Skeletal muscle monocarboxylate transporter content ... - Springer Link

Eur J Appl Physiol (2009) 105:623–632 DOI 10.1007/s00421-008-0942-0

ORIGINAL ARTICLE

Skeletal muscle monocarboxylate transporter content is not different between black and white runners Yolande X. R. Harley Æ Tertius A. Kohn Æ Alan St Clair Gibson Æ Timothy D. Noakes Æ Malcolm Collins

Accepted: 10 November 2008 / Published online: 22 November 2008 Ó Springer-Verlag 2008

Abstract The superior performance of black African runners has been associated with lower plasma lactate concentrations at sub-maximal intensities compared to white runners. The aim was to investigate the monocarboxylate transporters 1 (MCT1) and MCT4 content in skeletal muscle of black and white runners. Although black runners exhibited lower plasma lactate concentrations after maximum exercise (8.8 ± 2.0 vs. 12.3 ± 2.7 mmol l-1, P \ 0.05) and a tendency to be lower at 16 km h-1 (2.4 ± 0.7 vs. 3.8 ± 2.4 mmol l-1, P = 0.07) than the white runners, there were no differences in MCT1 or MCT4 levels between the two groups. For black and white runners together, MCT4 content correlated significantly with 10 km personal best time (r = -0.74, P \ 0.01) and peak treadmill speed (r = 0.88, P \ 0.001), but MCT1 content did not. Although whole homogenate MCT content was not different between the groups, more research is required to explain the lower plasma lactate concentrations in black runners. Keywords Lactate Monocarboxylate transporters Fibre type Ethnicity Performance

Y. X. R. Harley T. A. Kohn (&) A. St Clair Gibson T. D. Noakes M. Collins Department of Human Biology, University of Cape Town, UCT/MRC Research Unit for Exercise Science Sports Medicine, PO Box 115, Newlands, Cape Town 7725, South Africa e-mail: [email protected] M. Collins South African Medical Research Council, UCT/MRC Research Unit for Exercise Science Sports Medicine, PO Box 115, Newlands, Cape Town 7725, South Africa

Introduction The world of endurance running is dominated by black athletes originating from the East African continent, with a few black runners from South Africa also performing well in distances varying from 800 m to the marathon (Baker and Horton 2003; Larsen 2003; Onywera et al. 2006). This superior performance of black runners has not yet been definitely related to any physiological, biochemical or genetic variable (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Saltin et al. 1995b; Scott et al. 2003, 2005a, b, Weston et al. 1999). Plasma lactate concentrations are lower in black runners compared to white Studies from the past two decades on black African runners have shown one consistent finding that might be associated with their superior performance and ability to resist fatigue—lower plasma lactate concentrations at sub-maximal running intensities compared to their white counterparts (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Saltin et al. 1995b; Weston et al. 1999, 2000), with one showing this after a maximal exercise test (Coetzer et al. 1993). Previous research has shown a relationship between muscle fatigue (and thereby performance) and lactate accumulation. More specifically, athletes that have a low, rather than high plasma lactate concentration at the same exercise intensity can withstand fatigue for a longer period of time (Tesch et al. 1978). It may, therefore, be argued that whatever causes the plasma lactate concentrations to be lower, may at least partly be the advantage black runners have over others. From the studies on South African black and white runners, no consistent finding on either a physiological or

123

624

biochemical level has been able to explain the lower plasma lactate concentrations (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Weston et al. 1999). In all these studies, no differences were observed in maximum _ 2 max ), or maximum heart rate oxygen consumption (VO (HRmax) (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Weston et al. 1999). While others showed no difference in respiratory exchange ratio (RER—an indicator of fuel metabolism) at maximum exercise intensity, only one showed this to be lower in black runners (Coetzer et al. 1993). Furthermore, two investigations showed that black runners were able to utilize a higher percentage of their _ 2 max during sub-maximal exercise tests (Bosch et al. VO 1990; Weston et al. 2000), with others showing no difference (Coetzer et al. 1993; Kohn et al. 2007; Weston et al. 1999). Although not measured in all studies, muscle fibre type and enzyme analyses in biopsies from these athletes revealed controversial results and could not explain the lower plasma lactate concentrations. One study showed that black runners have significantly more type IIA fibres (high mitochondrial content, high oxidative and glycolytic capacity, fast in contraction speed) and less type I fibres (very high mitochondrial content and oxidative capacity, but slow in contraction speed) than white runners (Kohn et al. 2007), whereas others found no difference (Coetzer et al. 1993; Weston et al. 1999). As type I fibres have a higher ability to oxidise fat and carbohydrate, and a lower ability to produce lactate than type IIA fibres (Esseń-Gustavsson and Henriksson 1984; Pette and Staron 1993), the fibre type results from the ethnic studies would suggest that black runners should produce equal or even more plasma lactate than their white counterparts. This suggests that another mechanism related to lactate kinetics differs in these two populations, resulting in the lower plasma lactate concentration in the black athletes. Weston et al. (1999) attributed the lower plasma lactate concentrations to a higher oxidative capacity for both carbohydrate and fat oxidation, as evidenced by greater citrate synthase and 3-hydroxyacetyl CoA dehydrogenase activities in muscle biopsies, whereas Kohn et al. (2007) found no differences in these enzymes. However, the latter authors found that the lactate dehydrogenase (LDH) activities in both muscle homogenate and single fibre pools were significantly higher in black compared to white runners. In all, the exact biochemical or physiological explanation for the lower plasma lactate concentrations in black runners remains unknown. Monocarboxylate transporters Lactate transport across plasma membranes is facilitated by monocarboxylate transporters (MCT). These symporters

