31P nuclear magnetic resonance study of a human colon adenocarcinoma cultured cell line.

31P nuclear magnetic resonance (NMR) spectroscopy has been used to monitor the energy metabolism in a human colon adenocarcinoma cell line (HT 29). NMR spectra were recorded at 80.9 MHz on approximately 2.5 X 10(8) cells continuously perfused with culture medium within a 20-mm NMR sample tube. Typical NMR spectra display a series of well-resolved resonances assigned to nucleoside triphosphates (mainly adenosine 5'-triphosphate), uridine diphosphohexose derivatives (uridine 5'-diphosphate-N-acetylglucosamine, uridine 5'-diphosphate-N-acetylgalactosamine, uridine 5'-diphosphate-glucose), intra- and extracellular inorganic phosphate, and phosphomonoesters (mainly phosphorylcholine and glucose 6-phosphate). Measurement of phosphorylated metabolite concentrations from the intensity of NMR signals is in good agreement with the results provided by conventional biochemical assays. 31P NMR allows to follow noninvasively the effect of anoxia on HT 29 cells. The results indicate that the cells are able to maintain about 60% of their initial nucleoside triphosphate level after 2 h of anaerobic perfusion. Cells accumulate inorganic phosphate during anoxia and the intracellular-extracellular pH gradient increases from 0.5 in well-oxygenated cells to more than 1 pH unit under anoxic conditions. The value of intracellular pH of well-oxygenated HT 29 cells is 7.1. The effect of glucose starvation upon energy metabolism has also been examined in real time by NMR: a rapid decline of adenosine 5'-triphosphate down to 10% of the initial value is observed over a period of 2 h. In contrast, the level in uridine diphosphohexoses reaches a new steady state value representing 60% of the initial one. Refeeding the cells with 25 mM glucose leads to a dramatic drop of internal pH reflecting the activation of the glycolytic pathway.

Conformation of colipase. Prediction of the secondary structure, circular dichroism and 360 MHz proton NMR studies of porcine colipase A.

The secondary structure of porcine colipase (93 residues) was established according to the predictive method of Chou and Fasman (Chou, P.Y. and Fasman, G.D. (1974) Biochemistry 13, 211--222 and 222--245). The relative composition of the conformational regions was as follows: 5% alpha-helix (region 39--44), 25% beta-sheet (three regions, 7--11, 49--57 and 77--85) and eight beta-turns corresponding to 32% of the polypeptide. Colipase contains a large proportion (about 35%) of unordered structure. Estimated values for the alpha-helix and beta-sheet contents from the circular dichroism spectrum were in good accordance with the predicted model. A less satisfactory value was found for the beta-turns. A characteristic feature of the far ultraviolet dichroic spectrum is the presence of an unusual positive band at 225 nm that might be indicative of a particular spatial arrangement of the chromophores in the molecule. Two tyrosines (Tyr56 and Tyr57) and one histidine (His86) are at close vicinity in the three dimensional structure of the protein as shown by proton NMR studies. These residues are located at the end of two beta-sheet hydrophobic regions(49--57 and 77--85) which might play a role in the association of colipase with the lipid-water interface as indicated by results of the NMR studies of the taurodeoxycholate-colipase complex.

Cytosolic pH regulation in perfused rat liver: role of intracellular bicarbonate production.

The contribution of metabolic bicarbonate to cytosolic pH (pHcyto) regulation was studied on isolated perfused rat liver using phosphorus-31 NMR spectroscopy. Removal of external HCO3- decreased proton efflux from 18.6+/-5.0 to 1.64+/-0.29 micromol/min per g liver wet weight (w.w.) and pHcyto from 7.17+/-0.06 to 6.87+/-0.06. In the nominal absence of bicarbonate, inhibition of carbonic anhydrase by acetazolamide induced a further decrease of proton efflux of 0.69+/-0.26 micromol/min per g liver w.w. reflecting a reduction in metabolic CO2 hydration, and hence a decrease of H+ and HCO3- supplies. Even though 27% of the proton efflux was amiloride-sensitive under bicarbonate-free conditions, amiloride did not change pHcyto, revealing the contribution of additional regulatory processes. Indeed, pH regulation was affected by the combined use of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) and amiloride since pHcyto decreased by 0.16+/-0.05 and proton efflux by 0.60+/-0.14 micromol/min per g liver w.w. The data suggest that amiloride-sensitive or SITS-sensitive transport activities could achieve, by themselves, pHcyto regulation. The involvement of two mechanisms, most likely Na+/H+ antiport and Na+:HCO3 symport, was confirmed in the whole organ under intracellular and extracellular acidosis. The evidence of Na-dependent transport of HCO3- in the absence of exogenous bicarbonate implies that the amount of metabolic bicarbonate is sufficient to effectively participate to pHcyto regulation.

