Evidence for the mitochondrial lactate oxidation complex in rat neurons: demonstration of an essential component of brain lactate shuttles

To evaluate the presence of components of a putative Intracellular Lactate Shuttle (ILS) in neurons, we attempted to determine if monocarboxylate (e.g. lactate) transporter isoforms (MCT1 and -2) and lactate dehydrogenase (LDH) are coexpressed in neuronal mitochondria of rat brains. Immunohistochemical analyses of rat brain cross-sections showed MCT1, MCT2, and LDH to colocalize with the mitochondrial inner membrane marker cytochrome oxidase (COX) in cortical, hippocampal, and thalamic neurons. Immunoblotting after immunoprecipitation (IP) of mitochondria from brain homogenates supported the histochemical observations by demonstrating that COX coprecipitated MCT1, MCT2, and LDH. Additionally, using primary cultures from rat cortex and hippocampus as well as immunohistochemistry and immunocoprecipitation techniques, we demonstrated that MCT2 and LDH are coexpressed in mitochondria of cultured neurons. These findings can be interpreted to mean that, as in skeletal muscle, neurons contain a mitochondrial lactate oxidation complex (mLOC) that has the potential to facilitate both intracellular and cell-cell lactate shuttles in brain.     

MCT1 confirmed in rat striated muscle mitochondria.

We sought to test the hypothesis that monocarboxylate transporter isoform 1 (MCT1) is the inner mitochondrial membrane lactate/pyruvate transporter, and, as such, contributes to functioning of the intracellular lactate shuttle. However, presence of a mammalian mitochondrially localized MCT1 (mMCT1) has been contested. We sought to confirm by Western blotting the mitochondrial localization of MCT1 in rat cardiac, soleus, and extensor digitorum longus muscles utilizing three different cell fractionation methods and three different antibodies. We performed Western blotting using antibodies to cell membrane glucose transporter isoform GLUT1, inner mitochondrial constituent cytochrome oxidase, the monocarboxylate transporter protein chaperone CD147, as well as custom and commercially available MCT1 antibodies. Western blots demonstrated similar results with each MCT1 antibody and two of three methods of fractionation. MCT1 was found in the mitochondria, as well as in the sarcolemmal membrane and whole muscle homogenates. Probing with GLUT1 and CD147 demonstrated that mitochondrial fractions were not contaminated with sarcolemmal remnants. Probing with cytochrome oxidase showed mitochondrial localization of MCT1. Comparison of these results to the findings of others indicates that the most likely source of discrepancy is the cell fractionation procedure utilized.     

Cardiac and skeletal muscle mitochondria have a monocarboxylate transporter MCT1.

To evaluate the potential role of monocarboxylate transporter-1 (MCT1) in tissue lactate oxidation, isolated rat subsarcolemmal and interfibrillar cardiac and skeletal muscle mitochondria were probed with an antibody to MCT1. Western blots indicated presence of MCT1 in sarcolemmal membranes and in subsarcolemmal and interfibrillar mitochondria. Minimal cross-contamination of mitochondria by cell membrane fragments was verified by probing for the sarcolemmal protein GLUT-1. In agreement, immunolabeling and electron microscopy showed mitochondrial MCT1 in situ. Along with lactic dehydrogenase, the presence of MCT1 in striated muscle mitochondria permits mitochondrial lactate oxidation and facilitates function of the "intracellular lactate shuttle."     

Exercise-induced changes of MCT1 in cardiac and skeletal muscles of diabetic rats induced by high-fat diet and STZ

We hypothesized that a part of therapeutic effects of endurance training on insulin resistance is mediated by increase in cardiac and skeletal muscle mitochondrial lactate transporter, monocarboxylate transporter 1 (MCT1). Therefore, we examined the effect of 7 weeks endurance training on the mRNA and protein expression of MCT1 and MCT4 and their chaperon, CD147, on both sarcolemmal and mitochondrial membrane, separately, in healthy and type 2 diabetic rats. Diabetes was induced by injection of low dose of streptozotocin and feeding with high-fat diet. Insulin resistance was confirmed by homeostasis model assessment-estimated insulin resistance index and accuracy of two membranes separation was confirmed by negative control markers (glucose transporter 1 and cytochrome c oxidase. Real-time PCR and western blotting were used for mRNA and protein expression, respectively. Diabetes dramatically reduced MCT1 and MCT4 mRNA and their expression on sarcolemmal membrane whereas the reduction in MCT1 expression was less in mitochondrial membrane. Training increased the MCT1 mRNA and protein expression in both membranes and decreased insulin resistance as an adaptive consequence. In both tissues increase in CD147 mRNA was only parallel to MCT1 expression. The response of MCT1 on sarcolemmal and mitochondrial membranes was different between cardiac and skeletal muscles which indicate that intracellular lactate kinetic is tissue specific that allows a tissue to coordinate whole organism metabolism.     

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

Regulating effect of mitochondrial lactate dehydrogenase on oxidation of cytoplasmic NADH via an "external" pathway in skeletal muscle mitochondria.

1. The specific activity of lactate dehydrogenase of skeletal muscle mitochondria was found to be 2.5 times lower than specific activity of total NADH-cytochrome c reductase. 2. The specific activity of mitochondrial LDH in skeletal muscle mitochondria was almost equal to the activity of rotenone-insensitive NADH-cytochrome c reductase. 3. Mitochondrial LDH acting as an oxidase of lactate to pyruvate may feed an "external" pathway, but the activity of the mitochondrial enzyme is a limiting factor in oxidation of lactate-derived NADH. 4. Mitochondrial LDH acting as a reductase of pyruvate to lactate successfully competes with an "external" pathway for cytoplasmic NADH. 5. Exogenous NADH oxidation via an "external" pathway was inhibited by pyruvic acid. This inhibition was overcome by addition of oxamic acid or hydrazine.     

