14C Glucose Research Articles

Effect of cyclosporine A on hepatic carbohydrate metabolism and hepatic gene expression in rat

Objective: Cyclosporine A (CsA)-based therapy has been implicated in the development of diabetes. Hence, its effects on hepatic carbohydrate metabolism and gene expression will be investigated.Methods: Sprague-Dawley rats given 15 mg/kg body weight/day of CsA for 20 days, as well as healthy untreated animals, received a glucose load enriched with [U-13C]glucose and deuterated water to resolve load and endogenous contributions to plasma glucose. Blood glucose and plasma insulin levels were assayed and at 60-min post-load, plasma glucose 13C and 2H-enrichments were analyzed by nuclear magnetic resonance spectroscopy and liver tissue analyzed for hepatic gene expression.Results: CsA-treated rats were glucose intolerant relative to controls (AUCglucose = 21,297 ± 857 versus 14,183 ± 1094, p < 0.01). Contributions from endogenous glucose production (EGP) were significantly elevated in CsA-treated rats (179 ± 16 versus 123 ± 13 mg/dl, p < 0.05). The increased endogenous contributions were attributable to glycogenolysis or glucose-G6P cycling and not to gluconeogenesis. Significantly higher expressions of fatty acid synthase and acetyl-CoA carboxylase 1 and 2 genes were observed in CsA-treated rats.Conclusions: CsA-altered glucose metabolism and gene expression could reflect increased hepatic insulin resistance. In the liver of CsA-treated animals, EGP suppression is impaired whereas hepatic de novo lipogenesis is enhanced contributing to dysregulated glucose and lipid metabolism.

Fueling and Imaging Brain Activation

Metabolic signals are used for imaging and spectroscopic studies of brain function and disease and to elucidate the cellular basis of neuroenergetics. The major fuel for activated neurons and the models for neuron–astrocyte interactions have been controversial because discordant results are obtained in different experimental systems, some of which do not correspond to adult brain. In rats, the infrastructure to support the high energetic demands of adult brain is acquired during postnatal development and matures after weaning. The brain's capacity to supply and metabolize glucose and oxygen exceeds demand over a wide range of rates, and the hyperaemic response to functional activation is rapid. Oxidative metabolism provides most ATP, but glycolysis is frequently preferentially up-regulated during activation. Underestimation of glucose utilization rates with labelled glucose arises from increased lactate production, lactate diffusion via transporters and astrocytic gap junctions, and lactate release to blood and perivascular drainage. Increased pentose shunt pathway flux also causes label loss from C1 of glucose. Glucose analogues are used to assay cellular activities, but interpretation of results is uncertain due to insufficient characterization of transport and phosphorylation kinetics. Brain activation in subjects with low blood-lactate levels causes a brain-to-blood lactate gradient, with rapid lactate release. In contrast, lactate flooding of brain during physical activity or infusion provides an opportunistic, supplemental fuel. Available evidence indicates that lactate shuttling coupled to its local oxidation during activation is a small fraction of glucose oxidation. Developmental, experimental, and physiological context is critical for interpretation of metabolic studies in terms of theoretical models.

Hypoxanthine–xanthine oxidase down-regulates GLUT1 transcription via SIRT1 resulting in decreased glucose uptake in human placenta

Appropriate foetal growth and development is dependent on adequate placental glucose uptake. Oxidative stress regulates glucose uptake in various tissues. The effect of oxidative stress on placental glucose transport is not known. Thus, the aim of this study was to determine the effect of oxidative stress on glucose uptake and glucose transporters (GLUTs) in human placenta. Human placenta was incubated in the absence or presence of 0.5 mM hypoxanthine+15 mU/ml xanthine oxidase (HX/XO) for 24 h. Gene and protein expressions of the GLUTs were analysed by quantitative RT-PCR and western blotting respectively. Glucose uptake was measured using radiolabelled ((14)C) glucose. HX/XO significantly decreased GLUT1 gene and protein expression and resultant glucose uptake. There was no effect of the antioxidants N-acetylcysteine, catalase and superoxide dismutase or the NF-κB inhibitor BAY 11-0782 on HX/XO-induced decrease in glucose uptake. However, HX/XO treatment significantly decreased both gene and protein expression of SIRT1. In the presence of the SIRT1 activator resveratrol, the decrease in GLUT1 expression and glucose uptake mediated by HX/XO was abolished. Collectively, the data presented here demonstrate that oxidative stress reduces placental glucose uptake and GLUT1 expression by a SIRT1-dependent mechanism.

