Isomeric and anomeric specificity of the metabolic and secretory response of rat pancreatic islets to glucose pentaacetate.

In the presence of 2.8 mM D-glucose, beta-D-glucose pentaacetate (1. 7 mM) augmented insulin release from isolated rat pancreatic islets more than alpha-D-glucose pentaacetate. Likewise, the further increment in insulin output evoked by nateglinide (0.01 mM) was higher in islets exposed to beta- rather than alpha-D-glucose pentaacetate. Inversely, in the presence of 2.8 mM unesterified D-glucose, alpha-L-glucose pentaacetate, but not beta-L-glucose pentaacetate, significantly augmented insulin output. The higher insulinotropic potency of the beta-anomer of D-glucose pentaacetate coincided with the fact that it significantly increased the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, whereas alpha-D-glucose pentaacetate failed to do so. These findings are interpreted to support the concept that the stimulation of insulin release by these esters is largely attributable to their direct interaction with a stereospecific receptor, with preference for the configuration of the C1 common to beta-D-glucose pentaacetate and alpha-L-glucose pentaacetate.     

Metabolism of D-[3-3H]glucose, D-[5-3H]glucose, D-[U-14C]glucose, D-[1-14C]glucose and D-[6-14C]glucose in pancreatic islets in an animal model of type-2 diabetes

This study aims at exploring specific aspects of D-glucose metabolism, so far not yet investigated, in pancreatic islets from adult control rats and animals (STZ rats) injected with streptozotocin during the neonatal period. The latter animals, which represent a current model of type-2 diabetes, displayed a lower body weight, higher plasma D-glucose concentration and lower insulinogenic index than control rats. The protein, DNA and insulin content were all also lower in islets prepared from STZ, rather than control rats. In the presence of 10.0 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was also decreased in the islets from STZ rats. No significant difference between control and STZ rats was observed, however, in terms of the ratios between D-[3-3H]glucose and D-[5-3H]glucose utilization, between the generation of radioactive lactate from 14C-labelled D-glucose and tritiated D-glucose utilization and between D-[1-14C]glucose and D-[6-14C]glucose oxidation. These findings reinforce the view that the previously documented preferential impairment of the oxidative modality of glycolysis in islets from STZ rats contrasts with the absence of any major anomaly in other variables of D-glucose catabolism.     

Metabolism of 13C-enriched D-fructose in hepatocytes from Goto-Kakizaki rats

This study aims at assessing the conversion of exogenous D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]-fructose (10 mM) to 13C-enriched and either hydrogenated or deuterated D-glucose, L-lactate and L-alanine released by rat liver cells prepared from Goto-Kakizaki rats and incubated for 120 min in the presence of unlabelled D-glucose (also 10 mM) and D2O. The results of this study are relevant to the relative contribution of fructokinase and hexokinase isoenzyme to the phosphorylation of D-fructose, the capacity of D-glucose to confer to glucokinase positive cooperativity towards D-fructose, the circulation of D-fructose 6-phosphate in the pentose phosphate pathway, the regulation of the cytosolic NADD/NADH ratio, the respective fate of D-fructose-derived D-glyceraldehyde and dihydroxyacetone phosphate, the deuteration of fructose-derived glycolytic intermediates at the phosphoglucoisomerase, phosphomannoisomerase, enolase, pyruvate kinase and glutamate-alanine transaminase levels, and the unequal generation of L-[1-13C]lactate by cells exposed to D-[1-13C]fructose or D-[6-13C]fructose versus D-[2-13C]-fructose.     

Metabolism of D-[1-(13)C]fructose, D-[2-(13)C]fructose, and D-[6-(13)C]fructose in rat hepatocytes incubated in the presence of H(2)O or D(2)O

Isolated hepatocytes from fed rats were exposed for 120 min to D-[1-(13)C]fructose, D-[2-(13)C]fructose, or D-[6-(13)C]fructose in the presence of H(2)O or D(2)O. The identification and quantification of (13)C-enriched metabolites (D-glucose, L-lactate) in the incubation medium and the measurement of their deuterated isotopomers indicated that the ketohexose was phosphorylated predominantly at the intervention of fructokinase and that the majority of the D-glyceraldehyde molecules generated from d-fructose 1-phosphate were further metabolized, e.g., after phosphorylation to D-glyceraldehyde 3-phosphate. It is proposed that the present procedure may help to further characterize the regulation of D-fructose metabolism in both hepatocytes and other cell types.     

Metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C]succinate) in hepatocytes from Goto-Kakizaki rats

The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.     

