The mitochondrial lactate oxidation complex: endpoint for carbohydrate carbon disposal

Abstract

The lactate shuttle concept has revolutionized our understanding and study of metabolism in physiology, biochemistry, intermediary metabolism, nutrition, and medicine. Seminal findings of the mitochondrial lactate oxidation complex (mLOC) elucidated the architectural structure of its components. Here, we report that the mitochondrial pyruvate carrier (mPC) is an additional member of the mLOC in mouse muscle and C2C12 myoblasts and myotubes. Immunoblots, mass spectrometry, and co-immunoprecipitation experiments of mitochondrial preparations revealed abundant amounts of mitochondrial lactate dehydrogenase (mLDH), monocarboxylate transporter (mMCT), basigin (CD147), cytochrome oxidase (COx), and pyruvate carriers 1 and 2 (mPC1 and 2). In addition, using confocal laser scanning microscopy (CLSM) and in situ proximity ligation, we also demonstrated planar and three-dimensional (3-D) colocalization of pyruvate and lactate transporters with COx in fixed mouse skeletal muscle sections and C2C12 myoblasts and myotubes skeletal muscle sections, mouse muscle and C2C12 myoblasts and myotubes myotubes, and C2C12 myoblasts. This work serves as a landmark for configuring the final pathway of carbohydrate oxidation.NEW & NOTEWORTHY We expand on knowledge of the architectural design of the mitochondrial lactate oxidation complex (mLOC); key members are: mitochondrial lactate dehydrogenase (mLDH), monocarboxylate transporter 1 (mMCT1), cytochrome oxidase (COx), basigin scaffolding protein (CD147), and the mitochondrial pyruvate carrier (mPC). The mLOC is key in creating the lower end of the concentration gradient for disposal of lactate and pyruvate.

Keywords: lactate; lactate shuttle; mitochondrial reticulum; pyruvate; skeletal muscle.

Figure 1.

Immuno (Western) blot separation of mitochondrial and cytosolic compartment proteins in National Institutes of Aging (NIA) mouse hind limb muscles. Little contamination of the mitochondrial (MI) fraction by cytosolic (CY) proteins in muscle (MU) is indicated: Columns represent subcellular fractions of mitochondrial (MI) and cytosolic (CY) compartments from 20 μg of mouse skeletal muscle (MU). We used classic loading controls for compartment purity including (cytochrome oxidase, COx) for mitochondria and (GAPDH) for the cytosol. Blot replicated on tissues of two other male and two female NIA C57Bl/6J mice (n = 5).

Figure 2.

Immunoblots from mitochondrial preparations of limb muscles from five different mice are depicted. The muscle mitochondrial preparations contain both lactate and pyruvate transporters that support the final pathway of carbohydrate oxidative disposal: immunoblots showing expression of pyruvate carriers (mitochondrial pyruvate carrier 1, mPC1) and (mPC2), lactate transporters (monocarboxylate transporter, mMCT1) and (mMCT4), the MCT chaperone (CD147), mitochondrial marker (cytochrome oxidase, COx), and the oxidoreductases (lactate dehydrogenase A, LDHA) and (LDHB). Total Protein Stain (TPS) was used to illustrate uniform loading of the gel between muscle samples. Mitochondrial fractions from skeletal muscle (1–5) and liver mitochondria (+) contained all the components of the lactate oxidation complex. Red blood cell membrane ghost lysates (−) contained concentrated amounts of MCT4 and LDH and showed no traces of mitochondrial proteins.

Figure 3.

Colocalization of lactate and pyruvate transporters and cytochrome oxidase highlight the rich mitochondrial networks of the skeletal muscle. Colocalization of monocarboxylate transporter (mMCT1)-mitochondrial pyruvate carrier 1 (mPC1)-cytochrome oxidase (COx) in fixed cross-sections from the skeletal muscle of mice (×63 magnification) demonstrate extensive colocalization. Individual fluorescence of mMCT1, mPC1, and COx draw attention to the mitochondrial reticulum. Yellow arrows indicate sarcolemmal fluorescence via MCT1 which cannot be seen by markers confined to mitochondrial proteins such as COx and mPC shown as green arrows. Orange arrows indicate colocalization. An n = 3 mice were used for correlation analysis with one representative image. Scale bar is 20 μm.

Figure 4.

Proximity ligation assays show that mitochondrial lactate and pyruvate transporters form a dynamic link required for balanced translocation across mitochondrial membranes in situ: A: red dots illustrate associations of proteins in C2C12 cells including the well-established monocarboxylate transporter (mMCT)1:CD147 interaction. Proximity ligation instituting the association between mitochondrial pyruvate carrier 1 (mPC1)-mMCT1. Negative controls between Normal IgG-mPC and Normal IgG-mMCT1 showing no interactions. Dots represent a positive interaction between the two proteins. Scale bar is 25 μm. B: proximity ligation showing merged figure (gold color, bottom right) of MitoTracker Red (top left), and mPC1-mMCT1 associations (green, top right), nuclei (blue, bottom left), in differentiated C2C12 myotubes in situ. Scale bar is 20 μm.

Figure 5.

Immunocoprecipitation of mitochondrial pyruvate carrier 1 (mPC1) and mitochondrial monocarboxylate transporter 1 (mMCT1) exhibits multiple interactions among each other and other members of the lactate oxidation complex immunoblots of (n = 3 technical replicates from 5 biological replicates of each muscle) coprecipitated proteins using protein-G magnetic beads. Antibodies for mPC1, mPC2, MCT1, MCT4, cytochrome oxidase (COx), CD147, lactate dehydrogenase (LDH), and IgG were used to pull down intact protein complexes. Mitochondrial preparations (100 μg) from the skeletal muscle of mice were used as the vehicle. Data collected showed interactions between multiple components of the mitochondrial lactate oxidation complex (mLOC). There were no significant pulldowns when normal IgG was used as the precipitating antibody; (B) Beads and (L) Lysate from bead pulldown.

Figure 6.

Enzyme activities of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) from isolated mitochondrial fragments. Specific activity of mitochondrial enzymes involved in the TCA cycle and ETC in mitochondrial (MI) and whole muscle homogenates (MU) from five mice. There were no differences between LDH and PDH activities. Data are presented as means ± SD. Statistical significance was assessed using a paired T test. Significance was determined at P < 0.05. • = PDH; ○ = LDH.