Physiology & Biochemistry
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Endurance Training Alters Skeletal Muscle MCT Contents in T2DM Men
Affiliations
Key words ▶ MCT ● ▶ exercise ● ▶ diabetes ● ▶ lactate ●
accepted after revision February 06, 2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371838 Published online: July 10, 2014 Int J Sports Med 2014; 35: 1065–1071 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Christian Brinkmann Department of Molecular and Cellular Sport Medicine Institute of Cardiovascular Research and Sport Medicine German Sport University Cologne Am Sportpark Müngersdorf 6 50933 Cologne Germany Tel.: + 49/221/4982 5220 Fax: + 49/221/4982 8370 [email protected]
D. Opitz1, E. Lenzen1, T. Schiffer2, R. Hermann1, M. Hellmich3, W. Bloch1, K. Brixius1, C. Brinkmann1 1
Department of Molecular and Cellular Sport Medicine, Institute of Cardiovascular Research and Sport Medicine, German Sport University Cologne, Cologne, Germany 2 Outpatient Clinic for Sports Traumatology and Public Health Consultation, German Sport University Cologne, Cologne, Germany 3 University of Cologne, Institute of Medical Statistics, Informatics and Epidemiology, Cologne, Germany
Abstract
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Patients suffering from type 2 diabetes mellitus (T2DM) often exhibit chronic elevated lactate levels which can promote peripheral insulin resistance by disturbing skeletal muscle insulinsignaling. Monocarboxylate transporter (MCT) proteins transfer lactate molecules through cellular membranes. MCT-1 and MCT-4 are the main protein isoforms expressed in human skeletal muscle, with MCT-1 showing a higher affinity (lower Km) for lactate than MCT-4. T2DM patients have reduced membranous MCT-1 proteins. Consequently, the lactate transport between muscle cells and the circulation as well as within an intracellular lactate shuttle, involving mitochondria (where lactate can be further metabolized),
Introduction
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Chronic elevated blood lactate levels have been found in patients with type 2 diabetes mellitus (T2DM) [7]. This might be a consequence of a reduced aerobic-oxidative capacity in skeletal muscle and other lactate-releasing cells and tissues (e. g., adipocytes) due to dysfunctions of the mitochondrial respiratory chain, as well as microcirculatory deficits with a concomitant diminished oxygen supply. Furthermore, a high amount of free fatty acids in T2DM could lead to an increased transformation of lipids to acetyl-coenzyme A (CoA), which inhibits the pyruvate dehydrogenase enzyme complex converting pyruvate to acetyl-CoA. Thus, pyruvate accumulates and is reduced to lactate [1]. Elevated lactate levels might contribute to the development and progression of peripheral insulin resistance in T2DM. It has been demonstrated that long-term lactate infusion in rats reduces glucose uptake of skeletal muscle by a down-regulation of the glucose transporter-4 (GLUT-4) protein expression [19] and disturbances within
can be negatively affected. This study investigates whether moderate cycling endurance training (3 times per week for 3 months) can change skeletal muscle MCT contents in T2DM men (n = 8, years = 56 ± 9, body mass index (BMI) = 32 ± 4 kg/m2). Protein content analyses (immunohistochemical stainings) were performed in biopsies taken from the vastus lateralis muscle. Intracellular MCT-1 proteins were up-regulated (relative increase + 89 %), while intracellular MCT-4 contents were down-regulated (relative decrease − 41 %) following endurance training. Sarcolemmal MCT-1 and MCT-4 did not change. The question of whether the training-induced up-regulation of intracellular MCT-1 leads to an improved lactate transport (and clearance) in T2DM patients requires further research.
the insulin signaling cascade [8], which normally ends with the translocation of GLUT-4 proteins from the cytoplasm to the sarcolemma. “Intercellular and intracellular lactate shuttle concepts” describe the possibility to specifically transport lactate to cells/cellular components where it can be further metabolized [6]. Skeletal muscle plays an important role in the lactate metabolism of the human body and cannot only produce and release lactate, but also consume it [3]. Lactate can pass the skeletal muscle plasma membrane via simple diffusion and, to a higher extent, via the 2 monocarboxylate transporter (MCT) proteins MCT-1 and MCT-4 which both allow the co-transport from lactate-/H + into or out of the cell. MCTs are also located in the membranes of mitochondria. Dubouchaud et al. [10] found MCT-1, but not MCT-4 proteins in mitochondria-enriched fractions of human muscle preparations. Hashimoto et al. [14] demonstrated the co-localization of MCT-1 proteins and an internal lactate dehydrogenase in the mitochondria of L6 muscle cells, which could facilitate lactate oxidation in these organelles.
Opitz D et al. Endurance Training Alters Skeletal … Int J Sports Med 2014; 35: 1065–1071
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Authors
Juel et al. [17] report that membranous MCT-1 protein contents are drastically decreased in the skeletal muscle of T2DM men compared to healthy control subjects, while they found no difference for skeletal muscle MCT-4 contents. It can thus be concluded that T2DM patients show a diminished lactate transport capacity in skeletal muscle and a restricted lactate exchange between skeletal muscle and the circulation system. Lactate can probably less effectively escape from a lactate-rich skeletal muscle cell and find its way to other lactate consuming cells (e. g., heart muscle or lactate-poor skeletal muscle cells). Vice versa, the uptake of lactate from a lactate-rich cell environment by lactate-poor muscle cells might also be more difficult in T2DM. It can furthermore be assumed that internal lactate oxidation in mitochondria is reduced in T2DM patients due to decreased intracellular MCT contents in skeletal muscle cells. Studies using western blot analyses have demonstrated that MCT proteins can be up-regulated in healthy subjects through endurance training [4, 10]. This study aims to examine whether similar adaptations to endurance training occur in T2DM subjects. A training-induced up-regulation of MCT proteins could normalize down-regulated MCT contents and improve lactate transport in these patients. The analysis of skeletal muscle slices in the present study through immunohistochemical staining methods should identify possible changes in sarcolemmal and intracellular MCT contents. In addition, muscle fiber typing should help to determine whether the protein expression of MCT-1 and MCT-4 is fibertype specific in T2DM men, and whether possible traininginduced changes in muscle fiber composition could affect MCT protein contents.
