14C Glucose Research Articles

Bacterial Substrate Transformation Tracked by Stable-Isotope-Guided NMR Metabolomics: Application in a Natural Aquatic Microbial Community.

The transformation of organic substrates by heterotrophic bacteria in aquatic environments constitutes one of the key processes in global material cycles. The development of procedures that would enable us to track the wide range of organic compounds transformed by aquatic bacteria would greatly improve our understanding of material cycles. In this study, we examined the applicability of nuclear magnetic resonance spectroscopy coupled with stable-isotope labeling to the investigation of metabolite transformation in a natural aquatic bacterial community. The addition of a model substrate (13C6–glucose) to a coastal seawater sample and subsequent incubation resulted in the detection of >200 peaks and the assignment of 22 metabolites from various chemical classes, including amino acids, dipeptides, organic acids, nucleosides, nucleobases, and amino alcohols, which had been identified as transformed from the 13C6–glucose. Additional experiments revealed large variability in metabolite transformation and the key compounds, showing the bacterial accumulation of glutamate over the incubation period, and that of 3-hydroxybutyrate with increasing concentrations of 13C6–glucose added. These results suggest the potential ability of our approach to track substrate transformation in aquatic bacterial communities. Further applications of this procedure may provide substantial insights into the metabolite dynamics in aquatic environments.

A 2D & 3D fluorescent toxicity pathway reporter platform for high throughput imaging-based evaluation of hepatotoxicity liabilities

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C–labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1 min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16 min, glycogen plummeted, lactate became the largest 13C–glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C–glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3 h EC50 of 19 mM ± 8.9 mM and 17.4 mM ± 3.7 mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2 ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy.

Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations, are not well understood. To determine the mechanisms underlying cardiac substrate use during pregnancy. We use here 13C glucose, 13C lactate, and 13C fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not seem altered. Insulin signaling seems intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 (cluster of differentiation 36) and GLUT4 (glucose transporter type 4), are also unchanged. Rather, we find that the pregnancy hormone progesterone induces PDK4 (pyruvate dehydrogenase kinase 4) in cardiomyocytes and that elevated PDK4 levels in late pregnancy lead to inhibition of PDH (pyruvate dehydrogenase) and pyruvate flux into the tricarboxylic acid cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy. Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling.

Dose-response in a high density three-dimensional liver device with real-time bioenergetic and metabolic flux quantification

Real-time dose-response curves for fructose have been non-invasively determined in primary rat hepatocyte alginate spheroids cultured in a NMR-compatible fluidized-bed bioreactor. Using 13C–labeled glucose and glycine culture medium, fructose dose was compared to glucose uptake and glycogen synthesis rate using 13C NMR spectroscopy, and to ATP and fructose-1-phosphate concentration using 31P NMR spectroscopy. A highly efficient multicoaxial perfusion system maintains high density 3-D hepatocyte cultures, permitting 13C and 31P NMR spectral time courses with 1min time points. The perfusion system was turned off to demonstrate its efficiency and effect on the metabolites. Within 16min, glycogen plummeted, lactate became the largest 13C–glucose metabolite via anaerobic glycolysis, while glutathione was the largest 13C–glycine metabolite. ATP depletion and fructose-1-phosphate formation demonstrated a dose response with a 3h EC50 of 19mM±8.9mM and 17.4mM±3.7mM, respectively. Computational modeling of mass transfer corroborated experimental results and helped determine the optimal bioreactor loading densities, oxygen concentration, and perfusion rates to maintain physiologically-relevant nutrient levels. The total bioreactor plus perfusion loop has a dead volume of 2ml, and contains 5 million hepatocytes. Due to the non-invasive measurements, there is a reduction of animal tissue by an order-of-magnitude, depending on the number of time points in an experiment. This dynamic flux approach may have generic utility for dose-response studies monitoring multiple metabolic reactions in other primary mammalian cells, such as human, that have strict oxygen demands.