123

Eur J Appl Physiol (2009) 105:623–632

allow lactate to move (in conjunction with an H? ion) from a high to low concentration, either from skeletal muscle to the circulation, or from the latter to various active or inactive skeletal muscles or other tissues where it may be metabolized (Bergman et al. 1999; Brooks 2007; Halestrap and Price 1999). The MCT family of transporters is currently known to consist of 14 isoforms. The MCT1 and MCT4 isoforms are found abundantly in skeletal muscle, but may be expressed in other tissues (Benton et al. 2004; Halestrap and Price 1999; Hashimoto and Brooks 2008). Skeletal muscle MCTs are found in the plasma membrane and mitochondrial membranes, and in both oxidative and glycolytic fibres (Dubouchaud et al. 2000; Hashimoto et al. 2005). The predominant expression of MCT1 in oxidative fibres and MCT4 in glycolytic fibres suggests that, although the transporters are bidirectional in function, MCT4 is well positioned to assist the efflux of lactate produced in cells with high rates of glycolysis, while MCT1 is well placed to allow the influx of lactate into oxidative fibres (Fishbein et al. 2002; Hashimoto et al. 2005; Pilegaard et al. 1999; Wilson et al. 1998). This redistribution of lactate ties in with the theories of the cell–cell and intracellular lactate shuttles, which hold that lactate is a key intermediate in fuel utilization during exercise. According to these hypotheses, lactate produced by glycolytic muscle fibres becomes an energy source at other sites, and so can be consumed by oxidative fibres, where it is taken up by the mitochondria (Brooks 1998). Lactate kinetics and MCTs During high intensity exercise, the rate of lactate production may surpass the rate of lactate clearance from blood, thus elevating the overall plasma lactate concentration. Studies investigating the effect of endurance training on lactate kinetics have shown an improved lactate clearance after training and lower lactate production by skeletal muscle (Bergman et al. 1999; MacRae et al. 1992). The production, removal and oxidation of lactate by skeletal muscle are complex processes and their detailed review falls beyond the scope of this manuscript [for recent reviews, see Gladden (2008) and Hashimoto and Brooks (2008)]. Exercise training results in a multitude of metabolic adaptations. Along with a decrease in lactate production and improved lactate clearance, an increase in the MCT content of skeletal muscle is evident (Burgomaster et al. 2007; Dubouchaud et al. 2000). Lowering training intensity has also been shown to lead to a decrease in MCT content (Evertsen et al. 2001). The content of MCTs plays an important role in overall blood lactate concentrations. Higher concentrations of MCT1 are associated with


increased blood lactate removal and lower net lactate concentrations (Thomas et al. 2005). Furthermore, muscle MCT content has been found to correlate (MCT1), or tend to correlate (MCT4), positively with supramaximal work capacity (Messonnier et al. 2007). In light of recent findings that skeletal muscle LDH activity was higher in black runners compared to their white counterparts (in theory being able to produce more lactate for the same level of glycolysis), and given that they have lower plasma lactate concentrations at the same absolute workloads, the possibility arises that factors involved in the lactate shuttle system may differ between black and white runners, and this may involve the MCT isoform content (Kohn et al. 2007). The aim No study currently exists on the MCT content of skeletal muscle of runners of different ethnicities. The aim was to investigate the MCT1 and MCT4 content in homogenate skeletal muscle samples of black and white endurance runners, matched for performance, with a view to elucidate whether this may, at least in part, explain the lower plasma lactate concentrations observed in black runners. The hypothesis is that black runners could have a higher abundance of MCT1 and/or MCT4 in their skeletal muscle, which may facilitate a more rapid release and uptake of lactate during exercise resulting in the lower plasma lactate concentrations observed in these athletes.

Materials and methods Subjects The study was approved by the Ethics and Research Committee of the Faculty of Health Sciences at the University of Cape Town. Seven white and eight black male sub-elite athletes were recruited from local athletics clubs in the Western Cape region, South Africa, and each signed a written informed consent. Each athlete filled in a questionnaire where personal details and recent performance in races were recorded. Athletes were excluded if they had any injuries during the previous six months. For the purpose of the study, athletes were selected according to their recent 10 km personal best (PB), and excluded if their time was greater than 40 min. All athletes were familiarized with running on a treadmill (Quinton Instruments, Seattle, USA) before actual testing commenced. Before each running test, they were allowed a 5 minute warm-up on the treadmill. Athletes were encouraged to be well rested