Material and Methods
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Subjects All subjects were recruited via a newspaper advertisement. The inclusion criteria required the subjects to be non-insulindependent type 2 diabetic, overweight/obese (BMI > 25 kg/m2), untrained and to belong to an age group of around 50 years. A total of 23 men, who declared being free of diabetic nephropathy, neuropathy, retinopathy and/or any other cardio-vascular complications (apart from well-controlled hypertension, n = 16) took part in the study and ultimately completed the program prescribed by the study. The duration of the disease had been 6 ± 6 years (self-report). Some of the subjects were taking medications during the investigation period (self-report T2DM patients: 19 men took anti-diabetic drugs, 16 men anti-hypertensive and 4 men anti-hyperlipidemia/anti-hypercholesterolemia drugs, 7 men took other drugs (drugs for reducing stomach acid, anti-gout drugs, drugs prescribed for symptoms of an enlarged prostate, anti-hypothyroidism drugs, anti-arthritis drugs, antidepressants)). It was determined by questionnaire that none of the subjects had regularly exercised during the last 3 years prior to the commencement of the study.
Study design 8 diabetic men (56 ± 9 years) participated in a cycle ergometer endurance training (endurance training = ET group) and 15 men (58 ± 10 years) in a control training program, consisting primarily of relaxing, flexibility and coordination exercises (control = C group) for 3 months. Subjects of the C and ET group did not significantly differ in age, any cardio-metabolic variable or muscle
▶ Table 1). All subjects fiber type composition at baseline (T1) (● confirmed by questionnaire at the end of the study that they did not change their dietary habits during the intervention as they were initially instructed. Before (T1), 6 weeks after initiation (T2) and following completion of the 3-month training intervention (T3), venous blood was collected after a 12-h overnight fast and before medication intake in the early morning. The Cobas Mira plus analyzer (Hoffmann La Roche AG, Basel, Switzerland) was used to determine blood glucose, triglycerides and total cholesterol in the blood serum. Plasma HbA1c was determined in an external laboratory. Shortly thereafter, body mass index (BMI) was calculated. Weight and height were always measured in underwear without shoes and by the same investigator. Blood pressure was measured after a 5-min rest period in the seated position. Furthermore, muscle tissue was obtained from the vastus lateralis muscle, and physical performance was tested on a cycle ergometer 1–2 days later. The protocol for the research project was approved by a suitably constituted Ethics Committee of the German Sport University prior to the investigation. It conformed to the provisions of the Declaration of Helsinki and to the ethical guidelines of the journal [13]. Written informed consent was obtained from all subjects.
Training interventions Training was performed on non-consecutive days 3 times/week for 3 months and supervised by professional coaches. In the ET group, the training bouts (cycle ergometer training) were gradually increased, from 25 min in the first week to 50 min in the last. The endurance practice intensity (heart rate measured) was individually adapted to the heart rate corresponding to 65–75 % of the VO2peak based on the endurance test conducted before the training period and during the training intervention 6 weeks after its initiation. In the C group, the training bouts consisted of a 1-h group training session mainly involving relaxing, flexibility and coordination exercises (e. g., progressive muscle relaxation by Jacobsen, stretching exercises for the whole body, balance exercises), excluding systematic endurance stimuli. All diabetic subjects participated in at least 90 % of the training units.
Muscle biopsy Biopsies were obtained from the superficial part of the vastus lateralis muscle under resting conditions with a biopsy needle as described by Evans et al. [11]. The muscle tissue (approximately 100 mg), covered with “Tissue Tek” (Sakura, Zoeterwoude, the Netherlands), was first cooled down for a few seconds in a glass of isopentane surrounded by liquid nitrogen. Subsequently, it was frozen in liquid nitrogen and then stored in a freezer at − 80 °C. Cryosectioned 10 μm slices of frozen muscle tissue were cut for further analyses.
Performance diagnostics An endurance test was performed on the upright bike “Ergometrics900” (Ergoline, Bitz, Germany) coupled to an ECG (“Ergoscript EK3012”, Ergoline). During exercise, respiratory gas measurement was carried out using the “ZAN 600 USB” system (nSpire Health, Longmont, Colorado, USA). Subjects were tested with the following stopping criteria: muscular exhaustion, angina pectoris, ischemia, paleness, cyanosis, arrhythmia, respiratory insufficiency, hypertension (systolic blood pressure > 250 mmHg or diastolic blood pressure > 115 mmHg),
Opitz D et al. Endurance Training Alters Skeletal … Int J Sports Med 2014; 35: 1065–1071
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aberration, dizziness and/or coordination problems. Starting at 25 W, intensity gradually increased by 25 W every 2 min. Capillarized blood was extracted from the ear lobe immediately after each incremental workload. Blood lactate was analyzed using the “EBIO plus” system (Eppendorf, Hamburg, Germany). The highest attained O2 consumption during exercise (average value from the beginning of the last minute to the end of the test) was defined as VO2peak. The subjects were always tested at the same time of day, and they were instructed not to engage in physically exhausting activities 24 h prior to measurement.
Immunohistochemistry Immunohistochemistry is a standard procedure used in cell biology that has proven successful in the localization and semiquantitative analysis of proteins [5, 9]. Immunohistochemistry and densitometry were performed as described previously [9]. The following primary antibodies were used for the immunohistochemical staining of the MCT-isoforms: MCT-1 polyclonal (Chemicon, Temecula, California, USA) with a dilution of 1:1 000 and MCT-4 polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA) with a dilution of 1:500. In addition, a polyclonal goat antirabbit secondary antibody (Dako, Hamburg, Germany) was used with a dilution of 1:400. All primary antibodies were tested for their specific binding by a negative immunohistochemical control experiment during each staining (the muscle tissue was treated by the same immunohistochemistry procedure, without, however, using the first antibody). For the intensity analysis of immunostaining, the gray values of at least 40 muscle cells (total number) from 3 randomly selected areas of each muscle slice were measured. The intensity was reported as arbitrary gray value [DU = density unit] of the mean of measured cells’ gray value minus the background gray value. The background gray value was measured in a cell-free area of the sample between the muscle cells (cellular interstice).
Fiber typing For muscle fiber typing, immunohistochemical staining was performed using the A4.840 monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA) with a dilution of 1:200. It can recognize slow myosin heavy chains, indicating slow-twitch type I muscle fibers [16]. 100 cells per muscle slice were randomly analysed under the light microscope.
