14C Glucose Research Articles

TM-07 * 13C MRS DETECTS METABOLIC FLUX ADAPTATION IN IDH MUTANT GLIOMA CELLS

The isocitrate dehydrogenase (IDH)1 mutation is associated with accumulation of 2-hydroxyglutarate (2-HG), but the metabolic fluxes associated with 2-HG production and cellular reprogramming of mutant IDH1 cells are not fully understood. The objective of this study was to use 13C magnetic resonance spectroscopy (MRS) to probe the fate of 13C-labeled metabolites (at thermal polarization and hyperpolarized by dynamic nuclear polarization) and to monitor the glycolytic pathway, the TCA cycle, and glutamine metabolism in wild-type and mutant IDH1 glioma cells. To achieve this goal, U87 cells expressing mutant IDH1 and wild-type IDH1 were generated by transduction with a lentiviral vector coding for mutant or wild-type IDH1 respectively. MRS studies were performed using a cell perfusion (bioreactor) system. Live cells were exposed to hyperpolarized 1-13C or 2-13C-labeled pyruvic acid, 1-13C glucose or 3-13C glutamine and cell metabolism was probed using 13C MRS. We found that in mutant IDH1 cells pyruvate flux to lactate was increased relative to wild-type cells, and pyruvate flux to glutamate and 2-HG was decreased compared to pyruvate flux to glutamate in wild-type IDH1 cell. Glutamine flux to glutamate in wild-type cells, or to glutamate and 2-HG in mutant IDH1 cells was comparable. Accordingly, the total intracellular glutamate pool was reduced in mutant IDH1 cells. The drop in pyruvate flux to the TCA cycle was mediated by a significant drop in pyruvate dehydrogenase (PDH) activity, which was due to significantly increased inhibitory PDH phosphorylation. When considering 2-HG synthesis, our data also indicate that, consistent with previous work, the majority of 2-HG is produced from glutamine (82%). However in our cells a significant portion (18%) was also derived from glucose. Our findings thus point to metabolic reprogramming in IDH1 mutant cells beyond 2-HG production and highlight the value of MRS for characterizing the metabolic changes associated with the IDH1 mutation.

Abstract 1424: Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism

Abstract Proliferating cancer cells require an active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. The two major anaplerotic pathways are the deamidation of glutamine to form glutamate catalyzed by glutaminase (GLS) or glutaminolysis, and the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (PC). We determined the expression of PC and GLS proteins in fresh tumor tissue and paired adjacent benign tissue from 86 human NSCLC patients. PC was elevated (median 8-10 fold) in 94% of the tumor tissues, whereas GLS expression did not differ significantly between tumor and paired non tumorous lung tissue. Using 13C6 glucose as tracer and stable isotope-resolved metabolomics (SIRM), we also observed elevated PC activity in vivo in human patients with early stage NSCLC. To examine the importance of PC in the growth and survival of NSCLC, 5 NSCLC cell lines were transduced with a lentiviral vector containing an shRNA against PC. PC knockdown slowed proliferation, induced multinucleation, and reduced colony formation in soft agar. To validate attenuated PC activity and to profile the metabolic effect of PC knockdown, A549 cells were grown in 13C6 glucose or 13C5 glutamine and the incorporation of 13C into various metabolic pathways was measured by NMR and MS by SIRM. We found reduced entry of both glucose and glutamine carbon into the Krebs cycle metabolites, fatty acyl chains of lipids, and nucleotides, suggesting that both energy production and anabolic pathways were hindered and blocking the PC pathway was not compensated by GLS activity. In addition, glutathione biosynthesis and homeostasis were perturbed by PC suppression, leading to compromised anti-oxidation capacity. We further found that PC knockdown in A549 cells reduced their growth rate in mouse xenografts, and recapitulate the metabolic perturbations seen both in cell culture and in human patients. Together, these results suggest that PC is indispensible for the growth and anabolism of NSCLC. This work was funded by 5P20RR018733, 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, and 3R01CA118434-02S1; and the KLCRP, CTSPGP, and the KY Challenge for Excellence. Citation Format: Katherine E. Sellers, Matthew P. Fox, Michael Bousamra, Jun Yan, Mariia Yuneva, Richard M. Higashi, Andrew N. Lane, Teresa WM Fan. Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2014-1424

Resolving confounding enrichment kinetics due to overlapping resonance signals from 13C-enriched long chain fatty acid oxidation and uptake within intact hearts.

