C6 cells were used to establish a glioma-bearing rat model by stereotaxic injection in the left caudate nucleus. The tumor status was evaluated by magnetic resonance imaging and conventional histology. The glioma-bearing rats were infused for 1 h with a [1-13 C]glucose solution. Perchloric acid extracts of the tumor and the ipsilateral and contralateral hemispheres were analyzed by 13 C-NMR spectroscopy. The 13 C-labeling patterns in compounds, mainly amino acids, indicated no drastic modification of carbon metabolism in both ipsilateral and contralateral hemispheres, as compared with control rats, whereas profound metabolic differences between brain tissue and tumor were observed. Glutamine C4 enrichment was lower in the glioma than in the brain [mean Ϯ SD values, 5.4 Ϯ 2.3 (n ϭ 5) and 15.0 Ϯ 0.8% (n ϭ 10), respectively] and also lower than the glutamate C4 enrichment in the glioma (mean Ϯ SD value, 22.6 Ϯ 4.2%; n ϭ 5), indicating that tumor glutamine was neither synthesized inside the glioma nor taken up from the surrounding brain. The glutamine C4 enrichment in the serum (6.7 Ϯ 0.5%; n ϭ 10) suggested that the glioma imported glutamine from the blood, a process probably connected with angiogenesis.
13C and 1 H NMR spectroscopy was used to investigate the metabolism of L-lactate and D-glucose in C6 glioma cells. The changing of lactate and glucose concentration in the extracellular medium of C6 glioma cells incubated with 5.5 mM glucose and 11 mM lactate indicated a net production of lactate as the consequence of an active aerobic glycolysis. The 13 C enrichments of various metabolites were determined after 4-h cell incubation in media containing both substrates, each of them being alternatively labeled in the form of either [3-13 C]L-lactate or [1-13 C]D-glucose. Using 11 mM [3-13 C]L-lactate, the enrichment of glutamate C4, 69%, was found higher than that of alanine C3, 32%, when that of acetyl-CoA C2 was 78%. These results indicated that exogenous lactate was the major substrate for the oxidative metabolism of the cells. Nevertheless, an active glycolysis occurred, leading to a net lactate production. This lactate was, however, metabolically different from the exogenous lactate as both lactate species did not mix into a unique compartment. The results were actually consistent with the concept of the existence of two pools of both lactate and pyruvate, wherein one pool was closely connected with exogenous lactate and was the main fuel for the oxidative metabolism, and the other pool was closely related to aerobic glycolysis.Neoplastic cells preferentially utilize aerobic glycolysis for their energy needs rather than oxidative phosphorylations, that leads to an important lactate production (1). The reason for this behavior is not yet clearly understood. It has been proposed recently that it would be a means to minimize oxidative stress, in particular during the cell phases linked to biosynthesis and cell division (2). With regard to a tumor, aerobic glycolysis may increase lactate concentration inside the tumor itself and in its close vicinity (3, 4). Consequently, local changes in metabolic activities could be generated as an increased local lactate concentration provides a possible source for cell energy requirements in competition with glucose or other substrates. There is no data concerning the competition between lactate and glucose as fuel for the oxidative metabolism in glioma cells. It has been only reported that, in vitro, glioma cells utilize glucose, but that once it is depleted, they will take up pyruvate and lactate generated by aerobic glycolysis for further metabolism (5).To study the possible competition between glucose and lactate metabolism in glioma cells, we investigated the fate of each of these substrates when both were present in cell medium by using either [3-13 C]L-lactate or [1-13 C]D-glucose. We used the C6 glioma cell clone for which the metabolism of [1-13 C]glucose has been investigated already (6, 7). The initial glucose concentration (5.5 mM) was chosen higher than the apparent K m for transport (1.7 mM) (8), whereas the different lactate concentrations used (11, 5.5, and 1.1 mM) were higher or in the same range than the apparent K m for transport (1 mM) (9). The 13 C enrich...
Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO43− from [γ‐32P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32P‐phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181. In the second peptide, Ser197, Ser199, and Ser204 were phosphorylated. All these serines are clustered in the C‐terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0. We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197, Ser199, Ser204, Ser208, and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.
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