Malate-aspartate Shuttle

Glutaminolysis Pathway
Figure 4 The glutaminolysis pathway and malate shunt, indicating alternative routes and main transport systems around the mitochondria. The enzymes involved in the pathway are indicated in italics and the corresponding full names are given in the list of abbreviations in Fig. 1.

through the cytosolic enzymes malate dehydrogenase (MDH) and PEPCK or through the cytosolic ME. Vriezen and van Dijken (45) have reported the detection of PEPCK in a myeloma cell line. However, various studies have shown the lack of PEPCK and also PC in hybridoma, CHO, and BHK cell lines (12,13,15,16). Bonar-ius et al. (21) have used 13C-labeled compounds tracing and metabolite balance to characterize the metabolic pathway distribution of a hybridoma cell line showing that the PC activity was negligible, while the ME was active in the malate shunt, which had a rate of 8% of the glucose uptake rate.

In the TA pathway for glutamine metabolism (shown in Fig. 3), glutamate is also converted in a-ketoglurate, but the released ammonium ion is transferred in a stoichiometric proportion to oxaloacetate to form aspartate (in the so-called aspTA pathway), or to pyruvate to form alanine (in the so called alaTA pathway) as follows:

Because the molecules of oxaloacetate or pyruvate being transaminated are obtained from the glutamate metabolism through the TCA, these TA pathways have a loop structure. The TA pathways can be used by the cell as an internal mechanism to balance ammonium overflow due to the rapid glutamine consumption, as will be discussed in the next section. In addition, alanine and aspartate are important precursors, in particular for the biosynthesis of purine, pyrimidine, and asparagine. In Fig. 5 the two main possibilities for the localization of the TA pathways are considered: cytosolic or mitochondrial TA. The experimental evidence is still not conclusive enough on this aspect, as discussed in detail by Haggstrom (3). However, for aspartate formation, mitochondrial TA is considered the main active pathway, which could be explained by two facts: the equilibrium of the reaction between malate and oxaloacetate is favored toward malate formation and, in addition, this reaction would generate NADH in the cytosol, a compound already in excess due to the glycolysis activity. The aspTA pathway uses the glutamate-aspartate shuttle to incorporate glutamate into the mitochondria in an exclusive way, since other pathways would deplete aspartate in the mitochondria. In the case of alanine formation, both cytosolic and mitochondrial TAs may be active. For the cytosolic alanine formation, the a-ketoglutarate/malate exchange system in the mitochondrial membrane (48-50) plays a central role for the balance between cytolosic and mitochon-drial reactions. In this exchange carrier, the a-ketoglutarate produced in the TA reaction is incorporated into the mitochondria and TCA, and in the exchange malate, is exported out of the mitochondria. In the cytosol, malate can be further converted to pyruvate by the cytosolic ME present in most cell lines, as previously discussed. Although a higher alaTA activity has been detected in the cytosol with respect to the mitochondria (45,51), this seems contradictory to the fact that ME is dependent on NADP+, and therefore the malate shunt should be confined in the mitochondria (42,52). Thus, the specific localization of the enzymatic activities of the malate shunt remains to be clarified. The second possibility, alanine generation in the mitochondria, has been observed experimentally in different cell lines (53-56) when pyruvate is available. Lipid synthesis is also related to alanine generation in the mitochondria. In growing cells, acetyl CoA is required in the cytosol for the synthesis of cholesterol and fatty acids. Citrate, produced as an intermediate of TCA, is exported from the mitochondria through the citrate/malate exchanger, then it is converted in the cytosol to acetyl CoA for lipid synthesis and oxaloacetate, and

Citrate Malate Pyruvate Shuttle

Figure 5 Illustration of the different glutamine metabolism possibilities, indicating the main transport systems around the mitochondria: cytosolic transamination pathways, mitochondrial transamination to aspartate, and interaction between glutamine metabolism and lipids cycle. The main enzymes involved in the pathway are indicated in italics and the corresponding full names are given in the list of abbreviations in Fig. 1.