Further characterization of bovine pancreatic lipase.

1. The amino acid composition of bovine pancreatic lipase is very similar to that of porcine lipase. 2. Bovine lipase possesses a residue of lysine at the N-terminal position and a half cystine or a cysteine at the C-terminal position. 3. Bovine lipase contains two free sulfhydryl groups of different reactivities to 5, 5'-dithiobis-(2-nitrobenzoic) acid. One of these groups is buried in the native conformation of the enzyme and is fully titrated in 1.5 M urea when reaction is performed in the presense of 1 mM EDTA.

Limited trypsinolysis of porcine and equine colipases. Spectroscopic and kinetic studies.

Porcine and equine colipases have been submitted to mild tryptic digestion. Proteolysis occurs at the Arg5-Gly6 bond with the loss of the N-terminal pentapeptide. Studies of native and trypsin-treated colipases by circular dichroism and laser chemically induced dynamic nuclear polarization indicate that proteolysis induces conformational changes in the region of the tyrosine cluster. Experiments in the presence of phospholipid provide further evidence showing that these residues are in or close to the region of the protein interacting with aggregated lipids. Kinetic studies of the reaction of bile salt-inhibited lipase with emulsified triolein in the absence and in the presence of lecithin show that tryptic hydrolysis of the protein cofactor increases its affinity for the enzyme in the presence of lipid substrate. In both cases, it was found that the apparent dissociation constant of the lipase-colipase complex is decreased by one order of magnitude. Our results confirm that the biological activity of the lipase cofactor is enhanced by specific tryptic cleavage in the amino terminal region of the polypeptide and support the suggestion by Borgström et al. (Borgström, B., Wieloch, T., Erlanson-Albertsson (1981) FEBS. Lett. 108, 407-410) that the secreted form of colipase is a precursor.

Isolation and partial structural characterization of chicken pancreatic colipase.

Colipase has been isolated from acidic extracts of chicken pancreatic tissue homogenized with Triton X-100. The cofactor fully activates bile salt inhibited mammalian lipases. The amino terminal sequence of the avian protein has been determined up to position 39 and compared to the homologous region of the mammalian colipases (pig, horse, man) previously studied. From this comparison, it appears that a high degree of homology exists between the proteins.

13C nuclear magnetic resonance study of glycogen resynthesis in muscle after glycogen-depleting exercise in healthy men receiving an infusion of lipid emulsion.

In healthy individuals, glycogen recovery after a strong depletion is known to be rapid and insulin independent during the initial phase, and subsequently, slow and insulin dependent. Free fatty acids (FFAs) as a putative source of insulin resistance (IR) could thus impair glycogen recovery during the second period. Using in vivo 13C nuclear magnetic resonance (NMR), we studied the effect of long-chain triglyceride emulsion on gastrocnemius glycogen resynthesis during a 3-h recovery period after 90 min of moderate exercise consisting of plantar flexion on overnight-fasted healthy men (n = 8). In separate experiments, each subject was infused with 10% Ivelip (0.015 ml x kg(-1) x min(-1)) or 10% glycerol (0.13 mg x kg(-1) x min(-1)). NMR spectra were acquired before and at the end of the exercise and during the recovery period. Whole-body glucose and lipid oxidation rates (indirect calorimetry), plasma insulin, C-peptide, glucose, lactate, beta-hydroxybutyrate, triglycerides, and FFAs were determined. Glycogen consumption was 47.6 +/- 4.5% (glycerol) and 49.7 +/- 4.8% (Ivelip) of the initial glycogen. An acquired IR in the Ivelip group was significant at the onset of the recovery period by homeostasis model assessment (P = 0.002). Glycogen resynthesis in the glycerol group appeared faster during the 1st h than during the subsequent 2nd h of the postexercise period. The glycogen resynthesis level was significantly lower in the Ivelip group than in the glycerol group during the recovery period (P = 0.04 during the 1st h and P = 0.001 during the next 2 h). During the recovery, plasma lactate and whole-body oxidation rates were similar in the two groups, whereas glycemia was significantly higher in the Ivelip group. A decreased cellular uptake of glucose as a substrate for glycogenosynthesis, rather than a competition between oxidation of carbohydrate and FFA, is discussed.