Statistical analyses Data are presented as mean values ± standard deviations (SD). Statistical analyses were carried out using the “SPSS 19.0” program (SPSS Incorporation, Chicago, Illinois, USA). Non-parametric (rank-based) hypotheses tests were used throughout as normality of continuous data distributions seemed to be questionable (skewness, outliers). The Mann-Whitney U-test was performed for the comparative analyses of data between the ET and C group. Training-induced effects on MCT contents, as well as on muscle morphology (fiber type composition) and cardiometabolic variables were checked within each group using the Friedman analysis of variance (ANOVA) for repeated measurements. If found statistically significant, implemented post-hoc tests for multiple pairwise comparisons were conducted (Bonferroni corrected). Significance was considered at p-value ≤ 0.05.
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p ≤ 0.05 p ≤ 0.05 n. s. n. s. 54 ± 4 46 ± 4 51 ± 6 49 ± 6 46 ± 9 54 ± 9 46 ± 8 54 ± 8 42 ± 9 58 ± 9 44 ± 12 56 ± 12
n. s. n. s.
24.63 ± 5.27 21.35 ± 5.25 n. s. 22.59 ± 4.04 21.63 ± 4.13 22.41 ± 4.26
131 ± 26
Values are means ± SD. T1 = before training; T2 = 6 weeks after the initiation of training; T3 = after 3 months of training. C = control group, ET = endurance training group, BMI = body mass index, HbA1c = glycated hemoglobin, VO2peak = maximal oxygen uptake, n.s = not significant
n. s. n. s. T1 vs. T3, adjusted p ≤ 0.05
n. s. n. s.
n. s. n. s. n. s. 162 ± 38 148 ± 30 n. s. 133 ± 25 135 ± 33
26.37 ± 4.49
n. s. n. s. p ≤ 0.05 n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. 6.7 ± 0.6 135 ± 22 162 ± 34 158 ± 82 122 ± 12 88 ± 13 94 ± 36 6.8 ± 0.7 145 ± 41 172 ± 27 185 ± 67 131 ± 13 84 ± 15 86 ± 33 6.7 ± 0.8 156 ± 40 177 ± 28 166 ± 53 137 ± 21 85 ± 19 86 ± 24 6.6 ± 1.1 149 ± 42 197 ± 37 185 ± 91 135 ± 20 88 ± 13 107 ± 30
plasma HbA1c [ %] serum fasting glucose [mg/dl] serum cholesterol [mg/dl] serum triglycerides [mg/dl] systolic blood pressure [mmHg] diastolic blood pressure [mmHg] workload corresponding to the 2 mmol/l blood lactate concentration [W] workload corresponding to the 4 mmol/l blood lactate concentration [W] VO2peak [ml/min/kg] Muscle fiber composition type I muscle fibers [ %] type II muscle fibers [ %]
n. s. n. s. n. s. n. s. n. s. n. s. n. s. 6.8 ± 1.1 153 ± 53 197 ± 32 173 ± 97 128 ± 17 84 ± 13 122 ± 44 6.7 ± 1.1 148 ± 48 196 ± 35 190 ± 153 135 ± 15 85 ± 14 115 ± 43
161 ± 38
n. s.
C T3–ET T3
n. s.
T1 vs. T3, T2 vs. T3, adjusted p ≤ 0.05 n. s. n. s. n. s. n. s. n. s. n. s. n. s. 31.7 ± 3.9 31.7 ± 4.1 32.4 ± 3.9 BMI [kg/m2]
n. s. 32.1 ± 3.6 32.2 ± 3.7
30.9 ± 3.8
Significance Significance
C T1–ET T1 T3
ET
T2 T1 C: T1–T2–T3 T3
C
ET ET Significance C
T2 T1
C Cardio-metabolic variable
Table 1 Data of cardio-metabolic variables and muscle fiber composition.
ET: T1–T2–T3
Significance
Physiology & Biochemistry
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Endurance training-induced alterations in skeletal muscle monocarboxylate transporter contents in T2DM patients We investigated whether the basal contents of skeletal muscle monocarboxylate transporters in the skeletal muscle of T2DM patients change following a 3-month endurance training program. Immunohistochemical stainings revealed that intracellular MCT-1 in the skeletal muscle cells significantly increased over time (relative increase T1–T3: + 89 %), while there were no changes in basal MCT-1 contents in the sarcolemmas. Training of the C group, involving primarily relaxing, flexibility and coordination exercises, led to an increase in sarcolemmal MCT-1 contents (relative increase T1–T3: + 3 %), but did not affect ▶ Fig. 1). Intracellular MCT-4 conintracellular MCT-1 proteins (● tents in the skeletal muscle cells were significantly down-regulated in T2DM patients following endurance training (relative decrease T1–T3: − 41 %). Although there was overall significance for intracellular MCT-4 contents in the C group, Bonferroni
corrected pairwise comparisons did not indicate significant changes between the times of measurement in this group. Sar▶ Fig. 2). colemmal MCT-4 contents changed in neither group (● ▶ Fig. 3). MCT contents obviously vary depending on fiber type (● Sarcolemmal as well as intracellular MCT-1 contents were predominantly detected in type I muscle fibers. Intracellular MCT-4 proteins were relatively homogeneously distributed among type I and type II fibers, while sarcolemmal MCT-4 proteins tended to have a stronger immunoreactivity in type II muscle fibers.
Training-induced alterations in cardio-metabolic variables and skeletal muscle fiber composition in T2DM patients Cardio-metabolic variables and muscle fiber composition were measured in order to analyze their possible effect on MCT proteins in the skeletal muscle of T2DM men. Physical fitness, measured as VO2peak, significantly increased in the ET group, but not so in the C group. BMI significantly changed in T2DM patients following endurance training, but did not change in the C group.
Sarcolemmal MCT-1
Intracellular MCT-1 250
Arbitrary Gray Value: Baseline (100%) +/– Change from Baseline [%]
150
* *
200
100 150
100 50 50
0
T1
T2
T3
T1
T2
0
T3 C
T1
T2
T3
T1
T2
T3
ET
C
ET
T1
T2
T3
Fig. 1 Immunohistochemical results: Monocarboxylate transporter-1 (MCT-1) proteins in the M. vastus lateralis of men suffering from non-insulindependent type 2 diabetes mellitus before training (T1), 6 weeks after the initiation of training (T2) and after 3 months of training (T3). C = control group, ET = endurance training group. Values are means ± SD. *Significantly different: T1 vs. T3 (adjusted p ≤ 0.05). Adjusted p-values in the ET group did not indicate significance for T1 vs. T2 or for T2 vs. T3.