Human retinal pigment epithelial cells prefer proline as a nutrient and transport metabolic intermediates to the retinal side

Metabolite transport is a major function of the retinal pigment epithelium (RPE) to support the neural retina. RPE dysfunction plays a significant role in retinal degenerative diseases. We have used mass spectrometry with 13C tracers to systematically study nutrient consumption and metabolite transport in cultured human fetal RPE. LC/MS-MS detected 120 metabolites in the medium from either the apical or basal side. Surprisingly, more proline is consumed than any other nutrient, including glucose, taurine, lipids, vitamins, or other amino acids. Besides being oxidized through the Krebs cycle, proline is used to make citrate via reductive carboxylation. Citrate, made either from 13C proline or from 13C glucose, is preferentially exported to the apical side and is taken up by the retina. In conclusion, RPE cells consume multiple nutrients, including glucose and taurine, but prefer proline, and they actively synthesize and export metabolic intermediates to the apical side to nourish the outer retina.

Abstract 120: The CoExpressed 1a (Liver) Isoform of Carnitine Palmitoyl Transferase 1 is Critical to Cardiac Function and Transcriptional Activation of Metabolic Genes in Aging Hearts

Carnitine palmitoyl transferase (CPT) 1 is the rate-limiting enzyme controlling long chain fatty acid (LCFA) oxidation in the heart. The muscle isoform, CPT1b, is predominantly expressed in heart along with the liver isoform, CPT1a, that is co-expressed at comparatively low levels. However, CPT1a content increases in response to pathological stress. Both neonatal and pathologically hypertrophied hearts display elevated CPT1a, despite low LCFA oxidation. To understand the role of cardiac CPT1a, cpt1a fl/fl (fl/fl) mice were crossed with mice heterozygous for cre recombinase under the control of the cardiac specific α-MHC promoter to generate cardiac specific cpt1a null mice (cpt1a null). Echocardiography revealed that CPT1a deletion induces age-related declines in systolic function and reduced left ventricular wall thickness. At 5 mos, cpt1a null showed no significant functional defects. At 10 mos, cpt1a null hearts displayed reduced ejection fraction (EF) and fractional shortening (FS) vs fl/fl mice (EF 63±1 fl/fl vs. 21±5 cpt1a null; FS 52±1 fl/fl vs. 18±2 cpt1a null, p<0.0001) with elevated systolic and diastolic volumes. Also at 10 mos, PPARα and PGC1α gene expression declined in CPT1a null hearts by 44% and 22% respectively, vs f/f mice (from arbitrary units referenced to f/f/ control signal, AU). The reduced activation of PPARα and PGC1α genes is the potential consequence of reduced ligand from ATGL-dependent triacylglycerol lipolysis, due to low ATGL gene expression (38% decrease vs. 5 mos f/f, p<0.01, AU). CPT1b gene expression was reduced by 26% (AU) at 10 mos vs f/f/, which is consistent with changes in PPARα, but was not sufficient to alter CPTb protein content. As with CPT1b content, the fractional contribution of exogenous LCFA to oxidative metabolism in hearts perfused with media containing 13C palmitate, glucose, and lactate, was similar among groups, ranging from 0.70-0.73. Therefore, CPT1a does not impact the contribution of LCFA oxidation to mitochondrial energy metabolism in unstressed hearts. However, as hearts age, CPT1a plays a critical role in maintaining transcriptional activation of genes for LCFA metabolism enzymes, via PGC-1α and PPARα and in maintaining normal cardiac function and pathophysiology.

The Dual Activity Responsible for the Elongation and Branching of β-(1,3)-Glucan in the Fungal Cell Wall.