Figure 5 Illustration of the different glutamine metabolism possibilities, indicating the main transport systems around the mitochondria: cytosolic transamination pathways, mitochondrial transamination to aspartate, and interaction between glutamine metabolism and lipids cycle. The main enzymes involved in the pathway are indicated in italics and the corresponding full names are given in the list of abbreviations in Fig. 1.

further into malate, as shown in Fig. 5. Malate is incorporated back into the mitochondria through the citrate/malate exchanger, where it can be converted to pyruvate by the ME, and then transaminated to alanine by glutamate. In this case, the glutamate is either generated inside the mitochondria or incorporated through the proton symport system. In any case, it should be mentioned that part of the acetyl CoA requirements in the cell are also provided from glucose metabolism. These two pathways could occur in a simultaneous way in the cell. Indeed, Bonarius et al. (21) have recently described, using isotope labeling determinations and mass balancing, how the ME is participating simultaneously in the generation of extramito-chondrial malate, both through the malate shunt and through the pyruvate/malate shuttle linked to the lipid synthesis.

As can be observed, the glutamine metabolism can be diverted to a number of different possible pathways that in turn share a number of intermediate metabolites and transport systems around the mitochondria. From the energy yield perspective, the GDH pathway is more efficient than TA (27 ATP instead of 9) (57), and usually is the favored pathway when glutamine metabolism must be accelerated in the cell, for example, as a consequence of glucose depletion (58). Also, glutamine and glucose metabolism are related to each other, as will be discussed specifically in the section on flexibility of animal cell metabolism. As a consequence of this metabolic flexibility, different cell lines will adjust the metabolic rates through GDH, alaTA, or aspTA pathways, depending on the specific culture conditions.

Deregulated Glutaminolysis in Mammalian Cells

Similar to glucose, glutamine is also consumed by most mammalian cell lines at high rates, normally associated with the general use of excess glutamine concentrations in the culture media. As is well known, this leads to the undesired accumulation of ammonium ions, in addition to an inefficient use of glutamine. The increase in glu-tamine consumption when glutamine concentration is raised, either as a pulse or a step change in a chemostat culture of a hybridoma cell line, was reported by Miller et al. (59), confirming early results seen in batch cultures (24,28,60,61). Vriezen et al. (62) performed a series of chemostat experiments with two different cell lines, a hybridoma and a myeloma, at glutamine concentrations in the range of 0.5-4 mM at a dilution rate of 0.03 per hour. For the limiting conditions, up to 2mM glutamine in the feed, no residual glutamine was detected in the culture, and the specific consumption rate of glutamine was in the order of 20 nmol 10~6cells per hour. This value increased to 36 nmol 10~6cells per hour when excess glutamine was fed at a concentration of 4 mM without any further increase in cell concentration, and a parallel increase in the specific ammonium generation rate. Sanfeliu et al. (27) performed a series of continuous experiments with a hybridoma cell line, decreasing stepwise the glutamine concentration in the feed from 5 to 0.75 mM; they obtained a constant level of cell concentration with a gradual decrease of specific glutamine consumption, and a fourfold lower ammonium generation. They estimated a minimum specific requirement for glutamine of 30.72 nmol 10~6cells per hour. Mancuso et al. (63) observed a significant reduction of specific glutamine consumption in a hybridoma cell line, when glutamine concentration in the medium was reduced from 2.4 to 1.2 mM in a continuous hollow fiber reactor. Under these conditions, they reached a glutamine concentration of 0.08 mM at the reactor outlet that was not limiting for energy metabolism. Similar results were obtained by Sharfstein (14). Cruz et al. (33) observed a similar pattern in continuous cultures of BHK cells: specific glutamine consumption was reduced from 33 to 16.8 nmol 10~6cells per hour when the feed concentration was reduced from 0.52 to 0.14 mM.

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Responses

  • HIWET DAWIT
    Which cells use malateaspartate shuttle?
    4 years ago
  • BELBA
    What is the abbreviation TA in malate aspartate shuttle?
    2 years ago

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