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Results
Physiology & Biochemistry
Sarcolemmal MCT-4
Intracellular MCT-4
150
* 100
100
50
50
0
T1
T2
T3
T1
T2
0
T3 C
T1
T2
T3
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C
ET
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T2
T3
Fig. 2 Immunohistochemical results: Monocarboxylate transporter-4 (MCT-4) proteins in the M. vastus lateralis of men suffering from non-insulindependent type 2 diabetes mellitus before training (T1), 6 weeks after the initiation of training (T2) and after 3 months of training (T3). C = control group, ET = endurance training group. Values are means ± SD. *Significantly different: T1 vs. T3 (adjusted p ≤ 0.05). Adjusted p-values in the ET group did not indicate significance for T1 vs. T2 or for T2 vs. T3.
Muscle fiber type composition was affected in neither group ▶ Table 1). (●
Discussion
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The present study investigated training-induced alterations in MCT protein contents in biopsies taken from the M. vastus lateralis of overweight/obese men suffering from non-insulindependent T2DM before endurance training, 6 weeks following commencement thereof and after 3 months of training. Sarcolemmal MCT-1 and MCT-4 contents did not change in T2DM men following endurance training, while intracellular MCT-1 proteins significantly increased and MCT-4 proteins decreased. Other studies involving healthy subjects also revealed alterations in MCT proteins in skeletal muscle following short- and long-term endurance training. Bonen et al. [4] observed MCT-1 increases in total skeletal muscle after 7 days of cycling training (65 % VO2peak), while Dubouchaud et al. [10] found MCT-1 increases in total skeletal muscle in mitochondria-enriched as well as in sarcolemma-enriched muscle fractions after 9 weeks of cycling training (75 % VO2peak). In addition, Dubouchaud et al.
[10] found that MCT-4 proteins increased in sarcolemmaenriched muscle fractions, but to a lesser extent than MCT-1. Obviously, MCT-1 and MCT-4 can both be regulated by aerobic endurance training with greater adaptations for MCT-1. Moreover, it seems that the training-induced regulation of MCT proteins could be different in healthy and T2DM subjects, especially regarding the regulation of MCT-4. The increases in total intracellular MCT-1 proteins in skeletal muscle in our study could be explained by subcellular increases in mitochondrial MCT-1 [10]. Mitochondrial MCT-1 increases could lead to a better lactate oxidation in the mitochondria and thus to a better intracellular lactate clearance. However, Benton et al. [2] have demonstrated that MCT-1 is present only in isolated subsarcolemmal but not in intermyofibrillar mitochondria obtained from rat skeletal muscle. Another notion is that increases in intracellular MCT-1 proteins could improve the cells’ ability to react to changing conditions: more intracellular MCT-1 proteins could translocate to the plasma membrane – similar to GLUT-4 proteins – when the difference between cellular and extracellular lactate concentrations is high. However, there are no explicit indications yet for a temporary transloca-
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Arbitrary Gray Value: Baseline (100%) +/– Change from Baseline [%]
150
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tion of MCT proteins triggered by any mechanical or metabolic stimulus in humans. The down-regulation of intracellular MCT-4 proteins in our study might be a compensatory effect to the up-regulation of MCT-1 proteins. Furthermore, it is known that the transport activities of MCT-1 and MCT-4 for lactate are quite different (KM = 3.5 mM, KM = 17–34 mM) [3]. It can thus be assumed that MCT-4 proteins are particularly important at very high lactate concentrations. Due to the fact that the T2DM patients in our study only cycled at relatively moderate training intensities (65– 75 % VO2peak), and lactate concentrations probably did not reach very high levels during the training bouts, we presume that intracellular MCT-4 proteins were down-regulated in the skeletal muscle cells because they were less important for the transport of lactate produced at these intensities than MCT-1 proteins. The assumption that the up-regulation of MCT-1 proteins leads to a better lactate transport and clearance during exercise could be supported by the lactate data from the performance tests suggesting delayed lactate increases during incremental workload. However, these results were not significant. Increases in MCT-1, which shows a higher affinity for lactate than MCT-4, could also be useful for better lactate transport at non-exercising conditions, considering that T2DM patients often exhibit chronic elevated lactate levels [7]. In general, it is difficult to prove the effects of changes in MCT proteins on lactate levels, as basal lactate levels underlie day-to-day variations [21]. Changes in MCT contents may depend on muscle fiber composition given that we have shown that there is a fiber type-specific MCT distribution in the skeletal muscle cells of T2DM men. While we were able to detect changes in MCT contents, traininginduced muscle fiber shifts were not observed. Endurance training reduced BMI in type 2 diabetic men in our study as has already been evidenced in previous work [18]. The reduction in intracellular MCT-4 proteins may be related to the weight loss of the subjects in our study, since Metz et al. [20] demonstrated that MCT-4 proteins were significantly reduced
after weight loss in obese subjects. It is noteworthy that there was no difference in MCT-1 expression in their subjects before and after weight loss. As expected, there were hardly any significant changes in variables measured in the control group. Sarcolemmal MCT-1 increased significantly from T1 to T3, but to a minor extent. We cannot adequately explain this result based on our current state of knowledge. In general, there is a lack of information on signaling pathways which induce the protein expression of MCT proteins. It is likely that variables which are altered in the pathophysiology of T2DM and chronic inflammation [15] may affect MCT protein expression on the transcriptional or translational level. Further studies on this issue are necessary. As a limitation of this study, the measured variables may have been influenced by the intake of medications. However, the patients did not change their medication intake over the course of the study to illustrate the training effects to the best possible extent. Furthermore, we used only one method to quantify protein expression. Future studies should include further assessment methods (e. g., western blot analyses) to confirm the presented results.
Conclusions
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It can be concluded that the endurance training performed in our study alters MCT contents in the skeletal muscle of T2DM men. The long-term up-regulation of skeletal muscle MCT-1 may lead to more effective lactate transport between muscle cells and the circulation (considering the speculative possibility of a temporary translocation of MCT proteins to the sarcolemma) following an acute bout of exercise. Furthermore, increases in intracellular MCT-1 could improve lactate transport within an intracellular lactate shuttle when exercising as well as under resting conditions. This may contribute to an improved lactate clearance in T2DM patients.
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Fig. 3 Immunohistochemical results: Fiber type-specific distribution of monocarboxylate transporter (MCT) proteins in the M. vastus lateralis of men suffering from non-insulin-dependent type 2 diabetes mellitus. Type I muscle fibers were identified using the A4.840 antibody against slow myosin heavy chains. 1–8, 15–22 = type I muscle fibers; 9–14, 23–27 = type II muscle fibers.