Rat brain slices oxidize glucose at high rates: A 13C NMR study

Since glucose is the main cerebral substrate, we have characterized the metabolism of various 13C glucose isotopomers in rat brain slices. For this, we have used our cellular metabolomic approach that combines enzymatic and carbon 13 NMR techniques with mathematical models of metabolic pathways. We identified the fate and the pathways of the conversion of glucose carbons into various products (pyruvate, lactate, alanine, aspartate, glutamate, GABA, glutamine and CO2) and determined absolute fluxes through pathways of glucose metabolism. After 60min of incubation, lactate and CO2 were the main end-products of the metabolism of glucose which was avidly metabolized by the slices. Lactate was also used at high rates by the slices and mainly converted into CO2. High values of flux through pyruvate carboxylase, which were similar with glucose and lactate as substrate, were observed. The addition of glutamine, but not of acetate, stimulated pyruvate carboxylation, the conversion of glutamate into succinate and fluxes through succinate dehydrogenase, malic enzyme, glutamine synthetase and aspartate aminotransferase. It is concluded that, unlike brain cells in culture, and consistent with high fluxes through PDH and enzymes of the tricarboxylic acid cycle, rat brain slices oxidized both glucose and lactate at high rates.

Cutting Off Functional Loops from Homodimeric Enzyme Superoxide Dismutase 1 (SOD1) Leaves Monomeric β-Barrels

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.

Variation in bulk tissue, fatty acid and monosaccharide δ13C values between autotrophic and heterotrophic plant organs

The flowers of 23 species of grass and herb plants were collected from a mesotrophic grassland to assess natural variability in bulk, monosaccharide and fatty acid δ 13C values from one plant community and were compared with previous analyses of leaves from the same species. The total mean bulk δ 13C value of flower tissues was −28.1‰, and there was no significant difference between the mean δ 13C flower values for grass (−27.8‰) and herb (−28.2‰) species. On average bulk δ 13C flower values were 1.1‰ higher than bulk δ 13C leaf values, however, the δ 13C flower and δ 13C leaf values of grasses did not differ between organs suggesting that carbon isotope discrimination is different in grass and herb species. The abundance of different monosaccharides abundance varied between plant types, i.e. xylose concentrations in the grass flowers were as high as 40%, compared with up to 15% in the herb species, but the general relationship δ 13C arabinose > δ 13C xylose > δ 13C glucose > δ 13C galactose which had been observed in leaves was similar in flowers (total mean δ 13C values = −25.9‰, −27.2‰, −28.8‰ and −28.1‰, respectively). However, the average 5.4‰ depletion in the δ 13C values of the C 16:0, C 18:2 and C 18:3 fatty acids in flowers compared to bulk tissue was significantly greater than observed for leaves. The trend C 16:0 < C 18:2 < C 18:3 previously observed in leaves was also observed in grass flowers ( δ 13C C16:0 = −33.8‰; δ 13C C18:2 = −33.1‰; δ 13C C18:3 = −34.2‰) but not herb flowers ( δ 13C C16:0 = −34.1‰; δ 13C C18:2 = −32.4‰; δ 13C C18:3 = −34.5‰). We conclude: (i) that the biological processes influencing carbon isotope discrimination in grass flowers are different from herbs flowers; and, (ii) that a range of post-photosynthetic fractionation effects caused the observed differences between flower and leaf δ 13C values, especially the significant 13C-depletion in flower fatty acid δ 13C values.