Long chain fatty acid (LCFA) oxidation measurements in the intact heart from 13C-NMR rely on detection of 13C-enriched glutamate. However, progressive increases in overlapping resonance signal from LCFA can confound detection of the glutamate 4-carbon (GLU-C4) signal. We evaluated alternative 13C labeling for exogenous LCFA and developed a simple scheme to distinguish kinetics of LCFA uptake and storage from oxidation. Sequential 13C-NMR spectra were acquired from isolated rat hearts perfused with 13C LCFA and glucose. Spectra were evaluated from hearts supplied: U 13C LCFA, [2,4,6,8,10,12,14,16-(13) C8 ] palmitate, [2,4,6,8,10,12,14,16,18-(13) C9 ] oleate, [4,6,8,10,12,14,16-(13) C7 ] palmitate, or [4,6,8,10,12,14,16,18-(13) C8 ] oleate. 13C signal reflected the progressive enrichment at 34.6 ppm from GLU-C4, confounded by additional signal with distinct kinetics attributed to 13C-enriched LCFA 2-carbon (34.0 ppm). Excluding 13C at the 2-carbon of both palmitate and oleate eliminated signal overlap and enabled detection of the exponential enrichment of GLU-C4 for assessing LCFA oxidation. Eliminating enrichment at the 2-carbon of 13C LCFA resolved confounding kinetics between GLU-C4 and LCFA 2-carbon signals. With this enrichment scheme, oxidation of LCFA, the primary fuel for cardiac ATP synthesis, can now be more consistently examined in whole organs with dynamic mode, proton-decoupled (13C-NMR

Stability of Carbon Coating Surrounding LiMPO4 (M=Fe, Co) Particles in Water Free and Water Containing Electrolytes

LiMPO4 (M=Fe, Co) materials are known as possible high voltage materials leading to high energy density batteries. LiFePO4 is electrochemically active below 4V vs. Li/Li+ with a theoretical capacity of 170 mAh/g, and it is well known for its good stability upon cycling [1]. Conversely, LiCoPO4, which works at higher potential (4.85 V), exhibits poor performance on cycling in common alkyl carbonate electrolytes [2]. In the literature, two hypotheses can be found which try to explain the low stability of LiCoPO4: the Li salt is corrosive for the material [3] and/or the electrolyte oxidation at high voltage plays a role. Tsiouvaras et. al. [4] have shown that CO2 is the main gas evolved by the anodic oxidation of water free alkyl carbonate based electrolytes (e.g. PC) at high electrode potentials (≥ 4.5 V vs. Li/Li+). Moreover, a recent study by our group [5], has demonstrated that CO2 is already evolved at lower potentials if the electrolyte contains traces of water.In this work we will investigate the evolution of CO2 at high electrode potentials (≥ 4.5 V vs. Li/Li+) for LiFePO4/C and LiCoPO4/C by on-line electrochemical mass spectrometry (OEMS). It can be due to the electrochemical oxidation of the electrolyte and/or the carbon, according to the well-known reaction:C + 2H2O → CO2 + 4H+ + 4e- (1)Both materials are synthetized in house by a solid state route for LiCoPO4 and a solvothermal route for LiFePO4.Firstly, a preliminary experiment on LiFePO4/C is done in order to study the stability of the carbon coating of LiFePO4 at high potentials. Glucose 13C is used as carbon precursor in the LiFePO4 synthesis to obtain an isotopically labelled carbon coating. In this case, the signal from the electrolyte decomposition and from the carbon of the electrode can be deconvoluted. By adding a defined amount of water (4000 ppm) to the electrolyte one can mimic the effect of trace water that could unintentionally be introduced to the cell through the active materials.Secondly, in order to compare the stability of carbon coating from LiFePO4 and LiCoPO4, isotopically labeled 13C electrolyte (1M LiPF6 in labelled DMC) is used to deconvolute the formation of CO2 from the carbon and the electrolyte. To distinguish carbon from the electrode and from the coating of particles, the electrode is designed with 13C carbon instead of the common Super P (Timcal) as conductive additive. The potential limit of the charge is fixed to 5.5 V to allow comparison on carbon coating stability between both, LiCoPO4 and LiFePO4.