Acknowledgements
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We thank A. Voss, M. Ghilav and B. Collins for their excellent technical assistance.
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1065
Endurance Training Alters Skeletal Muscle MCT Contents in T2DM Men
Affiliations
Key words ▶ MCT ● ▶ exercise ● ▶ diabetes ● ▶ lactate ●
accepted after revision February 06, 2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1371838 Published online: July 10, 2014 Int J Sports Med 2014; 35: 1065–1071 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0172-4622 Correspondence Dr. Christian Brinkmann Department of Molecular and Cellular Sport Medicine Institute of Cardiovascular Research and Sport Medicine German Sport University Cologne Am Sportpark Müngersdorf 6 50933 Cologne Germany Tel.: + 49/221/4982 5220 Fax: + 49/221/4982 8370 [email protected]
D. Opitz1, E. Lenzen1, T. Schiffer2, R. Hermann1, M. Hellmich3, W. Bloch1, K. Brixius1, C. Brinkmann1 1
Department of Molecular and Cellular Sport Medicine, Institute of Cardiovascular Research and Sport Medicine, German Sport University Cologne, Cologne, Germany 2 Outpatient Clinic for Sports Traumatology and Public Health Consultation, German Sport University Cologne, Cologne, Germany 3 University of Cologne, Institute of Medical Statistics, Informatics and Epidemiology, Cologne, Germany
Abstract
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Patients suffering from type 2 diabetes mellitus (T2DM) often exhibit chronic elevated lactate levels which can promote peripheral insulin resistance by disturbing skeletal muscle insulinsignaling. Monocarboxylate transporter (MCT) proteins transfer lactate molecules through cellular membranes. MCT-1 and MCT-4 are the main protein isoforms expressed in human skeletal muscle, with MCT-1 showing a higher affinity (lower Km) for lactate than MCT-4. T2DM patients have reduced membranous MCT-1 proteins. Consequently, the lactate transport between muscle cells and the circulation as well as within an intracellular lactate shuttle, involving mitochondria (where lactate can be further metabolized),
Introduction
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Chronic elevated blood lactate levels have been found in patients with type 2 diabetes mellitus (T2DM) [7]. This might be a consequence of a reduced aerobic-oxidative capacity in skeletal muscle and other lactate-releasing cells and tissues (e. g., adipocytes) due to dysfunctions of the mitochondrial respiratory chain, as well as microcirculatory deficits with a concomitant diminished oxygen supply. Furthermore, a high amount of free fatty acids in T2DM could lead to an increased transformation of lipids to acetyl-coenzyme A (CoA), which inhibits the pyruvate dehydrogenase enzyme complex converting pyruvate to acetyl-CoA. Thus, pyruvate accumulates and is reduced to lactate [1]. Elevated lactate levels might contribute to the development and progression of peripheral insulin resistance in T2DM. It has been demonstrated that long-term lactate infusion in rats reduces glucose uptake of skeletal muscle by a down-regulation of the glucose transporter-4 (GLUT-4) protein expression [19] and disturbances within
can be negatively affected. This study investigates whether moderate cycling endurance training (3 times per week for 3 months) can change skeletal muscle MCT contents in T2DM men (n = 8, years = 56 ± 9, body mass index (BMI) = 32 ± 4 kg/m2). Protein content analyses (immunohistochemical stainings) were performed in biopsies taken from the vastus lateralis muscle. Intracellular MCT-1 proteins were up-regulated (relative increase + 89 %), while intracellular MCT-4 contents were down-regulated (relative decrease − 41 %) following endurance training. Sarcolemmal MCT-1 and MCT-4 did not change. The question of whether the training-induced up-regulation of intracellular MCT-1 leads to an improved lactate transport (and clearance) in T2DM patients requires further research.
the insulin signaling cascade [8], which normally ends with the translocation of GLUT-4 proteins from the cytoplasm to the sarcolemma. “Intercellular and intracellular lactate shuttle concepts” describe the possibility to specifically transport lactate to cells/cellular components where it can be further metabolized [6]. Skeletal muscle plays an important role in the lactate metabolism of the human body and cannot only produce and release lactate, but also consume it [3]. Lactate can pass the skeletal muscle plasma membrane via simple diffusion and, to a higher extent, via the 2 monocarboxylate transporter (MCT) proteins MCT-1 and MCT-4 which both allow the co-transport from lactate-/H + into or out of the cell. MCTs are also located in the membranes of mitochondria. Dubouchaud et al. [10] found MCT-1, but not MCT-4 proteins in mitochondria-enriched fractions of human muscle preparations. Hashimoto et al. [14] demonstrated the co-localization of MCT-1 proteins and an internal lactate dehydrogenase in the mitochondria of L6 muscle cells, which could facilitate lactate oxidation in these organelles.
Opitz D et al. Endurance Training Alters Skeletal … Int J Sports Med 2014; 35: 1065–1071
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Authors
Juel et al. [17] report that membranous MCT-1 protein contents are drastically decreased in the skeletal muscle of T2DM men compared to healthy control subjects, while they found no difference for skeletal muscle MCT-4 contents. It can thus be concluded that T2DM patients show a diminished lactate transport capacity in skeletal muscle and a restricted lactate exchange between skeletal muscle and the circulation system. Lactate can probably less effectively escape from a lactate-rich skeletal muscle cell and find its way to other lactate consuming cells (e. g., heart muscle or lactate-poor skeletal muscle cells). Vice versa, the uptake of lactate from a lactate-rich cell environment by lactate-poor muscle cells might also be more difficult in T2DM. It can furthermore be assumed that internal lactate oxidation in mitochondria is reduced in T2DM patients due to decreased intracellular MCT contents in skeletal muscle cells. Studies using western blot analyses have demonstrated that MCT proteins can be up-regulated in healthy subjects through endurance training [4, 10]. This study aims to examine whether similar adaptations to endurance training occur in T2DM subjects. A training-induced up-regulation of MCT proteins could normalize down-regulated MCT contents and improve lactate transport in these patients. The analysis of skeletal muscle slices in the present study through immunohistochemical staining methods should identify possible changes in sarcolemmal and intracellular MCT contents. In addition, muscle fiber typing should help to determine whether the protein expression of MCT-1 and MCT-4 is fibertype specific in T2DM men, and whether possible traininginduced changes in muscle fiber composition could affect MCT protein contents.
