Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle

Abstract

To evaluate the potential role of mitochondrial lactate dehydrogenase (LDH) in tissue lactate clearance and oxidation in vivo, isolated rat liver, cardiac, and skeletal muscle mitochondria were incubated with lactate, pyruvate, glutamate, and succinate. As well, alpha-cyano-4-hydroxycinnamate (CINN), a known monocarboxylate transport inhibitor, and oxamate, a known LDH inhibitor were used. Mitochondria readily oxidized pyruvate and lactate, with similar state 3 and 4 respiratory rates, respiratory control (state 3/state 4), and ADP/O ratios. With lactate or pyruvate as substrates, alpha-cyano-4-hydroxycinnamate blocked the respiratory response to added ADP, but the block was bypassed by addition of glutamate (complex I-linked) and succinate (complex II-linked) substrates. Oxamate increased pyruvate (approximately 10-40%), but blocked lactate oxidation. Gel electrophoresis and electron microscopy indicated LDH isoenzyme distribution patterns to display tissue specificity, but the LDH isoenzyme patterns in isolated mitochondria were distinct from those in surrounding cell compartments. In heart, LDH-1 (H4) was concentrated in mitochondria whereas LDH-5 (M4) was present in both mitochondria and surrounding cytosol and organelles. LDH-5 predominated in liver but was more abundant in mitochondria than elsewhere. Because lactate exceeds cytosolic pyruvate concentration by an order of magnitude, we conclude that lactate is the predominant monocarboxylate oxidized by mitochondria in vivo. Mammalian liver and striated muscle mitochondria can oxidize exogenous lactate because of an internal LDH pool that facilitates lactate oxidation.

Figure 1

Agarose gel electrophoresis of LDH in mitochondria from rat liver and heart. LDH isoenzyme patterns differ between cytosol and mitochondria in both tissues.

Figure 2

Electron micrograph of high-pressure frozen rat liver showing mitochondria, rough endoplasmic reticulum, and cytosol. Immunolocalization of anti-LDH-5 (M4) antibodies is indicated by the 15 nm gold particles. Note presence of LDH-5 in mitochondria and surrounding matrix and organelles. (Magnification, ×58,300; scale bar = 400 nm.)

Figure 3

Electron micrograph of conventionally processed rat left ventricle. Immunolocalization of anti-LDH-5 (M4) antibodies is shown by the 15 nm gold particles. Note presence of LDH-5 in mitochondria and surrounding matrix and myofibrils and cytosol. (Final magnification, ×60,500; scale bar = 400 nm.)

Figure 4

Electron micrograph of conventionally processed rat left ventricle. Immunolocalization of anti-LDH-1 (H4) antibodies is shown by the 15 nm gold particles. Note abundance of LDH-1 in mitochondria. (Final magnification, ×67,000; scale bar = 300 nm.)

Figure 5

Depiction of the functional relationship between mitochondrial LDH and mMCT in operation of the intracellular lactate shuttle. The predominant monocarboxylate entering the mitochondrial intermembrane space is lactate. Entry of lactate and pyruvate into the mitochondrial matrix is facilitated by mMCT. Thus, lactate enters mitochondria; lactate is oxidized to pyruvate via mitochondrial LDH when mitochondrial Redox decreases, and pyruvate is oxidized via the tricarboxylic acid cycle and electron transport chain (ETC).