Study to Evaluate Molecular Mechanics behind Synergistic Chemo-Preventive Effects of Curcumin and Resveratrol during Lung Carcinogenesis

BackgroundThe combination approach is the future of the war against cancer and the present study evaluated molecular mechanics behind the synergistic effects of curcumin and resveratrol during lung carcinogenesis.MethodsThe mice were segregated into five groups which included normal control, Benzo[a]pyrene[BP] treated, BP+curcumin treated, BP+resveratrol treated and BP+curcumin+resveratrol treated.ResultsThe morphological analyses of tumor nodules confirmed lung carcinogenesis in mice after 22 weeks of single intra-peritoneal[i.p] injection of BP at a dose of 100 mg/Kg body weight. The BP treatment resulted in a significant increase in the protein expressions of p53 in the BP treated mice. Also, a significant increase in the protein expression of phosphorylated p53[ser15] confirmed p53 hyper-phosphorylation in BP treated mice. On the other hand, enzyme activities of caspase 3 and caspase 9 were noticed to be significantly decreased following BP treatment. Further, radiorespirometric studies showed a significant increase in the 14C-glucose turnover as well as 14C-gulcose uptake in the lung slices of BP treated mice. Moreover, a significant rise in the cell proliferation was confirmed indirectly by enhanced uptake of 3H-thymidine in the lung slices of BP treated mice. Interestingly, combined treatment of curcumin and resveratrol to BP treated animals resulted in a significant decrease in p53 hyper-phosphorylation, 14C glucose uptakes/turnover and 3H-thymidine uptake in the BP treated mice. However, the enzyme activities of caspase 3 and caspase 9 showed a significant increase upon treatment with curcumin and resveratrol.ConclusionThe study, therefore, concludes that molecular mechanics behind chemo-preventive synergism involved modulation of p53 hyper-phosphorylation, regulation of caspases and cellular metabolism enzymes.

Glucose decomposition and its incorporation into soil microbial biomass depending on land use in Mt. Kilimanjaro ecosystems

Land use change can affect terrestrial C stocks, resulting in increased CO2 flux from soil to the atmosphere. In Africa, conversion of natural ecosystems to agricultural lands is the most common land use change. This study investigated the effects of six land use types occurring in Mt. Kilimanjaro ecosystems i.e. (1) lower montane forest (2) grassland and (3) savannah (natural ecosystems) (4) Chagga homegardens (semi-natural ecosystem) and (5) maize fields and (6) coffee plantation (agroecosystems) on microbial biomass carbon (MBC) and dynamics of 14C labelled glucose added into soil. Decomposition of 14C labelled glucose and its incorporation into microbial biomass in surface soils (0–10 and 10–20 cm) were determined. MBC decreased significantly with increased intensity of land use. Mineralization of the 14C labelled glucose occurred in two phases with contrasting rates: 0–10 days (6–18% of 14C d−1) and 15–65 days (<0.1% of 14C d−1). Land use intensification in agroecosystems led to an average increase of glucose decomposition of 14%. The decay rates of the labile C pool in intensively used agricultural lands were up to three times higher compared to natural ecosystems. The incorporation of 14C glucose into microorganisms ranged between 1 and 7% of 14C input in all soils, and was highest in savannah. Agricultural intensification decreased C content in soil through increased mineralization of organic substances and negatively impacted the upper soil layer more compared to the lower one. Based on these results we conclude that semi-natural ecosystems (e.g. Chagga homegardens) are more sustainable in Mt. Kilimanjaro ecosystems compared to intensive agroecosystems.

Low and High Dietary Protein:Carbohydrate Ratios during Pregnancy Affect Materno-Fetal Glucose Metabolism in Pigs1–3