ABSTRACTβ-(1,3)-Glucan, the major fungal cell wall component, ramifies through β-(1,6)-glycosidic linkages, which facilitates its binding with other cell wall components contributing to proper cell wall assembly. Using Saccharomyces cerevisiae as a model, we developed a protocol to quantify β-(1,6)-branching on β-(1,3)-glucan. Permeabilized S. cerevisiae and radiolabeled substrate UDP-(14C)glucose allowed us to determine branching kinetics. A screening aimed at identifying deletion mutants with reduced branching among them revealed only two, the bgl2Δ and gas1Δ mutants, showing 15% and 70% reductions in the branching, respectively, compared to the wild-type strain. Interestingly, a recombinant Gas1p introduced β-(1,6)-branching on the β-(1,3)-oligomers following its β-(1,3)-elongase activity. Sequential elongation and branching activity of Gas1p occurred on linear β-(1,3)-oligomers as well as Bgl2p-catalyzed products [short β-(1,3)-oligomers linked by a linear β-(1,6)-linkage]. The double S. cerevisiae gas1Δ bgl2Δ mutant showed a drastically sick phenotype. An ScGas1p ortholog, Gel4p from Aspergillus fumigatus, also showed dual β-(1,3)-glucan elongating and branching activity. Both ScGas1p and A. fumigatus Gel4p sequences are endowed with a carbohydrate binding module (CBM), CBM43, which was required for the dual β-(1,3)-glucan elongating and branching activity. Our report unravels the β-(1,3)-glucan branching mechanism, a phenomenon occurring during construction of the cell wall which is essential for fungal life.

Priming effects in biochar enriched soils using a three-source-partitioning approach: 14C labelling and 13C natural abundance

The changes to soil properties due to biochar addition may affect both the direction and magnitude of priming effects. However, the mechanisms involved in biochar induced priming effects still remain largely unknown due to the limitation of methods to separate more than two carbon (C) sources (e.g. soil, biochar, substrate). We combined 14C labeling with 13C natural abundance to separate the total CO2 from i) native soil organic C (SOC, C3 signature), ii) added glucose (14C labelled) and iii) biochar (C4 signature). The primed soil CO2 emissions following a large addition of glucose (1000 mg glucose kg−1 soil) to one Chinese and one German Luvisol soil were much larger (140% and 53% respectively) in a soil recently amended with maize derived biochar (pyrolyzed at 400 °C), compared to non amended soil. Glucose addition at a lower rate (100 mg C kg−1 soil) produced no significant differences in priming effects of native soil organic matter between the biochar amended and non-amended soils. Glucose also caused priming of biochar decomposition, with an additional C4 biochar loss of between 270 μg CO2-C g−1 and 540 μg CO2-C g−1 depending on soils and glucose concentrations. Approaches using two stable isotopes (13C and12C) have previously been limited to partitioning two sources (biochar C and soil organic C). Here, for the first time, 14C labeling was combined with 13C natural abundance to partition three C sources in a biochar amended soil. By partitioning soil CO2 emissions derived from SOC, added biochar and glucose decomposition, this study provides a better understanding of the priming effects following addition of substrates to biochar amended soil, to approximate to the true complexity of biochar enriched soils.

Soil carbon dioxide emissions controlled by an extracellular oxidative metabolism identifiable by its isotope signature

Abstract. Soil heterotrophic respiration is a major determinant of the carbon (C) cycle and its interactions with climate. Given the complexity of the respiratory machinery, it is traditionally considered that oxidation of organic C into carbon dioxide (CO2) strictly results from intracellular metabolic processes. Here we show that C mineralization can operate in soils deprived of all observable cellular forms. Moreover, the process responsible for CO2 emissions in sterilized soils induced a strong C isotope fractionation (up to 50 ‰) incompatible with respiration of cellular origin. The supply of 13C glucose in sterilized soil led to the release of 13CO2 suggesting the presence of respiratory-like metabolism (glycolysis, decarboxylation reaction, chain of electron transfer) carried out by soil-stabilized enzymes, and by soil mineral and metal catalysts. These findings indicate that CO2 emissions from soils can have two origins: (1) from the well-known respiration of soil heterotrophic microorganisms and (2) from an extracellular oxidative metabolism (EXOMET) or, at least, catabolism. These two metabolisms should be considered separately when studying effects of environmental factors on the C cycle because the likelihood is that they do not obey the same laws and they respond differently to abiotic factors.