625

before each test and to abstain (where possible) from hard training sessions and competitions. Anthropometric measurements Each subject underwent a thorough anthropometric analysis that included height, weight and seven skinfolds (biceps, triceps, subscapular, supra-iliac, abdominal, midthigh and calf sites) (Ross and Marfell-Jones 1991). Body fat was expressed as a percentage (%BF) based on the method of Durnin and Womersley (1974). Forearm, subgluteal, mid-thigh, above-knee and calf girths along with the sub-gluteal to above-knee height were used for the calculation of muscle mass (%MM) (Martin et al. 1990). Lean thigh volume (LTV) was calculated using the assumption that the thigh was the shape of a truncated cone (Coetzer et al. 1993; Katch and Katch 1974). All measurements were performed by the same investigator to remove inter-observer error. Body mass index (BMI) was calculated from the weight divided by the (height)2 of the subject. Maximum exercise test Each athlete performed a maximal exercise test according to Noakes et al. (1990) with slight modifications. Briefly, athletes commenced the test at a treadmill speed of 10 km h-1 for 30 s, whereafter the speed was increased by _ 2; 0.5 km h-1 every 30 s until voluntary exhaustion. VO RER, and HR were recorded throughout the test (Jaeger Oxycon, The Netherlands). Peak treadmill speed (PTS) was defined as the fastest speed the athlete was able to complete the full 30 second workload at before voluntary exhaustion. An intravenous catheter and three-way stopcock were fitted to the athletes’ antecubital vein, and flushed with saline containing 0.04% heparin (Heparin Novo, Novo Nordisk). Prior to and directly after the test, a blood sample was collected, centrifuged at 3,0009g and the plasma stored at -20°C until analyses. Lactate concentrations (in mmol l-1) were determined using a commercially available enzymatic spectrophotometric assay kit (Lactate PAP, Bio Merieux, France). Sub-maximal exercise test In order to assess sub-maximum running ability, each athlete completed four workloads of 5 min each at set speeds of 10, 12, 14, and 16 km h-1, with recording of _ 2 ; RER and HR. Between these workloads, athletes VO rested for 5 min and a blood sample was collected straight after cessation of the workload, processed and analyzed as described above. All athletes were able to complete the four prescribed workloads.

123

626

Muscle biopsy A suction-assisted muscle biopsy was performed by a qualified medical practitioner from the Vastus lateralis as previously described by Bergstro¨m (1962) and Evans et al. (1982). Where possible, the biopsy was divided into two parts, one part frozen in embedding medium (Tissue-Tek, Miles Laboratories Inc., Naperville, IL, USA) and rapidly frozen in isopentane, precooled with liquid nitrogen for fibre type analyses, and the remaining part frozen in liquid nitrogen for MCT analyses. All samples were stored at -80°C. Fibre type Muscle fibre type was determined using a modification of the ATPase staining method described by Brooke and Kaiser (1970) on serial cross-sections. Fibres were classified according to their staining intensities at pH 4.30, 4.60, and 9.40, and identified as either type I, type IIA or type IIX (Brooke and Kaiser 1970). The different types were then expressed as a percentage of the total number of fibres counted.


(Beckman DU-62) using the bicinchoninic acid assay according to the manufacturer’s instructions (Pierce, IL, USA) with bovine serum albumin as a standard. A total of 5 lg protein from each sample was combined with sample buffer to yield a total volume of 20 ll, boiled for 5 min and loaded onto a gel (10% separating gel, 4% stacking gel). Electrophoresis was carried out for ±2 h at 150 V. The proteins were transferred to a PVDF membrane at 100 V for 1 h. The membranes were briefly washed in Tris-buffered saline containing 0.1% Tween (TBS-T) and blocked over night at 4°C in 10% non-fat dried milk-TBS-T. Thereafter, the membranes were incubated with primary antibodies against MCT1 or MCT4 (kindly donated by G. Brooks, University of California at Berkley), subsequent secondary antibodies and visualized using enhanced chemiluminescence (KPL LumiGLO, MA, USA) exposed to X ray film. Membranes were stained with Ponceau S to verify equal total protein loading. Films were photographed using a photodocumentation system (UVItec Ltd, Cambridge, UK) and band intensities determined using UVIDocMw software (UVIDocMw version 99.04, UVISoft). Values were normalized against a control sample run on each gel and expressed as arbitrary units ± SD.

Monocarboxylate transporters Statistical analyses Identification of the MCT isoforms was performed using a modification of the methods described by McCullagh et al. (1996) and Dubouchaud et al. (2000). Each muscle sample (±127 mg) was homogenized with a manual glass homogenizer at 4°C in 3 ml Buffer A [210 mM sucrose, 2 mM EGTA, 40 mM NaCl, 30 mM HEPES, pH 7.4 and 4.5 ll protease inhibitor cocktail (Sigma, St Louis, USA)], transferred to a 15 ml polypropylene tube and the homogenizing tube rinsed with 1 ml Buffer A to make a final volume of 4 ml in the 15 ml polypropylene tube. The homogenate was centrifuged using a desktop centrifuge (Mistral 2000R, MSE, UK) at 6009g for 10 min at 4°C to eliminate red blood cell material. The resulting supernatant was transferred to a new centrifuge tube (10.4 ml polycarbonate, Beckman, CA, USA) with 0.75 volume of Buffer B (1.167 M KCl, 58.3 mM sodium pyrophosphate, pH 7.4) and centrifuged (Beckman Optima L-70 Ultracentrifuge; 40Ti fixed angle rotor) at 145,000 9g for 2 h at 4°C. The resulting pellet was washed with 1 ml Buffer C (1 mM EDTA, 10 mM Tris, pH 7.4) and then resuspended in 200 ll Buffer C by passing the mixture through a pipette followed by a needle (21 gauge needle, 1 ml syringe). To this suspension, 66 ll of 16% SDS was added, mixed and centrifuged (Beckman J2-21; JA14 rotor) at 1,1009g for 20 min at room temperature. The supernatant was collected, divided into aliquots (±30 ll) and stored at -80°C for subsequent Western blotting. Protein concentrations were determined spectrophotometrically

