Tissue-specific and isoform-specific changes in MCT1 and MCT4 in heart and soleus muscle during a 1-yr period

Hideo Hatta1, Mio Tonouchi2, Dragana Miskovic2, Yuxiang Wang2, John J. Heikkila3, and Arend Bonen2

1 Department of Life Sciences (Sports Sciences), University of Tokyo, Tokyo 153, Japan; and Departments of 2 Kinesiology and 3 Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the postnatal changes (days 10, 36, 84, 160, 365) of monocarboxylate transporters (MCT)1 and MCT4 in rat heart and soleus muscle. In the heart, MCT1 protein and mRNA remained unaltered from day 10 until 1 yr of age. Both MCT4 protein and mRNA in the heart were detected at 10 days of age, but the MCT4 protein and transcript were not detected thereafter. In the soleus muscle, MCT1 protein (+38%) and mRNA (+136%) increased during the first 84 days and remained stable until 1 yr of age. In contrast, soleus MCT4 protein decreased by 90% over the course of 1 yr, with the most rapid decrease (-60%) occurring by day 84 (P < 0.05). At the same time, MCT4 mRNA was increased by 74% from days 10 to 84 (P < 0.05), remaining stable thereafter. In conclusion, developmental changes in MCT transport proteins are tissue specific and isoform specific. Furthermore, it appears that MCT1 expression in the heart and MCT1 and MCT4 expression in the soleus are regulated by pretranslational processes, whereas posttranscriptional processes regulate MCT4 expression in the soleus muscle.

monocarboxylate transporters; protein; messenger ribonucleic acid; phosphofructokinase; citrate synthase; glucose transporter 4


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOVEMENT OF LACTATE across the plasma membrane of heart and skeletal muscle occurs via a transport system involving carrier proteins (16, 19). There appears to be a family of seven monocarboxylate transporter (MCT) isoforms (MCT1-7) that can transport a variety of monocarboxylates (see Ref. 16 for review). Tissue-specific expression has been shown for some of these MCTs, although several are coexpressed in heart and skeletal muscle (27, 38). The transport kinetics of the various MCT proteins for different monocarboxylates as well as the regulation of MCT isoform expression remain to be clarified.

MCT1 is present in heart and skeletal muscles, as well as in other tissues (2, 18, 24, 25). In general, in adult rats, MCT1 is present in greater quantities in heart than in skeletal muscles (2, 4, 25), whereas among different types of rat skeletal muscles, the expression of MCT1 is highly correlated with their oxidative capacities (25). Increasing the oxidative capacities of muscles by chronic electrical stimulation increases their MCT1 content (4, 24). Exercise training increases MCT1 in both heart and muscle (2). In these studies, the changes in lactate uptake rates parallel the changes in MCT1 in heart and muscles (2, 24). Indeed, we have recently shown (5) that an increase in lactate uptake by muscle occurs when only MCT1, but not MCT4, protein levels are increased.

Thus far, much less is known about MCT4. In human heart, MCT4 is detectable, but in hearts obtained from rats it is not (38). Skeletal muscle expresses both MCT1 and MCT4 (4, 5, 38); however, MCT4 is present in very low concentrations in the slow-twitch oxidative soleus muscle from mature animals, whereas it is very abundant in fast-twitch oxidative glycolytic muscles (4, 38). MCT4 concentrations are also positively correlated with the content of fast-twitch glycolytic muscle fibers in different rat muscles (4), and among rat hindlimb muscles with different metabolic capacities there is an inverse relationship between the MCT1 and MCT4 content (4). Finally, MCT1 has a greater affinity for lactate [Michaelis-Menten constant (Km) ~4 mM] (6) than does MCT4 (Km ~30 mM) (11).

On the basis of the relationships between MCT1 and the oxidative capacity of muscle fibers, we have proposed that MCT1 expression may reflect the capability to oxidize lactate (24, 25). Along similar lines, we have proposed that the relationship between MCT4 and the glycolytic capacity of muscle fibers may reflect the muscle's requirement for lactate efflux (4, 38). This view is also supported by others (11).