Sugar-Starvation-Induced Changes of Carbon Metabolism in Excised Maize Root Tips.

Excised maize (Zea mays L.) root tips were used to study the early metabolic effects of glucose (Glc) starvation. Root tips were prelabeled with [1-13C]Glc so that carbohydrates and metabolic intermediates were close to steady-state labeling, but lipids and proteins were scarcely labeled. They were then incubated in a sugar-deprived medium for carbon starvation. Changes in the level of soluble sugars, the respiratory quotient, and the 13C enrichment of intermediates, as measured by 13C and 1H nuclear magnetic resonance, were studied to detect changes in carbon fluxes through glycolysis and the tricarboxylic acid cycle. Labeling of glutamate carbons revealed two major changes in carbon input into the tricarboxylic acid cycle: (a) the phosphoenolpyruvate carboxylase flux stopped early after the start of Glc starvation, and (b) the contribution of glycolysis as the source of acetyl-coenzyme A for respiration decreased progressively, indicating an increasing contribution of the catabolism of protein amino acids, fatty acids, or both. The enrichment of glutamate carbons gave no evidence for proteolysis in the early steps of starvation, indicating that the catabolism of proteins was delayed compared with that of fatty acids. Labeling of carbohydrates showed that sucrose turnover continues during sugar starvation, but gave no indication for any significant flux through gluconeogenesis.

Glutamate-glutamine metabolism in the perfused rat liver. 13C-NMR study using (2-13C)-enriched acetate.

13C-NMR has been used to follow the metabolism of 13C-enriched substrates in isolated perfused rat liver. The fate of 90% enriched [2-13C]acetate has been studied in the perfused liver in order to investigate 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 13C into glutamine can be linked to the operation of the glutamate-glutamine antiport and to the high activity of cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is evidence of the existence of an active glutamine-glutamate futile cycle.

Glutathione, but not glutamine, is detected in 13C-NMR spectra of perchloric acid extracts from C6 glioma cells.

Glutamine, which is expected to be produced by C6 glioma cells, is not detected in both amino-acid analyses and 13C-NMR spectra of perchloric acid extracts of cells incubated for 4 h with [1-13C]glucose in the absence of extracellular glutamine. However, the resonances of a glutamate-linked product are observed in these spectra. The analysis of the pH dependence of chemical shifts from various glutamate-derived compounds shows that the observed resonances came from glutathione. Glutamine and glutathione signals are in close proximity on the frequency scale, leading to possible misinterpretation of the spectra.

Compartmentation of inorganic phosphate in perfused rat liver. Can cytosol be distinguished from mitochondria by 31P NMR?

Compartmentation of inorganic phosphate was studied in intact perfused rat liver at 4 degrees C by 31P NMR. It was shown that decreases in cytosolic pH or cytosolic Pi concentration induced the appearance of an additional Pi resonance at low field which was assigned to Pi from an alkaline compartment. Valinomycin (K+ ionophore) induced a further splitting of the lines whereas nigericin (K+/H+ antiport) or potassium cyanide (inhibitor of cytochrome oxidase) had opposite effects. As valinomycin acts mainly on the cytosolic/mitochondrial K+ gradient and KCN on the mitochondrial respiratory chain, it was deduced that the alkaline compartment as revealed by the second Pi resonance was probably mitochondria. Single Pi lines observed on perchloric extracts of livers exhibiting two resonances during cold perfusion confirmed that the split peaks in the intact liver indeed arose from the same molecular species.

Functional and metabolic early changes in calf muscle occurring during nutritional repletion in malnourished elderly patients.