Inadequate dietary protein during pregnancy causes intrauterine growth retardation. Whether this is related to altered maternal and fetal glucose metabolism was examined in pregnant sows comparing a high-protein:low-carbohydrate diet (HP-LC; 30% protein, 39% carbohydrates) with a moderately low-protein:high-carbohydrate diet (LP-HC; 6.5% protein, 68% carbohydrates) and the isoenergetic standard diet (ST; 12.1% protein, 60% carbohydrates). During late pregnancy, maternal and umbilical glucose metabolism and fetal hepatic mRNA expression of gluconeogenic enzymes were examined. During an i.v. glucose tolerance test (IVGTT), the LP-HC–fed sows had lower insulin concentrations and area under the curve (AUC), and higher glucose:insulin ratios than the ST– and the HP-LC–fed sows (P < 0.05). Insulin sensitivity and glucose clearance were higher in the LP-HC sows compared with ST sows (P < 0.05). Glucagon concentrations during postabsorptive conditions and IVGTT, and glucose AUC during IVGTT, were higher in the HP-LC group compared with the other groups (P < 0.001). 13C glucose oxidation was lower in the HP-LC sows than in the ST and LP-HC sows (P < 0.05). The HP-LC fetuses were lighter and had a higher brain:liver ratio than the ST group (P < 0.05). The umbilical arterial inositol concentration was greater in the HP-LC group (P < 0.05) and overall small fetuses (230–572 g) had higher values than medium and heavy fetuses (≥573 g) (P < 0.05). Placental lactate release was lower in the LP-HC group than in the ST group (P < 0.05). Fetal glucose extraction tended to be lower in the LP-HC group than in the ST group (P = 0.07). In the HP-LC and LP-HC fetuses, hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC) was higher than in the ST fetuses (P < 0.05). In conclusion, the HP-LC and LP-HC sows adapted by reducing glucose turnover and oxidation and having higher glucose utilization, respectively. The HP-LC and LP-HC fetuses adapted via prematurely expressed hepatic gluconeogenic enzymes.

In Vivo Pyro-SIP Assessing Active Gut Microbiota of the Cotton Leafworm, Spodoptera littoralis

The gut microbiota is of crucial importance for the host with considerable metabolic activity. Although great efforts have been made toward characterizing microbial diversity, measuring components' metabolic activity surprisingly hasn't kept pace. Here we combined pyrosequencing of amplified 16S rRNA genes with in vivo stable isotope probing (Pyro-SIP) to unmask metabolically active bacteria in the gut of cotton leafworm (Spodoptera littoralis), a polyphagous insect herbivore that consumes large amounts of plant material in a short time, liberating abundant glucose in the alimentary canal as a most important carbon and energy source for both host and active gut bacteria. With 13C glucose as the trophic link, Pyro-SIP revealed that a relatively simple but distinctive gut microbiota co-developed with the host, both metabolic activity and composition shifting throughout larval stages. Pantoea, Citrobacter and Clostridium were particularly active in early-instar, likely the core functional populations linked to nutritional upgrading. Enterococcus was the single predominant genus in the community, and it was essentially stable and metabolically active in the larval lifespan. Based on that Enterococci formed biofilm-like layers on the gut epithelium and that the isolated strains showed antimicrobial properties, Enterococcus may be able to establish a colonization resistance effect in the gut against potentially harmful microbes from outside. Not only does this establish the first in-depth inventory of the gut microbiota of a model organism from the mostly phytophagous Lepidoptera, but this pilot study shows that Pyro-SIP can rapidly gain insight into the gut microbiota's metabolic activity with high resolution and high precision.

Evaluation of antidiabetic property of Andrographis paniculata powder in high fat and sucrose-induced type-2 diabetic adult male rat

ObjectiveTo evaluate the antidiabetic effect of the aerial part of Andrographis paniculata (A. paniculata) powder (500 mg/kg body weight) in high fat and sucrose-induced type-2 diabetic rat model. MethodsThe fasting blood glucose, oral glucose tolerance test, serum insulin, lipid profile, mRNA and protein levels of insulin signaling molecules, 14C-2 deoxy glucose uptake and 14C glucose oxidation in liver were checked. ResultsIn the type-2 diabetes-induced group, the fasting blood glucose, oral glucose tolerance, serum insulin, lipid profile, glucose uptake and oxidation, Akt and glucose transporter 2 mRNA, insulin receptor and glucose transporter 2 protein (both cytosolic and plasma membrane) and phosphorylation of insulin receptor substrate 1 and Akt were impaired. A. paniculata was able to successfully reinstate this impairment. In addition to this, A. paniculata did not cause a hypoglycemic condition in normal rat, affirming its activity in hyperglycemic state alone. ConclusionsA. paniculata possesses significant antidiabetic and antihyperlipidemic activities in high fat and sucrose-induced type-2 diabetic rat and the molecular actions at the level of insulin signaling molecules in liver reinforce it.

