Carsten Juel

Lactate transport in skeletal muscle — role and regulation of the monocarboxylate transporter

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibres also use lactic acid as a respiratory fuel. The stereoselective transport of L‐lactic acid across the plasma membrane of muscle fibres has been shown to involve a proton‐linked monocarboxylate transporter (MCT) similar to that described in erythrocytes and other cells. This transporter plays an important role in the pH regulation of skeletal muscle. A family of eight MCTs has now been cloned and sequenced, and the tissue distribution of each isoform varies. Skeletal muscle contains both MCT1 (the only isoform found in erythrocytes but also present in most other cells) and MCT4. The latter is found in all fibre types, although least in more oxidative red muscles such as soleus, whereas expression of MCT1 is highest in the more oxidative muscles and very low in white muscles that are almost entirely glycolytic. The properties of MCT1 and MCT2 have been described in some detail and the latter shown to have a higher affinity for substrates. MCT4 has been less well characterized but has a lower affinity for L‐lactate (i.e. a higher Km of 20 mM) than does MCT1 (Km of 5 mM). MCT1 expression is increased in response to chronic stimulation and either endurance or explosive exercise training in rats and humans, whereas denervation decreases expression of both MCT1 and MCT4. The mechanism of regulation is not established, but does not appear to be accompanied by changes in mRNA concentrations. However, in other cells MCT1 and MCT4 are intimately associated with an ancillary protein OX‐47 (also known as CD147). This protein is a member of the immunoglobulin superfamily with a single transmembrane helix, whose expression is known to be increased in a range of cells when their metabolic activity is increased.

Anaerobic energy production and O2 deficit-debt relationship during exhaustive exercise in humans.

1. Eight subjects performed one‐legged, dynamic, knee‐extensor exercise, first at 10 W followed by 10 min rest, then at an intense, exhaustive exercise load (65 W) lasting 3.2 min. After 60 min recovery, exercise was performed for 8‐10 min each at 20, 30, 40 and 50 W. Measurements of pulmonary oxygen uptake, heart rate, blood pressure, leg blood flow, and femoral arterial‐venous differences of oxygen content and lactate were performed as well as determination of ATP, creatine phosphate (CP) inosine monophosphate (IMP) and lactate concentrations on biopsy material from the quadriceps muscle before and immediately after the intense exercise, and at 3, 10 and 60 min into recovery. 2. Individual linear relations (r = 0.95‐1.00) between the power outputs for submaximal exercise and oxygen uptakes (leg and pulmonary) were used to estimate the energy demand during intense exercise. Pulmonary and leg oxygen deficits determined as the difference between energy demand and oxygen uptake were 0.46 and 0.48 l (kg active muscle)‐1, respectively. Limb and pulmonary oxygen debts (oxygen uptake during 60 min of recovery ‐ pre‐exercise oxygen uptake) were 0.55 and 1.65 l (kg active muscle)‐1, respectively. 3. During the intense exercise, muscle [ATP] decreased by 30% and [CP] by 60% from resting concentrations of 6.2 and 22.4 mmol (kg wet wt)‐1, respectively, and [IMP] increased to 1.1 mmol (kg wet wt)‐1. Muscle [lactate] increased from 2 to 28.1 mmol (kg wet wt)‐1, and the concomitant net lactate release was 14.8 mmol (kg wet wt)‐1 or about 1/3 of the total net lactate production. During recovery 70% of the accumulated lactate was released to the blood, and the nucleotides and CP returned to about 40 and 85% of pre‐exercise values at 3 and 10 min of recovery, respectively. 4. Total reduction in ATP and CP (and elevation of IMP) during the intense exercise amounted to 16.4 mmol ATP (kg wet wt)‐1, which together with the lactate production accounted for 83.1 mmol ATP (kg wet wt)‐1. In addition 6‐8 mmol ATP (kg wet wt)‐1 are made available related to accumulation of glycolytic intermediates including pyruvate (and alanine). Estimated leg oxygen deficit corresponded to an ATP production of 94.7 mmol ATP kg‐1; this value included 3.1 mmol kg‐1 related to unloading of HbO2 and MbO2.(ABSTRACT TRUNCATED AT 400 WORDS)

Lactic Acid Efflux from White Skeletal Muscle Is Catalyzed by the Monocarboxylate Transporter Isoform MCT3

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40–60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2–3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Effect of high-intensity exercise training on lactate/H+ transport capacity in human skeletal muscle.

The present study examined the effect of high-intensity exercise training on muscle sarcolemmal lactate/H+ transport and the monocarboxylate transporters (MCT1 and MCT4) as well as lactate and H+ release during intense exercise in humans. One-legged knee-extensor exercise training was performed for 8 wk, and biopsies were obtained from untrained and trained vastus lateralis muscle. The rate of lactate/H+ transport determined in sarcolemmal giant vesicles was 12% higher ( P < 0.05) in the trained than in untrained muscle ( n = 7). The content of MCT1 and MCT4 protein was also higher (76 and 32%, respectively; n = 4) in trained muscle. Release of lactate and H+ from the quadriceps muscle at the end of intense exhaustive knee-extensor exercise was similar in the trained and untrained leg, although the estimated muscle intracellular-to-interstitial gradients of lactate and H+ were lower ( P < 0.05) in the trained than in the untrained muscle. The present data show that intense exercise training can increase lactate/H+transport capacity in human skeletal muscle as well as improve the ability of the muscle to release lactate and H+ during contractions.

Formation of hydrogen peroxide and nitric oxide in rat skeletal muscle cells during contractions.