Altered metabolic priming by D-glucose in pancreatic islets from Goto-Kakizaki rats

The possible priming by D-glucose of metabolic events in islets from control rats and Goto-Kakizaki rats (GK rats) was investigated by first incubating the islets for 120 min either in the absence of any exogenous nutrient or presence of 16.7 mM D-glucose. The islets were then incubated for a second period of 120 min either at 2.8 mM or 16. 7 mM D-glucose, the hexose being now mixed with tracer amounts of D-[U-14C]glucose and D-[5-3H]glucose. In islets from control rats first incubated in the absence of exogenous nutrient the hierarchy in the 16.7 mM/2.8 mM ratio for metabolic variables was as follows: D-[U-14C]glucose oxidation > D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to amino acids > D-[U-14C]glucose conversion to acidic metabolites. When the islets from control rats were first incubated in the presence of 16.7 mM D-glucose, the preferential stimulation of mitochondrial oxidative events at high hexose concentration, as documented by the increase in the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, was further enhanced. The 16.7 mM/2.8 mM ratio for the conversion of D-[U-14C]glucose to amino acids, relative to that for D-[U-14C]glucose conversion to acidic metabolites, was much lower, however, after a first incubation in the presence of D-glucose, rather than in its absence, probably as a result of the progressive exhaustion of endogenous amino acids considered as transamination partners. The major differences between these results and those obtained in islets from GK rats consisted, in the latter animals, in i) higher absolute values for all metabolic fluxes, ii) lower 16.7 mM/ 2.8 mM ratios, iii) lower paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization, and iv) absence of a priming effect of D-glucose (16.7 mM) upon such a paired ratio in the islets incubated at 16.7 mM D-glucose during the second incubation. Taken as a whole, these observations confirm that the preferential stimulation of mitochondrial oxidative events, in response to a rise in D-glucose concentration, is impaired in islets from GK rats and extend this knowledge to the priming action of D-glucose, in high concentration, on the catabolism of the hexose during a subsequent incubation.     

Impairment by cytochalasin B of the inhibitory action of D-mannoheptulose upon D-glucose metabolism in rat pancreatic islets

D-mannoheptulose was recently found to inhibit D-glucose metabolism in hepatocytes and pancreatic islets, whilst failing to do so in parotid cells, erythrocytes and the exocrine pancreas. In the latter three systems, however, the hexaacetate ester of D-mannoheptulose efficiently inhibits D-glucose metabolism. It was proposed, therefore, that the transport of unesterified D-mannoheptulose into cells may be mediated by GLUT2. Since cytochalasin B is known to inhibit D-glucose transport into pancreatic islet cells, it was now investigated whether the mould metabolite (0.02 mM) also impairs the inhibitory action of D-mannoheptulose (1.0 mM) upon D-glucose metabolism in rat pancreatic islets. The relative extent of D-mannoheptulose inhibitory action on D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to 14CO2, as well as radioactive amino acids and acidic metabolites, was indeed much less marked in the presence of cytochalasin B (13+/-4% inhibition) than in its absence (40+/-3% inhibition). A comparable situation was not observed, however, in the case of glucose-stimulated insulin secretion, cytochalasin B augmenting insulin output to the same relative extent in the absence or presence of D-mannoheptulose. These findings support the view that the entry of D-mannoheptulose into cells may be mediated by a cytochalasin B-sensitive transport system, such as the GLUT2 carrier.     

Effects of gliquidone on D-glucose metabolism in rat pancreatic islets depend on hexose concentration

The hypoglycemic sulfonylurea gliquidone, used at a 10 microM concentration, failed to affect the metabolism of D-glucose in rat pancreatic islets incubated in the presence of 5.6 mM, 8.3 mM or 16.7 mM D-glucose. However, at 2.8 mM D-glucose, gliquidone increased D-[U-14C]glucose oxidation while decreasing the utilization of D-[5-3H]glucose and generation of radioactive acidic metabolites and amino acids from D-[U-14C]glucose. These dissociated effects could conceivably be attributable, respectively, to activation of FAD-linked glycerophosphate dehydrogenase as a result of an increase in cytosolic Ca2+ concentration and to a subsequent inhibition of phosphofructokinase as a result of an increase in cytosolic ATP concentration. The effect of gliquidone on the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was indeed duplicated by repaglinide and suppressed in the absence of extracellular Ca2+ or at low temperature. The present findings thus provide a further illustration of the often contrasting effects of pharmacological and physiological insulinotropic agents on selected metabolic, cationic and functional variables in pancreatic islet cells.     