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

Re-examination of metabolic fluxes in Escherichia coli during anaerobic fermentation of glucose using 13C labeling experiments and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy

Improved design of metabolic flux estimation using mixed label 13C labeling experiments and identifiability analysis motivated re-examination of metabolic fluxes during anaerobic fermentation in the Escherichia coli. Comprehensive metabolic flux maps were determined by using a mixture of differently labeled glucose and compared to conventional flux maps obtained using extracellular measurements and comprehensive metabolic flux maps obtained using only U-13C glucose as the substrate. As expected, conventional flux analysis performs poorly in comparison to 13C-MFA, especially in the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways. Identifiability analysis indicated and experiments confirmed that a mixture of 10% U-l3C glucose, 25% 1-13C glucose, and 65% naturally labeled glucose significantly improved the statistical quality of all calculated fluxes in the PP pathway, the EMP pathway, the anaplerotic reactions, and the tricarboxylic acid cycle. Modifying the network topology for the presence and absence of the Entner-Doudoroff pathway and the glyoxylate shunt did not affect the value or quality of estimated fluxes significantly. Extracellular measurement of formate production was necessary for the accurate estimation of the fluxes around the formate node.

Modeling soil metabolic processes using isotopologue pairs of position-specific 13C-labeled glucose and pyruvate

Most organic carbon (C) in soils eventually turns into CO 2 after passing through microbial metabolic pathways, while providing cells with energy and biosynthetic precursors. Therefore, detailed insight into these metabolic processes may help elucidate mechanisms of soil C cycling processes. Here, we describe a modeling approach to quantify the C flux through metabolic pathways by adding 1- 13C and 2,3- 13C pyruvate and 1- 13C and U- 13C glucose as metabolic tracers to intact soil microbial communities. The model calculates, assuming steady-state conditions and glucose as the only substrate, the reaction rates through glycolysis, Krebs cycle, pentose phosphate pathway, anaplerotic activity through pyruvate carboxylase, and various biosynthesis reactions. The model assumes a known and constant microbial proportional precursor demand, estimated from literature data. The model is parameterized with experimentally determined ratios of 13CO 2 production from pyruvate and glucose isotopologue pairs. Model sensitivity analysis shows that metabolic flux patterns are especially responsive to changes in experimentally determined 13CO 2 ratios from pyruvate and glucose. Calculated fluxes are far less sensitive to assumptions concerning microbial chemical and community composition. The calculated metabolic flux pattern for a young volcanic soil indicates significant pentose phosphate pathway activity in excess of pentose precursor demand and significant anaplerotic activity. These C flux patterns can be used to calculate C use efficiency, energy production and consumption for growth and maintenance purposes, substrate consumption, nitrogen demand, oxygen consumption, and microbial C isotope composition. The metabolic labeling and modeling methods may improve our ability to study the biochemistry and ecophysiology of intact and undisturbed soil microbial communities.

Effect of Acute Hypoglycemia on Human Cerebral Glucose Metabolism Measured by 13C Magnetic Resonance Spectroscopy

OBJECTIVETo investigate the effect of acute insulin-induced hypoglycemia on cerebral glucose metabolism in healthy humans, measured by 13C magnetic resonance spectroscopy (MRS).RESEARCH DESIGN AND METHODSHyperinsulinemic glucose clamps were performed at plasma glucose levels of 5 mmol/L (euglycemia) or 3 mmol/L (hypoglycemia) in random order in eight healthy subjects (four women) on two occasions, separated by at least 3 weeks. Enriched [1-13C]glucose 20% w/w was used for the clamps to maintain stable plasma glucose labeling. The levels of the 13C-labeled glucose metabolites glutamate C4 and C3 were measured over time in the occipital cortex during the clamp by continuous 13C MRS in a 3T magnetic resonance scanner. Time courses of glutamate C4 and C3 labeling were fitted using a one-compartment model to calculate metabolic rates in the brain.RESULTSPlasma glucose 13C isotopic enrichment was stable at 35.1 ± 1.8% during euglycemia and at 30.2 ± 5.5% during hypoglycemia. Hypoglycemia stimulated release of counterregulatory hormones (all P < 0.05) and tended to increase plasma lactate levels (P = 0.07). After correction for the ambient 13C enrichment values, label incorporation into glucose metabolites was virtually identical under both glycemic conditions. Calculated tricarboxylic acid cycle rates (VTCA) were 0.48 ± 0.03 μmol/g/min during euglycemia and 0.43 ± 0.08 μmol/g/min during hypoglycemia (P = 0.42).CONCLUSIONSThese results indicate that acute moderate hypoglycemia does not affect fluxes through the main pathways of glucose metabolism in the brain of healthy nondiabetic subjects.

