Effect of glucose on glutamine metabolism in rat brain slices: a cellular metabolomic study with Effect of glucose ¹³C NMR.

To examine the effect of glucose on the cerebral metabolism of glutamine, rat brain slices were incubated with 5mM [3-(13)C]glutamine without and with 5mM unlabeled glucose. Tissue plus medium extracts were analyzed by using enzymatic and (13)C NMR techniques and fluxes through the enzymatic steps involved were calculated with a mathematical model. We demonstrate that glucose increased alanine, pyruvate and glutamate accumulations and decreased ammonium ions accumulation, aspartate accumulation and labeling, and GABA labeling. In order to determine the participation of glutamine synthetase when glucose was added to the incubation medium, we incubated rat brain slices with 5mM [3-(13)C]glutamine plus 5mM unlabeled glucose without and with 2mM methionine sulfoximine (MSO). The results indicate that 77% of the newly appeared glutamine was formed via glutamine synthetase and 23% from endogenous sources; the stimulation of [3-(13)C]glutamine removal by MSO also strongly suggests the existence of a cycle between [3-(13)C]glutamine and [3-(13)C]glutamate. This work also demonstrates that glucose increased fluxes through hexokinase, pyruvate kinase, lactate dehydrogenase, alanine aminotransferase, pyruvate carboxylase, pyruvate dehydrogenase, citrate synthase, flux from α-ketoglutarate to glutamate and flux through glutamine synthetase whereas it inhibited fluxes through aspartate aminotransferase, glutamic acid decarboxylase and GABA aminotransferase.

High energy compound stability during experimental brain death.

The aim of this study was to examine the effect of sudden brain death (BD) on myocardial function and high energy phosphate (HEP) stores. BD was induced by cerebral vessel ligation in six swine (BD group) that were compared to six control swine. At the end of the BD period (3 hours), harvested hearts were stored at 4 degrees C. Myocardial tissue HEP were assessed by: (i) (31)P-NMR spectroscopy of left ventricle for phosphocreatine (PCr), adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH (pHi), and by (ii) HPLC for ATP, ADP, and AMP levels in left ventricle biopsies. Brain death resulted in a instantaneous major increase in catecholamines (>50-fold, P < .001) and paradoxically a significant progressive decrease in the regional contractility of the left ventricle. After cardioplegia, no significant differences on HEP compounds (ATP/Pi, PCr/Pi, ATP, energetic index) or in pHi were observed between BD and control groups. These data suggest that early heart injury occurring during BD does not seem to be an ischemic phenomenon.

A simple and reliable method to assess heart viability after hypothermic procurement.

Hearts from brain dead pigs (n = 18) were submitted to 0 (group I), 10 (group II), or 20 (group III) minutes of in situ warm ischemia (animal exsanguination). After harvesting, cold cardioplegia solution was perfused in retrograde fashion and initial coronary flow (ICF) measured. After left ventricular energetic indices were measured using NMR spectroscopy, the hearts were transplanted orthotopically. Follow-up was performed over 120 minutes after cardiopulmonary bypass. We observed a progressive decrease in ICF with increased warm ischemia times: 50 +/- 3.4 mL/min per 100 g of tissue in the group I, 36 +/- 7 and 30 +/- 3.5 in groups II and III, respectively (P < .05 and P < .01 versus group I). The ICF strongly correlated with the energetic index (r = 0.76, P < .001) and with posttransplant function of the transplanted heart. These data showed that measurement of initial coronary flow after cardioplegia was a reliable test to evaluate cardiac graft viability before transplantation.

Physiological differences of elite and professional road cyclists related to competition level and rider specialization.