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

Cerebral Glucose Metabolism in an Immature Rat Model of Pediatric Traumatic Brain Injury

Altered cerebral metabolism and mitochondrial function have been identified in experimental and clinical studies of pediatric traumatic brain injury (TBI). Metabolic changes detected using (1)H (proton) magnetic resonance spectroscopy correlate with long-term outcomes in children after severe TBI. We previously identified early (4-h) and sustained (24-h and 7-day) abnormalities in brain metabolites after controlled cortical impact (CCI) in immature rats. The current study aimed to identify specific alterations of cerebral glucose metabolism at 24 h after TBI in immature rats. Rats (postnatal days 16-18) underwent CCI to the left parietal cortex. Sham rats underwent craniotomy only. Twenty-four hours after CCI, rats were injected (intraperitoneally) with [1,6-(13)C]glucose. Brains were removed, separated into hemispheres, and frozen. Metabolites were extracted with perchloric acid and analyzed using (1)H and (13)C-nuclear magnetic resonance spectroscopy. TBI resulted in decreases in N-acetylaspartate in both hemispheres, compared to sham contralateral. At 24 h after TBI, there was significant decrease in the incorporation of (13)C label into [3-(13)C]glutamate and [2-(13)C]glutamate in the injured brain. There were no differences in percent enrichment of [3-(13)C]glutamate, [4-(13)C]glutamate, [3-(13)C]glutamine, or [4-(13)C]glutamine. There was significantly lower percent enrichment of [2-(13)C]glutamate in both TBI sides and the sham craniotomy side, compared to sham contralateral. No differences were detected in enrichment of (13)C glucose label in [2-(13)C]glutamine, [2-(13)C]GABA (gamma-aminobutyric acid), [3-(13)C]GABA, or [4-(13)C]GABA, [3-(13)C]lactate, or [3-(13)C]alanine between groups. Results suggest that overall oxidative glucose metabolism in the immature brain recovers at 24 h after TBI. Specific reductions in [2-(13)C]glutamate could be the result of impairments in either neuronal or astrocytic metabolism. Future studies should aim to identify pathways leading to decreased metabolism and develop cell-selective "metabolic rescue."

Decomposition of added and native organic carbon from physically separated fractions of diverse soils

There have been increasing efforts to understand the dynamics of organic carbon (OC) associated with measurable fractions of bulk soil. We compared the decomposition of native OC (native C) with that of an added substrate (glucose) on physically separated fractions of a diverse suite of soils. Five soil orders were selected from four contrasting climate zones (Mollisol from temperate, Ultisol and Oxisol from tropics, Andisol from sub-arctic, and Gelisol from arctic region). Soils from the A horizon were fractionated into particulate OC (POC) and mineral-associated OC (MOC) by a size-based method. Fractions were incubated at 20 °C and 50 % water-holding capacity in the dark after the addition of unlabeled d-glucose (0.4 mg C g−1 fraction) and U–14C glucose (296 Bq g−1 fraction). Respiration of glucose 14C indicated 64 to 84 % of added glucose 14C which was respired from POC and 62 to 70 % from MOC within 150 days of incubation, with more than half of the cumulative respiration occurring within 4 days. Native C respiration varied widely across fractions: 12 to 46 % of native C was respired from POC and 3 to 10 % was respired from MOC fractions. This suggested that native C was more stabilized on the MOC than on the POC, but respiration from the added glucose was generally similar for MOC and POC fractions. Our study suggests a fundamental difference between the behavior of freshly added C and native C from MOC and POC fractions of soils.

Experimental evidence and isotopomer analysis of mixotrophic glucose metabolism in the marine diatom Phaeodactylum tricornutum.