During early postnatal life, marked alterations occur in substrate metabolism in heart and soleus muscle. In the rabbit and rat hearts, there is a marked reduction in the rates of glycolysis and a sharp increase in palmitate oxidation in the first 7-20 days of life (13, 22). In soleus muscle during the first 12 wk of life, there are changes in oxidative capacity, muscle fiber composition, muscle fiber area, and muscle capillarization (31, 32). However, all of these changes in muscle do not occur at the same time. For example, soleus muscle succinate dehydrogenase activity attains a maximum at 28 days of age, which is maintained at least up to 84 days of age, whereas muscle capillarization attains a maximum 2 wk later, at 6 wk, and soleus muscle fiber composition and the areas of type I and type IIa fibers increase for at least 12 wk, albeit at different rates (31, 32). Thus, clearly, muscle architecture, substrate delivery, and metabolic capacities do not change in a tightly coordinated pattern during the first 12 wk of life in the rat.

During the life span of the rat (<= 25 mo), changes in the substrate transporters in muscle and heart have been examined only for GLUT-1 and GLUT-4. These studies have shown that, in the early postnatal period (days 1-16), GLUT-1 decreased rapidly in skeletal muscle and heart (30), whereas GLUT-4 increased in both tissues (30, 34). Importantly, the rates of change in GLUT-1 and GLUT-4 differed, and the rates of change of each glucose transporter isoform differed when these were compared in muscle and heart (30, 34). In some skeletal muscles, but not all, GLUT-4 content is reduced in old (24-25 mo) compared with young (2-3 mo) rats (3, 8, 15, 20), although these decreases, when observed, occurred somewhere between 1 and 10 mo of age (15). Collectively, the foregoing studies indicate that changes in substrate transporters such as GLUT-1 and GLUT-4 occur rapidly in postnatal life, and the more gradual changes in GLUT-4, when they are observed, are not closely related to changes in muscle metabolism or architecture.

Whether there are age-dependent changes in the MCT1 and MCT4 in heart and/or muscle is not known. In view of the changes in the metabolic capacities of both the heart and soleus muscle during the early postnatal period, it can be expected that there may also be marked changes in MCT1 and MCT4 in these tissues. However, beyond this period, there may also be further changes in the expression of these MCTs that are not necessarily tightly associated with changes in the metabolic capacities of heart and soleus muscle, as has been shown for GLUT-4 (15). We hypothesized that, as the heart and soleus muscle become more dependent on oxidative metabolism, particularly early in life, MCT1 would be increased, whereas MCT4 would concomitantly be reduced. We also hypothesized that MCT4 in muscle might be reduced further after the early postnatal period, because lactate release from muscle is reduced during the first year of life (14). Therefore, in the present study we have examined, in rat soleus muscle and heart, the tissue-specific changes in MCT1 and MCT4 proteins, as well as their transcripts, over a 1-yr period (days 10-365). We also compared changes in GLUT-4 protein and mRNA in these studies with previous reports (10, 34, 36) as a positive control for the present studies.


    METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Sprague-Dawley rats were used. Ethical approval was obtained for this work from the committee on animal care at the University of Waterloo. Animals were bred in the University of Waterloo animal care facilities. They were handled daily and were allowed free access to water and food ad libitum. Soleus muscles and the hearts of the rats were taken while they were anesthetized (Somnotol, ip). Tissues were obtained in neonatal rats (day 10) just after weaning at 28 days [i.e., days 33-40 (mean = 36)], after sexual maturity (>42 days) had occurred [i.e., days 80-88 (mean = 84)], and during adult life [i.e., days 143-170 (mean = 160) and at 1 yr of age].

Enzyme analyses. Activities of phosphofructokinase (PFK) and citrate synthase (CS) in heart and soleus muscles were analyzed using standard procedures (23).

Western blotting of MCT1 and MCT4. Proteins were isolated from soleus muscles and the heart and detected using Western blotting, as we have previously described in detail (4, 5, 24, 25, 38). MCT1 and MCT4 antibodies for these purposes were a gift from from Dr. A. P. Halestrap (Dept. of Biochemistry, University of Bristol, Bristol, UK). GLUT-4 antibodies were obtained from East-Acres Biologicals (Southbridge, MA). For each set of Western blots, data from hearts and soleus muscles at each time point were included, as well as a rat heart standard. This permitted normalization of the data to the rat heart standard across the different blots. MCT1, MCT4, and GLUT-4 protein band densities were obtained by scanning the blots on a densitometer connected to a Macintosh LC computer with appropriate software.