AIM: The aim of this investigation was to study the physiological response to laboratory tests in elite and professional cyclists, and to relate it to the level at which riders compete and their specialization. METHODS: A total of 71 cyclists were divided into two groups, elite and professional, and were assessed for physical measurements, a maximal graded test and a 30 s all-out test, both performed on a cycle ergometer. The sample included 24 uphill riders (UR), 32 flat terrain riders (FTR), 11 all terrain riders (ATR) and 4 sprinters (SP). RESULTS: Professional riders showed significantly higher gross mechanical efficiency (GME) than their elite counterparts (25.6+/-2.6 vs 24.4+/-2%), but otherwise no other physiological differences emerged from the comparison between these two groups. However, many differences exist as a function of rider specialization, especially between UR and FTR. Compared with FTR, UR showed a higher VO2max (78.2+/-5.5 vs 72.6+/-6.5 mL x min(-1) x kg(-1)) and a lower maximal aerobic power (438.5+/-40.8 vs 465.3+/-36.2 W). From the 30 s all-out test, SP presents the highest maximal power (P < 0.05) and maximal velocity (P < 0.05) compared with all the other groups. CONCLUSIONS: The results for GME indicate a better efficiency for professional riders and suggest the importance of technical aspects related to movement pattern in cycling. The sensitivity of the maximal graded test and the 30 s all-out test did not allowed other differentiations between elite and professional cyclists.

Physiological differences in professional basketball players as a function of playing position and level of play.

AIM: The aim of this investigation is to evaluate the physical and physiological characteristics of different first (ProA) and second division (ProB) professional basketball players, and to relate them to playing position and level of play. METHODS: A total of 58 players were divided into ProA and ProB groups and were assessed for physical characteristics, maximal treadmill test and a 30 s all-out test. The sample included 22 centers, 22 forwards and 14 guards. RESULTS: Centers were significantly taller and heavier (203.9+/-5.3 cm and 103.9+/-12.4 kg) than forwards (195.8+/-4.8 cm and 89.4+/-7.1 kg) and guards (185.7+/-6.9 and 82+/-8.8 kg) and also had higher body fat percentages than the other groups. Forwards were also significantly taller than guards. Centers presented a lower maximal aerobic velocity (kmxh-1) than guards (15.5+/-1.2 vs 16.8+/-1.5, P<0.05) on the maximal treadmill test and a lower maximal velocity (rpm) than forwards (156.5+/-18.4 vs 170.3+/-18.3, P<0.05) on the 30 s all-out test. VO2max (mlxmin-1xkg-1) was significantly lower for ProA (53.7+/-6.7) compared to ProB (56.5+/-7.7) players and the fatigue index on the 30 s all-out test was higher for the ProA group (P<0.05). CONCLUSION: Many physical differences, most notably size, exist between players as a function of their playing position. But these differences have no relationship to the level of play of professional players. General aerobic capacity is fairly homogeneous between playing position and level of play, even if there are observable VO2max differences due to inter-individual profiles. On the other hand, anaerobic capacity seems to be a better predictor of playing level even though it is not clear whether such capacity comes from specific training in ProA, or from an initial selection criteria.

Acute renal failure in a patient with phosphofructokinase deficiency.

A 16-year-old Caucasian girl was admitted to hospital with acute renal failure and hemolytic anemia due to rhabdomyolysis following a 3-km walk. (31)P-magnetic resonance spectroscopy provided characteristic spectra of type VII glycogen storage disease (phosphofructokinase deficiency).

Carbon 13 NMR spectroscopy: a powerful tool for studying renal metabolism.

Using precise examples, this paper shows that carbon 13 NMR spectroscopy in conjunction with radioactive and enzymatic methods as well as with adequate mathematical modeling of metabolic pathways allows not only to identify but also to quantify fluxes through enzymes involved in substrate and drug metabolism. Carbon 13 NMR spectroscopy is a tool of unprecedented power to unravel the complexity of renal metabolism. Currently it plays a major role in what is nowadays called metabolomics.

Gluconeogenesis from glutamine and lactate in the isolated human renal proximal tubule: longitudinal heterogeneity and lack of response to adrenaline.