(13)C glucose labelling studies using 2D NMR are a useful tool for determining ex vivo whole organ metabolism during hypothermic machine perfusion of kidneys.

BackgroundThe aim of this study is to determine the feasibility of using nuclear magnetic resonance (NMR) tracer studies (13C-enriched glucose) to detect ex vivo de novo metabolism in the perfusion fluid and cortical tissue of porcine kidneys during hypothermic machine perfusion (HMP).MethodsPorcine kidneys (n = 6) were subjected to 24 h of HMP using the Organ Recovery Systems LifePort Kidney perfusion device. Glucose, uniformly enriched with the stable isotope 13C ([U-13C] glucose), was incorporated into KPS-1-like perfusion fluid at a concentration of 10 mM. Analysis of perfusate was performed using both 1D 1H and 2D 1H,13C heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The metabolic activity was then studied by quantifying the proportion of key metabolites containing 13C in both perfusate and tissue samples.ResultsThere was significant enrichment of 13C in a number of central metabolites present in both the perfusate and tissue extracts and was most pronounced for lactate and alanine. The total amount of enriched lactate (per sample) in perfusion fluid increased during HMP (31.1 ± 12.2 nmol at 6 h vs 93.4 ± 25.6 nmol at 24 h p < 0.01). The total amount of enriched alanine increased in a similar fashion (1.73 ± 0.89 nmol at 6 h vs 6.80 ± 2.56 nmol at 24 h p < 0.05). In addition, small amounts of enriched acetate and glutamic acid were evident in some samples.ConclusionsThis study conclusively demonstrates that de novo metabolism occurs during HMP and highlights active metabolic pathways in this hypothermic, hypoxic environment. Whilst the majority of the 13C-enriched glucose is metabolised into glycolytic endpoint metabolites such as lactate, the presence of non-glycolytic pathway derivatives suggests that metabolism during HMP is more complex than previously thought. Isotopic labelled ex vivo organ perfusion studies using 2D NMR are feasible and informative.

Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing

Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled 13C6 glucose, 13C3 lactate, and 13C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture.

Identification and Characterization of DcUSAGT1, a UDP-Glucose: Sinapic Acid Glucosyltransferase from Purple Carrot Taproots.

Purple carrots accumulate abundant cyanidin-based anthocyanins in taproots. UDP-glucose: sinapic acid glucosyltransferase (USAGT) can transfer the glucose moiety to the carboxyl group of sinapic acid thereby forming the ester bond between the carboxyl-C and the C1 of glucose (1-O-sinapoylglucose). 1-O-sinapoylglucose can serve as an acyl donor in acylation of anthocyanins and generate cyanidin 3-xylosyl (sinapoylglucosyl) galactoside in purple carrots. This final product helps stabilize the accumulation of anthocyanins. In this study, a gene named DcUSAGT1 encoding USAGT was cloned from ‘Deep purple’ carrot taproots. Enzymatic activity was determined using high performance liquid chromatography (HPLC). The optimal temperature and pH value were 30°C and 7.0, respectively. Kinetic analysis suggested a Km (sinapic acid) of 0.59 mM. Expression profiles of DcUSAGT1 showed high expression levels in the taproots of all the three purple carrot cultivars but low expression levels in those of non-purple carrot cultivars. The USAGT activity of different carrots in vitro indicated that crude enzyme extracted from the purple carrot taproots rather than non-purple carrot taproots exhibited USAGT activity. These results indicated that DcUSAGT1 may influence anthocyanin biosynthesis of purple carrot taproots.