Abstract LB-255: A mass spectrometry platform to quantitatively profile cancer cell metabolism from cells, tumors, and fixed tissue

Abstract The metabolic requirements of cancer and proliferating cells are different from that of normal differential tissue and may have diverse applications in the treatment of cancers. However, many of the molecular mechanisms that reorganize metabolism to support cell proliferation are unknown. To study cancer cell metabolism, we implemented a mass spectrometry based platform to quantitatively profile endogenous metabolites from proliferating cell lines, tumor tissues and formalin fixed paraffin embedded (FFPE) tissue. Cell lines are derived from several cancers including lung, multiple myeloma, prostate, and gliobastoma (GBM). In some cases, these were compared to a drosophila reference cell line. Patients were also profiled for disease classification from their cerebrospinal fluid (CSF). We also were successful in extracting polar metabolites from FFPE tissue more than five years old stored at room temperature. For FFPE tissue samples, we show that we can observe differences in disease states involving PI3K-TSC-TOR pathway and we compared different extraction methods for acquiring metabolomics data from FFPE blocks. We show that we can cluster GBM versus normal patients from analyzing their CSF. We target more than 255 unique metabolites using selected reaction monitoring (SRM) based analyses with an AB/Sciex 5500 QTRAP mass spectrometer coupled to a Shimadzu UFLC using normal phase chromatography. For a single 18 min run, our platform allows for unprecedented sensitivity, quantitation and coverage of metabolites that comprise of diverse metabolic pathways from as little as a single 6 cm tissue culture dish of cells or approximately 2–3 million cells from tissue samples. We find that amide XBridge columns (Waters) at 275 uL/min perform well in both negative and positive ion switching mode and that the sampling rate of the instrument is sufficiently fast (cycle time of 1.6 sec with 3 msec dwell times) to effectively capture up to 300 metabolite targets without scheduled SRM runs. Peak areas of metabolites are integrated using MultiQuant 1.1 software (Applied Biosystems). Peak areas from triplicate runs are hierarchically clustered and statistical analyses are applied to generate P values for metabolite changes over different biological conditions. We also probed metabolic flux in pathways by targeting a set of 13C glucose labeled metabolites. In addition, we have also analyzed a model cell line after stimulation with Insulin and EGF to examine if growth factor induced metabolic changes are evolutionarily conserved. Using metabolic inhibitors, such as Iodoacetic acid, KCN, etc., we have been able to characterize the consequences of inhibiting glycolysis and oxidative phosphorylation, respectively. Finally, we considered kinase inhibitors and measured their effects on metabolism in proliferating cancer cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-255. doi:10.1158/1538-7445.AM2011-LB-255

Stem labeling results in different patterns of 14C rhizorespiration and 15N distribution in plants compared to natural assimilation pathways

AbstractTo investigate C and N rhizodeposition, plants can be 13C‐15N double‐labeled with glucose and urea using a stem‐feeding method (wick method). However, it is unclear how the 13C applied as glucose is released into the soil as rhizorespiration in comparison with the 13C applied as CO2 using a natural uptake pathway. In the present study, we therefore compared the short‐term fate of 14C and 15N in white lupine and pea plants applied either by the wick method or the natural pathways of C and N assimilation. Plants were pulse‐labeled in 14CO2‐enriched atmosphere and 15N urea was applied to the roots (atmosphere–soil) following the natural assimilation pathways, or plants were simultaneously labeled with 14C and 15N by applying a 14C glucose–15N urea solution into the stem using the wick method. Plant development, soil microbial biomass, total rhizorespiration, and distribution of N in plants were not affected by the labeling method used but by plant species. However, the 15N : N ratio in plant parts was significantly (p &lt; 0.05) affected by the labeling method, indicating more homogeneous 15N enrichment of plants labeled via root uptake. After 14CO2 atmosphere labeling of plants, the cumulated 14CO2 release from roots and soil showed the common saturation dynamics. In contrast, after 14C‐glucose labeling by the wick method, the cumulated 14CO2 release increased linearly. These results show that 14C applied as glucose using the wick method is not rapidly transferred to the roots as compared to a short‐term 14CO2 pulse. This is partly due to a slower 14C uptake and partly due to slow distribution within the plant. Consequently, 14C‐glucose application by the wick method is no pulse‐labeling approach. However, the advantages of the wick method for 13C‐15N double labeling for estimating rhizodeposition especially under field conditions requires further methodological research.