Recent studies in vivo have suggested that, in humans in the postabsorptive state, the kidneys contribute a significant fraction of systemic gluconeogenesis, and that the stimulation of renal gluconeogenesis may fully explain the increase in systemic gluconeogenesis during adrenaline infusion. Given the potential importance of human renal gluconeogenesis in various physiological and pathophysiological situations, we have conducted a study in vitro to further characterize this metabolic process and its regulation. For this, successive segments (S1, S2 and S3) of human proximal tubules were dissected and incubated with physiological concentrations of glutamine or lactate, two potential gluconeogenic substrates that are taken up by the human kidney in vivo, and glucose production was measured. The effects of adrenaline, noradrenaline and cAMP, a well established stimulator of gluconeogenesis in animal kidney tubules, were also studied in suspensions of human renal proximal tubules. The results indicate that the three successive segments have about the same capacity to synthesize glucose from glutamine; by contrast, the S2 and S3 segments synthesize more glucose from lactate than the S1 segment. In the S2 and S3 segments, lactate appears to be a better gluconeogenic precursor than glutamine. The addition of cAMP, but not of adrenaline or noradrenaline, led to the stimulation of gluconeogenesis from lactate and glutamine by human proximal tubules. These results indicate that, in the human kidney in vivo, lactate might be the main gluconeogenic precursor, and that the stimulation of renal gluconeogenesis observed in vivo upon adrenaline infusion may result from an indirect action on the renal proximal tubule.

Activation of the phosphatidylinositol 3-kinase/Akt pathway protects against interleukin-3 starvation but not DNA damage-induced apoptosis.

Baf-3 cells are dependent on interleukin-3 (IL-3) for their survival and proliferation in culture. To identify anti-apoptotic pathways, we performed a retroviral-insertion mutagenesis on Baf-3 cells and selected mutants that have acquired a long term survival capacity. The phenotype of one mutant, which does not overexpress bcl-x and proliferates in the absence of IL-3, is described. We show that, in this mutant, Akt is constitutively activated leading to FKHRL1 phosphorylation and constitutive glycolytic activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells from DNA damage-induced cell death. Indeed, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does not prevent the ability of IL-3 to protect cells against gamma-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor starvation-induced apoptosis but is not required for IL-3 inhibition of DNA damage-induced cell death.

Metabolism of rat skeletal muscle after spinal cord transection.

We investigated the energy metabolism of the gastrocnemius muscle of the rat after spinal cord transection, using in vivo (31)P magnetic resonance spectroscopy (MRS). Spectra were obtained at rest and during exercise and recovery before, and at different time-points after, spinal cord transection. At rest, the adenosine triphosphate (ATP) level was not altered and the intracellular pH became permanently more alkaline. In electrically stimulated muscle, cord transection caused a greater phosphocreatine depletion than in control animals, and the maximum rate of oxidative ATP synthesis was significantly diminished; at days 30 and 60 after transection, an intracellular acidification was observed at the end of exercise. These effects indicate that, as in humans, spinal cord transection in rats leads to a decrease in mitochondrial oxidative metabolism and probably to an increase in anaerobic metabolism. This experimental model may prove useful for evaluating various approaches to improve muscle function in paraplegia.

Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study.

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.

Glutamine synthesis is heterogeneous and differentially regulated along the rabbit renal proximal tubule.

Glutamine synthesis, a major process for ammonia detoxification and the control of acid-base balance, occurs from various precursors in suspensions of rabbit proximal tubules. However, no data are currently available on the distribution of glutamine synthesis along the rabbit proximal tubule, and its modulation by changes of substrate concentration. Therefore we have microdissected and incubated the three parts (S1, S2 and S3) of rabbit proximal tubules and measured glutamine synthesis from alanine and aspartate. With a physiological concentration of alanine (0.25 mM) or aspartate (0.05 mM), glutamine synthesis in the S1 segment was about half of that in the S2 and S3 segments, and was greater from alanine than from aspartate along the entire proximal tubule. Elevation of alanine and aspartate concentrations to 5 mM increased glutamine synthesis in both a substrate- and segment-dependent manner. It is concluded that glutamine synthesis occurs from alanine and aspartate along the entire rabbit proximal tubule; however, contrary to what might have been expected on the basis of measurement of glutamine synthetase activity, the basal rate of glutamine synthesis and its adaptation to increased substrate availability are heterogeneous along this nephron segment.

