Sequence variations and two levels of MCT1 and CD147 expression in red blood cells and gluteus muscle of horses.

MCT1-CD147 complex is the prime lactate transporter in mammalian plasma membranes. In equine red blood cells (RBCs), activity of the complex and expression of MCT1 and CD147 is bimodal; high in 70% and low in 30%. We studied whether sequence variations contribute to the bimodal expression of MCT1 and CD147. Samples of blood and cremaster muscle were collected in connection of castration from 24 horses. Additional gluteus muscle samples were collected from 15 Standardbreds of which seven were known to express low amounts of CD147 in RBCs. The cDNA of MCT1 and CD147 together with a promoter region of CD147 was sequenced. The amounts of MCT1 and CD147 expressed in RBC and muscle membranes were measured by Western blot and mRNA levels in muscles by qPCR. MCT1 and CD147 were expressed in 20 castrates, and in four only were traces found. Sequence variations found in MCT1 were not linked to MCT1 expression. In CD147 linked heterozygous single nucleotide polymorphisms (SNPs) 389A>G (Met(125)Val) and 990C>T (3'-UTR) were associated to low expression of CD147. Also a mutation 168A>G (Ile(51)Val) in CD147 was associated to low MCT1 and CD147 expression. Low MCT1 and CD147 mRNA levels in gluteus were found in Standardbreds with low CD147 expression in RBCs. The results suggest that sequence variations affect the expression level of CD147, but do not explain its bimodality. The levels of MCT1 and CD147 mRNA correlated with the expression of CD147 and suggest that bimodality of their expression is regulated at transcriptional level.

MCT1, MCT4 and CD147 gene polymorphisms in healthy horses and horses with myopathy.

Polymorphisms in human lactate transporter proteins (monocarboxylate transporters; MCTs), especially the MCT1 isoform, can affect lactate transport activity and cause signs of exercise-induced myopathy. Muscles express MCT1, MCT4 and CD147, an ancillary protein, indispensable for the activity of MCT1 and MCT4. We sequenced the coding sequence (cDNA) of horse MCT4 for the first time and examined polymorphisms in the cDNA of MCT1, MCT4 and CD147 of 16 healthy horses. To study whether signs of myopathy are linked to the polymorphisms, biopsy samples were taken from 26 horses with exercise-induced recurrent myopathy. Two polymorphisms that cause a change in amino acid sequence were found in MCT1 (Val(432)Ile and Lys(457)Gln) and one in CD147 (Met(125)Val). All polymorphisms in MCT4 were silent. Mutations in MCT1 or CD147 in equine muscle were not associated with myopathy. In the future, a functional study design is needed to evaluate the physiological role of the polymorphisms found.

Effects of age and concentrate feeding on the expression of MCT 1 and CD147 in the gastrointestinal tract of goats and Hereford finishing beef bulls.

Monocarboxylate transporter 1 (MCT 1) necessary for the absorption of short chain fatty acids in the gastrointestinal tract, was measured in ventral wall of rumen, abomasum and duodenum of kids at age of 1 day and 1, 2 and 8 weeks. Samples from rumen, abomasum and duodenum were also taken from finishing beef bulls fed concentrate either ad libitum (A) or restrictively (R). Increased expression of MCT 1 was observed during the first week and parallel increases were found in its ancillary protein, CD147 in the rumen of kids. In duodenum, MCT 1 decreased with age and a similar tendency was seen in abomasum. In bulls, MCT 1 was higher in ventral wall and atrium than in other parts of gastrointestinal tract. However, in ventral wall of rumen MCT 1 was higher in A than in R. These findings show that MCT 1 increases with the development of rumen function and also in adult animals MCT 1 may change with the feeding.

Expression of lactate transporters MCT1, MCT2 and CD147 in the red blood cells of three horse breeds: Finnhorse, Standardbred and Thoroughbred.