BACKGROUND: Metabolic alterations in skeletal muscle associated with malnutrition and the potential reversibility of such alterations during refeeding are not fully understood. OBJECTIVE: We characterized early changes in muscle during refeeding in malnourished, hospitalized elderly subjects. DESIGN: Muscle function, metabolism, and mass were evaluated in 24 clinically stable patients (11 were malnourished) by using isokinetic plantar flexor torque measurements and nuclear magnetic resonance (NMR) imaging for medial gastrocnemius mass assessment and 31P and 13C NMR spectroscopy for inorganic phosphate (Pi), phosphocreatine, and glycogen quantitation. RESULTS: Malnourished subjects had lower muscle mass (P < 0.02) and tended to have lower strength than did control subjects. In malnourished subjects, muscle strength increased after refeeding (P < 0.01) whereas muscle mass was unchanged. The ratio of Pi to ATP was lower in malnourished than in control subjects (P < 0.001) and increased during refeeding (P < 0.01). The mean ratio of phosphocreatine to ATP was lower in malnourished than in control subjects (P < 0.01) and increased to control values after refeeding. Muscle glycogen showed a scattered distribution for malnourished subjects; the mean value did not differ significantly from that of control subjects, either at baseline or after refeeding. CONCLUSIONS: The lower ratio of phosphocreatine to ATP in malnourished subjects could have resulted from either lower total muscle creatine or reduced oxidative capacities. High or normal glycogen associated with a low Pi-to-ATP ratio in malnourished subjects suggested preferential use of lipid over carbohydrate for energy supply, which is known to reduce muscle performance. The data suggest that normalization of muscle metabolite content after refeeding improves muscle strength in malnourished subjects.

Proton N.M.R. study of the conformational dynamics of porcine pancreatic colipase. Titration of aromatic residues.

The low-field portion of the 360 MHz proton N.M.R. spectrum of native porcine pancreatic colipase has been studied as a function of pH over the pH range 2-12. Resonances associated with the 26 protons of the aromatic rings of the two histidines, two phenylalanines and three tyrosines have been identified and tentatively assigned to specific residues. Titrations of pH yielded apparent pKa's of 7.9, 6.9, 10.4, 10.3 and 11.3 for His I (His 30), His II (His 86), Tyr I (Tyr 56 or 57), Tyr II (Tyr 56 or 57) and Tyr III (Tyr 53) respectively (tentative assignments). The high pKa value of His 30 is attributed to the vicinity of Asp 31. The mobility of the aromatic ring of Tyr 53 is hindered and an upper bound of 500 s-1 on the rate of rotation can be estimated. The aromatic rings of the 2 other tyrosine residues and of the 2 phenylalanine residues can rotate freely on the N.M.R. time scale. The study of perturbations in titration profiles and chemical shift values reveals a specific interaction of His 86 with Tyr I and, to a lesser extent, Tyr II. The existence of this interaction indicates that the protein folding brings in close spatial vicinity two distant regions of the covalent structure to form a "hydrophobic-aromatic" site which might be involved in the binding of bile salt micelles to pancreatic colipase.

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture.

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

[Study of in vivo cellular metabolism by phosphorus 31 nuclear magnetic resonance]. / Etude du métabolisme cellulaire in vivo par résonance magnétique nucléaire du phosphore-31.

Phosphorus-31 nuclear magnetic resonance spectroscopy has been recently increasingly used to study cellular metabolism in a manner respecting the cell integrity. Intrinsic advantages of the phosphorus nucleus for in vivo NMR studies are discussed in this review together with some selected applications. A particular emphasis is layed on metabolite identification and quantitation (relative and absolute concentrations), the measurement of intracellular pH and the problem of cellular compartmentation. The determination of metabolite fluxes under normal and abnormal biological and physiological conditions, and the in vivo direct measurement by saturation transfer techniques of kinetic parameters for enzymatic reactions at equilibrium, are illustrated by several examples taken from the available literature and work carried out in this laboratory. Whenever possible, and appropriate, the NMR approach has been compared with other more classical techniques of investigation. The future and the potentialities of phosphorus-31 NMR study of intact biological systems, the clinical applications and the foreseeable interfacing with imaging techniques are evaluated. The concept of "functional imaging" versus "anatomic imaging" is proposed to illustrate the impact of this new technology in the understanding of cellular mechanisms, not only in the intact cell but also in whole tissues or organs after excision or in living animals and human.