Astrocyte Oxidative Metabolism and Metabolite Trafficking after Fluid Percussion Brain Injury in Adult Rats

Despite various lines of evidence pointing to the compartmentation of metabolism within the brain, few studies have reported the effect of a traumatic brain injury (TBI) on neuronal and astrocyte compartments and/or metabolic trafficking between these cells. In this study we used ex vivo ¹³C NMR spectroscopy following an infusion of [1-¹³C] glucose and [1,2-¹³C₂] acetate to study oxidative metabolism in neurons and astrocytes of sham-operated and fluid percussion brain injured (FPI) rats at 1, 5, and 14 days post-surgery. FPI resulted in a decrease in the ¹³C glucose enrichment of glutamate in neurons in the injured hemisphere at day 1. In contrast, enrichment of glutamine in astrocytes from acetate was not significantly decreased at day 1. At day 5 the ¹³C enrichment of glutamate and glutamine from glucose in the injured hemisphere of FPI rats did not differ from sham levels, but glutamine derived from acetate metabolism in astrocytes was significantly increased. The ¹³C glucose enrichment of the C3 position of glutamate (C3) in neurons was significantly decreased ipsilateral to FPI at day 14, whereas the enrichment of glutamine in astrocytes had returned to sham levels at this time point. These findings indicate that the oxidative metabolism of glucose is reduced to a greater extent in neurons compared to astrocytes following a FPI. The increased utilization of acetate to synthesize glutamine, and the acetate enrichment of glutamate via the glutamate-glutamine cycle, suggests an integral protective role for astrocytes in maintaining metabolic function following TBI-induced impairments in glucose metabolism.

Assessment of insulin resistance by a 13C glucose breath test: a new tool for early diagnosis and follow-up of high-risk patients

Background/AimsInsulin resistance (IR) plays an important role in the pathogenesis of diabetes and non-alcoholic fatty liver disease (NAFLD). Current methods for insulin resistance detection are cumbersome, or not sensitive enough for early detection and follow-up. The BreathID® system can continuously analyse breath samples in real-time at the point-of-care. Here we determined the efficacy of the BreathID® using the 13C-Glucose breath test (GBT) for evaluation of insulin resistance.MethodsTwenty healthy volunteers were orally administered 75 mg of 13C-glucose 1-13C. An oral glucose tolerance test (OGTT) was performed immediately; followed by serum glucose and insulin level determinations using GBT. GBT and OGTT were repeated following exercise, which alters insulin resistance levels.ResultsWithin-subject correlations of GBT parameters with serum glucose and serum insulin levels were high. Before and after exercise, between-subjects correlations were high between the relative insulin levels and the % dose recoveries at 90 min (PDR 90), and the cumulative PDRs at 60 min (CPDR 60). Pairwise correlations were identified between pre-exercise Homeostasis Model Assessment (HOMA) IR at 90 min and PDR 90; HOMA B (for beta cell function) 120 and CPDR 30; HOMA IR 60 and peak time post-exercise; and HOMA B 150 with PDR 150.ConclusionsThe non-invasive real-time BreathID® GBT reliably assesses changes in liver glucose metabolism, and the degree of insulin resistance. It may serve as a non-invasive tool for early diagnosis and follow up of patients in high-risk groups.

Study of the antidiabetic capacity of the VO(dmpp) 2 complex

In this work we report biochemical ex vivo studies with a vanadium compound containing a pyridinone ligand, the bis(1,2-dimethyl-3-hydroxy-4-pyridinonate)oxovanadium (IV), V IVO(dmpp) 2, which has shown to have promising antidiabetic activity. The experiments were carried out on primary adipocytes of 6–8 week old Wistar rats. Insulin-stimulated glucose uptake studies were performed using a radioactive assay by measuring the (U)– 14C–glucose taken up by the isolated adipocytes for 30 min. Adipocytes were incubated with and without insulin and in the presence and absence of different concentrations of V IVO(dmpp) 2 (100–500 μM) for 45 min. We observed that in a nontoxic concentration, as demonstrated by the Alamar Blue test, V IVO(dmpp) 2 significantly increases glucose uptake, in the absence of insulin, by 5-folds higher than basal, and it has a significant inhibitory effect of 78% on free fatty acid release in isolated adipocytes from normal rats. We also demonstrated that it promotes the phosphorylation of Akt1, a key protein in the insulin signaling cascade. These results were compared with those obtained with another vanadium compound reported in the literature, with a similar structure, the bis(maltolato)oxovanadium (IV) (BMOV), which is now in clinical trials. Our ex vivo results clearly indicate that V IVO(dmpp) 2 is a good candidate to be a promising drug for the treatment of diabetes and other metabolic disorders.