Changes in MCT 1, MCT 4, and LDH expression are tissue specific in rats after long-term hypobaric hypoxia.

Little is known about the effect of chronic hypobaric hypoxia on the enzymes and transporters involved in lactate metabolism. We looked at the protein expression of monocarboxylate transporters MCT 1, MCT 2, and MCT 4, along with total lactate dehydrogenase (LDH) and LDH isozymes in skeletal muscle, cardiac muscle, and liver. Expression of these components of the lactate shuttle affects the ability to transport and oxidize lactate. We hypothesized that the expression of MCTs and LDH would increase after acclimation to high altitude (HA). The response to acclimation to HA was, however, tissue specific. In addition, the response was different in whole muscle (Mu) and mitochondria-enriched (Mi) fractions. Heart, soleus, and plantaris muscles showed the greatest response to HA. Acclimation resulted in a 34% increase in MCT 4 in heart and a decrease in MCT 1 (-47%) and MCT 4 (-47%) in plantaris Mu. In Mi fractions, the heart had an increase (+40%) and soleus a decrease (-40%) in LDH. HA also had a significant effect on the LDH isozyme composition of both the Mu and Mi fractions. Mitochondrial density was decreased in both the soleus (-17%) and plantaris (-44%) as a result of chronic hypoxia. We conclude that chronic hypoxia had a tissue-specific effect on MCTs and LDH (that form the lactate shuttle) but did not produce a consistent increase in these components in all tissues.     

Fluorescence resonance energy transfer studies on the interaction between the lactate transporter MCT1 and CD147 provide information on the topology and stoichiometry of the complex in situ.

The monocarboxylate (lactate) transporters MCT1 and MCT4 require the membrane-spanning glycoprotein CD147 for their correct plasma membrane expression and function. We have successfully expressed CD147 and MCT1 tagged on their C or N termini with either the cyan (CFP) or yellow (YFP) variants of green fluorescent protein. The tagged proteins were correctly targeted to the plasma membrane of COS-7 cells and were functionally active. Measurements of fluorescence resonance energy transfer (FRET) between all combinations of the tagged proteins were made. FRET was observed when either the C or N terminus of MCT1 (intracellular) is tagged with CFP or YFP and co-expressed with CD147 tagged with YFP or CFP on the C terminus (intracellular) but not the N terminus (extracellular). FRET was also observed between two CD147 molecules when both YFP and CFP were on the C terminus but not when both were on the N terminus or one on either end. No FRET was observed between MCT1-YFP and MCT-CFP in any combination. A wide range of controls including photobleaching were employed to confirm that where FRET was observed, it was not an artifact of direct excitation of YFP by the CFP excitation laser. It was also shown that nonspecific overcrowding of proteins did not induce FRET. Because FRET only occurs between two fluorophores if they are less than 100 A apart and in a suitable orientation, our data provide important information on the topology of CD147 and MCT1 within the plasma membrane. The minimum configuration consistent with the data is a dimer of CD147 associating with two MCT1 molecules such that the C terminus of CD147 in the cytosol is close to the C terminus of its partner CD147 and to the C and N termini of an associated MCT1 molecule. FRET may provide a non-invasive technique for measuring changes in these interactions in living cells.     

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.     

Lactate shuttles at a glance: from physiological paradigms to anti-cancer treatments

Hypoxia and oncogene expression both stimulate glycolytic metabolism in tumors, thereby leading to lactate production. However, lactate is more than merely a by-product of glycolysis: it can be used as a metabolic fuel by oxidative cancer cells. This phenomenon resembles processes that have been described for skeletal muscle and brain that involve what are known as cell-cell and intracellular lactate shuttles. Two control points regulate lactate shuttles: the lactate dehydrogenase (LDH)-dependent conversion of lactate into pyruvate (and back), and the transport of lactate into and out of cells through specific monocarboxylate transporters (MCTs). In tumors, MCT4 is largely involved in hypoxia-driven lactate release, whereas the uptake of lactate into both tumor cells and tumor endothelial cells occurs via MCT1. Translating knowledge of lactate shuttles to the cancer field offers new perspectives to therapeutically target the hypoxic tumor microenvironment and to tackle tumor angiogenesis.     

CD147 regulates the expression of MCT1 and lactate export in multiple myeloma cells

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.     

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for β-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.     

Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4: the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70)

Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). Here we demonstrate by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolished co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhanced labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, was found to co-immunoprecipitate with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibited lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. We conclude that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane.     

The complete mitochondrial genome of the yeast Pichia sorbitophila

Here, we report the complete nucleotide sequence of the 39 107-bp mitochondrial genome of the yeast Pichia sorbitophila . This genome is closely related to those of Candida parapsilosis and Debaryomyces hansenii , as judged from sequence similarities and synteny conservation. It encodes three subunits of cytochrome oxidase ( COX1, COX2 and COX3 ), three subunits of ATP synthase ( ATP6, ATP8 and ATP9 ), the seven subunits of NADH dehydrogenase ( NAD1-6 and NAD4L ), the apocytochrome b ( COB ), the large and small rRNAs and a complete set of tRNAs. Although the mitochondrial genome of P. sorbitophila contains the same core of mitochondrial genes observed in the ascomycetous yeasts, those coding for the RNAse P and the ribosomal protein VAR1p are missing. Moreover, the mtDNA of P. sorbitophila contains several introns in its genes and has the particularity of possessing an intron, which is not linked to any upstream exon.