Northern blot analysis. mRNA determinations were made as we (4, 5) have described previously. Briefly, 3 µg of total RNA were used for electrophoresis on 1.2% formaldehyde agarose gels (29) and then transferred to positively charged nylon membrane (Boehringer Mannheim, Laval, QC, Canada). The Northern blots were ultraviolet cross-linked with a GS Gene linker (Bio-Rad, Mississauga, ON, Canada). Human GLUT-4 cDNA was obtained from American Type Culture Collection (Rockville, MD). MCT1 cDNA and MCT4 cDNA were a gift from from Dr. A. P. Halestrap.

A 1.9-kb fragment containing the coding sequence of MCT1 cDNA was isolated from the full length (3.3 kb) MCT1 cDNA by digestion with the EcoRI restriction enzyme (17) and subcloned into the EcoRI restriction enzyme site of pBluescript (KS). The orientation was checked by digestion with HindIII restriction endonuclease. Template DNA was linearized with XbaI restriction enzyme, and digoxigenin (DIG)-labeled antisense MCT1 riboprobe was generated by in vitro transcription with T3 RNA polymerase. MCT4 cDNA was originally subcloned into BamHI/ApaI restriction enzyme sites of pBluescript (38). DIG-labeled antisense MCT4 riboprobe was generated by digestion of the template DNA with XbaI restriction enzyme and in vitro transcription with T7 RNA polymerase. GLUT-4 cDNA was subcloned into pGEM-4Z. A 2-kb-long DIG-labeled GLUT-4 antisense riboprobe was generated by digestion of the template DNA with BamHI restriction endonuclease and in vitro transcription with T7 RNA polymerase.

The ingredients for RNA transcription included 1-2 µg of DNA template plus the NTP mix [in nM: 2.5 CTP, 2.5 GTP, 2.5 ATP, 1.625 UTP (Promega, Madison, WI) and 0.875 Dig-11 UTP (Boehringer Mannheim)], 20 dithiothreitol (Promega), and 1 U/µg template DNA of RNase Inhibitor (Promega) and 1× RNA polymerase buffer [5× buffer: 400 mM Tris · HCl pH 7.5, 60 mM MgCl2, and 20 mM spermidine-HCl (Promega)] maintained at room temperature. The appropriate RNA polymerase [T3 or T7 RNA polymerases (Boehringer Mannheim)] was added (>= 20 IU/µg of DNA template) and incubated for 2 h at 37°C. The DNA template was then digested for 10 min at 37°C with RNase-free DNase (1 IU/1 µg of DNA template; Promega). After precipitation in ethanol and centrifugation at 12,000 rpm for 15 min, the probe was resuspended in 10-20 ml DIG easy-hyb hybridization buffer (Boehringer Mannheim) or standard hybridization buffer with 50% formamide [5× standard sodium citrate, 50% formamide, 0.1% sodium lauroylsarcosine, 0.02% SDS, and 2% blocking reagent (Boehringer Mannheim)].

After prehybridization of the membrane for at least 4 h at 68°C, the prehybridization buffer was replaced with the same buffer containing DIG-labeled antisense RNA probe, and the membrane was incubated with the probe overnight at 68°C. High-stringency washes and chemiluminescent detection were performed in accordance with the protocol supplied by the manufacturer (Boehringer Mannheim), and the membrane was exposed to Kodak BioMax film. After exposure, the film was developed in Kodak developer and fixed in Kodak fixer. By using a 40-lane gel, we were able to load all of the data at one time. MCT1, MCT4, and GLUT-4 mRNA band densities were obtained by scanning the blots on a densitometer connected to a Macintosh LC computer with appropriate software.

Data analyses. The data were analyzed with analyses of variance. Significance was determined with Fischer's least significant differences post hoc test. All data are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

During the course of the study, body weight of the rats increased progressively from 21.7 ± 0.8 g at 10 days of age to 515.5 ± 9.0 g at 1 yr of age (P < 0.05; Fig. 1A). Soleus weights (P < 0.05) and heart weight (P < 0.05) were also increased during this period (Fig. 1B).


