Hasimoto, T., Hussien, R., & Brooks, G. A. (January 24, 2006). Co-localization of MCT1, CD147 and LDH in mitochondrial inner membrane of L6 skeletal muscle cells: Evidence of a mitochondrial lactate oxidation complex. American Journal of Physiology Endocrinology and Metabolism [on line at].

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This study clarified the role of mitochondria in intracellular and cell-cell lactate shuttles. Using a rat-derived L6 skeletal muscle cell line and confocal laser-scanning microscopy, the cellular locations of mitochondria, lactate dehydrogenase (LDH), the lactate/pyruvate transporter MCT1, and CD147, a purported chaperone protein for MCT1, were examined.

It was shown that LDH, MCT1, and CD147 are co-localized with the mitochondrial reticulum. Western blots showed that cytochrome oxidase (COX), NADH-dehydrogenase (NADH-dh), LDH, MCT1 and CD147 are abundant in mitochondrial fractions of L6 cells. Interactions among COX, MCT1, and CD147 in mitochondria were confirmed.

Implication. These findings support the presence of a mitochondrial lactate oxidation complex associated with the cytochrome oxidase end of the electron transport chain that may explain the oxidative catabolism of lactate and, hence, the mechanism of the Intracellular Lactate Shuttle.

The implications of this study were further explained in press release of an interview with Professor George Brooks [Sanders, R. (April 19, 2006). If you "feel the burn," you need to bulk up your mitochondria. UCBerkeleyNews, [].

"Endurance training teaches the body to efficiently use lactic acid as a source of fuel on par with the carbohydrates stored in muscle tissue and the sugar in blood. Efficient use of lactic acid, or lactate, not only prevents lactate build-up, but ekes out more energy from the body's fuel.

Lactate is the link between oxidative and glycolytic, or anaerobic metabolism. During normal exertion, lactate seeps out of the muscle cells into the blood to be used elsewhere. During intense exercise, oxidation increases rapidly to remove accumulating lactate and create more energy. The intense effort of interval training "generates big lactate loads, and body adapts by building up mitochondria to clear lactic acid quickly. If you use it up, it doesn't accumulate.

To move, muscles need energy in the form of ATP, adenosine triphosphate. Most people think glucose, a sugar, supplies this energy, but during intense exercise, it's too little and too slow as an energy source, forcing muscles to rely on glycogen, a carbohydrate stored inside muscle cells. For both fuels, the basic chemical reactions producing ATP and generating lactate comprise the glycolytic pathway, often called anaerobic metabolism because no oxygen is needed. This pathway was thought to be separate from the oxygen-based oxidative pathway, sometimes called aerobic metabolism, used to burn lactate and other fuels in the body's tissues.

Experiments with dead frogs in the 1920s seemed to show that lactate build-up eventually causes muscles to stop working. But Brooks in the 1980s and '90s showed that in living, breathing animals, the lactate moves out of muscle cells into the blood and travels to various organs, including the liver, where it is burned with oxygen to make ATP. The heart even prefers lactate as a fuel, Brooks found.

Brooks always suspected, however, that the muscle cell itself could reuse lactate, and in experiments over the past 10 years he found evidence that lactate is burned inside the mitochondria, an interconnected network of tubes, like a plumbing system, that reaches throughout the cell cytoplasm.

In 1999, for example, he showed that endurance training reduces blood levels of lactate, even while cells continue to produce the same amount of lactate. This implied that, somehow, cells adapt during training to put out less waste product. He postulated an 'intracellular lactate shuttle' that transports lactate from the cytoplasm, where lactate is produced, through the mitochondrial membrane into the interior of the mitochondria, where lactate is burned. In 2000, he showed that endurance training increased the number of lactate transporter molecules in mitochondria, evidently to speed uptake of lactate from the cytoplasm into the mitochondria for burning.

The new paper and a second paper to appear soon finally provide direct evidence for the hypothesized connection between the transporter molecules - the lactate shuttle - and the enzymes that burn lactate. In fact, the cellular mitochondrial network, or reticulum, has a complex of proteins that allow the uptake and oxidation, or burning, of lactic acid.

"This experiment is the clincher, proving that lactate is the link between glycolytic metabolism, which breaks down carbohydrates, and oxidative metabolism, which uses oxygen to break down various fuels," Brooks said.

Post-doctoral researcher Takeshi Hashimoto and staff research associate Rajaa Hussien established this by labeling and showing colocalization of three critical pieces of the lactate pathway: the lactate transporter protein; the enzyme lactate dehydrogenase, which catalyzes the first step in the conversion of lactate into energy; and mitochondrial cytochrome oxidase, the protein complex where oxygen is used. Peering at skeletal muscle cells through a confocal microscope, the two scientists saw these proteins sitting together inside the mitochondria, attached to the mitochondrial membrane, proving that the 'intracellular lactate shuttle' is directly connected to the enzymes in the mitochondria that burn lactate with oxygen.

"Our findings can help athletes and trainers design training regimens and also avoid overtraining, which can kill muscle cells," Brooks said. "

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