Increase in oxidative key enzymes in a case of muscle ubiquinol-cytochrome c reductase deficiency.

In a 29-year-old patient suffering from exertional muscle intolerance with a ubiquinol-cytochrome c reductase deficiency related to a cytochrome b gene point mutation of the mitochondrial DNA, we conducted a study of the aims of which were: (1) to test whether changes in the maximum activities of muscle key enzymes of the main energy-producing pathways occur, (2) to address the issue of whether fibers of different types are equally affected in their enzymatic machinery involved in energy production, and (3) to correlate the results obtained with histochemical and 31P NMR spectroscopy data. When compared to results obtained in six normal subjects, our study clearly shows that the type I fibers of the patient virtually all contained subsarcolemmal mitochondrial aggregates and increased activities of succinate dehydrogenase and cytochrome c oxidase; microdissected type I fibers also displayed a significant increase in both citrate synthase and beta-hydroxyacyl-CoA dehydrogenase, two key enzymes of mitochondrial oxidative metabolism. Despite these changes in the patient's muscle, its whole energy-producing machinery remained impaired as revealed by a slowed post-exercise recovery of phosphocreatine.

The rabbit kidney tubule simultaneously degrades and synthesizes glutamate. A 13C NMR study.

The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13C]- and [14C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO2, glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the "glutamate --> alpha-ketoglutarate --> glutamate" cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through alpha-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the "substrate cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.

Model applicable to NMR studies for calculating flux rates in five cycles involved in glutamate metabolism.

Based on the same principles as those utilized in a recent study for modeling glucose metabolism (Martin, G., Chauvin, M. F., Dugelay, S., and Baverel, G. (1994) J. Biol. Chem. 269, 26034-26039), a method is presented for determining metabolic fluxes involved in glutamate metabolism in mammalian cells. This model consists of five different cycles that operate simultaneously. It includes not only the tricarboxylic acid cycle, the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> oxaloacetate" cycle and the "oxaloacetate --> phosphoenolpyruvate --> pyruvate --> acetyl-CoA --> citrate --> oxaloacetate" cycle but also the "glutamate --> alpha-ketoglutarate --> glutamate" and the "glutamate --> glutamine --> glutamate" cycles. The fates of each carbon of glutamate, expressed as ratios of integrated transfer of this carbon to corresponding carbons in subsequent metabolites, are described by a set of equations. Since the data introduced in the model are micrograms of atom of traced carbon incorporated into each carbon of end products, the calculation strategy was determined on the basis of the most reliable parameters determined experimentally. This model, whose calculation routes offer a large degree of flexibility, is applicable to data obtained by 13C NMR spectroscopy, gas chromatography - mass spectrometry, or 14C counting in a great variety of mammalian cells.

Advantages of a two-chamber culture system to test drug nephrotoxicity: the example of cephaloridine.

Rabbit renal proximal tubular cells, cultured to confluency on a permeable collagen film in a two-chamber system, were exposed for 72 h to various concentrations of the nephrotoxic antibiotic, cephaloridine (CLD). A decrease in cellular proteins, leakage of lactate dehydrogenase and morphological changes appeared at CLD concentrations of 0.1, 1.0, and 0.5 mg/ml, respectively. The permeability of the monolayer to Lucifer yellow (LY), a dye that does not cross cell membranes, was increased by 1 or 2 mg/ml but not by lower concentrations of CLD. The large basolateral/apical glucose concentration gradient established by the cells was decreased by CLD. However, the fact that, at the CLD concentration of 1 mg/ml, LY totally equilibrated by diffusion across the monolayer, whereas the injured monolayer was still able to maintain a detectable glucose gradient, shows that damage caused by CLD to the diffusion barrier prevails over that affecting glucose uptake. Consistent with the data in the literature concerning the mechanism of CLD accumulation in renal cells, our results show that CLD was more toxic when it was added at the basolateral than at the apical side of the cultured cells. These results illustrate the advantages of using a two-chamber system of cell culture in nephrotoxicity studies.