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Fig. 1.   Changes in body weight (A), and the weights of heart and soleus muscle (B) from day 10 until 1 yr of age (mean ± SE); n = 20 at day 10, and n = 9-12 on days 36-365. In some cases, the error bars fall within the size of the symbol. *Day 36 vs. day 10, P < 0.05; **day 84 vs. day 36, P < 0.05; ***day 160 vs. day 84, P < 0.05; ****day 365 vs. day 160, P < 0.05.

Enzyme activities. In the heart, neither PFK nor CS activities were altered over the course of 1 yr (P > 0.05; Fig. 2A). In soleus muscle, CS activity was increased ~40% from days 10 to 36 (P < 0.05; Fig. 2B). In contrast, there was a marked decrease in PFK activity from days 10 to 36 (38% decrease, P < 0.05; Fig. 2A). Thereafter, PFK activity was not further altered in this muscle.


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Fig. 2.   Changes in phosphofructokinase (PFK; A) and citrate synthase (CS; B) activities in heart and soleus muscle (mean ± SE); n = 3-4 at each time point. PFK: *soleus, day 10 vs. day 36, P < 0.05. CS: *soleus, day 10 vs. day 36, P < 0.05.

Age-related changes in MCTs. Data in the present study were normalized to the results obtained on day 10 (100%) in both soleus muscle and the heart. Representative MCT1 and MCT4 Western and Northern blots obtained from animals ranging in age from 10 to 365 days of age are shown in Figs. 3 and 4.


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Fig. 3.   Representative Western blots of MCT1, MCT4, and GLUT-4 in rat heart and soleus muscles at selected ages, in days.



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Fig. 4.   Representative Northern blots of MCT1, MCT4, and GLUT-4 in rat heart and soleus muscles at selected ages, in days.

Heart MCT1 and MCT4. In the heart, the MCT1 protein content was not altered (<10% change) from day 10 until 1 yr of age (P > 0.05; Fig. 5A). Similarly, MCT1 mRNA abundance in the rat heart was not altered (P > 0.05; Fig. 5B). In contrast, MCT4 protein was detectable only at 10 days of age, but not thereafter (Fig. 6A). Similarly, MCT4 mRNA transcripts were also detected only at 10 days of age (Fig. 6B).


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Fig. 5.   Changes in monocarboxylase transporter (MCT)1 protein (A) and MCT1 mRNA (B) in heart and soleus muscle (mean ± SE); n = 5-10 at each time point for protein determinations, and n = 3 at each time point for mRNA determinations. *Day 36 vs. day 10; **days 84, 160, and 365 vs. day 36, P < 0.05.



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Fig. 6.   Changes in MCT4 protein (A) and MCT4 mRNA (B) in heart and soleus muscle (mean ± SE); n = 4 at each time point for protein determinations, and n = 3 at each time point for mRNA determinations. *Days 84, 160, and 365 vs. day 36; **day 36 vs. day 10; ***day 365 vs. day 160, all P < 0.05.

Soleus MCT1 and MCT4. In the soleus muscle, MCT1 increased 38% from day 10 until 84 days of age. (P < 0.05; Fig. 5A), with no changes thereafter. During this same 84-day period. MCT1 mRNA was also increased (136%; P < 0.05; Fig. 5B).

Soleus muscle MCT4 protein increased slightly (15%) from days 10 to 36 (P = 0.05). By 84 days, MCT4 protein was reduced by 60% from concentrations observed at day 10 (P < 0.05; Fig. 6A). There was then a further decrease until 1 yr of age when MCT4 fell to 10% of that observed on day 10 (P < 0.05). Thus, over the course of almost 1 yr (days 10-365), MCT4 concentrations were reduced by 90% (P < 0.05; Fig. 6A).

Although MCT4 protein content was decreasing in soleus muscle, MCT4 mRNA increased. From day 10 until 84 days of age, MCT4 mRNA was increased by 74% (P < 0.05; Fig. 6B). Thereafter, the MCT4 mRNA abundance remained constant until 1 yr of age (Fig. 6B).