BackgroundHeterotrophic fermentation using simple sugars such as glucose is an established and cost-effective method for synthesizing bioproducts from bacteria, yeast and algae. Organisms incapable of metabolizing glucose have limited applications as cell factories, often despite many other advantageous characteristics. Therefore, there is a clear need to investigate glucose metabolism in potential cell factories. One such organism, with a unique metabolic network and a propensity to synthesize highly reduced compounds as a large fraction of its biomass, is the marine diatom Phaeodactylum tricornutum (Pt). Although Pt has been engineered to metabolize glucose, conflicting lines of evidence leave it unresolved whether Pt can natively consume glucose.ResultsIsotope labeling experiments in which Pt was mixotrophically grown under light on 100% U-13C glucose and naturally abundant (~99% 12C) dissolved inorganic carbon resulted in proteinogenic amino acids with an average 13C-enrichment of 88%, thus providing convincing evidence of glucose uptake and metabolism. The dissolved inorganic carbon was largely incorporated through anaplerotic rather than photosynthetic fixation. Furthermore, an isotope labeling experiment utilizing 1-13C glucose and subsequent metabolic pathway analysis indicated that (i) the alternative Entner-Doudoroff and Phosphoketolase glycolytic pathways are active during glucose metabolism, and (ii) during mixotrophic growth, serine and glycine are largely synthesized from glyoxylate through photorespiratory reactions rather than from 3-phosphoglycerate. We validated the latter result for mixotrophic growth on glycerol by performing a 2-13C glycerol isotope labeling experiment. Additionally, gene expression assays showed that known, native glucose transporters in Pt are largely insensitive to glucose or light, whereas the gene encoding cytosolic fructose bisphosphate aldolase 3, an important glycolytic enzyme, is overexpressed in light but insensitive to glucose.ConclusionWe have shown that Pt can use glucose as a primary carbon source when grown in light, but cannot use glucose to sustain growth in the dark. We further analyzed the metabolic mechanisms underlying the mixotrophic metabolism of glucose and found isotopic evidence for unusual pathways active in Pt. These insights expand the envelope of Pt cultivation methods using organic substrates. We anticipate that they will guide further engineering of Pt towards sustainable production of fuels, pharmaceuticals, and platform chemicals.

Processing of 13C glucose in mineral soil from aspen, spruce and novel ecosystems in the Athabasca Oil Sands Region

Microbial composition is known, on similar soil types, to vary based on differing organic matter inputs, or stand composition. Fine-textured luvisolic soils, which dominate the upland boreal forests of Western Canada, support a canopy cover of aspen (Populus tremuloides Michx.), white spruce (Picea glauca (Moench) Voss) or a mixture of the two. These soils then reflect different belowground biogeochemical processing of organic matter. Novel, anthropogenic soils formed from a combination of peat litter and fine textured mineral soil, are now also a part of the landscape in the western boreal. This study set out to determine if a simple labeled compound (13C glucose) was processed differently by soils from the two dominant stand types (aspen and spruce) and from an anthropogenic (newly reclaimed) site. Results indicate that while all three soils rapidly incorporated and respired the labeled carbon, each maintained a distinct microbial community structure (as evidenced by phospholipid fatty acid analysis) throughout the 300hour experiment. Therefore soils with different microbial communities from varied organic matter inputs decompose organic carbon by different processes, even in the case of simple labile compounds.

Does exogenous carbon extend the realized niche of canopy lichens? Evidence from sub‐boreal forests in British Columbia

Foliose lichens with cyanobacterial bionts (bipartite and tripartite) form a distinct assemblage of epiphytes strongly associated with humid microclimatic conditions in inland British Columbia. Previous research showed that these cyano- and cephalolichen communities are disproportionately abundant and species-rich on conifer saplings beneath Populus compared to beneath other tree species. More revealing, lichens with cyanobacterial bionts were observed beneath Populus even in stands that did not otherwise support them. We experimentally test the hypothesis that this association is due to the interception of glucose-rich nectar that is exuded from Populus extra-floral nectaries (EFN). Using CO2 flux measurements and phospholipid fatty acid (PLFA) analysis with experimental applications of 13C6-labeled glucose, we demonstrate that cyano- and cephalolichens have a strong respiratory response to glucose. Lichens treated with glucose had lower net photosynthesis and higher establishment rates than control thalli. Furthermore, lichens with cyanobacterial bionts rapidly incorporate exogenous 13C into lichen fatty acid tissues. A large proportion of the 13C taken up by the lichens was incorporated into fungal biomarkers, suggesting that the mycobiont absorbed and assimilated the majority of applied 13C6 glucose. Our observations suggest that both cyanolichens and cephalolichens may utilize an exogenous source of glucose, made available by poplar EFNs. The exogenous C may enable these lichens to become established by providing a source of C for fungal respiration despite drought-induced inactivity of the cyanobacterial partner. As such, the mycobiont may adopt an alternative nutritional strategy, using available exogenous carbon to extend its realized niche.

Abstract 1866: Stable isotope resolved metabolomics of colorectal cancer cells treated with inhibitor of monocarboxylate transporter-1.

