The paper by Lemire and colleagues is aimed at adding to existing knowledge of the ability of cells to metabolize L-lactate. In particular, it is claimed that astrocytoma, i.e. cancer cells, can take up and metabolize externally added lactate in vitro. In the light of these results, the authors hypothesize that normal astrocytes might also metabolize lactate, with an additional role for these cells in the removal of lactate from the extracellular space. However since tumor cells are known to have an altered pH regulation with respect to normal cells [Stubbs M et al. Mol. Med. Today. 2000 6:15-9], to be highly glycolytic thus producing high amounts of lactate, and to have an aberrant mitochondrial phenotype [Oudard S et al. Anticancer Res. 1997, 17:1903-11; Ristow M. Curr. Opin. Clin. Nutr. Metab. Care. 2006, 9:339-45; Moreno-Sánchez R et al. FEBS J. 2007, 274:1393-418; Gogvadze V et al. Trends Cell Biol. 2008], further investigations on normal cells are strictly required to extend Lemire’s results to normal astrocytes.

There are, however, some issues that we would like to raise concerning this paper. Firstly, it is somewhat surprising that no reference is made to the pre-existing literature on mitochondrial LDH [Sacktor, B. Arch. Biochem. Biophys. 1959, 80:68-71; Baba N. and Sharma HM J. Cell. Biol. 1971, 51:621-635; Kline ES et al. Arch. Biochem. Biophys. 1986, 246:673-680; Brandt RB et al. Arch. Biochem. Biophys. 1987, 259:412-422] or to the controversy surrounding the existence of a mitochondrial enzyme for lactate [Rasmussen HN et al. J. Physiol. 2002, 541:575-580; Sahlin K et al. J. Physiol. 2002, 541:569-574; Yoshida Y et al. J. Physiol. 2007, 582:1317-1335]. Previous reports which seem to us to be pertinent in the present context include those in which coupled mitochondria isolated from rat heart [Valenti D. et al. Biochem J. 2002, 364:101-4] and liver [de Bari L. et al. Biochem J. 2004, 380:231-42.] were shown to import lactate from the extramitochondrial phase and oxidize it in the matrix, in the absence of externally added NAD+, in vitro. Very recently, by using similar experimental approaches, we have shown that rat neurons can take up lactate from the extracellular phase and oxidize it at the mitochondrial level for energy production and for shuttling NADH to the matrix, by virtue of the existence of LDH located in the inner mitochondrial compartments [Atlante A. et al. Biochim Biophys Acta. 2007, 1767:1285-99]. This agrees with Lemire’s finding that LDH1 (considered to be the mitochondrial LDH isoenzyme) preferentially localizes in the inner mitochondrial/matrix fraction (Figure 9I). Similarly lacking in the paper is any discussion of the existence of several mitochondrial carriers for the uptake of externally added lactate and for the efflux of newly synthesized metabolites from the matrix in exchange with lactate, as shown in mitochondria from heart [Valenti D. et al. Biochem J. 2002, 364:101-4]], liver [de Bari L. et al. Biochem J. 2004, 380:231-42.] and neurons [Atlante A. et al. Biochim Biophys Acta. 2007, 1767:1285-99]. Among these, the L-lactate/H+ symporter, which mediates lactate entrance into the matrix and differs from the mitochondrial MCT1 (pyruvate carrier) on the basis of the different inhibitor sensitivity, is of particular importance. A consideration of the roles of these carriers seems to us essential for a proper understanding of intramitochondrial lactate metabolism.

Our second area of concern relates to the somewhat unusual techniques that Lemire and colleagues have used to study mitochondrial metabolism of lactate. The authors report that in all the experiments mitochondria are suspended either in “phosphate buffer” or in “equilibration buffer” which contain 10 mM phosphate plus 5 mM MgCl2 and 25 mM Tris-HCl plus 5 mM MgCl2, respectively. If we interpret this correctly it means that the buffers were hypo-osmotic for mitochondria and would certainly lead to their rupture. If that were the case then it is hard to see how the authors manage to determine oxygen consumption and ATP production after addition of lactate (or other respiratory substrates) to mitochondria. Related to this, it is surprising that they were able to measure increasing NADH amounts after incubating mitochondria with NAD+ in the absence of any inhibitor of the NADH-dehydrogenase (complex I of the respiratory chain) given that complex I should oxidize NADH as fast as it forms after lactate oxidation. It is also reported that several analysis were carried out with mitochondria incubated for up to 240 min at 37°C; mitochondria incubated under these conditions are unlikely to remain intact and capable of synthesizing ATP.

A final methodological concern is the authors’ choice of NAD+ to test the ability of mitochondria to oxidize lactate given that they find the mitochondrial fraction on which the measurements were carried out to contain (at least) two LDH isoenzyme (Figure 7I) i.e. LDH1, localized in the inner compartments (Figure 9I) and another LDH probably located in the outer mitochondrial compartments; the latter might be the cytosolic isoenzyme bound to the outer mitochondrial membrane, as previously reported [Brandt R.B. et al. Arch Biochem Biophys. 1987, 259:412-22]. Hence, in the presence of NAD+, externally added lactate could be oxidized in the outer mitochondrial compartments thus producing pyruvate which in turn could enter mitochondria and be oxidized therein. We would be forced to conclude, therefore, that under the stated conditions no information can be obtained on the ability of mitochondria to take up lactate and use it as a respiratory substrate for energy production and for the shuttling of cytosolic NADH to the matrix, as graphically described in Figure 11 of the paper.

In short, therefore, although we consider the system studied by Lemire and colleagues to be an interesting one, and capable of shedding new light on the mitochondrial metabolism of lactate, there are serious methodological problems that need to be addressed before the results presented can be accepted at their face value. We should be interested to have the authors’ response to the issues that we raise.



Salvatore Passarella, Anna Atlante, Lidia de Bari and Daniela Valenti