Heart and soleus muscle GLUT-4. GLUT-4 protein increased rapidly from days 10 to 36 in heart (P < 0.05) and soleus muscle (P < 0.05; Fig. 7). In heart, a plateau was attained by day 36, whereas in soleus this was not observed until day 84. Heart and soleus muscle GLUT-4 transcripts also increased rapidly from days 10 to 36 (P < 0.05). In soleus muscle, a plateau was attained by 160 days, but in the heart, GLUT-4 mRNA was seen to increase from days 36 to 88 (P < 0.05) and from day 88 to 1 yr of age (P < 0.05; Fig. 7).


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Fig. 7.   Changes in GLUT-4 protein (A) and GLUT-4 mRNA (B) in heart and soleus muscle (mean ± SE); n = 4 at each time point for protein determinations, and n = 3 at each time point for mRNA determinations. A: *days 36, 84, 160, and 365 vs. day 10; **days 84 and 160 vs. day 36. B: heart: *days 36, 84, 160, and 365 vs. day 10; **day 365 vs. day 36; ***day 365 vs. day 84; soleus: dagger days 88, 170, and 365 vs. day 36; dagger dagger day 365 vs. day 88. All significant at P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first study to examine changes in MCT1 and MCT4 in heart and soleus muscles over the course of a 1-yr period. This time frame included the early postnatal period (day 10), the immediate postweaning period (day 36), and the period of sexual maturity and adult life (days 84, 160 and 365). In the early postnatal period (<36 days), profound changes occur in substrate metabolism in the heart and muscle, whereas thereafter (>36 days), changes continue to occur in muscle architecture and capillarization (31, 32). We have presented new data that indicate that postnatal MCT protein expression is regulated in an isoform- and tissue-specific manner. Specifically, 1) over a 1-yr period, MCT1 protein content was not altered in the heart, whereas 2) MCT1 was increased in soleus muscle to adult levels by 36-84 days of age. In contrast, 3) in the heart, MCT4 protein expression was completely repressed within the first 36 days of life, and 4) MCT4 protein expression in soleus muscle was rapidly repressed (-60%) in the first 84 days of life and was almost fully repressed (-90%) by 1 yr of age.

For comparison purposes, we also measured the changes in GLUT-4 protein and mRNA in heart and muscle. In agreement with other studies (30, 34, 36), we also observed that GLUT-4 protein and mRNA were increased in soleus muscle and in the heart (Fig. 7). Thus the data obtained on GLUT-4 protein and GLUT-4 mRNA provided a positive control for our studies.

In the present studies, MCT1 was not altered in the heart but was increased in soleus muscle, whereas MCT4 was reduced in both tissues. Tissue- and isoform-specific changes have previously been observed in the glucose transporter proteins GLUT-1 and GLUT-4. From birth until 16 days of age, GLUT-1 is repressed ~50-fold, whereas GLUT-4 is increased about sixfold during this same period in heart and muscle (30). Others have shown similar increases in heart and muscle GLUT-4 during postnatal development (days 0-41), whereas adipose tissue GLUT-4 is concomitantly decreased (34). Thus the tissue- and isoform-specific changes in MCT1 and MCT4 are consistent with observations made previously for GLUT-1 and GLUT-4 transport proteins.

The mechanisms for these tissue- and isoform-specific changes in MCT1 and MCT4 over the course of 1 yr are difficult to identify. It is known that GLUT-4 expression in perinatal life is regulated by thyroid hormone in the heart but not in brown adipose tissue (9), but whether this hormone also regulates MCT1 and MCT4 is not known. It has also been suggested that the dietary changes in the suckling-to-weaning transition period may account for differences in tissue-specific expression of GLUT-4 during development (see Ref. 34 for discussion). However, this is speculative, and to date, the regulation of MCT transporter expression by diet has not been examined.

In the heart, no changes were observed in PFK or CS activity, whereas the activities of both of these enzymes were altered in the soleus muscles from the same animals (i.e., CS was increased, whereas PFK was decreased). The lack of changes in these enzymes in the heart seems to be at odds with the known reductions in glycolysis and the concomitant increase in fatty acid oxidation in the heart (13, 22). For example, in rabbit hearts, from days 1 to 7 of life, rates of glycolysis and glucose oxidation were decreased 80 and 50%, respectively, whereas palmitate and lactate oxidation rates were increased 1,200 and 170%, respectively (22). Fatty acid metabolism in the rat heart is also increased during development and attains adult oxidation rates 20 days after birth (13). Thus it may be that measuring the maximal activities of PFK and CS does not provide a good index of changes in substrate metabolism in the heart. Perhaps other markers (e.g., beta -hydroxyacyl-CoA dehydrogenase or fatty acid transporters) would have provided a better index of the changes in substrate metabolism in the heart.

