Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.     

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.     

Noninvasive Measurements of [1-13C] Glycogen Concentrations and Metabolism in Rat Brain In Vivo

Using a specific 13C NMR localization method, 13C label incorporation into the glycogen C1 resonance was measured while infusing [1-(13)C]glucose in intact rats. The maximal concentration of [1-(13)C]glycogen was 5.1 +/- 0.6 micromol g(-1) (mean +/- SE, n = 8). During the first 60 min of acute hyperglycemia, the rate of 13C label incorporation (synthase flux) was 2.3 +/- 0.7 micromol g(-1) h(-1) (mean +/- SE, n = 9 rats), which was higher (p < 0.01) than the rate of 0.49 +/- 0.14 micromol g(-1) h(-1) measured > or = 2 h later. To assess whether the incorporation of 13C label was due to turnover or net synthesis, the infusion was continued in seven rats with unlabeled glucose. The rate of 13C label decline (phosphorylase flux) was lower (0.33 +/- 0.10 micromol g(-1) h(-1)) than the initial rate of label incorporation (p < 0.01) and appeared to be independent of the duration of the preceding infusion of [1-(13)C]glucose (p > 0.05 for correlation). The results implied that net glycogen synthesis of approximately 3 micromol g(-1) had occurred, similar to previous reports. When infusing unlabeled glucose before [1-(13)C]glucose in three studies, the rate of glycogen C1 accumulation was 0.46 +/- 0.08 micromol g(-1) h(-1). The results suggest that steady-state glycogen turnover rates during hyperglycemia are approximately 1% of glucose consumption.     

Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts

The objective of this study was to determine the contribution of myocardial triglycerides to overall ATP production in isolated working rat hearts. Endogenous lipid pools were initially prelabeled (pulsed) by perfusing hearts for 60 min with Krebs-Henseleit buffer containing 1.2 mM [1-14C]palmitate. During a subsequent 60-min period (chase), hearts were perfused with either no fat, low fat (0.4 mM [9,10-3H] palmitate), or high fat (1.2 mM [9,10-3H]palmitate). All buffers contained 11 mM glucose. During the "chase," 14CO2 production (a measure of endogenous fatty acid oxidation) and 3H2O production (a measure of exogenous fatty acid oxidation) were determined. Oxidative rates of endogenous fatty acids during the chase were 279 +/- 50, 88 +/- 14, and 88 +/- 8 nmol of [14C]palmitate oxidized per g dry weight.min in the no fat, low fat, and high fat groups, respectively, compared to exogenous palmitate oxidation rates of 0, 361 +/- 68, and 633 +/- 60 nmol of [3H]palmitate/g dry weight.min, in the no fat, low fat, and high fat groups, respectively. Endogenous [14C]palmitate oxidation rates were matched by loss of [14C]palmitate from endogenous myocardial triglycerides. Overall triglyceride content decreased during the no fat and low fat chase perfusion but did not change during the high fat chase. Loss of triglyceride [14C]palmitate during the high fat chase was matched by incorporation of exogenous [3H]palmitate in triglycerides. In a second series of perfusions, three groups of hearts were perfused under similar conditions, except that unlabeled palmitate was used during the "pulse" and that 11 mM [2-3H/U-14C]glucose and unlabeled palmitate was present during the chase. During the chase, both glycolysis (3H2O production) and glucose oxidation (14CO2 production) rates were measured. Rates of glucose oxidation were inversely related to the fatty acid concentration in the perfusate (1257 +/- 158, 366 +/- 40, and 124 +/- 26 nmol of glucose oxidized per min.g dry weight in the no fat, low fat, and high fat groups, respectively), while rates of glycolysis were not significantly different between these groups. Calculation of overall ATP production from both oxidative and glycolytic sources determined that even in the presence of high concentrations of fatty acids, myocardial triglyceride turnover can provide over 11% of steady state ATP production in the aerobically perfused heart. In the absence of fatty acids, myocardial triglyceride fatty acids can become the major energy substrate of the heart.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of muscle glycogen autoregulation in humans

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.     

Isotopic discrimination between D-[1-(13)C]fructose and D-[2-(13)C]fructose in rat liver cells

When liver cells from either normal or hereditarily diabetic rats are exposed to (13)C-enriched D-fructose (10 mM) and unlabelled D-glucose (also 10 mM) in the presence of D(2)O, the output of (13)C-enriched D-glucose generated from D-[1-(13)C]fructose is significantly lower than that from D-[2-(13)C]fructose. This coincides with a higher generation of (13)C-enriched L-lactate and L-alanine from D-[1-(13)C]fructose, as compared to D-[2-(13)C]fructose. In absolute terms, the mean paired difference in the output of (13)C-enriched D-glucose generated from D-[1-(13)C]fructose versus D-[2-(13)C]fructose is not significantly different from the mean paired difference in the production of (13)C-enriched L-lactate and L-alanine from the same precursors, with an overall mean value of 7.01 +/- 1.59 micromol (n = 8; P < 0.005). It is proposed that these findings indicate isotopic discrimination at the phosphoglucoisomerase level between (12)C and (13)C for the carbon atom in position 1 (as compared to that in position 2) of D-fructose 6-phosphate.     

