabolite concentrations and fluxes in response to changes in various substrate concentrations in the perfusion medium. The model simulates gluconeogenesis in the liver perfused separately with lactate and pyruvate and the combination of these substrates with fatty acids (oleate). The model consists of key reactions representing gluconeogenesis, glycolysis, fatty acid metabolism, tricarboxylic acid cycle, oxidative phosphorylation, and ketogenesis. Michaelis-Menten-type kinetic expressions, with control by ATP/ADP, are used for many of the reactions. For key regulated reactions (fructose-1,6-bisphosphatase, phosphofructokinase, pyruvate carboxylase, pyruvate dehydrogenase complex, and pyruvate kinase), rate expressions were developed that incorporate allosteric effectors, specific substrate relationships (e.g., cooperative binding), and/or phosphorylation/dephosphorylation using in vitro enzyme activity data and knowledge of the specific mechanisms. The model was independently validated by comparing model predictions with 10 sets of experimental data from 7 different published works, with no parameter adjustments. The simulations predict the same trends, in terms of stimulation of substrate uptake by fatty acid addition, as observed experimentally. In general, the major metabolic indicators calculated by the model are in good agreement with experimental results. For example, the simulated glucose/pyruvate mass yield is 43% compared with the average of 45% reported in the literature. The model accurately predicts the specific time constants of the glucose response (2.5-4 min) and the dynamic behavior of substrate and product fluxes. It is expected that this model will be a useful tool for analyzing the complex relationships between carbohydrate and fat metabolism.

Cold glycerol–saline: The promising quenching solution for accurate intracellular metabolite analysis of microbial cells

Microbial metabolomics has been seriously limited by our inability to perform a reliable separation of intra- and extracellular metabolites with efficient quenching of cell metabolism. Microbial cells are sensitive to most (if not all) quenching agents developed to date, resulting in leakage of intracellular metabolites to the extracellular medium during quenching. Therefore, as yet we are unable to obtain an accurate concentration of intracellular metabolites from microbial cell cultures. However, knowledge of the in vivo concentrations of intermediary metabolites is of fundamental importance for the characterization of microbial metabolism so as to integrate meaningful metabolomics data with other levels of functional genomics analysis. In this article, we report a novel and robust quenching method for microbial cell cultures based on cold glycerol–saline solution as the quenching agent that prevents significant leakage of intracellular metabolites and, therefore, permits more accurate measurement of intracellular metabolite concentrations in microbial cells.

Effect of alterations in the quantity and composition of the pre-mating diet on embryo survival and foetal growth in the pig

This trial examined the effects of feeding six diets, which varied in either amount or composition, during the oestrous cycle prior to insemination on embryo survival and foetal development on day 27 ± 2 of pregnancy in gilts. Ten or 11 gilts per group received either a maintenance (M) diet, 1.8 × M, 2.6 × M or nutritionally balanced diets in which the content of fibre, protein or starch was increased. Of the six diets tested, only the high fibre diet significantly increased embryo survival when compared to its 1.8 × M isoenergetic control (88.20 ± 1.96% versus 81.25 ± 2.67%; P < 0.05). More litters from gilts fed the 1.8 × M and the starch diets had foetuses defined as intra-uterine growth retarded (IUGR; 50% and 62.5 of litters, respectively), compared to the other four groups in which 0–12.5% of litters contained IUGR foetuses ( P < 0.05). There was no effect of dietary treatment on foetal or placental size or on the within-litter variability in foetal and placental size. Plasma concentrations of oestradiol and progesterone on days 4–8 of the oestrous cycle and on day 27 ± 2 of pregnancy were unaffected by treatment. Feed intake was positively related to mean plasma IGF-1 concentrations on days 4–8 of the cycle ( P < 0.01) and to mean leptin concentrations on days 4 and 5 ( P < 0.001). Leptin concentrations were unaffected by alterations in the composition of the diet, whereas IGF-1 concentrations were higher in gilts fed the starch diet compared to the M control (159 ± 9.52 versus 127 ± 7.65 ng/ml; P < 0.05). These data demonstrate that alteration to the composition of the feed consumed during the cycle before insemination can affect both embryo survival and the distribution of foetal size within the litter. The underlying mechanism(s) remain to be determined, but probably involve dietary-induced changes in concentrations of reproductive hormones and/or intermediary metabolites that in turn affect ovarian follicular and oocyte development.