Despite the lack of changes in PFK and CS activities in the heart, the reduction in glycolysis, along with a concomitant increase in fatty acid oxidation in the developing heart (13, 22), would seem to provide a partial explanation for the reduction in MCT4 in this tissue, because we (4, 38) and others (11) have previously suggested that the MCT4 isoform may have a specialized role to facilitate the extrusion of lactate from the tissue. Thus, as the heart becomes more reliant on fatty acid metabolism and glycolysis is reduced (13, 22), the need for lactate extrusion is also reduced. Consequently, the need for MCT4, a low-affinity lactate transporter (Km ~30 mM) (11) that exports lactate out of the tissue (4, 11, 38), is also reduced.

MCT1 levels were not altered in the heart despite the changes in fuel metabolism that occur in the early postnatal period. We did not observe any changes in the activity of the mitochondrial marker enzyme CS over the course of 1 yr. The recent reports that MCT1 in heart is present in the plasma membrane (18) and mitochondria (7) support our suggestions (2, 5, 24, 25, 38) that the MCT1 transporter is located to favor the uptake of lactate and to facilitate the oxidative disposal of lactate. The lack of change in MCT1 suggests that the lactate uptake and oxidation capacities in the rat heart are not altered during the first year of life (days 10-365). However, in the rabbit heart, lactate oxidation is increased (+170%) in the 1st wk of life (22). Our first measurements in rat hearts were obtained at 10 days of age. There is some kinetic evidence to suggest that rat hearts express another MCT protein (37), which has not yet been cloned. It may well be that this MCT is more closely linked to changes in the substrate metabolism in the neonatal heart.

In contrast to the heart MCT1, soleus muscle MCT1 was increased during the early postnatal period (<36 days). This increase in MCT1 parallels the increase in oxidative metabolism in the soleus muscle during the same time period (32). This is likely due to the increased postural work being performed by the soleus muscle in the rapidly growing neonate (12). In the first few weeks of life (days 10-36) there was a 40% increase in CS activity, which is similar to the increase in succinate dehydrogenase activity observed in the 7- to 30-day postnatal period in rat soleus muscle (32). Previously, we have shown, in adult muscle, that MCT1 expression is closely correlated with the muscles' oxidative capacities (24, 25); therefore, we (24, 25) have proposed that MCT1 expression may reflect the ability of muscle to oxidize lactate. Recently, MCT1 has been localized to the plasma membrane (4) and mitochondrion in skeletal muscle (7). This subcellular distribution of MCT1 would favor the uptake and oxidative disposal of lactate. When we (5) increased the oxidative capacity of muscle via chronic electrical stimulation, MCT1, but not MCT4, was increased, and at the same time, lactate uptake was also increased. In addition, in similar studies, we found that lactate uptake in control and experimental muscles was highly correlated with the MCT1 content of these muscles (r = 0.93) (24). Thus our studies (5, 24) indicated that an increase in MCT1 alone is sufficient to increase lactate uptake by muscle. Consequently, we can surmise that the increase in soleus muscle MCT1 in the present study enhanced the capacity for lactate uptake by this muscle.

The decrement in soleus muscle MCT4 is likely attributable to the reduction in fast-twitch muscle fibers in the soleus muscle (12, 32), because MCT4 is expressed primarily in these fast-twitch muscle fibers (4, 38). Along with the reduction in fast-twitch fibers is a reduced capacity for glycolytic metabolism in this muscle. We (4, 38) have previously suggested that the MCT4 isoform may have a specialized role in muscle, namely, to facilitate the extrusion of lactate from the tissue. This suggestion is given further credence in the present study, because the greatest reduction in MCT4 protein roughly parallels the reduced glycolytic capacity of the soleus muscle, as indicated by the reduction in PFK activity. Given that MCT4 is confined to the fast-twitch muscle fibers (4, 38), the MCT4 in the 1-yr-old soleus muscle is presumably attributable to the few remaining fast-twitch oxidative fibers (1, 25).

