Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat plantaris muscle

Abstract

We addressed the need for histological assessment of myocellular domains occupied by monocarboxylate transporters (MCT1, MCT2 and MCT4). From the perspective of lactate shuttle hypotheses we posited that MCT1 would be highly expressed in oxidative fibres, whereas MCT4 would be found in highly glycolytic fibres. Furthermore, we hypothesized that MCT1 would be detected at interfibrillar as well as at subsarcolemmal and sarcolemmal cell domains, whereas MCT2 and MCT4 abundances would be most prominent at the sarcolemma. To test these hypotheses, we examined cellular locations of MCT1, MCT2 and MCT4 transporter proteins in different fibre types (slow oxidative, SO; fast oxidative glycolytic, FOG; fast glycolytic, FG) in rat plantaris muscles by the avidin-biotin complex (ABC) as well as other methods. The plantaris was used as it is a mixed fibre skeletal muscle. MCTs, glucose transporter (GLUT4) protein, and mitochondrial constituent cytochrome oxidase (COX) abundances were assessed by immunohistochemistry and Western blotting using affinity-purified antibodies. The staining method was specific and stable, which allowed for semiquantitative assessment of MCT expression. As well, confocal laser scanning microscopy assessed MCT isoform localizations. Findings of the present study were: (1) MCT1 is located at the sarcolemma and throughout the cell interior in SO and FOG fibres where the mitochondrial reticulum was present; (2) in contrast, MCT4 was highly expressed in the sarcolemmal domain of FG and FOG fibres but poorly expressed in SO fibres; and (3) confocal laser-scanning microscopy demonstrated that MCT1 and COX are co-localised at both interfibrillar and subsarcolemmal cell domains, whereas MCT2 is only faintly detected at the sarcolemma of oxidative fibres. MCTs and associated proteins are positioned to facilitate the function of the lactate shuttles.

Figure 1. Serial cryostat sections stained for myosin ATPase

A, acid preincubation; B, alkaline preincubation, C, SDH; D, α-GPD; E, MCT1; F, MCT4; and G, GLUT 4. Fibre-type identifications were made on the bases of myosin ATPase, SDH, and α-GPD staining (A–D). No primary antibody was reacted in control (H). Thin arrows in E indicate MCT1 abundance near the sarcolemma of oxidative (SO and FOG) fibres. Very little MCT1 is visible in FG fibres, either at sarcolemmal or interfibrillar cell domains. MCT4 was concentrated at the cell surface in fast (FOG and FG) fibres (thin arrows in F). Note that in SO fibres MCT1 is strongly stained (thick arrows in E), while little MCT4 is detected (arrowheads in F). Sections 10 μm; scale bar = 50 μm. Data obtained on four rats (Table 2), all panels shown from the same animal.

Figure 2. The quantitative analysis of MCT1, MCT4, and GLUT4 protein expression and SDH and α-GPD contents in terms of optical density (OD) in different fibre types are shown

Values are expressed means ±s.e.m.*P < 0.05, **P < 0.01.

Figure 3. Cellular locations of MCT1 and MCT2 lactate transporter isoforms and the mitochondrial reticulum (cytochrome oxidase, COX) determined using confocal laser scanning microscopy (CLSM) and fluorescent probes for the respective proteins

A–C, comparisons for MCT1, and D–F, for MCT2. The localization of COX was detected in rat plantaris muscle (A and D). MCT1 was detected throughout the cells including subsarcolemmal (arrowheads) and interfibrillar (arrows) domains (B). MCT1 abundance was greatest in oxidative fibres where COX is abundant and the signal strong. When these MCT1 (green) and COX (red) were merged, superposition of the two probes was clear (yellow), a finding prominent at interfibrillar (arrows) as well as sarcolemmal (arrowheads) cell domains (C). In contrast, the signal for MCT2 (E) was weak, relatively more noticeable in fibres denoted by strong staining for COX (D and F, broken line is delineated around oxidative fibre to distinguish the faint signal for MCT2). Overlap of MCT2 and COX is insignificant, denoted by absence of yellow in F. Sections 10 μm scale bar = 50 μm. Sections from the same animal.

Figure 4. The co-localization of MCT1 (green) and COX (red) was observed as a yellow colour at interfibrillar as well as sarcolemmal cell domains

Note that approximately half of the subsarcolemmal regions show yellow colour where MCT1 and COX are co-localized (arrows). Sections 10 μm; scale bar = 20 μm. Section from the same muscle as shown in Fig. 3.