REASONS FOR PERFORMING STUDY: In exercising horses, up to 50% of blood lactate is taken up into red blood cells (RBCs). Lactate transporter proteins MCT1, MCT2 and CD147 (an ancillary protein for MCT1) are expressed in the equine RBC membrane. In Standardbreds (SB), lactate transport activity is bimodally distributed and correlates with the amount of MCT1 and CD147. About 75% of SB studied have high lactate transport activity in RBCs. In other breeds, the distribution of lactate transport activity is unknown. OBJECTIVES: To study whether similar bimodal distribution of MCT1 and CD147 is present also in the racing Finnhorse (FH) and Thoroughbred (TB) as in the SB and to study the distribution of MCT2 in all 3 breeds and to determine if there is a connection between MCT expression and performance markers in TB racehorses. METHODS: Venous blood samples were taken from 118 FHs, 98 TBs and 44 SBs. Red blood cell membranes were purified and MCT1, MCT2 and CD147 measured by western blot. The amount of transporters was compared with TB performance markers. RESULTS: In TBs, the distribution of MCT1 was bimodal and in all breeds distribution of MCT2 unimodal. The amount of CD147 was clearly bimodal in FH and SB, with 85 and 82% expressing high amounts of CD147. In TBs, 88% had high expression of CD147 and 11% low expression, but one horse showed intermediate expression not apparent in FH or SB. Performance markers did not correlate with the amount of MCT1, MCT2 or CD147. CONCLUSIONS: High lactate transport activity was present in all 3 racing breeds, with the greatest proportion in the TB, followed by the racing FH, then SB. There was no significant statistical correlation found between lactate transporters in RBC membrane and markers of racing performance in the TB.

Effects of training on equine muscle fibres and monocarboxylate transporters in young Coldblooded Trotters.

REASONS FOR PERFORMING STUDY: Muscular changes caused by training are breed-specific and studies on the Norwegian-Swedish Coldblooded Trotter (NSCT) are limited. Knowledge about lactate-transporters in muscle in this light draught breed used for harness racing is lacking. OBJECTIVES: To identify muscular changes associated with training in young NSCTs and investigate muscular distribution of the monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147, which facilitate lactate transport across membranes. METHODS: Nine horses were followed from the start of their training period until the end of their 3-year-old season. A biopsy sample of the middle gluteal muscle was collected on 4 occasions. On the last 3 sampling occasions, individual V(La4)-values (the speed corresponding to a blood lactate concentration of 4 mmol/l) were determined in an incremental exercise test on a high-speed treadmill. One horse was excluded due to lameness. Histochemical and immunohistochemical analyses were performed on all muscle samples to determine fibre types (I, IIA, IIAX, IIX), oxidative capacity (NADH) and the expression of MCT1 and CD147. The activity of selected metabolic enzymes in the muscle before and after training was determined. RESULTS: The percentage of type IIX fibres decreased with training while the percentage of type IIAX fibres increased. The activity of citrate synthase and 3-OH-acyl-CoA-dehydrogenase increased with training. The expression of MCT1 was lower in membranes and cytoplasm of type IIX fibres compared to all other fibre types both before and after training. The antibody against CD147 stained membranes and cytoplasm of all fibres. The first V(La4)-value was lower than the last 2 in all horses. CONCLUSIONS: Muscular changes with training of NSCTs were similar to those reported in Standardbreds, indicating fibre type transitions and increased oxidative capacity. Expression of MCT1 differed among fibre types and was related to the oxidative capacity of the fibres.

Immunohistochemical analysis of MCT1 and CD147 in equine skeletal muscle fibres.

Monocarboxylate transporter 1 (MCT1) and its ancillary protein CD147 facilitate efflux of lactate from the muscle. Expression of MCT1 and CD147 were studied with immunohistochemistry in type I, IIA, IIAB and IIB fibres of equine gluteal muscle. Staining intensity of MCT1 in the cytoplasm as well as in the membranes of fibre types decreased in the order I=IIA>IIAB>IIB and correlated with the oxidative capacity. Capillaries were pronounced in the MCT1 staining. CD147 antibody stained plasma membranes of all fibre types evenly, whereas the staining in the cytoplasm followed that of MCT1. In the middle gluteal muscle the expression of MCT1 follows the oxidative capacity of muscle fibres, but the expression of CD147 in sarcolemma does not vary among fibre types. The use of horse specific MCT1 and CD147 antibodies can in future studies help to evaluate lactate efflux from different muscle fibre types.