123

All values are expressed as means ± standard deviation (SD) and the statistical analyses of data were performed using the Statistica software package (StatSoft, USA). Between-group analyses were conducted using an unpaired Student’s t-test. Relationships between variables were investigated using the two-tailed Pearson’s correlation test. Significance for all analyses was set at P \ 0.05. Due to small biopsy size, only six of the black athletes had data for muscle fibre type.

Results Physiological parameters The physiological characteristics and recent 10 km PB of the black and white runners are presented in Table 1. Although the white runners were significantly heavier and taller than the black runners, there was no significant difference in BMI. Furthermore, %BF, %MM and LTV were also not different between the two groups. Maximum exercise test No significant differences were found between black and _ 2 max ; HRmax, RERmax, and PTS white runners for VO (21.4 ± 1.2 vs. 21.5 ± 1.4 km h-1, respectively) (Table 2).


627

Table 1 Subject characteristics and recent 10 km personal best times of white and black runners White runners (n = 7)

Black runners (n = 8)

Age (years)

23 ± 2

21 ± 3

Height (cm)

180 ± 7

167 ± 4*

Weight (kg)

70 ± 6

60 ± 6*

-2

Table 2 Sub-maximal and maximum exercise test results of white and black runners

BMI (kg m )

21.6 ± 1.5

21.4 ± 1.6

%BF

12.8 ± 4.6

11.2 ± 1.8

%MM

53.6 ± 1.9

55.1 ± 2.3

LTV (l) 10 km time (min)

3.49 ± 0.68 35.5 ± 3.0

3.68 ± 0.33 34.2 ± 2.8

Values are means ± SD BF Body fat, BMI body mass index, LTV lean thigh volume, MM muscle mass *Different from white runners, P \ 0.01

On the other hand, plasma lactate concentrations were significantly lower directly after the maximum exercise test in the black runners compared to their white counterparts (8.8 ± 2.0 vs. 12.3 ± 2.7 mmol l-1, P \ 0.05) (Fig. 1). Sub-maximal exercise test No significant differences were found between the two _ 2 expressed as either per kg or percentage of groups for VO maximum, or for RER or HR at all four sub-maximal intensities (Table 2). Plasma lactate concentrations showed a tendency to be lower in the black runners after the 16 km h-1 workload compared to the white runners at the same intensity (2.4 ± 0.7 vs. 3.8 ± 2.4 mmol l-1, P = 0.07) (Fig. 1), with no differences at the lower running intensities.

White runners (n = 7) 10 km h-1 _ 2 (ml min-1 kg-1) VO RER HR (bpm) 12 km h-1 _ 2 (ml min-1 kg-1) VO


36.4 ± 2.4

36.0 ± 1.7

0.82 ± 0.04 130 ± 11

0.84 ± 0.04 124 ± 7

43.1 ± 1.6

43.3 ± 2.2

RER

0.86 ± 0.03

0.84 ± 0.02

HR (bpm)

143 ± 11

136 ± 6

14 km h-1 _ 2 (ml min-1 kg-1) VO

51.0 ± 2.3

50.2 ± 2.4

RER

0.88 ± 0.04

0.86 ± 0.03

HR (bpm)

158 ± 13

151 ± 10

58.6 ± 2.6

57.1 ± 3.4

RER

0.93 ± 0.06

0.90 ± 0.04

HR (bpm)

171 ± 10

166 ± 10

HRmax (bpm)

68.9 ± 4.3 192 ± 7

66.7 ± 5.4 189 ± 6

RERmax

1.17 ± 0.05

1.15 ± 0.06

PTS (km h-1)

21.5 ± 1.4

21.4 ± 1.2

16 km h-1 _ 2 (ml min-1 kg-1) VO

Maximum exercise test _ 2 max (ml min-1 kg-1) VO

Values are means ± SD _ 2 Oxygen consumption, HR heart rate, RER respiratory exchange VO ratio, PTS peak treadmill speed

Muscle fibre type and MCT content No differences in muscle fibre type from the V. lateralis were found between black and white runners (Table 3). MCT1 (Fig. 2a) and MCT4 (Fig. 2b) isoform content were also not different between the two groups. A negative correlation between the MCT4 content and 10 km PB of runners (Fig. 3c, r = -0.74, P \ 0.01) was found, with a stronger positive correlation between MCT4 content and PTS (Fig. 3d, r = 0.88, P \ 0.001). A relationship was not found between MCT1 and either 10 km PB (Fig. 3a) or PTS (Fig. 3b). No relationships were found between muscle fibre type and either of the two MCT isoforms (data not shown).