Digging in the dirt – Inadequacy of belowground plant biomass quantification

Accurate quantification of belowground plant biomass (BGP) is crucial to account for the carbon (C) and nitrogen (N) stored by plants. As soil sieving to recover roots leaves a large proportion of root borne compounds defined as rhizodeposits (fine roots, root fragments, exudates) unaccounted for, isotope-labelling approaches have frequently been used. The aim of the present study was to compare two approaches that estimate BGP-N from isotope labelling experiments and assess their potential error. Pisum sativum was grown in a pot experiment and repeatedly pulse labelled with a 13C glucose and 15N urea solution using a cotton wick method. Additionally, data from a previous study using the same labelling approach were used for comparative BGP-N calculations. In both experiments, the amount of BGP-N calculated with a mass balance approach was significantly lower compared with the classical calculation, indicating substantial overestimation of N rhizodeposition in previous studies. Multiple pulse labelling of plants with 15N can result in homogeneous label distribution, which allows both calculation approaches to be used. However, when label distribution is heterogeneous, the classical approach overestimates N rhizodeposition and BGP-N.

Critical Level of 13C Enrichment for the Successful Isolation of 13C Labeled DNA

In the stable-isotope probing (SIP) technology, the 13C-labelled substrate is normally used to incorporate 13C into nucleic acids. A proper abundance of 13C in substrate is critical to the success of SIP, because the 13C level not only determines whether 13C in nucleic acids is sufficient to be detective, but also affects the enrichment bias in the labeled microbes. However, such information is very rare. In this study, a serial of 13C-labelled glucose from 0 to 50 atom% 13C was used to incubate with Escherichia coli and then performed DNA-SIP. Our results showed that the detective level of 13C-DNA could be reduced to 2 atom% 13C of glucose (1.30 atom% 13C in DNA extract), while the ideal level was 10 atom% 13C glucose (2.25 atom% 13C in DNA). The critical level of 13C for the separation of 13C-DNA provides a new reference of DNA-SIP in order to trace active microbial populations utilizing specific C substrates in environments.

Assessment of Metabolism of the Eastern Oyster and Eastern Elliptio Using NMR Spectroscopy

Metabolites are known to characterize the functional responses of a cell. Therefore, a quantitative measurement of metabolite profiles can provide insight into the underlying effect of genetic or environmental actions on cell metabolism. It follows then that the study of the metabolite profiles of bivalve mollusks, such as oysters, can be particularly worthwhile in assessing changes in physiology, pharmacology and toxicology that result from their adaptation to changing environments. We have determined the metabolic profiles of three different organ groups in freshwater mussel and oysters by using 1H (proton) and 13C (carbon) NMR (nuclear magnetic resonance) spectroscopy following the infusion of 13C glucose or 13C glycine, respectively. The result shows infused glucose formed glycogen by glycogen synthesis, alanine by glycolysis, and glutamate and aspartate through the Krebs cycle and glycine formed serine by the glycine cleavage system in oysters. Decrease in adenosine triphosphate (ATP), glucogen, putrescine and ornithine were observed in the fasting state freshwater mussel. Our result opens the possibility that organ specific metabolic fingerprints can be established to interpret functional adaptations to environmental and nutritional challenges using NMR spectroscopy.

Microbial physiology and necromass regulate agricultural soil carbon accumulation

Strategies for mitigating soil organic carbon (SOC) losses in intensively managed agricultural systems typically draw from traditional concepts of soil organic matter formation, and thus emphasize increasing C inputs, especially from slowly decomposing crop residues, and reducing soil disturbance. However these approaches are often ineffective and do not adequately reflect current views of SOC cycling, which stress the important contributions of microbial biomass (MB) inputs to SOC. We examined microbial physiology as an alternate mechanism of SOC accumulation under organic (ORG) compared to conventional (CT) agricultural management practices, where ORG is accumulating C despite fewer total C inputs and greater soil tillage. We hypothesized that microbial communities in ORG have higher growth rates (MGR) and C use efficiencies (CUE) and that this relates to greater MB production and ultimately higher retention of new C inputs. We show that ORG had 50% higher CUE (±8 se) and 56% higher MGR (±22 se) relative to CT (p < 0.05). From in situ13C substrate additions, we show that higher CUE and MGR are associated with greater rates and amounts of 13C glucose and phenol assimilation into MBC and mineral-associated SOC pools in ORG up to 6 mo after field substrate additions (p < 0.05). ORG soils were also enriched in proteins and lipids and had lower abundances of aromatic compounds and plant lipids (p < 0.05). These results illustrate a new mechanism for SOC accumulation under reduced C inputs and intensive soil disturbance and demonstrate that agricultural systems that facilitate the transformation of plant C into MB may be an effective, often overlooked strategy for building SOC in agricultural soils.