Uptake of D-mannoheptulose by normal and tumoral pancreatic islet cells

D-mannoheptulose was recently proposed as a possible tool to label preferentially insulin-producing cells in the pancreatic gland. In the present study, D-[3H]-mannoheptulose uptake by rat pancreatic islets or dispersed islet cells was found to represent a time-related and temperature-sensitive process inhibited by cytochalasin B. This mould metabolite also inhibited the efflux of D-[3H]-mannoheptulose from prelabelled islets. After 60 min incubation at 37 degrees C, the apparent intracellular distribution space of the tritiated heptose was close to or somewhat higher than that of D-[5-3H]glucose and close to 50% of the intracellular 3HOH space. It was further enhanced by D-glucose and a high concentration of 10 mM of D-mannoheptulose. The uptake of D-[3H]mannoheptulose was much lower however than that of D-[3H]mannoheptulose hexaacetate. As judged from the fate of D-mannoheptulose hexa[2-14C]acetate, the latter ester was efficiently hydrolyzed in the islet cells. The internalization of D-[3H]mannoheptulose (or its ester) coincided with the generation of tritiated acidic metabolites, reflecting phosphorylation of the heptose. The situation found in normal islet cells sharply differed from that found in tumoral islet cells of either the RINm5F or INS-1 line, in which the apparent distribution space of D-[3H]mannoheptulose represented only about 3 and 9%, respectively, of the intracellular 3HOH space. These results indicate that the entry of D-mannoheptulose into islet cells represents a carrier-mediated process, possibly mediated at the intervention of GLUT2 and, hence, provide further support to the possible use of a suitable D-mannoheptulose analog as a tool for the preferential labelling of insulin-producing cells in the pancreatic gland.     

The use of the [1,2-13C]acetate recovery factor in metabolic research

To provide guidelines on the correct application of the acetate recovery factor in metabolic research, we investigated the influence of exercise intensity and infusion protocol on [1,2-¹³C]acetate label recovery during exercise. Eight cyclists were studied during [1,2-¹³C]acetate infusion for 1 h at rest followed by three 30-min stages of cycling exercise at a workload of 40, 55 and 75% maximal workload (W _max), respectively (protocol 1). Four cyclists were subsequently studied following [1,2-¹³C]acetate infusion in three separate trials while cycling at the same workloads but in the absence of any pre-exercise infusion period (protocol 2). Finally, we observed the cyclists during [1,2-¹³C]acetate infusion at a 40% W _max workload after 4 h of pre-exercise infusion (protocol 3). Acetate recovery increased from 13.7 (0.4)%, after 1 h of rest, to a plateau value of 75.1 (2), 91.2 (0.7) and 101 (2)% during exercise at 40, 55 and 75% W _max workloads, respectively. In protocol 2, without prior infusion time, fractional label recovery was substantially lower at each separate workload. In contrast, when applying an extensive pre-exercise infusion period of 4 h, acetate recovery rates were substantially increased compared to the values observed in protocols 1 and 2 during exercise at a 40% W _max workload. In conclusion, in contrast to resting conditions, acetate recovery reaches a plateau value during exercise. Though this plateau value is repeatedly used to correct for label recovery in various exercise studies, our data clearly show that acetate label recovery during exercise not only depends on the exercise intensity but also on the applied infusion protocol. Therefore, theoretical acetate recovery factors taken from previous literature are not generally applicable. Electronic Publication     

Estimating gluconeogenesis by NMR isotopomer distribution analysis of [13C]bicarbonate and [1-13C]lactate

The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h was determined by 13C-NMR isotopomer distribution analysis of secreted glucose enriched from 99% [13C]bicarbonate (n = 4) and 99% [1-13C]lactate (n = 4). Experiments with 3% 2H2O were also performed, allowing the gluconeogenic contribution to be measured by the relative 2H enrichments at positions 5 and 2 of glucose. From 13C-NMR analyses, the contribution of gluconeogenesis to glucose output was estimated to be 93 +/- 3% for [13C]bicarbonate perfusion and 91 +/- 3% for [1-13C]lactate perfusion, in good agreement with the 2H-NMR analysis of the gluconeogenic contribution to glucose production (100 +/- 1% and 99 +/- 1%, respectively) and consistent with the expected negligible contribution from glycogenolysis. These results indicate that 13C-NMR analysis of glucose 13C-isotopomer distribution from either [13C]bicarbonate or [1-13C]lactate precursor provides realistic estimates of the gluconeogenic contribution to hepatic glucose output.     