Direct observation of glycogenesis and glucagon-stimulated glycogenolysis in the rat liver in vivo by high-field carbon-13 surface coil NMR

High-field 13C surface coil nuclear magnetic resonance has been employed to investigate glucose and glycogen metabolism in rat liver in vivo. Natural abundance and isotopically enriched proton-decoupled 13C NMR experiments were conducted at 90.56 MHz on a standard commercial spectrometer utilizing a laboratory-built high-sensitivity double-resonance coaxial coil probe. At variance with a previous preliminary report, natural abundance spectra of the liver in vivo from a rat fed ad libitum reveal resonances of substantial intensity from hepatic glycogen with approximately 10 min of signal averaging. The response of hepatic glycogen levels to an intravenous injection of the hormone glucagon was continuously monitored through the glycogen C-1 carbon resonance intensity; this revealed an average 60% depletion of hepatic glycogen stores in vivo within approximately 1 h. In a complementary study utilizing fasted rats, 100 mg of D-[1-13C]glucose (90% enriched) was administered via a peripheral vein injection and continuously monitored by 13C NMR with 3-min time resolution as it was incorporated into hepatic glycogen. The C-1 carbon resonances of hepatic glucose and glycogen are well-resolved in vivo enabling the time course for the relative change in concentration for both metabolites to be established simultaneously. The 13C label incorporated into the glycogen pool reaches a steady-state level in approximately 40 min.     

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.     

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.     

Alterations in substrate utilization in the reperfused myocardium: a direct analysis by 13C NMR.

An alternative 13C NMR method which allows direct determination of substrate oxidation in tissue for up to three competing 13C-enriched substrates is presented. Oxidation of competing substrates can be measured by 13C NMR spectroscopy under non-steady-state conditions if the relative areas of the glutamate C3 and C4 resonances can be determined. The accuracy of this measurement is limited during brief exposure to 13C-enriched substrates because of the low enrichment in the C3 carbon. The glutamate C4 resonance from a tissue sample which has oxidized a combination of [1,2-13C]acetate (or a uniformly enriched fatty acid mixture) and [3-13C]lactate appears as a nine-line resonance consisting of four multiplet components: a singlet (C4S), two doublets with differing one-bond coupling constants (C4D34 and C4D45), and a quartet (C4Q). It is shown that the sum of the C4S + C4D34 resonance areas versus the C4D45 + C4Q resonance areas directly reports the relative utilization of [3-13C]lactate versus [1,2-13C]acetate, respectively, regardless of citric acid cycle intermediate pool sizes or carbon flux through anaplerotic reactions. We also show that homonuclear 13C decoupling of the glutamate C2 resonance collapses the C3 resonance multiplet into an apparent triplet (actually, a singlet plus a doublet); the relative area of the singlet component reflects the amount of unlabeled acetyl-CoA entering the cycle. The method has been used to determine the contribution of lactate/acetate/glucose to acetyl-CoA in normoxic and reperfused rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between carbon and nitrogen metabolism in Fibrobacter succinogenes S85: a 1H and 13C nuclear magnetic resonance and enzymatic study

The effect of the presence of ammonia on [1-13C]glucose metabolism in the rumen fibrolytic bacterium Fibrobacter succinogenes S85 was studied by 13C and 1H nuclear magnetic resonance (NMR). Ammonia halved the level of glycogen storage and increased the rate of glucose conversion into acetate and succinate 2.2-fold and 1.4-fold, respectively, reducing the succinate-to-acetate ratio. The 13C enrichment of succinate and acetate was precisely quantified by 13C-filtered spin-echo difference 1H-NMR spectroscopy. The presence of ammonia did not modify the 13C enrichment of succinate C-2 (without ammonia, 20.8%, and with ammonia, 21.6%), indicating that the isotopic dilution of metabolites due to utilization of endogenous glycogen was not affected. In contrast, the presence of ammonia markedly decreased the 13C enrichment of acetate C-2 (from 40 to 31%), reflecting enhanced reversal of the succinate synthesis pathway. The reversal of glycolysis was unaffected by the presence of ammonia as shown by 13C-NMR analysis. Study of cell extracts showed that the main pathways of ammonia assimilation in F. succinogenes were glutamate dehydrogenase and alanine dehydrogenase. Glutamine synthetase activity was not detected. Glutamate dehydrogenase was active with both NAD and NADP as cofactors and was not repressed under ammonia limitation in the culture. Glutamate-pyruvate and glutamate-oxaloacetate transaminase activities were evidenced by spectrophotometry and 1H NMR. When cells were incubated in vivo with [1-13C]glucose, only 13C-labeled aspartate, glutamate, alanine, and valine were detected. Their labelings were consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.     

Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy

[99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically and used to determine the anomeric specificity of the CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition of CMP-KDO synthetase to reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid CMP-KDO formation which was accompanied by a substantial decrease in the 13C-enriched resonances of the beta-pyranose form of KDO relative to the resonances of other KDO species in solution, demonstrating that the beta-pyranose is the preferred substrate. Concomitant with the production of CMP-KDO was the appearance of peaks at 174.3 and 101.4 ppm when [1-13C]- and [2-13C]KDO, respectively, were used as substrates. The correspondence of these resonances to the enriched carbons in CMP-KDO was confirmed by the expected 3-bond (3JP,C-1 = 6.9 Hz) and 2-bond coupling (2JP,C-2 = 8.3 Hz) between the labeled carbons and the ketosidically linked phosphoryl group. A large coupling (3J = 5.7 Hz) was observed in proton-coupled spectra of CMP-[1-13C]KDO between carbon 1 and the axial proton at carbon 3 of KDO. The magnitude of this coupling constant supports a diaxial relationship between these two groups and, along with chemical shift data, indicates that KDO retains the beta-configuration when linked in CMP-KDO.     

13C enrichment of extracellular neurotransmitter glutamate in rat brain--combined mass spectrometry and NMR studies of neurotransmitter turnover and uptake into glia in vivo.

13C-enrichment analysis of glutamate in the extracellular fluid (GLU(ECF): 2-3 microM) by gas-chromatography/mass-spectrometry (GCMS) was combined with in vivo NMR observation of whole-brain GLU (approximately10 mM) to study neurotransmitter uptake. Brain GLU C5 was 13C-enriched by intravenous [2,5-13C]glucose infusion. GLU(ECF) was collected by microdialysis from the cortico-striatal region of awake rats. The 13C-enrichment of basal dialysate GLU C5 during 0.75-1.25 hr of infusion was 0.263 +/- 0.01, very close to the enrichment of whole-brain GLU C5. The result strongly suggests that dialysate GLU consists predominantly of neurotransmitter GLU. For selective 13C-enrichment of neurotransmitter GLU, the whole-brain 13C-enrichment was followed by [12C]glucose infusion to chase 13C from the small glial GLU pool. This leaves [5-13C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. The uptake of synaptic [5-13C]GLU(ECF) into glia and metabolism to glutamine (GLN) were monitored in vivo by NMR observation of [5-13C,15N]GLN formed during 15NH4Ac infusion. The rate of GLN synthesis, derived from neurotransmitter GLU(ECF) (which provided 80-90% of the substrate) was 6.4 +/- 0.44 micromol/g/hr. Hence, the observed rate represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Production of specific site probes of tRNA structure by enrichment with carbon 13 at particular locations.

Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-(13)C] adenine, and [2-(13)C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.     

Metabolic interactions between glucose, glycerol, alanine and acetate in Leishmania braziliensis panamensis promastigotes

13C-nuclear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeled substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-1/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [1,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose phosphorylation is not rate limiting for accumulation of glycogen from glucose in perfused livers from fasted rats

Incorporation of Glc and Fru into glycogen was measured in perfused livers from 24-h fasted rats using [6-3H]Glc and [U-14C]Fru. For the initial 20 min, livers were perfused with low Glc (2 mM) to deplete hepatic glycogen and were perfused for the following 30 min with various combinations of Glc and Fru. With constant Fru (2 mM), increasing perfusate Glc increased the relative contribution of Glc carbons to glycogen (7.2 +/- 0.4, 34.9 +/- 2.8, and 59.1 +/- 2.7% at 2, 10, and 20 mM Glc, respectively; n = 5 for each). During perfusion with substrate levels seen during refeeding (10 mM Glc, 1.8 mumol/g/min gluconeogenic flux from 2 mM Fru), Fru provided 54.7 +/- 2.7% of the carbons for glycogen, while Glc provided only 34.9 +/- 2.8%, consistent with in vivo estimations. However, the estimated rate of Glc phosphorylation was at least 1.10 +/- 0.11 mumol/g/min, which exceeded by at least 4-fold the glycogen accumulation rate (0.28 +/- 0.04 mumol of glucose/g/min). The total rate of glucose 6-phosphate supply via Glc phosphorylation and gluconeogenesis (2.9 mumol/g/min) exceeded reported in vivo rates of glycogen accumulation during refeeding. Thus, in perfused livers of 24-h fasted rats there is an apparent redundancy in glucose 6-phosphate supply. These results suggest that the rate-limiting step for hepatic glycogen accumulation during refeeding is located between glucose 6-phosphate and glycogen, rather than at the step of Glc phosphorylation or in the gluconeogenic pathway.     