Abstract The rationale of this cell based study was to examine the metabolic effects of a synthetic monocarboxylate transporter 1 (MCT1) inhibitor (AR-C155858) on multiple cellular metabolites in colorectal tumor cells using [1,2,3,4,5,6-13C6]-D-glucose or [1,2,3,4,5-13C5]-D-glutamine as the single precursor metabolic tracer. DiFi colorectal cancer cells were cultured for 24 hrs in the presence of 0.1% DMSO (vehicle control) or 100 nM of MCT-1 inhibitor. Intracellular glycolysis intermediates measured by liquid chromatography/mass spectrometry (LC/MS) included phosphoenolpyruvate (PEP), phosphoglycerate (PG), fructose 1,6 bisphosphate (FBP), Glucose-6-phosphate (G6P), Gluconate-6-phosphate, glyceraldehydes 3-phosphate (G3P), ribose-5-phosphate (R5P), seduheptulose-7-phosphate (S7P), pyruvate, and lactate. TCA cycle intermediates measured included malate, α-ketoglutarate, succinate, fumarate, and citrate. Both glutamine and glutamate were also measured. Increases in PEP, PG3, G6P, Gluconate-6-phosphate, G3P, S7P, pyruvate, and lactate were induced by MCT1 inhibitor when traced with 13C6 glucose. When traced with 13C5 glutamine, PEP, G6P, S7P, pyruvate, and lactate were increased by MCT1 inhibitor. In contrast, decreases in α-ketoglutarate and glutamate were seen with both tracers. Citation Format: Bo Tan, Ming-Shang Kuo, Matthew D. Breyer, Ling Liu, Yang Zhao. Stable isotope resolved metabolomics of colorectal cancer cells treated with inhibitor of monocarboxylate transporter-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1866. doi:10.1158/1538-7445.AM2013-1866

Human melanoma metabolic network analysis with combined 13C NMR/bioreactor technique: testing the Warburg effect

The bioreactor techniques are becoming an important tool to study cancer cell metabolism(1). The goal of the present work was to develop a 13C metabolic “bonded cumomer” modeling approach adapted for fitting bioreactor data obtained with 13C glucose as substrate and to calculate the extent of the Warburg effect and how much energy comes from aerobic glycolysis vs oxidative phosphorylation.ResultsFigure 1 shows experimental time courses for labeled glutamate obtained during [1,6‐13C2] glucose perfusion of melanoma cells cultured in a CST bioreactor. Using extracted fluxes we estimated that 54% of the energy coming from oxidative phosphorylation (from which 44% are from glucose) and 46% from aerobic glycolysis and the extent of the Warburg effect (aerobic glycolysis) was 87%.ConclusionsUnder aerobic conditions glycolysis provides nearly as much ATP as oxidative phosphorylation. The model is validated by excellent agreement between model predicted and experimentally measured values of CMRO2 and cytosolic glutamate pool size. Dynamic high‐resolution MR spectra is very sensitive to changing/adding biochemical pathways and flux values, and bonded cumomer modeling allows one to check precisely the feasibility of assumed general bionetworks and particular metabolic pathways.This work was supported by NIH grants 2U24‐CA083105 and 5R01CA129544–02

LACTATING MOUSE MAMMARY GLAND EXPRESSES SECRETED MGAM WHICH ENABLES UNWEANED SUCKLING PUP STARCH DIGESTION

All starch digesting activities are reported absent in suckling mice by jejunal mucosal enzyme assay, imunohistochemistry or Western blots. We confirmed that there was no maltase activity or brush border staining with Mgam antibodies at 13d and that these were detected at 25d only in WT. Last year we reported that at 13 d both Mgam null and WT suckling mice can digest amylase treated 13C‐starch derived α‐limit dextrins (LDx) to increase blood 13C glucose paradoxically in 13d Mgam null suckling mice lumenal contents were stained by specific Mgam antibodies.ObjectiveTest lactating mouse mammary glands for Mgam and Si gene expression.MethodsRNA was extracted from the mammary gland of 10 different strains of WT 30d pp lactating mice. After RT, the cDNA was amplified for Mouse Si (amplicons for exons 1 to 6 or exons 16 to 18) and Mgam (amplicons for exons 1 to 7 or exons 3 to 7).ResultsOnly the Mgam Exon 3 to 7 primers produced 516 bp bands on the Gel.Conclusionwe suggest that maltase activity is secreted in mouse milk which enables unweaned pup starch digestion well before brush border bound enzyme development. Splicoforms of the Mgam message, lacking the membrane binding exon 2 coding region, in lactating mouse mammary tissue suggest synthesis of a soluble enzyme activity. This was confirmed by the expression of the exon 2 deleted Mgam message in lactating mammary glands of 10 different mouse strains.