Oxidation of [1,12-14C]dodecanedioic acid by rat pancreatic islets

Several aliphatic dioic acids were recently reported to stimulate insulin release in isolated rat pancreatic islets incubated at close-to-physiological D-glucose concentrations. In order to gain insight into the mode of action of these acids in pancreatic islet B-cells, the oxidation of [1,12-14C]dodecanedioic acid (5.0 mM) was now measured in rat islets. Expressed as pmol of [1, 12-14C]dodecanedioic acid equivalent, the production of 14CO2 was close to 1.0 pmol/islet per 120 min, representing about 8% of that attributable to the oxidation of D-[U-14C]-glucose (8.3 mM). The dioic acid and the hexose failed to exert any significant reciprocal effect upon their respective oxidation rate. These findings support the view that the insulinotropic action of dodecanedioic acid, and presumably other aliphatic dioic acids, is causally linked to their capacity to act as nutrients in pancreatic islet cells.     

Topography of basal glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose

The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [1-14C]glucose and [6-14C]glucose and their 14C-labelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of 14C occurred in the granule cell layer of the dentate gyrus and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosum-molecular. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [1-14C]glucose and [6-14C]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [1-14C]glucose. Although there was significantly more radioactivity at 30 min than at 10 min, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-14C]glucose utilized in different hippocampal regions is oxidized to 14CO2 under these conditions. Most regions of high glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study on [1-13C]glucose metabolism in the rat brain during hypoxia

The metabolism of [1-13C]glucose in the rat brain during hypoxia was investigated by 13C NMR spectroscopy. Male Wistar rats, weighing 100-120g, were anesthetized with ketamine (50 approximately 75 mg/kg i.p.) and ventilated mechanically with a mixture of 30% oxygen, 69.5% nitrogen and 0.5% halothane. [1-13C]glucose (250 mg/kg) was infused twice, at 10 minute intervals, through the femoral vein. For the control group (n = 4), the oxygen concentration of the inspiratory gas was maintained at 30% by vol throughout the experiments. For the hypoxia group (n = 6), the oxygen concentration in the inspiratory gas was reduced to 6-7% (93-94% nitrogen) and maintained for 30 min following [1-13C]glucose infusion. 13C NMR spectra were measured by a gated proton-decoupling method without a nuclear Overhauser effect. The [1-13C]glucose infusion gave apparent signals of the C1 carbon in the alpha- and beta-anomers of [1-13C]glucose at 92.7 and 96.7 ppm, respectively. Signals of the C2, C3 and C4 carbon atoms in glutamate and/or glutamine (glx) also appeared at 55, 27 and 34 ppm, respectively. The intensity of glx-C2 and glx-C3 signals increased later than that of glx-C4. The time lag between the different glx signals may reflect the turnover rate of the TCA cycle. Under the hypoxic condition, the signal of C3 carbon in lactate appeared at 21 ppm and increased. The alpha-glucose signal diminished during hypoxia, whereas the beta-glucose signal kept its intensity. The difference in changes of the signal intensity between alpha- and beta-glucose suggests that alpha-glucose is consumed more than beta-glucose in the hypoxic brain.     

Effects of D-mannoheptulose upon D-glucose metabolism in pancreatic B and non-B islet cells.

D-mannoheptulose was recently proposed to be transported into cells mainly at the intervention of GLUT2. In the present study, the heptose (10 mM) decreased the steady state content of dispersed rat pancreatic islet cells in D-[U-(14)C]glucose, and inhibited to a greater relative extent the utilization of D-[5-(3)H]glucose, the oxidation of D-[U-(14)C]-glucose and its conversion to radioactive amino acid when the dispersed islet cells were incubated at 16.7 mM rather than 2.8 mM D-glucose. A comparable situation was found in purified islet B-cells, whereas D-mannoheptulose only exerted minor to negligible effects upon the metabolism of D-glucose in non-B islet cells. This coincided with a much higher uptake of D-[(3)H]mannoheptulose by B, as distinct from non-B, islet cells. These findings indicate that the unexpectedly greater relative inhibitory action of D-mannoheptulose upon D-glucose metabolism by isolated islets (or dispersed islet cells) observed at high rather than low hexose concentration cannot be accounted for solely by differences in the relative contribution of non-B cells to total D-glucose metabolism by islets incubated at increasing concentrations of D-glucose. A comparable metabolic response to D-mannoheptulose is indeed observed in purified B cells. It could be attributable, in part at least, to D-glucose and D-mannoheptulose countertransport, resulting inter alia in a greater net uptake of the heptose by B cells exposed to a high concentration of the hexose.     