Influence of glycogen storage on vascular smooth muscle metabolism

The role of glycogen as an oxidative substrate for vascular smooth muscle (VSM) remains controversial. To elucidate the importance of glycogen as an oxidative substrate and the influence of glycogen flux on VSM substrate selection, we systematically altered glycogen levels and measured metabolism of glucose, acetate, and glycogen. Hog carotid arteries with glycogen contents ranging from 1 to 11 micromol/g were isometrically contracted in physiological salt solution containing 5 mM [1-(13)C]glucose and 1 mM [1, 2-(13)C]acetate at 37 degrees C for 6 h. [1-(13)C]glucose, [1, 2-(13)C]acetate, and glycogen oxidation were simultaneously measured with the use of a (13)C-labeled isotopomer analysis of glutamate. Although oxidation of glycogen increased with the glycogen content of the tissue, glycogen oxidation contributed only approximately 10% of the substrate oxidized by VSM. Whereas [1-(13)C]glucose flux, [3-(13)C]lactate production from [1-(13)C]glucose, and [1, 2-(13)C]acetate oxidation were not regulated by glycogen content, [1-(13)C]glucose oxidation was significantly affected by the glycogen content of VSM. However, [1-(13)C]glucose remained the primary ( approximately 40-50%) contributor to substrate oxidation. Therefore, we conclude that glucose is the predominate substrate oxidized by VSM, and glycogen oxidation contributes minimally to substrate oxidation.     

Effects of D-glucose upon D-fuctose metabolism in rat hepatocytes: A 13C NMR study

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-¹³C]fructose, D-[2-¹³C]fructose or D-[6-¹³C]fructose (also 10 mM) in the presence of D₂O. The identification and quantification of ¹³C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of ¹³C-enriched D-fructose, decrease the production of ¹³C-enriched D-glucose, restrict the deuteration of the ¹³C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-¹³C]fructose, and to accentuate the lesser deuteration of the C₂ (as compared to C₅) of ¹³C-enriched D-glucose derived from D-[2-¹³C]fructose. The ratio between C2-deuterated and C₂-hydrogenated L-lactate, as well as the relative amounts of the CH₃-, CH₂D-, CHD₂ and CD₃- isotopomers of ¹³C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C₁ of D-[1-¹³C]glucose generated by hepatocytes exposed to D-[1-¹³C]fructose or D-[6-¹³C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-¹³C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.     

C Isotopomer Analysis of Glutamate by Tandem Mass Spectrometry

Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with (13)C NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-(13)C]acetate, 5 mM [1-(13)C]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-(13)C]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 +/- 2, 53 +/- 2, and 84 +/- 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 +/- 3, 25 +/- 8, and 16 +/- 8%. Similar results were obtained with (13)C NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using (13)C-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR.     

13C nuclear magnetic resonance study of trehalose mobilization in yeast spores.

Using high-resolution 13C nuclear magnetic resonance, we examined the mobilization of endogenous trehalose in suspensions of yeast asci. Sporulation of yeast cells in [1-13C]acetate resulted in incorporation of label into the C-3 and C-4 positions of trehalose within the asci. During germination of these asci with [1-13C]glucose, the consumption of both endogenous trehalose and exogenous glucose were followed simultaneously by 13C nuclear magnetic resonance, as was the formation of glycerol and ethanol, their glycolytic and products. Time courses for carbohydrate consumption indicated that trehalose, although it decreased to 25% of its initial value upon germination, was not preferentially catabolized and did not provide the primary energy supply for germination with glucose. The ratio of trehalose to glucose catabolized was 0.09. Exogenous glucose levels appeared to regulate trehalose mobilization since trehalose was only consumed when sufficiently high levels (more than 2 mM) of glucose were present. Upon glucose depletion newly synthesized [1-13C]trehalose was observed. Nuclear magnetic resonance spectra of extracts confirmed the trehalose peak assignments and showed products of [1-13C]glucose catabolism. In addition by quantitating trehalose consumption and 2-deoxyglucose incorporation in dormant yeast asci, we found that 3.8 +/- 0.l4 molecules of 2-deoxyglucose were incorporated for each trehalose molecule consumed. Trehalose can therefore function as a carbohydrate source for ATP formation during dormancy.