Material and Methods
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Subjects All subjects were recruited via a newspaper advertisement. The inclusion criteria required the subjects to be non-insulindependent type 2 diabetic, overweight/obese (BMI > 25 kg/m2), untrained and to belong to an age group of around 50 years. A total of 23 men, who declared being free of diabetic nephropathy, neuropathy, retinopathy and/or any other cardio-vascular complications (apart from well-controlled hypertension, n = 16) took part in the study and ultimately completed the program prescribed by the study. The duration of the disease had been 6 ± 6 years (self-report). Some of the subjects were taking medications during the investigation period (self-report T2DM patients: 19 men took anti-diabetic drugs, 16 men anti-hypertensive and 4 men anti-hyperlipidemia/anti-hypercholesterolemia drugs, 7 men took other drugs (drugs for reducing stomach acid, anti-gout drugs, drugs prescribed for symptoms of an enlarged prostate, anti-hypothyroidism drugs, anti-arthritis drugs, antidepressants)). It was determined by questionnaire that none of the subjects had regularly exercised during the last 3 years prior to the commencement of the study.
Study design 8 diabetic men (56 ± 9 years) participated in a cycle ergometer endurance training (endurance training = ET group) and 15 men (58 ± 10 years) in a control training program, consisting primarily of relaxing, flexibility and coordination exercises (control = C group) for 3 months. Subjects of the C and ET group did not significantly differ in age, any cardio-metabolic variable or muscle
▶ Table 1). All subjects fiber type composition at baseline (T1) (● confirmed by questionnaire at the end of the study that they did not change their dietary habits during the intervention as they were initially instructed. Before (T1), 6 weeks after initiation (T2) and following completion of the 3-month training intervention (T3), venous blood was collected after a 12-h overnight fast and before medication intake in the early morning. The Cobas Mira plus analyzer (Hoffmann La Roche AG, Basel, Switzerland) was used to determine blood glucose, triglycerides and total cholesterol in the blood serum. Plasma HbA1c was determined in an external laboratory. Shortly thereafter, body mass index (BMI) was calculated. Weight and height were always measured in underwear without shoes and by the same investigator. Blood pressure was measured after a 5-min rest period in the seated position. Furthermore, muscle tissue was obtained from the vastus lateralis muscle, and physical performance was tested on a cycle ergometer 1–2 days later. The protocol for the research project was approved by a suitably constituted Ethics Committee of the German Sport University prior to the investigation. It conformed to the provisions of the Declaration of Helsinki and to the ethical guidelines of the journal [13]. Written informed consent was obtained from all subjects.
Training interventions Training was performed on non-consecutive days 3 times/week for 3 months and supervised by professional coaches. In the ET group, the training bouts (cycle ergometer training) were gradually increased, from 25 min in the first week to 50 min in the last. The endurance practice intensity (heart rate measured) was individually adapted to the heart rate corresponding to 65–75 % of the VO2peak based on the endurance test conducted before the training period and during the training intervention 6 weeks after its initiation. In the C group, the training bouts consisted of a 1-h group training session mainly involving relaxing, flexibility and coordination exercises (e. g., progressive muscle relaxation by Jacobsen, stretching exercises for the whole body, balance exercises), excluding systematic endurance stimuli. All diabetic subjects participated in at least 90 % of the training units.
Muscle biopsy Biopsies were obtained from the superficial part of the vastus lateralis muscle under resting conditions with a biopsy needle as described by Evans et al. [11]. The muscle tissue (approximately 100 mg), covered with “Tissue Tek” (Sakura, Zoeterwoude, the Netherlands), was first cooled down for a few seconds in a glass of isopentane surrounded by liquid nitrogen. Subsequently, it was frozen in liquid nitrogen and then stored in a freezer at − 80 °C. Cryosectioned 10 μm slices of frozen muscle tissue were cut for further analyses.
Performance diagnostics An endurance test was performed on the upright bike “Ergometrics900” (Ergoline, Bitz, Germany) coupled to an ECG (“Ergoscript EK3012”, Ergoline). During exercise, respiratory gas measurement was carried out using the “ZAN 600 USB” system (nSpire Health, Longmont, Colorado, USA). Subjects were tested with the following stopping criteria: muscular exhaustion, angina pectoris, ischemia, paleness, cyanosis, arrhythmia, respiratory insufficiency, hypertension (systolic blood pressure > 250 mmHg or diastolic blood pressure > 115 mmHg),
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aberration, dizziness and/or coordination problems. Starting at 25 W, intensity gradually increased by 25 W every 2 min. Capillarized blood was extracted from the ear lobe immediately after each incremental workload. Blood lactate was analyzed using the “EBIO plus” system (Eppendorf, Hamburg, Germany). The highest attained O2 consumption during exercise (average value from the beginning of the last minute to the end of the test) was defined as VO2peak. The subjects were always tested at the same time of day, and they were instructed not to engage in physically exhausting activities 24 h prior to measurement.
Immunohistochemistry Immunohistochemistry is a standard procedure used in cell biology that has proven successful in the localization and semiquantitative analysis of proteins [5, 9]. Immunohistochemistry and densitometry were performed as described previously [9]. The following primary antibodies were used for the immunohistochemical staining of the MCT-isoforms: MCT-1 polyclonal (Chemicon, Temecula, California, USA) with a dilution of 1:1 000 and MCT-4 polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA) with a dilution of 1:500. In addition, a polyclonal goat antirabbit secondary antibody (Dako, Hamburg, Germany) was used with a dilution of 1:400. All primary antibodies were tested for their specific binding by a negative immunohistochemical control experiment during each staining (the muscle tissue was treated by the same immunohistochemistry procedure, without, however, using the first antibody). For the intensity analysis of immunostaining, the gray values of at least 40 muscle cells (total number) from 3 randomly selected areas of each muscle slice were measured. The intensity was reported as arbitrary gray value [DU = density unit] of the mean of measured cells’ gray value minus the background gray value. The background gray value was measured in a cell-free area of the sample between the muscle cells (cellular interstice).
Fiber typing For muscle fiber typing, immunohistochemical staining was performed using the A4.840 monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, Iowa, USA) with a dilution of 1:200. It can recognize slow myosin heavy chains, indicating slow-twitch type I muscle fibers [16]. 100 cells per muscle slice were randomly analysed under the light microscope.