Isolation and partial characterization of bovine pancreatic colipase.

Three molecular forms of colipase (colipases A, B and C) with the same specific activity have been isolated from an acid extract of bovine pancreas. Purification includes ammonium sulfate precipitation, ethanol treatment, chromatography on SP-Sephadex, chromatography on DEAE-cellulose and chromatography on QAE-Sephadex. The most basic form of bovine colipase (colipase A) has a molecular weight of 11,000-12,000 daltons and contains 104 residues. Its aminoacid composition is very similar to that of the intact form of porcine colipase isolated by Borgström et al. Colipases from both species have the same N-terminal residue (valine). It is likely that bovine colipases B and C represent partially degraded forms of colipase A. Their cofactor activity, however, is the same.

Glucose and glutamine metabolism in C6 glioma cells studied by carbon 13 NMR.

The question as to whether glutamine and glucose are both required for optimal growth of glioma cells is studied through the role of these substrates on the metabolism of the cells. C6 rat glioma cells grow only very slowly when glutamine is omitted from the culture medium. The rates of glucose consumption and lactate production on confluent cells in glutamine-free medium were 0.88 +/- 0.09 and 1.06 +/- 0.25 mumol/h/mg protein, respectively. In the presence of 4 mM glutamine, glucose utilization increase to 60% leading to a 45% increase of lactate production. We have studied the kinetics of enrichment of intracellular glutamate at C2, C3 and C4 positions on cells incubated with 5 mM 99% enriched [1-(13)C]glucose in the presence or the absence of glutamine in the incubation medium. The specific enrichments at metabolic steady state of all carbon positions were the same under both conditions, but we observed a significantly reduced rate of 13C incorporation in the presence of glutamine, showing an isotopic dilution of tricarboxylic acid cycle intermediates and indicating the use of this amino acid as an anaplerotic substrate. The fact that no dilution occurred at the level of pyruvate suggests strongly the lack of glutaminolysis in these cells. The main conclusion from this work is that glutamine metabolism in C6 cells appears complementary to that of glucose as far as energy production and carbon sources for the growing of the cells are concerned: glutamine is mainly utilized for anaplerosis as carbon donor to replenish the tricarboxylic acid cycle; it is not a substrate for energy metabolism. In contrast, glucose is poorly anaplerotic and is essentially used as energetic fuel by the C6 cells.

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase.

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.

Characterization of Triton X 100 extracted colipase from porcine pancreas.

Colipase was isolated from porcine pancreas homogenate prepared in the presence of detergent (Triton X 100). After precipitation by ammonium sulfate and ethanol, the cofactor was purified by chromatography on SP-Sephadex in the presence of Triton X 100 and on DEAE-cellulose in the absence of detergent. Two molecular forms of porcine colipase were obtained. They represent 80 per cent (colipase A) and 20 per cent (colipase B), respectively, of the total colipase. Valine is the N-terminal residue of both proteins. Their aminoacid composition is similar to that found by Borgstrom for the two forms of porcine colipase. Determination of the sequence of the first sixteen residues at the N-terminal end of colipase A indicates that the cofactor undergoes no proteolytic degradation in this region of the molecule when extraction is carried out in the presence of detergent. The recovery of colipase is about 30 per cent.

Lactate involvement in neuron-glia metabolic interaction: (13)C-NMR spectroscopy contribution.

Glucose is commonly admitted to be the main substrate for brain energy requirement. However, it has been recently proposed that lactate, generated from glucose via glycolysis, would be the oxidative substrate for neurons, particularly during neuronal activation, according to a mechanism called the astrocyte-neuron lactate shuttle hypothesis (ANLSH). In that mechanism, glutamate released in the synaptic cleft during brain activation is taken up by astrocytes. This uptake, via the glutamate/Na(+) transporter, induces the entry of sodium, which is then excluded from the astrocytes via the Na(+)/K(+) ATPase. This exclusion consumes ATP, which stimulates glycolysis and thus lactate formation in astrocytes. This lactate is then transferred to neurons where it is utilized as oxidative substrate. This review tries to gather the recent evidences that support this hypothesis and presents the contribution of NMR to this matter.