Expression of CD147 and monocarboxylate transporters MCT1, MCT2 and MCT4 in porcine small intestine and colon.

Lactate, formed mainly in the stomach and small intestines, and short-chain fatty acids (SCFAs) formed in the colon, are ionised and require transporter proteins such as monocarboxylate transporters (MCTs) for absorption. The amounts of MCT1, MCT2, MCT4 and CD147, an ancillary protein for MCT1 and MCT4, were measured by immunoblotting the small intestine and colon of 40 pigs (Landrace, Yorkshire and LandracexYorkshire). MCT1 and MCT4 were found in both small intestine and colon, but MCT2 only in the small intestine. In both small intestine and colon, Yorkshire pigs had more CD147 than Landrace pigs, while no interbreed differences were found in MCT isoforms. Since CD147 is essential for the activity of MCT1 and MCT4, the breed difference suggests that MCT activity is higher in Yorkshire than in Landrace pigs. The absence of MCT2 in the colon suggests that it is mainly a lactate transporter, while MCT1 and MCT4 facilitate the transport of both lactate and SCFA.

The inducible form of heat shock protein 70 in the serum, colon and small intestine of the pig: comparison to conventional stress markers.

Modern rearing conditions may cause stress to pigs. At the cellular level all animals respond to stress by synthesizing heat shock proteins (HSP), which protect cells from injury. The objective of this study was to examine the concentrations of stress-inducible HSP72 in porcine small intestine and colon, known to be stress sensitive tissues, and to compare the findings with HSP72 concentrations in serum and with conventional markers of stress, namely blood lactate and serum cortisol, glucose, free fatty acids and acute phase proteins. HSP72 in the colon correlated with serum HSP72 but there was a negative correlation with carcass weight (growth). The results suggest that the colon may be a significant source of serum HSP72, the concentration of which may reflect changes in the permeability of intestinal epithelium due to stressors, such as transport and handling.

MCT1 and CD147 gene polymorphisms in standardbred horses.

REASONS FOR PERFORMING STUDY: Transport of lactate across membranes is facilitated by proton-monocarboxylate transporters (MCT). The most widely distributed isoform is MCT1, which needs an ancillary protein CD147. Studies on erythrocytes have shown that high activity of MCT1 is inherited as the dominant allele and that activity is regulated through CD147. Mutations of human MCT1 have been described that appear to impair lactate transport in muscles and cause exertional rhabdomyolysis. There are no reports of this potential relationship in the horse. OBJECTIVES: To obtain sequences of equine MCT1 and CD147 to examine differences between horses with high and low lactate transport activity in their erythrocytes. METHODS: Muscle biopsy samples were taken from 3 healthy Standardbred horses and from 7 horses which according to the owners had signs of myopathy after intense exercise. DNA and RNA were isolated and PCR analysis and sequencing performed. RESULTS: Currently, PCR fragments covering 100% of MCT1 and 70% of CD147 coding region are retained and sequence analysis has demonstrated one single nucleotide polymorphism (SNP) in the C-terminal area of MCT1 and one SNP in the extracellular domain of CD147. Both cause an amino acid change. The SNPs found are not related to lactate transport activity in erythrocytes or signs of myopathy. CONCLUSIONS: More samples need to be analysed to make conclusions on the significance of the polymorphisms found. Furthermore, full sequence coverage of the coding region of CD147 is needed. POTENTIAL RELEVANCE: The molecular probes produced could be used as tools to study gene regulation of lactate transport.

Monocarboxylate transporters (MCT) as lactate carriers in equine muscle and red blood cells.