Discussion This is the first study to compare skeletal muscle MCT content in runners from two distinct ethnic groups. The

Fig. 1 Plasma lactate concentrations during the sub-maximal exercise test and maximum exercise test in white (open bars, n = 7) and black (solid bars, n = 8) runners. Values are means ± SD. Different from white runners: * P \ 0.05, P = 0.07

main finding was that no difference existed in MCT1 or MCT4 content in V. lateralis muscle samples between subelite black and white runners. Furthermore, there was a strong correlation between MCT4 content and both 10 km PB and PTS, which is consistent with similar findings by Messonnier et al. (2007). This study has also confirmed

123

628


Table 3 Fibre type determined from biopsies of the Vastus lateralis muscle in white and black runners White runners (n = 7)


Type I (%)

49 ± 16

56 ± 17

Type IIA (%)

49 ± 18

39 ± 15

Type IIX (%)

1±2

5 ± 12

Values are means ± SD

Physiological characteristics of athletes Consistent with similar previous studies, black runners were lighter and shorter in stature than their white counterparts (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Noakes et al. 2004; Saltin et al. 1995a; Weston et al. 1999). The differences in these two factors may be genetic, but can also result from nutrition during growth, which would involve socio-economic factors. This complication is challenging to avoid, as it is exceedingly difficult to match the two ethnic groups for mass and still retain viable subject numbers, due to the natural size difference that occurs between black and white South African males. On the other hand, it could also be argued that it is perhaps better not to match the ethnic groups for mass, as the chosen groups would then not be representative of their races, as a typical group of black runners would be smaller in size than a typical group of white runners. No differences were also found for %BF, %MM and LTV between the two groups. While it has been suggested that ethnicityspecific equations are needed when calculating percentage body fat in populations of different ethnic backgrounds (Nindl et al. 1998), there are, to our knowledge, no such ethnicity-specific equations or correction factors to apply differently to the two populations studied. Despite the weight and height differences, the two _ 2 max values, 10 km PB, and PTS, in groups had similar VO agreement with previous studies (Coetzer et al. 1993; Kohn et al. 2007; Weston et al. 1999). Noakes et al. (1990) have shown that PTS is a better and accurate predictor of 10 km _ 2 max : It is therefore concluded that the 10 km PB than VO PB times provided by the athletes were a true representation of their current performance level and was validated by their respective PTS during the maximal exercise test. Muscle fibre type

Fig. 2 MCT1 (a) and MCT4 (b) content in skeletal muscle samples of white (open bars, n = 7) and black (solid bars, n = 8) runners. Values are means ± SD

previous observations that black runners have lower plasma lactate concentrations at sub-maximal and maximal running intensities than their white counterparts (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Saltin et al. 1995b; Weston et al. 1999).

123

The present study found no difference in fibre type between black and white runners (Table 3), in accordance with previous studies (Coetzer et al. 1993; Saltin et al. 1995a; Weston et al. 1999). This is in contrast to a recent study by Kohn et al. (2007) where black runners had significantly more type IIA fibres compared to their white counterparts. It is not clear why the findings of this study were different, however, these same authors also showed that type I and IIA single fibre pools from black runners had higher LDH activities than those from white runners, suggesting that there might be differences in muscle physiological characteristics between the ethnic groups. In a previous study, the MCT1 content correlated positively with the percentage type I fibres of human Soleus, Triceps brachii, and V. lateralis muscles, analysed as one group. On the other hand, no relationship was


629

Fig. 3 Relationship between 10 km PB or PTS and MCT content in skeletal muscle samples of white (open bars, n = 7) and black (solid bars, n = 8) runners. a 10 km PB versus MCT1 (Pearson’s r = -0.12, not significant). b PTS versus MCT1 (Pearson’s r = 0.01, not significant). c 10 km PB versus MCT4 (Pearson’s r = -0.74, P \ 0.01). d PTS versus MCT4 (Pearson’s r = 0.88, P \ 0.001)

found between the MCT4 content and fibre type (Pilegaard et al. 1999). A similar correlation with oxidative fibre content was found when red and white muscle groups from rats were analysed for MCT1 (McCullagh et al. 1996). No relationships between the fibre types and the two MCT isoforms were found in the present study. This is supported by a study by Evertsen et al. (2001) who found no relationship between either MCT1 or MCT4 and the three fibre types. The difference in outcome with respect to MCT1 between the studies might be attributed to the training status of the subjects and muscle groups analysed. For example, the subjects in the study by Pilegaard et al. (1999) comprised a range of individuals that varied substantially in fitness level. On the other hand, the subjects in the present study and that of Evertsen et al. (2001) were all well trained, specialized athletes. Furthermore, only one muscle group was analysed in the present study, whereas those of Pilegaard et al. (1999) and McCullagh et al. (1996) used multiple muscle groups. Interestingly, Pilegaard et al. (1999) also showed that within a given muscle section the MCT1 content might be relatively fibre type independent, whereas the MCT4 content may be dependent on fibre type. Although the present study only looked at MCT content in homogenate muscle samples, more research is required to clarify the relationship between MCT content and the different fibre types, and how training affects the expression.