Effect of anion channel blockers on l-arginine action in spermatozoa from asthenospermic men

In earlier studies, we have established that l-arginine enhances motility and metabolic rate in spermatozoa of goat, bull and mouse. In the present study this work was extended to human sperm cells obtained from the semen samples of asthenospermic patients, which are characterised by low motility. The metabolic rate was followed by monitoring the glucose consumption (1-(13)C glucose as substrate) and the production of lactate in sperm cells, using (13)C NMR. The stimulatory effect of l-arginine was neutralised on adding an NO-synthase inhibitor like N(omega)-nitro-L-arginine methyl ester. On the other hand, the inactive d-enantiomorph did not affect the stimulatory effect of l-arginine. This strongly suggests that L-arginine acts through the NO signal pathway. We also demonstrated that the stimulatory effect of L-arginine was inhibited in the presence of anion channel inhibitors like 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Furthermore, bicarbonate supplementation was found to be essential for the action of L-arginine. These observations indicate that L-arginine induces NO synthesis and stimulates motility and metabolism only when an active anion transport system is present.

Seasonal variations in bulk tissue, fatty acid and monosaccharide δ13C values of leaves from mesotrophic grassland plant communities under different grazing managements

Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ 13C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ 13C values could be determined. The total mean leaf bulk δ 13C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ 13C values was only determined in October ( P = <0.001) when δ 13C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields ( P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ 13C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ 13C values had a major influence on bulk δ 13C values. An average depletion of ca. 1‰ in the bulk δ 13C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ 13C values, as glucose constituted >50% total leaf monosaccharides. In October, δ 13C values of all monosaccharides varied between species, with significant variation in δ 13C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ 13C values observed in October 1999. The fatty acids C 16:0, C 18:2 and C 18:3 were highly abundant in all plant species. Fatty acid δ 13C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C 16:0), −37.0‰ (C 18:2) and −36.5‰ (C 18:3) were determined. There was significant interspecific variation in the δ 13C values of all individual fatty acids during October and July, but only for C 18:2 in May ( P = <0.05). This indicated that seasonal trends observed in the δ 13C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ 13C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends ( P = <0.0001) for fatty acid and monosaccharide δ 13C values: δ 13C 16:0 < δ 13C 18:2 < δ 13C 18:3 and δ 13C arabinose > δ 13C xylose > δ 13C glucose > δ 13C galactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.

Validation of pentaacetylaldononitrile derivative for dual 2H gas chromatography/mass spectrometry and 13C gas chromatography/combustion/isotope ratio mass spectrometry analysis of glucose

A reference method to accurately define kinetics in response to the ingestion of glucose in terms of total, exogenous and endogenous glucose is to use stable-isotope-labelled compounds such as 2H and 13C glucose followed by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis. The use of the usual pentaacetyl (5Ac) derivative generates difficulties in obtaining accurate and reproducible results due to the two chromatographic peaks for the syn and anti isomers, and to the isotopic effect occurring during acetylation. Therefore, the pentaacetylaldononitrile derivative (Aldo) was validated for both isotopes, and compared with the 5Ac derivative. A correction factor including carbon atom dilution (stoichiometric equation) and the kinetic isotopic effect (KIE) was determined. Analytical validation results for the 2H GC/MS and 13C GC/C/IRMS measurements produced acceptable results with both derivatives. When 2H enrichments of plasma samples were < or = 1 mol % excess (MPE), the repeatability (RSD(Aldo Intra assay and Intra day) <0.94%, RSD(5Ac Intra assay and Intra day) <3.29%), accuracy (Aldo <3.4%, 5Ac <29.0%), and stability of the derivatized samples were significantly better when the Aldo derivatives of the plasma samples were used (p < 0.05). When the glucose kinetics were assessed in nine human subjects, after glucose ingestion, the plasma glucose 2H enrichments were identical with both derivatives, whereas the 13C enrichments needed a correction factor to fit together. Due to KIE variation, this correction factor was not constant and had to be calculated for each batch of analyses, to obtain satisfactory results. Mean quantities of exogenous glucose exhibit marked difference (20.9 +/- 1.3g (5Ac) vs. 26.7 +/- 2.5g (Aldo)) when calculated with stoichiometric correction, but fit perfectly when calculated after application of the correction factor (22.1 +/- 1.3g (5Ac) vs. 22.9 +/- 1.9g (Aldo)). Finally, the pentaacetylaldononitrile derivative, used here in GC/C/IRMS for the first time, enables measurement of 2H and 13C enrichments in plasma glucose with a single sample preparation.

Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [ 13C, 1H] COSY NMR spectroscopy

The physiological state of CHO cells in perfusion culture was quantified by determining fluxes through the bioreaction network using 13C glucose and 2D-NMR spectroscopy. CHO cells were cultivated in a 2.5 L perfusion bioreactor with glucose and glutamine as the primary carbon and energy sources. The reactor was inoculated at a cell density of 8×10 6 cells/mL and operated at ∼10×10 6 cells/mL using unlabeled glucose for the first 13 days. The second phase lasted 12 days and the medium consisted of 10% [U- 13C]glucose, 40% labeled [1- 13C]glucose with the balance unlabeled. After the culture attained isotopic steady state, biomass samples from the last 3 days of cultivation were considered representative and used for flux estimation. They were hydrolyzed and analyzed by 2D [ 13C, 1H] COSY measurements using the heteronuclear single quantum correlation sequence with gradients for artifacts suppression. Metabolic fluxes were determined using the 13C-Flux software package by minimizing the residuals between the experimental and the simulated NMR data. Normalized residuals exhibited a Gaussian distribution indicating good model fit to experimental data. The glucose consumption rate was 5-fold higher than that of glutamine with 41% of glucose channeled through the pentose phosphate pathway. The fluxes at the pyruvate branch point were almost equally distributed between lactate and the TCA cycle (55% and 45%, respectively). The anaplerotic conversion of pyruvate to oxaloacetate by pyruvate carboxylase accounted for 10% of the pyruvate flux with the remaining 90% entering the TCA cycle through acetyl-CoA. The conversion of malate to pyruvate catalyzed by the malic enzyme was 70% higher than that for the anaplerotic reaction catalyzed by pyruvate carboxylase. Most amino acid catabolic and biosynthetic fluxes were significantly lower than the glycolytic and TCA cycle fluxes. Metabolic flux data from NMR analysis validated a simplified model where metabolite balancing was used for flux estimation. In this reduced flux space, estimates from these two methods were in good agreement. This simplified model can routinely be used in bioprocess development experiments to estimate metabolic fluxes with much reduced analytical investment. The high resolution flux information from 2D-NMR spectroscopy coupled with the capability to validate a simplified metabolite balancing based model for routine use make 13C-isotopomer analysis an attractive bioprocess development tool for mammalian cell cultures.

Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells

13C metabolic flux analysis (MFA) is the most comprehensive means of characterizing cellular metabolic states. Uniquely labeled isotopic tracers enable more focused analyses to probe specific reactions within the network. As a result, the choice of tracer largely determines the precision with which one can estimate metabolic fluxes, especially in complex mammalian systems that require multiple substrates. Here we have experimentally determined metabolic fluxes in a tumor cell line, successfully recapitulating the hallmarks of cancer cell metabolism. Using these data, we computationally evaluated specifically labeled 13C glucose and glutamine tracers for their ability to precisely and accurately estimate fluxes in central carbon metabolism. These methods enabled us to identify the optimal tracer for analyzing individual fluxes, specific pathways, and central carbon metabolism as a whole. [1,2- 13C 2]glucose provided the most precise estimates for glycolysis, the pentose phosphate pathway, and the overall network. Tracers such as [2- 13C]glucose and [3- 13C]glucose also outperformed the more commonly used [1- 13C]glucose. [U- 13C 5]glutamine emerged as the preferred isotopic tracer for the analysis of the tricarboxylic acid (TCA) cycle. These results provide valuable, quantitative information on the performance of 13C-labeled substrates and can aid in the design of more informative MFA experiments in mammalian cell culture.