Contrasting effects of aged and fresh biochars on glucose-induced priming and microbial activities in paddy soil

The understanding of soil organic matter (SOM) decomposition to biochar (BC) addition in paddy soil remains limited. This study was to examine the glucose-induced priming effects of paddy soil as affected by aged and/or fresh BCs. Soil samples were collected from the paddy fields without and with BC applied 1 year before the laboratory incubation. The control without BC, the soil with BC applied in the field (aged BC), aged BC and the additional fresh BC applied (A/F-BC), and the soil with fresh BC (fresh BC), each with glucose (14C) addition or not were incubated under either moist or flooding conditions. Totally, 16 treatments were established. The aged-BC group treatments had greater carbon dioxide (CO2) efflux (averagely 26 %) compared to the control group, while CO2 efflux was reduced following fresh BC addition by 23 and 21 % in the A/F-BC and fresh-BC groups, respectively. In the presence of aged BC, microbial biomass and the potential activities of s-glucosidase, xylanase, cellobiohydrolase, and chitinase were increased under both moist and flooding conditions, but they were decreased in the fresh-BC group under flooding conditions. For the control group, positive priming of 58 and 102 μg C g−1 were observed after glucose addition under moist and flooding conditions, respectively. Positive priming was also observed in the fresh-BC group amounting to 34 and 19 μg C g−1 under moist and flooding conditions, respectively. However, negative priming was found in the soil samples with aged BC, especially under moist conditions, which was possibly due to the preferential use of glucose over the more recalcitrant organic C in both SOM and BC and SOC stabilization via either organic matter sorption on BC or the BC-induced organic-mineral interaction. BC effects on CO2 efflux and microbial activities depend strongly on its age. Aged BC improves soil microbial parameters and also contributes to C sequestration in soil by negative priming. In contrast, fresh BC addition depresses microbial activities but stimulates the mineralization of SOM and itself following glucose addition.

Cardioprotection Resulting from Glucagon-Like Peptide-1 Administration Involves Shifting Metabolic Substrate Utilization to Increase Energy Efficiency in the Rat Heart.

Previous studies have shown that glucagon-like peptide-1 (GLP-1) provides cardiovascular benefits independent of its role on peripheral glycemic control. However, the precise mechanism(s) by which GLP-1 treatment renders cardioprotection during myocardial ischemia remain unresolved. Here we examined the role for GLP-1 treatment on glucose and fatty acid metabolism in normal and ischemic rat hearts following a 30 min ischemia and 24 h reperfusion injury, and in isolated cardiomyocytes (CM). Relative carbohydrate and fat oxidation levels were measured in both normal and ischemic hearts using a 1-13C glucose clamp coupled with NMR-based isotopomer analysis, as well as in adult rat CMs by monitoring pH and O2 consumption in the presence of glucose or palmitate. In normal heart, GLP-1 increased glucose uptake (↑64%, p<0.05) without affecting glycogen levels. In ischemic hearts, GLP-1 induced metabolic substrate switching by increasing the ratio of carbohydrate versus fat oxidation (↑14%, p<0.01) in the LV area not at risk, without affecting cAMP levels. Interestingly, no substrate switching occurred in the LV area at risk, despite an increase in cAMP (↑106%, p<0.05) and lactate (↑121%, p<0.01) levels. Furthermore, in isolated CMs GLP-1 treatment increased glucose utilization (↑14%, p<0.05) and decreased fatty acid oxidation (↓15%, p<0.05) consistent with in vivo finding. Our results show that this benefit may derive from distinct and complementary roles of GLP-1 treatment on metabolism in myocardial sub-regions in response to this injury. In particular, a switch to anaerobic glycolysis in the ischemic area provides a compensatory substrate switch to overcome the energetic deficit in this region in the face of reduced tissue oxygenation, whereas a switch to more energetically favorable carbohydrate oxidation in more highly oxygenated remote regions supports maintaining cardiac contractility in a complementary manner.