REASONS FOR PERFORMING STUDY: Monocarboxylate transporters (MCT) facilitate the transport of lactate across membranes. In red blood cells (RBC) the transport activity varies interindividually due to differences in the amount of an ancillary protein CD147. Similar variations in muscles could have a great influence on lactate accumulation during exercise. OBJECTIVES: To study the expression of MCT isoforms and CD147 in the middle gluteal muscle. METHODS: Venous blood and muscle biopsy samples were taken from 14 Standardbred horses. Lactate transport activity in RBC and the amounts of MCT1, 2, 4 and CD147 were measured. RESULTS: In muscle MCT1, MCT4 and CD147 were found. Amount of MCT1 was variable and not dependent on age or training. Expression of MCT4 increased with age and correlated positively with CD147. CD147 in muscle correlated with that in RBC. MCT4 in muscle and CD147/MCT1 in RBC were higher in race fit than in moderately trained horses. CONCLUSIONS: MCT isoform profile in equine muscle is similar to that in man. The correlation between CD147 in muscle and RBC supports the view that lactate transport activity in muscles may vary interindividually as with RBC. POTENTIAL RELEVANCE: A larger number of horses need to be analysed to confirm the relationship of CD147 in muscle and RBC; and to allow the use the lactate transport activity in RBC as an indicator of the respective activity in muscles.

Carbohydrate metabolism in meat animals.

Oxidative energy production is by far dominant in living animal muscles, with the exception the short periods of severe stress, where the aerobic capacity is exceeded, and formation of large amounts of lactate and protons will take place. Energy consumption in muscle cells continues post-mortem with formation of large amounts of lactate and protons, because the aerobic processes for energy production are not available. Post-mortem, the fall in pH is delayed only by buffering capacity of the muscle fibres. In living animals, in addition to buffering capacity, both respiration and transport of lactate and protons out of the muscle fibres by monocarboxylate transporters participate in the regulation of muscle fibre pH which never falls as low as the ultimate pH of the meat. Understanding the regulation of pH in muscle is important both for the welfare of living animals and from the technological point of view as a factor influencing meat quality.

Distribution of monocarboxylate transporter isoforms MCT1, MCT2 and MCT4 in porcine muscles.

AIM: Monocarboxylate transporters (MCT), which cotransport lactate anions and protons across cell membranes, are important for regulation of muscle pH. We measured amounts of MCT1, MCT2 and MCT4 by immunoblotting in five different porcine muscles, to study MCT-isoform distribution both in oxidative and highly glycolytic muscles. METHODS: Samples from the longissimus dorsi, gluteus superficialis, semimembranosus, infraspinatus and masseter were taken from 18 slaughtered pigs. RESULTS: Oxidative capacity, estimated on the basis of the activities of lactate dehydrogenase (LDH), citrate synthase (CS) and 3-OH-acyl-CoA dehydrogenase (HAD), was highest in the infraspinatus and masseter, and was very low in the gluteus, semimembranosus and longissimus dorsi. In all muscles, the amount of MCT1 was small but variable. The amount of MCT2 was more abundant in the glycolytic than in the oxidative muscles, while MCT4 was found in equal amounts in all muscles. MCT2, but not MCT4, correlated negatively with CS and HAD. CONCLUSIONS: The results together with measured concentrations of lactate suggest that MCT2 may function as the housekeeping lactate transporter, preventing acidification especially in highly glycolytic muscles in which the capacity to oxidize lactate is low. The results also support the view that, as in other species, MCT4 would be important at high lactate concentrations that occur during stress.

Effect of exercise on plasma ferritin concentrations: implications for the measurement of iron status.

Iron is of key importance for aerobic metabolism, and natural feeds of the horse are fairly rich sources of iron. Accordingly, the known incidence of iron deficiency anaemia is apparently rare in performance horses; despite this, iron deficiency in performance horses continues to be of concern to trainers and veterinarians. Effects of exercise on plasma ferritin concentrations were therefore studied in Standardbreds, Finnhorses and half-bred riding horses. Blood samples were taken after a moderate exercise test on a racetrack, a competition exercise test on a treadmill and a race. Even moderate exercise caused an increase in plasma ferritin concentration, with the increase being greater as the intensity and duration of exercise increased. Return to the basal level was slower after maximal-intensity exercise than after moderate exercise. In conclusion, although ferritin is a useful marker of low iron stores, samples should be taken only after at least 2 days rest following strenuous exercise.