Statistical analyses Data are presented as mean values ± standard deviations (SD). Statistical analyses were carried out using the “SPSS 19.0” program (SPSS Incorporation, Chicago, Illinois, USA). Non-parametric (rank-based) hypotheses tests were used throughout as normality of continuous data distributions seemed to be questionable (skewness, outliers). The Mann-Whitney U-test was performed for the comparative analyses of data between the ET and C group. Training-induced effects on MCT contents, as well as on muscle morphology (fiber type composition) and cardiometabolic variables were checked within each group using the Friedman analysis of variance (ANOVA) for repeated measurements. If found statistically significant, implemented post-hoc tests for multiple pairwise comparisons were conducted (Bonferroni corrected). Significance was considered at p-value ≤ 0.05.
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p ≤ 0.05 p ≤ 0.05 n. s. n. s. 54 ± 4 46 ± 4 51 ± 6 49 ± 6 46 ± 9 54 ± 9 46 ± 8 54 ± 8 42 ± 9 58 ± 9 44 ± 12 56 ± 12
n. s. n. s.
24.63 ± 5.27 21.35 ± 5.25 n. s. 22.59 ± 4.04 21.63 ± 4.13 22.41 ± 4.26
131 ± 26
Values are means ± SD. T1 = before training; T2 = 6 weeks after the initiation of training; T3 = after 3 months of training. C = control group, ET = endurance training group, BMI = body mass index, HbA1c = glycated hemoglobin, VO2peak = maximal oxygen uptake, n.s = not significant
n. s. n. s. T1 vs. T3, adjusted p ≤ 0.05
n. s. n. s.
n. s. n. s. n. s. 162 ± 38 148 ± 30 n. s. 133 ± 25 135 ± 33
26.37 ± 4.49
n. s. n. s. p ≤ 0.05 n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. n. s. 6.7 ± 0.6 135 ± 22 162 ± 34 158 ± 82 122 ± 12 88 ± 13 94 ± 36 6.8 ± 0.7 145 ± 41 172 ± 27 185 ± 67 131 ± 13 84 ± 15 86 ± 33 6.7 ± 0.8 156 ± 40 177 ± 28 166 ± 53 137 ± 21 85 ± 19 86 ± 24 6.6 ± 1.1 149 ± 42 197 ± 37 185 ± 91 135 ± 20 88 ± 13 107 ± 30
plasma HbA1c [ %] serum fasting glucose [mg/dl] serum cholesterol [mg/dl] serum triglycerides [mg/dl] systolic blood pressure [mmHg] diastolic blood pressure [mmHg] workload corresponding to the 2 mmol/l blood lactate concentration [W] workload corresponding to the 4 mmol/l blood lactate concentration [W] VO2peak [ml/min/kg] Muscle fiber composition type I muscle fibers [ %] type II muscle fibers [ %]
n. s. n. s. n. s. n. s. n. s. n. s. n. s. 6.8 ± 1.1 153 ± 53 197 ± 32 173 ± 97 128 ± 17 84 ± 13 122 ± 44 6.7 ± 1.1 148 ± 48 196 ± 35 190 ± 153 135 ± 15 85 ± 14 115 ± 43
161 ± 38
n. s.
C T3–ET T3
n. s.
T1 vs. T3, T2 vs. T3, adjusted p ≤ 0.05 n. s. n. s. n. s. n. s. n. s. n. s. n. s. 31.7 ± 3.9 31.7 ± 4.1 32.4 ± 3.9 BMI [kg/m2]
n. s. 32.1 ± 3.6 32.2 ± 3.7
30.9 ± 3.8
Significance Significance
C T1–ET T1 T3
ET
T2 T1 C: T1–T2–T3 T3
C
ET ET Significance C
T2 T1
C Cardio-metabolic variable
Table 1 Data of cardio-metabolic variables and muscle fiber composition.
ET: T1–T2–T3
Significance
Physiology & Biochemistry
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Endurance training-induced alterations in skeletal muscle monocarboxylate transporter contents in T2DM patients We investigated whether the basal contents of skeletal muscle monocarboxylate transporters in the skeletal muscle of T2DM patients change following a 3-month endurance training program. Immunohistochemical stainings revealed that intracellular MCT-1 in the skeletal muscle cells significantly increased over time (relative increase T1–T3: + 89 %), while there were no changes in basal MCT-1 contents in the sarcolemmas. Training of the C group, involving primarily relaxing, flexibility and coordination exercises, led to an increase in sarcolemmal MCT-1 contents (relative increase T1–T3: + 3 %), but did not affect ▶ Fig. 1). Intracellular MCT-4 conintracellular MCT-1 proteins (● tents in the skeletal muscle cells were significantly down-regulated in T2DM patients following endurance training (relative decrease T1–T3: − 41 %). Although there was overall significance for intracellular MCT-4 contents in the C group, Bonferroni
corrected pairwise comparisons did not indicate significant changes between the times of measurement in this group. Sar▶ Fig. 2). colemmal MCT-4 contents changed in neither group (● ▶ Fig. 3). MCT contents obviously vary depending on fiber type (● Sarcolemmal as well as intracellular MCT-1 contents were predominantly detected in type I muscle fibers. Intracellular MCT-4 proteins were relatively homogeneously distributed among type I and type II fibers, while sarcolemmal MCT-4 proteins tended to have a stronger immunoreactivity in type II muscle fibers.
Training-induced alterations in cardio-metabolic variables and skeletal muscle fiber composition in T2DM patients Cardio-metabolic variables and muscle fiber composition were measured in order to analyze their possible effect on MCT proteins in the skeletal muscle of T2DM men. Physical fitness, measured as VO2peak, significantly increased in the ET group, but not so in the C group. BMI significantly changed in T2DM patients following endurance training, but did not change in the C group.
Sarcolemmal MCT-1
Intracellular MCT-1 250
Arbitrary Gray Value: Baseline (100%) +/– Change from Baseline [%]
150
* *
200
100 150
100 50 50
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T2
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T1
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T3 C
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ET
C
ET
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T3
Fig. 1 Immunohistochemical results: Monocarboxylate transporter-1 (MCT-1) proteins in the M. vastus lateralis of men suffering from non-insulindependent type 2 diabetes mellitus before training (T1), 6 weeks after the initiation of training (T2) and after 3 months of training (T3). C = control group, ET = endurance training group. Values are means ± SD. *Significantly different: T1 vs. T3 (adjusted p ≤ 0.05). Adjusted p-values in the ET group did not indicate significance for T1 vs. T2 or for T2 vs. T3.