Plasma lactate concentrations and MCT content It is now well established that black African runners have lower plasma lactate concentrations at moderate to high intensity workloads (Bosch et al. 1990; Coetzer et al. 1993; Kohn et al. 2007; Weston et al. 1999). The present study showed that at 16 km h-1 and at maximum exercise intensity, the plasma lactate concentrations of the black runners were *1.6-fold (P = 0.07) and 1.4-fold (P \ 0.05) lower, respectively, than that of the white runners. Kohn et al. (2007) and Weston et al. (1999) showed lower concentrations in black runners at 80% PTS (*17.5 km h-1) and 88% PTS (*19.2 km h-1), respectively. Coetzer et al. (1993) only showed lower concentrations in black runners at a high intensity (21 km h-1). Therefore, the present study once again showed that black runners tend to exhibit lower plasma lactate concentrations at the higher sub-maximal running intensities. This is unlikely to be related to net oxygen _ 2 ; RER and HR did consumption or fuel utilization as VO not differ between the black and white runners at any of the five workloads. This finding was therefore consistent with the hypothesis that the black runners may differ from white runners in muscle lactate transport, and this may involve differences in MCT content. Despite the plasma lactate differences, this investigation found no difference in the level of MCT1 or MCT4

123

630

expression between the two groups (Fig. 2). This suggests that the total cellular MCT1 or MCT4 content in the muscle is unlikely to be responsible for the lower plasma lactate values observed in black compared to white South African runners with exercise. It also suggests that total muscle MCT1 or MCT4 content does not account for the observed superior performance of black runners. This finding does not, however, preclude the involvement of MCTs in the observed ethnic discrepancy in exercising plasma lactate concentrations. As MCTs are found in subcellular fractions within the muscle cell (Dubouchaud et al. 2000; Hashimoto et al. 2005), there could be a difference between the black and white runners in the subcellular contents of MCTs. For example, MCT1 is found in the mitochondria, and is thought to facilitate lactate uptake into the mitochondria for oxidation (Dubouchaud et al. 2000; Hashimoto et al. 2005). If one ethnic group had a greater mitochondrial MCT1 content than another they could theoretically have a metabolic advantage during exercise via enhanced cytosolic lactate clearance, more efficient fuel oxidation and greater cellular redox balance. Strong relationships were observed between MCT4 and both the 10 km PB and PTS (Fig. 3c, d). This suggests that the greater the MCT4 content in a well-trained runner’s muscle, the better the performance, at least during a 10 km race. Although bidirectional in function, MCT4 is well positioned to assist the efflux of lactate produced in cells with high rates of glycolysis (Hashimoto et al. 2005). Therefore, by facilitating the removal of lactate from exercising muscle and so assisting the continuation of glycogenolysis and glycolysis, MCT4 may delay the development of fatigue during physical activity. In addition, cellular pH homeostasis in muscle is partly regulated by MCT-mediated H? removal in conjunction with the efflux of lactate (Juel 1998). By facilitating the transport of H? ions out of the cell, a high sarcolemmal density of MCT4 could lessen perturbations in intracellular pH with exercise, delaying fatigue. Greater skeletal muscle MCT4 content could therefore contribute to better running performance, but does not explain the lower plasma lactate concentrations observed in black runners. Further investigation is necessary into this matter as Evertsen et al. (2001) showed no relationship between MCT4 and running performance variables or the velocity associated with the lactate threshold. Messonnier et al. (2007) also found a relationship between muscle MCT content and work capacity, however, this was for supramaximal exercise and the correlation was only significant for MCT1, with a trend evident for MCT4. While the present study did not show a relationship between either PTS or 10 km PB and MCT1 content (Fig. 3a, b), it is possible that analyses of MCT content

123


within different muscle fibre types, might yield different results. There was also no relationship between plasma lactate concentrations and either of the MCTs (data not shown). This is in contrast to previous findings where relationships between plasma lactate concentrations at maximum exercise intensity and both MCT1 and MCT4 were found (Messonnier et al. 2007; Thomas et al. 2005). This lack of relationship might be attributed to the training status and gender of the subjects used in the studies. The present study only used trained, male runners, whereas that of Messonnier et al. (2007) male and female untrained subjects, and Thomas et al. (2005) a mixed population of untrained subjects and well trained middle- and long distance athletes. By incorporating a mixed group of subjects with different training backgrounds, correlations may be more likely. However, plasma lactate concentrations are the result of a number of variables other than MCT content, including lactate production, removal and oxidation (and the many factors affecting these). As the present study was only investigating MCT content and not an extensive range of the mechanisms contributing to exercising plasma lactate concentrations, it is perhaps not surprising that a relationship was not found. Limitations of the study Due to the complex nature of lactate kinetics, it is difficult to clarify the relationship between MCT concentrations and lactate levels during exercise without measuring a number of lactate-related variables. Future studies should include techniques investigating the appearance and disappearance of plasma lactate, as well as the oxidation of lactate. In addition, while total muscle MCT concentrations were shown to be similar between the groups, it is possible that the expression profiles of these transporters may vary at a subcellular level or between different muscle groups or fibre types. Finally, due to limited muscle biopsy size, additional enzyme analyses were not performed, but should be included in future studies as they would be important in elucidating the mechanism behind the lower plasma lactate with exercise in black runners.