Induction of heat shock protein 72 mRNA in skeletal muscle by exercise and training.

In response to stress, cells synthesise heat shock proteins (HSP) to maintain protein homeostasis. To study whether exercise and training induce expression of HSP72 in the middle gluteal muscle, 10 Finnhorses performed a submaximal 60 min exercise test on a treadmill. Test A was performed after 3 months of training, and the other two tests 2 (B) and 5 (C) weeks later. Blood samples were taken during and after the tests, and biopsy samples before, immediately after and 23 h after each test. HSP72 mRNA was analysed using a digoxigenin-labelled probe. Blood lactate concentration in the 3 tests varied between 7.2 and 10.2 mmol/l. Training increased HSP72 mRNA, as indicated by increases in samples taken at rest (A

Lactate transport in red blood cells by monocarboxylate transporters.

The lactate transport activity of red blood cells (RBC) varies widely among different species; in equine RBC, the activity of the main lactate carrier, H+-monocarboxylate co-transporter (MCT), is distributed bimodally. The influence of lactate transport activity is measurable in vivo; after maximal exercise, the RBC lactate concentration in horses with high (HT) lactate transport activity is higher than in those with low (LT) activity. To study the expression of MCT in HT and LT horses, blood samples were taken from 10 horses at rest and after submaximal exercise. Blood and plasma lactate concentrations, lactate and pyruvate transport activities and the amounts of MCT1, MCT2 and MCT4 were measured. After exercise, RBC lactate concentration was higher in HT (n = 5) than in LT (n = 5) horses. At lactate concentrations of 0.25-30 mmol/l and at a pyruvate concentration of 1 mmol/l, transport activity was higher in HT horses. At a lactate concentration of 0.1 mmol/l, transport was similar. In Western blots, the signals for MCT1 and MCT2 were similar in both groups. The amount of CD147, a chaperone necessary for the activity of MCT1, was lower in LT horses. We suggest that MCT2 transports lactate at low concentrations, while MCT1 is needed at higher concentrations. MCT1 may be less active in LT horses and, therefore, during exercise their capacity to take up lactate is low. Further studies are needed to show whether the differences in lactate influx in RBC affect the function of erythrocytes or the performance capacity of horses.

Age-related changes and inheritance of lactate transport activity in red blood cells.

In red blood cell membranes, the activity of the main lactate carrier, H+-monocarboxylate co-transporter (MCT), varies interindividually and its distribution is bimodal. To show the repeatability of MCT activity, 2 to 5 blood samples were taken, at an interval of approximately 1 year, from 51 Standardbred horses, age 2 weeks-8 years, for a total of 128 observations. The horses could be divided into low (LT) and high (HT) lactate transport activity groups. Age significantly affected (P<0.05) MCT activity such that activity was highest in foals, reached a nadir at 2-3 years, and tended to increase again thereafter. Interindividual variation was not sufficiently high to allow a horse to switch from the LT-group to the HT-group, or vice versa. When MCT activity from 4 sires, 15 dams and their 52 offspring was analysed, the data showed that MCT activity is heritable and supported the hypothesis that low MCT activity was caused by a recessive allele in a single autosomal locus. Because MCT activity affects RBC lactate concentrations, the phenomenon may be physiologically significant.

Monocarboxylate transporters and lactate metabolism in equine athletes: a review.

Lactate is known as the end product of anaerobic glycolysis, a pathway that is of key importance during high intensity exercise. Instead of being a waste product lactate is now regarded as a valuable substrate that significantly contributes to the energy production of heart, non-contracting muscles and even brain. The recent cloning of monocarboxylate transporters, a conserved protein family that transports lactate through biological membranes, has given a new insight into the role of lactate in whole body metabolism. This paper reviews current literature on lactate and monocarboxylate transporters with special reference to horses.