Priming of the decomposition of ageing soil organic matter: concentration dependence and microbial control

Summary The amount of carbon (C) stored in soil is an important regulator for the global climate and soil fertility and is the balance between formation and decomposition of soil organic matter (SOM). Decomposition of SOM can be powerfully affected by labile carbon (C) supplements in, for example, the rhizosphere. A stimulation of SOM mineralisation induced by labile C additions is termed ‘priming’, and the mechanisms for this phenomenon remain elusive. The most widely held explanation assigns priming to successional dynamics in r‐ and K‐selected groups within the microbial community; groups which have also been connected with fungal (K‐selected) and bacterial (r‐selected) decomposers. New evidence has also suggested that recently formed SOM is particularly sensitive to priming. We investigated (i) the labile C concentration dependence of SOM mineralisation, (ii) the susceptibility of differently aged SOM to priming and (iii) if priming is due to bacterial or fungal growth dynamics. To create an age gradient of traceable SOM, we spiked a pasture soil using 14C glucose, and subsampled plots 1 day, 2 months, 5 months and 13 months after application (i.e. SOM aged 1 day – 13 months). Glucose (0–4000 μg C g−1) was added in subsequent laboratory experiments, and respiration, SOM mineralisation (14CO2 evolution), bacterial growth rates (leucine incorporation) and fungal biomass (ergosterol) were tracked during ca. 1 week. Mineralisation of SOM aged 2–13 months showed similar labile C concentration dependencies, and priming increased mineralisation of SOM systematically by up to 350%. The glucose treatments induced variable microbial growth responses for differently aged SOM, which were unrelated to the priming effect. That successional dynamics in microbial r‐ and K‐selected groups, or bacterial and fungal decomposers, respectively, underpinned priming was incompatible with the results obtained. An alternative explanation could be that SOM transformation by extracellular enzymes, for subsequent respiration, could be triggered by labile C. In conclusion, labile C primed the mineralisation of 2–13 months aged SOM, and the mechanism for this priming was unrelated to microbial growth dynamics.

Abstract 15571: Suppressing Increased Expression of NADPH-Dependent Malic Enzyme 1 in Hypertrophied Hearts Eliminates Maladaptive Anaplerosis and Improves Redox Conditions

The remodeled metabolic phenotype of hypertrophic hearts includes inefficient glucose oxidation, due in part to pyruvate carboxylation into anaplerosis. Increased carboxylation of pyruvate to malate via elevated malic enzme-1 (ME1) also consumes NADPH, which is necessary for maintaining glutathione reduction. Therefore, we studied the consequences of selective inhibition of ME1 expression via non-native miRNA targeted to the ME1 gene (miRME1) in hypertrophied rat hearts after transverse aortic constriction (TAC). TAC elevated ME1 content, but ME1 was lowered in hearts receiving miRME1 vs PBS infusion (*P&lt;0.01). The effects of ME1 suppression on anaplerosis and GSH content were studied in isolated hearts supplied 13C palmitate, glucose, and lactate. While Sham miRME1 hearts showed no further reduction of inherently low baseline ratios of anaplerosis to citrate synthase flux from normal hearts, miRME1 reduced anaplerosis in TAC to baseline: TAC miRME1=0.034±0.004; TAC PBS=0.081±0.005 (*P&lt;0.01, **P&lt;0.001). Importantly, ME1 suppression restored GSH in TAC (†P&lt;0.05). The findings demonstrate the maladaptive increase in anaplerosis via ME1 in TAC is associated with reduced GSH content. Suppressing increased ME1 expression in hypertrophied hearts, and thus consumption of NADPH for anaplerotic malate, produced favorable metabolic shifts, improving intracellular redox state.