There is good reason to believe that skeletal muscle activity is one factor regulating the expression of MCT1 and MCT4, although the type of muscle activity may be critical. When muscles are denervated, both MCT1 and MCT4 are decreased (38). However, when muscle activity is increased and the oxidative capacity of the muscles is enhanced, then MCT1 is also increased [i.e., 1) with increased postural work by soleus (present study), 2) with treadmill training (2), or 3) with increased contractile activity induced with chronic electrical stimulation (24)]. However, these types of aerobic muscle activity patterns can either reduce MCT4 in young animals (present study) or fail to alter MCT4 in mature animals [i.e., chronic muscle stimulation (5)]. With more intense contractile activity, both MCT1 (+70%) and MCT4 (+30%) have been shown to be increased (26). Thus the intensity of muscle contractile activity may be a critical determinant regulating the expression of MCT1 and MCT4.

The changes in the relative levels of MCT1 mRNA in heart and soleus muscle over the course of 1 yr paralleled the pattern observed for the MCT1 protein. This suggests that MCT1 gene expression is regulated at the transcriptional and/or posttranscriptional level. Similar processes may regulate MCT4 gene expression in heart, because both MCT4 mRNA and MCT4 protein were rapidly reduced in the heart (<36 days). More complex regulation of MCT4 gene expression may occur in the soleus muscle, because MCT4 mRNA levels were elevated at the same time that MCT4 protein was decreased. This suggests that MCT4 gene expression in the soleus muscle may be controlled at the posttranscriptional and/or translational level. It is possible that MCT4 mRNA may be masked and unavailable for translation, as found for the developmental expression of the small subunit of ribonucleotide reductase, cyclin A, and fibroblast growth factor receptor-1 (28, 33, 35). Alternatively, MCT4 mRNA may bind an inhibitory protein that prevents translation, as has been shown for the regulation of ferritin gene expression (21). At present, we have no data that can provide an explanation for the regulation of MCT4. Interestingly, in previous studies, we found that the relative levels of MCT1 protein and mRNA are correlated in different muscle types, whereas MCT4 protein and mRNA are not correlated (4). Elucidation of the mechanisms regulating MCT4 gene expression will require additional experiments.

In summary, we have shown that MCT protein expression over the course of 1 yr, in soleus muscle and the heart, is regulated in 1) an isoform-specific manner and 2) a tissue-specific manner. It appears, therefore, that in both the heart and soleus muscle, MCT4 expression is repressed at the same time that glycolysis is reduced in these tissues. Thus, in muscle and heart, MCT4 expression conforms to our suggestions that this MCT isoform may have a specialized role to facilitate the extrusion of lactate from the tissue (4, 38). Conversely, the increase in MCT1 in the soleus muscle parallels the increase in slow-twitch oxidative muscle fibers in this muscle in the first few months of life (12, 32). This is consistent with our suggestions (4, 24, 25, 38) that MCT1 expression may reflect the ability of muscle to oxidize lactate. In contrast to soleus muscle, in the heart, MCT1 expression is not coordinated with known changes in substrate metabolism. There may be an as-yet-undiscovered MCT in the heart that is more closely regulated with changes in cardiac substrate metabolism. Although the mechanisms regulating MCT1 and MCT4 expression are not known, the present study suggests that MCT1 expression in the heart and MCT1 and MCT4 expression in the soleus are regulated by pretranslational processes, whereas posttranscriptional processes regulate MCT4 expression in the soleus muscle.


    ACKNOWLEDGEMENTS

We thank Dr. A. P. Halestrap, Dept. of Biochemistry, University of Bristol, Bristol, UK, for providing the MCT1 and MCT4 antibody and the MCT1 cDNA and MCT4 cDNA for our studies.


    FOOTNOTES

These studies were supported by the Heart and Stroke Foundation of Ontario and by the Natural Sciences and Engineering Research Council of Canada.

Address for reprint requests and other correspondence: A. Bonen, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON N2L 3G1, Canada (E-mail: abonen{at}healthy.uwaterloo.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 October 2000; accepted in final form 3 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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