Exercise-induced changes in atrial peptides in relation to neuroendocrine responses and fluid balance in the horse.

Previous data show that, in horses, plasma atrial natriuretic peptides (ANP and NT-ANP) remain elevated for a long time after exercise. To study whether exercise-induced changes in hormonal and fluid balance explain this, we measured plasma concentrations of COOH- and NH2-terminal atrial natriuretic peptides (ANP(99-129) and NT-ANP(1-98) together with arginine vasopressin (AVP), adrenocorticotrophin (ACTH), beta-endorphin, cortisol, catecholamines, and indicators of fluid balance in six Finnhorses after a graded submaximal exercise test on a treadmill. After exercise, AVP and catecholamines diminished rapidly; atrial peptides, ACTH, beta-endorphin, and cortisol remained elevated longer. ANP reached its peak value at 5 min and NT-ANP at 30 min post-exercise. At 60 min, ANP was still significantly increased and NT-ANP even above its level at the end of exercise. The different temporal patterns of ANP and NT-ANP are most probably explained by differences in their plasma half-lives. The post-exercise increase in NT-ANP indicates that the release of atrial peptides is stimulated during recovery after exercise. The rapid decrease in AVP and catecholamines suggests that these hormones do not explain the long-lasting increase in atrial peptides. Cortisol remained elevated longer and it may have contributed to some extent. After exercise, the packed cell volume (PCV) decreased more slowly than plasma total protein and electrolytes, which refers to a slow post-exercise return in blood volume. Taken together, the present results show that the long-lasting post-exercise increase in plasma atrial peptides in horses is most probably explained by elevated central blood volume and that the role of vasoactive hormones is small.

Lactate-transport activity in RBCs of trained and untrained individuals from four racing species.

In red blood cells (RBC) of horses, both lactate-transport activity and lactate accumulation during races vary interindividually. To study whether similar variation in lactate transport is apparent also in RBCs of other racing species, blood samples were collected from 21 reindeer, 40 horses, 31 humans, and 38 dogs. Total lactate-transport activity was measured at 10 and 30 mM concentrations, and the roles of the monocarboxylate-transporter (MCT) and the inorganic anion-exchange transporter (band-3 protein) were studied with inhibitors. In the reindeer and in one-third of the horses, lactate transport was low and mediated mainly by band-3 protein and nonionic diffusion. In the humans, dogs, and the remaining two-thirds of the horses, lactate transport was high and MCT was the main transporter. No correlation existed between MCT activity and the athleticism of the species. In the horses and humans, training had no effect on lactate transport, but in the reindeer and sled dogs, training increased total lactate transport. These results show that among the racing species studied, only in horses was the distribution of lactate-transport activity bimodal, and the possible connection between RBC lactate and performance capacity, especially in this species, warrants further studies.

Muscle fibre growth in undernourished reindeer calves (Rangifer tarandus tarandus L.) during winter.

To study whether moderate under-nutrition causes muscle wasting, reindeer (Rangifer tarandus tarandus L.) calves were fed either pelleted reindeer feed ad libitum (n=8) or restricted amounts of lichens (n=8). The restricted amount was 60% of ad libitum intake of lichens, and the feeding period was 6 weeks preceded by a 2-week adjustment period. Biopsy samples from the middle gluteal muscle (M. gluteus medius) for the analysis of fibre composition and area, as well as for the activity of cathepsin B were taken before the restriction period in November and January, and after the restriction period in April. In all calves the muscle fibre composition remained unchanged during the winter. In the lichen group, the fibre size also remained unchanged, whereas in control calves the cross sectional area of type I and type IIA fibres increased significantly from November to April. Cathepsin B activity decreased in all calves from November to January and remained at that low level for the rest of the study period, which suggests an attenuated rate of protein degradation. These results can be taken as an indication that moderate under-nutrition causes no muscle wasting in reindeer calves, and the decreased availability of nitrogen is partially compensated for by adaptive decrease in protein degradation. Interestingly the adaptive changes in protein metabolism are equally well seen in the well-fed controls as in the undernourished lichen-fed reindeer.