AMP-activated protein kinase regulates L-arginine mediated cellular responses

BackgroundOur prior study revealed the loss in short-term L-Arginine (ARG) therapeutic efficacy after continuous exposure; resulting in tolerance development, mediated by endothelial nitric oxide synthase (eNOS) down-regulation, secondary to oxidative stress and induced glucose accumulation. However, the potential factor regulating ARG cellular response is presently unknown.MethodHuman umbilical vein endothelial cells were incubated with 100 μM ARG for 2 h in buffer (short-term or acute), or for 7 days in culture medium and challenged for 2 h in buffer (continuous or chronic), in the presence or absence of other agents. eNOS activity was determined by analyzing cellular nitrite/nitrate (NO2–/NO3–), and AMP-activated protein kinase (AMPK) activity was assayed using SAMS peptide. 13C6 glucose was added to medium to measure glucose uptake during cellular treatments, which were determined by LC-MS/MS. Cellular glucose was identified by o-toluidine method. Superoxide (O2•–) was identified by EPR-spin-trap, and peroxynitrite (ONOO–) was measured by flow-cytometer using aminophenyl fluorescein dye.ResultsShort-term incubation of cells with 100 μM ARG in the presence or absence of 30 μM L-NG-Nitroarginine methyl ester (L-NAME) or 30 μM AMPK inhibitor (compound C, CMP-C) increased cellular oxidative stress and overall glucose accumulation with no variation in glucose transporter-1 (GLUT-1), or AMPK activity from control. The increase in total NO2–/NO3– after 2 h 100 μM ARG exposure, was suppressed in cells co-incubated with 30 μM CMP-C or L-NAME. Long-term exposure of ARG with or without CMP-C or L-NAME suppressed NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity below control, and increased overall cellular glucose, O2•– and ONOO–. Gluconeogenesis inhibition with 30 μM 5-Chloro-2-N-2,5-dichlorobenzenesulfonamido-benzoxazole (CDB) during ARG exposure for 2 h maintained overall cellular glucose to control, but increased cellular glucose uptake. Continuous co-incubation with CDB and ARG increased NO2–/NO3–, glucose uptake, GLUT-1, AMPK expression and activity, and maintained overall cellular glucose, O2•– and ONOO– to control conditions.ConclusionThe present study provides the fundamental evidence for AMPK as the primary modulator of ARG cellular responses and for regulating the mode of glucose accumulation during short-term and continuous ARG treatments.

Framework and Extraframework Tin Sites in Zeolite Beta React Glucose Differently

Here, we show that framework tin sites in pure silica zeolite Beta (Sn-Beta) can isomerize glucose to fructose by a Lewis acid-mediated intramolecular hydride shift in aqueous solvent, but not in methanol solvent. Mechanistic studies using isotopically labeled (2H, 13C) glucose reactants show that in methanol, Sn-Beta instead epimerizes glucose to mannose by a Lewis acid-mediated intramolecular carbon shift mechanism known as the Bilik reaction. We also provide evidence that extraframework tin sites located within the hydrophobic channels of zeolite Beta can isomerize glucose to fructose in both water and methanol solvent, but through a base-catalyzed proton-transfer mechanism. SnO2 particles located at external zeolite crystal surfaces or supported on amorphous silica catalyze isomerization in methanol but not in water, suggesting that contact with bulk water inhibits isomerization at SnO2 surfaces. 119Sn MAS NMR spectroscopy was used to unambiguously identify framework Sn sites, which give resonances for octahedral Sn (−685 to −700 ppm) in hydrated Sn-Beta that disappear upon dehydration, with the concomitant appearance of resonances for tetrahedral Sn (−425 to −445 ppm). In sharp contrast, spectra of hydrated samples containing extraframework SnO2 show resonances for octahedral Sn centered at −604 ppm that do not change upon dehydration. These findings demonstrate that aldose–ketose isomerization reactivity on Sn-zeolite samples cannot be ascribed to the presence of framework Sn sites in the absence of isotopic labeling studies. They also indicate that any Sn-zeolite samples that initially convert glucose to fructose, instead of mannose, in methanol solvent contain Sn species that are structurally different from framework Sn centers.