[MELAS syndrome with pure vascular manifestation?]. / Syndrome "MELAS" à expression vasculaire pure?

Two stroke-like episodes then a grand mal seizure occurred within nine years in a 42-year-old patient. Neuroradiological findings (CT-scan and MRI) led to the diagnosis of incomplete MELAS syndrome. MRI with T2-weighted images (TR: 1000ms; TE: 35ms) showed two small asignal lesions possibly resulting from hemosiderine. Metabolic studies are required to help decide on muscular biopsies. Histological findings are needed for diagnosis of this form of mitochondrial cytopathy with only stroke-like manifestations.

Advantages and limitations of the use of isolated kidney tubules in pharmacotoxicology.

Among the cellular models used in in vitro renal pharmacotoxicology, isolated kidney tubules, used as suspensions mainly of proximal tubules, offer important advantages. They can be prepared in large amounts under nonsterile conditions within 1-2 h; thus, it is possible to employ a great number of experimental conditions simultaneously and to obtain rapidly many experimental results. Kidney tubules can be prepared from the kidney of many animal species and also from the human kidney; given the very limited availability of healthy human renal tissue, it is therefore possible to choose the most appropriate species for the study of a particular problem encountered in man. Kidney tubules can be used for screening and prevention of nephrotoxic effects and to identify their mechanisms as well as to study the renal metabolism of xenobiotics. When compared with cultured renal cell, a major advantage of kidney tubules is that they remain differentiated. The main limitations of the use of kidney tubules in pharmacotoxicology are (1) the necessity to prepare them as soon as the renal tissue sample is obtained; (2) their limited viability, which is restricted to 2-3 h; (3) the inability to expose them chronically to a potential nephrotoxic drug; (4) the inability to study transepithelial transport; and (5) the uncertainty in the extrapolation to man of the results obtained using animal kidney tubules. These advantages and limitations of the use of human and animal kidney tubules in pharmacotoxicology are illustrated mainly by the results of experiments performed with valproate, an antiepileptic and moderately hyperammonemic agent. The fact that kidney tubules, unlike cultured renal cells, retain key metabolic properties is also shown to be of the utmost importance in detecting certain nephrotoxic effects.

Alanine metabolism in isolated human kidney tubules--Use of a mathematical model.

To gain insight into the fate of alanine nitrogen and carbon taken up by the human kidney under certain conditions, isolated human kidney cortex tubules were incubated in Krebs-Henseleit medium with L-alanine as substrate. The tubules metabolized alanine at high rates and in a dose-dependent manner. Most of the alanine nitrogen removed was recovered as ammonia and to a lesser extent as glutamate. Glucose, lactate and glutamate were also found to be significant products of alanine carbon metabolism. A simple mathematical model allowing one to calculate flux of alanine carbon through the various metabolic steps involved is proposed and applied to data obtained in experiments in which 5 mM [U-14C]-,[1-14C]-, [2-14C]- and [3-14C]alanine were used as substrates in parallel. About 40% of the alanine carbon removed was recovered as CO2 and oxidation of C1 of alanine accounted for most of the CO2 released from alanine. Calculations reveal that the ATP produced exceeded 3.2-fold the ATP consumed in relation to alanine metabolism. It is concluded that, in human kidney, alanine may serve as an energy supplier and as a precursor of glucose and ammonia.