Activation of glycogen synthase a in hepatocytes exposed to alpha-D-glucose pentaacetate

The effects of alpha-D-glucose pentaacetate (1.7 mM) upon glycogen synthase a activity and lactate output were examined in rat hepatocytes incubated at increasing concentrations of D-glucose. The ester enhanced the activity of glycogen synthase a at all concentrations (2.8, 4.0 and 8.0 mM) of D-glucose, which itself provoked a concentration-related increase in enzymatic activity. Likewise, the output of lactate augmented at increasing concentrations of D-glucose. However, alpha-D-glucose pentaacetate failed to cause a further increase in lactate output, the trend being even towards a lower production of lactate in the presence than absence of the ester. These findings suggest that the activation of glycogen synthase a by alpha-D-glucose pentaacetate and the subsequent increase in glycogen synthesis are sufficiently pronounced to prevent the increase in glycolysis otherwise expected from the generation of unesterified D-glucose from the same ester. Such a situation, which differs from that previously documented in pancreatic islet cells, could be favourable in the perspective of using alpha-D-glucose pentaacetate as a novel insulinotropic, and hence hypoglycaemic, tool in the treatment of non-insulin-dependent diabetes mellitus.     

Immobilization of tumoral pancreatic islet cells on a two-dimensional microsupport

A new procedure for the immobilization of tumoral pancreatic islet cells to a two-dimensional microsupport is presented. Tumoral islet cells of the RINm5F line (0.7x10(6) cells/ml) were immobilized to two-dimensional microcarriers (16.6 cm2/ml). Within 24 h of culture, and as judged from the number of cells, their protein or insulin content, less than 10% of the cells escaped immobilization. The metabolic response of the immobilized cells to D-glucose was well preserved, the paired ratio between D-[U-14C] glucose oxidation and D-[5-3H]glucose utilization being even significantly higher in immobilized than free cells. The advantages of this novel approach in the perspective of islet cell transplantation are underlined.     

Integration of [U-13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.     

In vivo single-shot, proton-localized 13C MRS of rhesus monkey brain

A single-shot, proton-localized, polarization transfer (13)C spectroscopic method was proposed and implemented on a 4.7 T scanner for studying rhesus monkey brains. The polarization transfer sequence was mostly adiabatic, minimizing signal loss due to B(1) inhomogeneity. RF pulses in polarization transfer were also used for voxel selection of protons with gradient fields. The transferred (13)C magnetization was refocused by additional refocusing adiabatic pulses. With the intravenous infusion of D-[1-(13)C]glucose solution, (13)C NMR spectra from a 30 mL voxel were acquired for the resonances of C1 of glucose, C2,3,4 of glutamate and glutamine. The time-resolved turnover of glutamate, glutamine and aspartate from intravenously infused D-[1-(13)C]glucose at a temporal resolution of 12 min was demonstrated with excellent spectral resolution and signal-to-noise ratio. Typically, the half-height linewidth of the decoupled (13)C peaks was approximately 4 Hz. Data obtained with infusion of sodium [2-(13)C]acetate using the proposed polarization transfer method and data from the carboxylic carbon region using non-localized acquisition are also presented.     

Profiling human gut bacterial metabolism and its kinetics using [U‐13C]glucose and NMR

This study introduces a stable‐isotope metabolic approach employing [U‐¹³C]glucose that, as a novelty, allows selective profiling of the human intestinal microbial metabolic products of carbohydrate food components, as well as the measurement of the kinetics of their formation pathways, in a single experiment. A well‐established, validated in vitro model of human intestinal fermentation was inoculated with standardized gastrointestinal microbiota from volunteers. After culture stabilization, [U‐¹³C]glucose was added as an isotopically labeled metabolic precursor. System lumen and dialysate samples were taken at regular intervals. Metabolite concentrations and isotopic labeling were determined by NMR, GC, and enzymatic methods. The main microbial metabolites were lactate, acetate, butyrate, formate, ethanol, and glycerol. They together accounted for a ¹³C recovery rate as high as 91.2%. Using an NMR chemical shift prediction approach, several minor products that showed ¹³C incorporation were identified as organic acids, amino acids, and various alcohols. Using computer modeling of the ¹²C contents and ¹³C labeling kinetics, the metabolic fluxes in the gut microbial pathways for synthesis of lactate, formate, acetate, and butyrate were determined separately for glucose and unlabeled background substrates. This novel approach enables the study of the modulation of human intestinal function by single nutrients, providing a new rational basis for achieving control of the short‐chain fatty acids profile by manipulating substrate and microbiota composition in a purposeful manner. Copyright © 2009 John Wiley & Sons, Ltd.