Tracing the lactate shuttle to the mitochondrial reticulum

Abstract

Isotope tracer infusion studies employing lactate, glucose, glycerol, and fatty acid isotope tracers were central to the deduction and demonstration of the Lactate Shuttle at the whole-body level. In concert with the ability to perform tissue metabolite concentration measurements, as well as determinations of unidirectional and net metabolite exchanges by means of arterial-venous difference (a-v) and blood flow measurements across tissue beds including skeletal muscle, the heart and the brain, lactate shuttling within organs and tissues was made evident. From an extensive body of work on men and women, resting or exercising, before or after endurance training, at sea level or high altitude, we now know that Organ-Organ, Cell-Cell, and Intracellular Lactate Shuttles operate continuously. By means of lactate shuttling, fuel-energy substrates can be exchanged between producer (driver) cells, such as those in skeletal muscle, and consumer (recipient) cells, such as those in the brain, heart, muscle, liver and kidneys. Within tissues, lactate can be exchanged between white and red fibers within a muscle bed and between astrocytes and neurons in the brain. Within cells, lactate can be exchanged between the cytosol and mitochondria and between the cytosol and peroxisomes. Lactate shuttling between driver and recipient cells depends on concentration gradients created by the mitochondrial respiratory apparatus in recipient cells for oxidative disposal of lactate.

Fig. 1. First report of LDH in the myocardium of rat heart.

Reaction products of LDH seen in the mitochondria are primarily located within the inner mitochondrial membranes (C, cristae). M matrix. X 93,000. Figure 11 from Baba and Sharma.

Fig. 2. First report using agarose gel electrophoresis to separate LDH isoforms in mitochondria from rat liver and heart.

LDH isoenzyme patterns differ between the cytosol and mitochondria in both tissues. Note cytosolic and mitochondrial LDH isoform differences between the cytosol and mitochondria across tissues. From Brooks et al..

Fig. 3. Plasma and mitochondrial membrane locations of monocarboxylate transporters (MCTs).

Figures showing the cellular locations of MCT1 and MCT2 lactate transporter isoforms and the mitochondrial reticulum (cytochrome oxidase, COx) in adult rat plantaris muscle determined using confocal laser scanning microscopy (CLSM) and fluorescent probes for the respective proteins; comparisons for MCT1 in the first row (Plates A1–A3, respectively) and for MCT2 in the second row (Plates B1–B3, respectively). The localization of COx was detected in rat plantaris muscle (Plates A1 and B1). MCT1 was detected throughout the cells, including the subsarcolemmal (arrowheads) and interfibrillar (arrows) domains (Plate A2). MCT1 abundance was greatest in oxidative fibers where COx was abundant and the signal was strong. When signals from probes for MCT1 (green) and COx (red) were merged, the superimposition of the two probes was clear (yellow), a finding prominent at the interfibrillar (arrows) and sarcolemmal (arrowheads) cell domains (Plate A3). In contrast, the signal for MCT2 (Plate B2) was weak and relatively more noticeable in fibers denoted by strong staining for COx (Plates B1 and B3, broken line delineated around oxidative fibers to distinguish the faint signal for MCT2). The overlap of MCT2 and COx is insignificant, denoted by the absence of yellow in Plate B3. Scale bar = 50 μm. Sections are from the same animal. Reprinted from Hashimoto et al..

Fig. 4. Immunohistochemical images demonstrating some components of the Mitochondrial Lactate Oxidation Complex (mLOC) in cultured L6 muscle cells.

This complex involves the mitochondrial constituent cytochrome oxidase (COx), lactate-pyruvate transport protein (MCT1), lactate dehydrogenase (LDH), and other constituents. A Colocalization of MCT1 and the mitochondrial reticulum. MCT1 was detected at both sarcolemmal and intracellular domains (A-1). Using MitoTracker, the mitochondrial reticulum was extensively elaborated and detected at intracellular domains throughout L6 cells (A-2). When signals from probes for the lactate transporter (MCT1, green, A-1) and mitochondria (red, A-2) were merged, the superimposition of the signals (yellow) showed the colocalization of MCT1 and components of the mitochondrial reticulum, particularly at perinuclear cell domains (A-3). In Panel (B), lactate dehydrogenase (LDH) (B-1), and mitochondrial cytochrome oxidase (COx) (B-2) are imaged. The superimposition of signals for LDH (red, B-1) and COx (green, B-2) shows the colocalization of LDH in the mitochondrial reticulum (yellow) of cultured L6 rat muscle cells (D-3). Depth of field ~1 μm, scale bar = 10 μm. Reprinted from Hashimoto et al..