Conclusion In conclusion, this is the first study to have investigated MCT content in skeletal muscle biopsies in runners from distinct ethnic backgrounds. The present study demonstrated once again that black African runners have lower plasma lactate concentrations during exercise compared to their white counterparts. No differences were found in the levels of MCT1 or MCT4 isoform expression in whole


muscle samples from the two groups, and so these could not explain the lower plasma lactate concentrations found in black runners. However, MCT4 content was found to be related to running performance. Further studies are required to examine differences in lactate kinetics and transport between these two groups to better understand the relationship between the lower plasma lactate levels in black runners and their superior performance. Acknowledgments The research was supported by the Harry Crossley and Nellie Atkinson Research Funds of the University of Cape Town, the Medical Research Council of South Africa and Discovery Health. A sincere thank you is extended to Prof. George Brooks (University of California at Berkeley) for the kind donation of antibodies, Liesl Grobler for assistance with fibre typing, and Karen Sharwood, Sacha West, Jonathan Dugas, Lara Keytel and Amanda Claassen for assistance with subject testing.

References Baker J, Horton S (2003) East African running dominance revisited: a role for stereotype threat? Br J Sports Med 37:553–555. doi: 10.1136/bjsm.37.6.553 Benton CR, Campbell SE, Tonouchi M, Hatta H, Bonen A (2004) Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria. Biochem Biophys Res Commun 323:249– 253. doi:10.1016/j.bbrc.2004.08.084 Bergman BC, Wolfel EE, Butterfield GE, Lopaschuk GD, Casazza GA, Horning MA, Brooks GA (1999) Active muscle and whole body lactate kinetics after endurance training in men. J Appl Physiol 87:1684–1696 Bergstro¨m J (1962) Muscle electrolytes in man. Scand J Clin Lab Invest Suppl 68:1–110 Bosch AN, Goslin BR, Noakes TD, Dennis SC (1990) Physiological differences between black and white runners during a treadmill marathon. Eur J Appl Physiol 61:68–72. doi:10.1007/BF00236696 Brooke MH, Kaiser KK (1970) Three myosin ATPase systems: the nature of their pH lability and sulfhydryl dependence. J Histochem Cytochem 18:670–672 Brooks GA (1998) Mammalian fuel utilization during sustained exercise. Comp Biochem Physiol 120:89–107 Brooks GA (2007) Lactate: link between glycolytic and oxidative metabolism. Sports Med 37:341–343. doi:10.2165/00007256200737040-00017 Burgomaster KA, Cermak NM, Phillips SM, Benton CR, Bonen A, Gibala MJ (2007) Divergent response of metabolite transport proteins in human skeletal muscle after sprint interval training and detraining. Am J Physiol 292:R1970–R1976 Coetzer P, Noakes TD, Sanders B, Lambert MI, Bosch AN, Wiggins T, Dennis SC (1993) Superior fatigue resistance of elite black South African distance runners. J Appl Physiol 75:1822–1827 Dubouchaud H, Butterfield GE, Wolfel EE, Bergman BC, Brooks GA (2000) Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle. Am J Physiol 278:E571–E579 Durnin JV, Womersley J (1974) Body fat assessed from total body density and its estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years. Br J Nutr 32:77–97. doi:10.1079/BJN19740060 Esseń-Gustavsson B, Henriksson J (1984) Enzyme levels in pools of microdissected human muscle fibres of identified type. Adaptive response to exercise. Acta Physiol Scand 120:505–515