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Results
Physiology & Biochemistry
Sarcolemmal MCT-4
Intracellular MCT-4
150
* 100
100
50
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0
T1
T2
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T1
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T3 C
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C
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Fig. 2 Immunohistochemical results: Monocarboxylate transporter-4 (MCT-4) proteins in the M. vastus lateralis of men suffering from non-insulindependent type 2 diabetes mellitus before training (T1), 6 weeks after the initiation of training (T2) and after 3 months of training (T3). C = control group, ET = endurance training group. Values are means ± SD. *Significantly different: T1 vs. T3 (adjusted p ≤ 0.05). Adjusted p-values in the ET group did not indicate significance for T1 vs. T2 or for T2 vs. T3.
Muscle fiber type composition was affected in neither group ▶ Table 1). (●
Discussion
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The present study investigated training-induced alterations in MCT protein contents in biopsies taken from the M. vastus lateralis of overweight/obese men suffering from non-insulindependent T2DM before endurance training, 6 weeks following commencement thereof and after 3 months of training. Sarcolemmal MCT-1 and MCT-4 contents did not change in T2DM men following endurance training, while intracellular MCT-1 proteins significantly increased and MCT-4 proteins decreased. Other studies involving healthy subjects also revealed alterations in MCT proteins in skeletal muscle following short- and long-term endurance training. Bonen et al. [4] observed MCT-1 increases in total skeletal muscle after 7 days of cycling training (65 % VO2peak), while Dubouchaud et al. [10] found MCT-1 increases in total skeletal muscle in mitochondria-enriched as well as in sarcolemma-enriched muscle fractions after 9 weeks of cycling training (75 % VO2peak). In addition, Dubouchaud et al.
[10] found that MCT-4 proteins increased in sarcolemmaenriched muscle fractions, but to a lesser extent than MCT-1. Obviously, MCT-1 and MCT-4 can both be regulated by aerobic endurance training with greater adaptations for MCT-1. Moreover, it seems that the training-induced regulation of MCT proteins could be different in healthy and T2DM subjects, especially regarding the regulation of MCT-4. The increases in total intracellular MCT-1 proteins in skeletal muscle in our study could be explained by subcellular increases in mitochondrial MCT-1 [10]. Mitochondrial MCT-1 increases could lead to a better lactate oxidation in the mitochondria and thus to a better intracellular lactate clearance. However, Benton et al. [2] have demonstrated that MCT-1 is present only in isolated subsarcolemmal but not in intermyofibrillar mitochondria obtained from rat skeletal muscle. Another notion is that increases in intracellular MCT-1 proteins could improve the cells’ ability to react to changing conditions: more intracellular MCT-1 proteins could translocate to the plasma membrane – similar to GLUT-4 proteins – when the difference between cellular and extracellular lactate concentrations is high. However, there are no explicit indications yet for a temporary transloca-
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Arbitrary Gray Value: Baseline (100%) +/– Change from Baseline [%]
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tion of MCT proteins triggered by any mechanical or metabolic stimulus in humans. The down-regulation of intracellular MCT-4 proteins in our study might be a compensatory effect to the up-regulation of MCT-1 proteins. Furthermore, it is known that the transport activities of MCT-1 and MCT-4 for lactate are quite different (KM = 3.5 mM, KM = 17–34 mM) [3]. It can thus be assumed that MCT-4 proteins are particularly important at very high lactate concentrations. Due to the fact that the T2DM patients in our study only cycled at relatively moderate training intensities (65– 75 % VO2peak), and lactate concentrations probably did not reach very high levels during the training bouts, we presume that intracellular MCT-4 proteins were down-regulated in the skeletal muscle cells because they were less important for the transport of lactate produced at these intensities than MCT-1 proteins. The assumption that the up-regulation of MCT-1 proteins leads to a better lactate transport and clearance during exercise could be supported by the lactate data from the performance tests suggesting delayed lactate increases during incremental workload. However, these results were not significant. Increases in MCT-1, which shows a higher affinity for lactate than MCT-4, could also be useful for better lactate transport at non-exercising conditions, considering that T2DM patients often exhibit chronic elevated lactate levels [7]. In general, it is difficult to prove the effects of changes in MCT proteins on lactate levels, as basal lactate levels underlie day-to-day variations [21]. Changes in MCT contents may depend on muscle fiber composition given that we have shown that there is a fiber type-specific MCT distribution in the skeletal muscle cells of T2DM men. While we were able to detect changes in MCT contents, traininginduced muscle fiber shifts were not observed. Endurance training reduced BMI in type 2 diabetic men in our study as has already been evidenced in previous work [18]. The reduction in intracellular MCT-4 proteins may be related to the weight loss of the subjects in our study, since Metz et al. [20] demonstrated that MCT-4 proteins were significantly reduced
after weight loss in obese subjects. It is noteworthy that there was no difference in MCT-1 expression in their subjects before and after weight loss. As expected, there were hardly any significant changes in variables measured in the control group. Sarcolemmal MCT-1 increased significantly from T1 to T3, but to a minor extent. We cannot adequately explain this result based on our current state of knowledge. In general, there is a lack of information on signaling pathways which induce the protein expression of MCT proteins. It is likely that variables which are altered in the pathophysiology of T2DM and chronic inflammation [15] may affect MCT protein expression on the transcriptional or translational level. Further studies on this issue are necessary. As a limitation of this study, the measured variables may have been influenced by the intake of medications. However, the patients did not change their medication intake over the course of the study to illustrate the training effects to the best possible extent. Furthermore, we used only one method to quantify protein expression. Future studies should include further assessment methods (e. g., western blot analyses) to confirm the presented results.
Conclusions
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It can be concluded that the endurance training performed in our study alters MCT contents in the skeletal muscle of T2DM men. The long-term up-regulation of skeletal muscle MCT-1 may lead to more effective lactate transport between muscle cells and the circulation (considering the speculative possibility of a temporary translocation of MCT proteins to the sarcolemma) following an acute bout of exercise. Furthermore, increases in intracellular MCT-1 could improve lactate transport within an intracellular lactate shuttle when exercising as well as under resting conditions. This may contribute to an improved lactate clearance in T2DM patients.
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Fig. 3 Immunohistochemical results: Fiber type-specific distribution of monocarboxylate transporter (MCT) proteins in the M. vastus lateralis of men suffering from non-insulin-dependent type 2 diabetes mellitus. Type I muscle fibers were identified using the A4.840 antibody against slow myosin heavy chains. 1–8, 15–22 = type I muscle fibers; 9–14, 23–27 = type II muscle fibers.
Acknowledgements
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We thank A. Voss, M. Ghilav and B. Collins for their excellent technical assistance.
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