Fig. 5. Results of efforts to deduce the organization of the mLOC using immunoprecipitation (IP) technology.

In the upper panel, representative immunoblots (IB) are shown using anti-COX, NADH-dh, LDH, or nIgG as precipitating antibodies (IPs). COX, NADH-DH, LDH, and nIgG were immunoprecipitated from mitochondrial fractions of L6 cells resuspended in a suspension medium without detergent. COX IP proteins were probed with MCT1, CD147, and LDH antibodies. MCT1, CD147, and LDH were coprecipitated with COX. NADH-dh IP pellets were probed with MCT1, COX, CD147, and LDH antibodies. Neither MCT1, CD147, nor LDH coprecipitated with NADH-DH, whereas COX coprecipitated with anti-NADH-dh. LDH IP proteins were probed with MCT1 and COX antibodies. Both MCT1 and COX coprecipitated with LDH. No protein coprecipitated with nIgG from mitochondrial fractions of L6 cells resuspended in medium without detergent (negative control). In the lower panel, the degree of coprecipitation evaluated by comparing signals in the IP and the lysate is shown. The results were categorized into four levels: +++, 80% or more precipitated; ++, ∼50% precipitated; +, 20% or less precipitated; -, no precipitation. IB immunoblot, IP immunoprecipitation. From Hashimoto et al..

Fig. 6. Schematic showing the putative lactate oxidation complex.

Lactate is oxidized to pyruvate via mitochondrial LDH (mLDH) in association with COx. This endergonic lactate oxidation reaction is coupled to the exergonic redox change in COx during mitochondrial electron transport. The transport of pyruvate across the inner mitochondrial membrane is facilitated by MCT1. GP glycerol phosphate, Mal-Asp malate-aspartate, ETC electron transport chain, TCA tricarboxylic acid. Figures 4 and 5 show the results of mitochondrial respiration studies^,,. Modified from Hashimoto et al..

Fig. 7. Cellular colocalization of mitochpondrial lactate (VCTs) and pyruvate transprters (mPCs).

Images assessing the colocalization of MCT1 and mPCs in L6 cells, which show the localization of DAPI-positive nuclei (A), MCT1 (B), mPC1 (C), and MitoTracker-positive MR (D) in L6 cells. The merged images are shown in (E). Colocalization analysis of mPC1 (C) and mitochondria D showed a Pearson correlation coefficient (r²) value of 0.8. Colocalization analysis of MCT1 (B) and mPC1 (C) showed an r² of 0.3, largely because MCT1 occupies sarcolemmal, mitochondrial and peroxisomal compartments. A channel to represent the colocalization of MCT1 and mitochondria was created to image mMCT1; subsequent colocalization of mMCT1 with mPC1 resulted in an r² of 0.8 (F). White dots indicate the colocalization of mMCT1 and mPC1 as observed in ImageJ software. Whole images were contrast-enhanced in (A, B, C, D, and E). Similar results were observed for mPC2. Scale bar = 20 µm. It appears that both MCT1 and the putative mPC colocalized to the mitochondria (r² = 0.8). However, at the light microscopic level, it is impossible to know if the two proteins interact physically and functionally. Additionally, with the benefit of the Orbitrap LC/MS device, we could determine the fractional synthesis rates of mLOC and mPC proteins. From Brooks.

Fig. 8. Differential cellular localizations in mitochondrial Lactate Oxidation Complexes (mLOC) proteins in normal and cancer breast cell lines.

Immunohistochemical detection of LDH, MCT isoforms, and cytochrome oxidase (COx) in the control breast cell line HMEC-184, Plate A, left, and in the breast cancer cell line MCF-7, Plate B, right. LDH isoforms MCT2 and MCT4 colocalized with the mitochondrial protein marker COx (rows A, C, D) but not MCT1, which was localized mainly in the plasma membrane in the control HMEC cell line (Plate B, Row B, left). Thus, mMCT isoform expression in breast cell mitochondria differs from that in skeletal muscle, where MCT1 predominates (Figs. 4 and 5); in control and cancer breast cells, MCT-2 and -4 isoforms colocalized with the inner mitochondrial membrane component COx. The thickness of the optical sections, ~1 µm, scale bar = 1 µm. Images from Hussien and Brooks.