631 Evans WJ, Phinney SD, Young VR (1982) Suction applied to a muscle biopsy maximizes sample size. Med Sci Sports Exerc 14:101–102 Evertsen F, Medbo JI, Bonen A (2001) Effect of training intensity on muscle lactate transporters and lactate threshold of cross-country skiers. Acta Physiol Scand 173:195–205. doi:10.1046/j.1365201X.2001.00871.x Fishbein WN, Merezhinskaya N, Foellmer JW (2002) Relative distribution of three major lactate transporters in frozen human tissues and their localization in unfixed skeletal muscle. Muscle Nerve 26:101–112. doi:10.1002/mus.10168 Gladden LB (2008) A lactatic perspective on metabolism. Med Sci Sports Exerc 40:477–485 Halestrap AP, Price NT (1999) The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 343(Pt 2):281–299. doi:10.1042/0264-6021:3430281 Hashimoto T, Brooks GA (2008) Mitochondrial lactate oxidation complex and an adaptive role for lactate production. Med Sci Sports Exerc 40:486–494 Hashimoto T, Masuda S, Taguchi S, Brooks GA (2005) Immunohistochemical analysis of MCT1, MCT2 and MCT4 expression in rat plantaris muscle. J Physiol 567:121–129. doi:10.1113/ jphysiol.2005.087411 Juel C (1998) Muscle pH regulation: role of training. Acta Physiol Scand 162:359–366. doi:10.1046/j.1365-201X.1998.0305f.x Katch VL, Katch FI (1974) A simple antropometric method for calculating segmental leg limb volume. Res Q 45:211–214 Kohn TA, Esseń-Gustavsson B, Myburgh KH (2007) Do skeletal muscle phenotypic characteristics of Xhosa and Caucasian endurance runners differ when matched for training and racing distances? J Appl Physiol 103:932–940. doi:10.1152/japplphysiol. 01221.2006 Larsen HB (2003) Kenyan dominance in distance running. Comp Biochem Physiol 136:161–170. doi:10.1016/S1095-6433(03) 00227-7 MacRae HS, Dennis SC, Bosch AN, Noakes TD (1992) Effects of training on lactate production and removal during progressive exercise in humans. J Appl Physiol 72:1649–1656 Martin AD, Spenst LF, Drinkwater DT, Clarys JP (1990) Anthropometric estimation of muscle mass in men. Med Sci Sports Exerc 22:729–733. doi:10.1249/00005768-199010000-00027 McCullagh KJ, Poole RC, Halestrap AP, O’Brien M, Bonen A (1996) Role of the lactate transporter (MCT1) in skeletal muscles. Am J Physiol 271:E143–E150 Messonnier L, Kristensen M, Juel C, Denis C (2007) Importance of pH regulation and lactate/H? transport capacity for work production during supramaximal exercise in humans. J Appl Physiol 102:1936–1944. doi:10.1152/japplphysiol.00691.2006 Nindl BC, Kraemer WJ, Emmert WH, Mazzetti SA, Gotshalk LA, Putukian M, Sebastianelli WJ, Patton JF (1998) Comparison of body composition assessment among lean black and white male collegiate athletes. Med Sci Sports Exerc 30:769–776. doi: 10.1097/00005768-199805000-00020 Noakes TD, Myburgh KH, Schall R (1990) Peak treadmill running velocity during the VO2 max test predicts running performance. J Sports Sci 8:35–45 Noakes TD, Harley YXR, Bosch AN, Marino FE, St Clair Gibson A, Lambert MI (2004) Physiological function and neuromuscular recruitment in elite South African distance runners. Equine Comp Exerc Physiol 1:261–271. doi:10.1079/ECP200437 Onywera VO, Scott RA, Boit MK, Pitsiladis YP (2006) Demographic characteristics of elite Kenyan endurance runners. J Sports Sci 24:415–422. doi:10.1080/02640410500189033 Pette D, Staron RS (1993) The molecular diversity of mammalian muscle fibres. NIPS 8:153–157

123

632 Pilegaard H, Terzis G, Halestrap A, Juel C (1999) Distribution of the lactate/H? transporter isoforms MCT1 and MCT4 in human skeletal muscle. Am J Physiol 276:E843–E848 Ross WD, Marfell-Jones MJ (1991) Kinanthropometry. In: MacDougall JD, Wegner HA, Green HS (eds) Physiological testing of the high-performance athlete Human Kinetics Books, Champaign Saltin B, Kim CK, Terrados N, Larsen H, Svedenhag J, Rolf CJ (1995a) Morphology, enzyme activities and buffer capacity in leg muscles of Kenyan and Scandinavian runners. Scand J Med Sci Sports 5:222–230 Saltin B, Larsen H, Terrados N, Bangsbo J, Bak T, Kim CK, Svedenhag J, Rolf CJ (1995b) Aerobic exercise capacity at sea level and at altitude in Kenyan boys, junior and senior runners compared with Scandinavian runners. Scand J Med Sci Sports 5:209–221 Scott RA, Georgiades E, Wilson RH, Goodwin WH, Wolde B, Pitsiladis YP (2003) Demographic characteristics of elite Ethiopian endurance runners. Med Sci Sports Exerc 35:1727– 1732. doi:10.1249/01.MSS.0000089335.85254.89 Scott RA, Moran C, Wilson RH, Onywera V, Boit MK, Goodwin WH, Gohlke P, Payne J, Montgomery H, Pitsiladis YP (2005a) No association between Angiotensin Converting Enzyme (ACE) gene variation and endurance athlete status in Kenyans. Comp Biochem Physiol 141:169–175. doi:10.1016/j.cbpb.2005.05.001

123

Eur J Appl Physiol (2009) 105:623–632 Scott RA, Wilson RH, Goodwin WH, Moran CN, Georgiades E, Wolde B, Pitsiladis YP (2005b) Mitochondrial DNA lineages of elite Ethiopian athletes. Comp Biochem Physiol 140:497–503. doi:10.1016/j.cbpc.2004.11.014 Tesch P, Sjo¨din B, Thorstensson A, Karlsson J (1978) Muscle fatigue and its relation to lactate accumulation and LDH activity in man. Acta Physiol Scand 103:413–420 Thomas C, Perrey S, Lambert K, Hugon G, Mornet D, Mercier J (2005) Monocarboxylate transporters, blood lactate removal after supramaximal exercise, and fatigue indexes in humans.J Appl Physiol 98:804–809. doi:10.1152/japplphysiol.01057.2004 Weston AR, Karamizrak O, Smith A, Noakes TD, Myburgh KH (1999) African runners exhibit greater fatigue resistance, lower lactate accumulation, and higher oxidative enzyme-activity. J Appl Physiol 86:915–923 Weston AR, Mbambo ZH, Myburgh KH (2000) Running economy of African and Caucasian distance runners. Med Sci Sports Exerc 32:1130–1134. doi:10.1097/00005768-200006000-00015 Wilson MC, Jackson VN, Heddle C, Price NT, Pilegaard H, Juel C, Bonen A, Montgomery I, Hutter OF, Halestrap AP (1998) Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3. J Biol Chem 273:15920–15926. doi:10.1074/jbc.273.26.15920