We examined intra- and extracellular H(2)O(2) and NO formation during contractions in primary rat skeletal muscle cell culture. The fluorescent probes DCFH-DA/DCFH (2,7-dichlorofluorescein-diacetate/2,7-dichlorofluorescein) and DAF-2-DA/DAF-2 (4,5-diaminofluorescein-diacetate/4,5-diaminofluorescein) were used to detect H(2)O(2) and NO, respectively. Intense electrical stimulation of muscle cells increased the intra- and extracellular DCF fluorescence by 171% and 105%, respectively, compared with control nonstimulated cells (p <.05). The addition of glutathione (GSH) or Tiron prior to electrical stimulation inhibited the intracellular DCFH oxidation (p <.05), whereas the addition of GSH-PX + GSH inhibited the extracellular DCFH oxidation (p <.05). Intense electrical stimulation also increased (p <.05) the intra- and extracellular DAF-2 fluorescence signal by 56% and 20%, respectively. The addition of N(G)-nitro-L-arginine (L-NA) completely removed the intra- and extracellular DAF-2 fluorescent signal. Our results show that H(2)O(2) and NO are formed in skeletal muscle cells during contractions and suggest that a rapid release of H(2)O(2) and NO may constitute an important defense mechanism against the formation of intracellular (*)OH and (*)ONOO. Furthermore, our data show that DCFH and DAF-2 are suitable probes for the detection of ROS and NO both intra- and extracellularly in skeletal muscle cell cultures.

Effects of high-intensity intermittent training on potassium kinetics and performance in human skeletal muscle

A rise in extracellular potassium concentration in human skeletal muscle may play an important role in development of fatigue during intense exercise. The aim of the present study was to examine the effect of intense intermittent training on muscle interstitial potassium kinetics and its relationship to the density of Na+,K+‐ATPase subunits and KATP channels, as well as exercise performance, in human skeletal muscle. Six male subjects performed intense one‐legged knee‐extensor training for 7 weeks. On separate days the trained leg (TL) and the control leg (CL) performed a 30 min exercise period of 30 W and an incremental test to exhaustion. At frequent intervals during the exercise periods interstitial potassium ([K+]I) was determined by microdialysis, femoral arterial and venous blood samples were drawn and thigh blood flow was measured. Time to fatigue for TL was 28% longer (P < 0.05) than for CL (10.6 ± 0.7 (mean ±s.e.m.) versus 8.2 ± 0.7 min). The amounts of Na+,K+‐ATPase α1 and α2 subunits were, respectively, 29.0 ± 8.4 and 15.1 ± 2.7% higher (P < 0.05) in TL than in CL, while the amounts of β1 subunits and ATP‐dependent K+ (KATP) channels were the same. In CL [K+]I increased more rapidly and was higher (P < 0.05) throughout the 30 W exercise bout, as well at 60 and 70 W, compared to TL, whereas [K+]I was similar at the point of fatigue (9.9 ± 0.7 and 9.1 ± 0.5 mmol l−1, respectively). During the 30 W exercise bouts and at 70 W during the incremental exercise femoral venous potassium concentration ([K+]v) was higher (P < 0.05) in CL than in TL, but identical at exhaustion (6.2 ± 0.2 mmol l−1). Release of potassium to the blood was not different in the two legs. The present data demonstrated that intense intermittent training reduce accumulation of potassium in human skeletal muscle interstitium during exercise, probably through a larger re‐uptake of potassium due to greater activity of the muscle Na+,K+‐ATPase pumps. The lower accumulation of potassium in muscle interstitium in the trained leg was associated with delayed fatigue during intense exercise, supporting the hypothesis that interstitial potassium accumulation is involved in the development of fatigue.

Potassium and sodium shifts during in vitro isometric muscle contraction, and the time course of the ion-gradient recovery.

Intracellular potassium ([K+]i), interstitial potassium ([K+]inter), intracellular sodium ([Na+]i), and resting membrane potential (RMP) were measured before and after repetitive stimulation of mouse soleus and EDL (extensor digitorum longus) muscles. At rest, RMP was −69.8 mV for soleus and −74.9 mV for EDL (37°C). [K+]i was 168 mM and 182 mM, respectively. In soleus, free [Na+]i was 12.7 mM. After repetitive stimulation (960 stimuli) RMP had decreased by 11.9 mV for soleus and by 18.2 mV for EDL. [K+]i was reduced by 32 mM and 48 mM, respectively, whereas [K+]inter was doubled. In soleus [Na+]i had increased by 10.6 mM, demonstrating that the [K+]i-decrease is three times higher than the [Na+]i-increase. It is concluded that this difference reflects different activity induced movements of Na and K, and that the difference is not due to the Na/K pumping ratio. The possible involvement of the potassium loss in muscle fatigue is discussed. After stimulation RMP recovered with a time constant of 0.9 min for soleus and 1.5 min for EDL. Within the first minutes after stimulation the intracellular potassium concentration increased by 20.4 mM/min for soleus and 21.7 mM/min for EDL. Free [Na+]i decreased with less than 10 mM/min. The mechanisms underlying the different rate of changes are discussed.

Distribution of the lactate/H+ transporter isoforms MCT1 and MCT4 in human skeletal muscle.

The profiles of the lactate/H+ transporter isoforms [monocarboxylate transporter isoforms (MCT)] MCT1 and MCT4 (formerly MCT3 of Price, N. T., V. N. Jackson, and A. P. Halestrap. Biochem. J. 329: 321-328, 1998) were studied in the soleus, triceps brachii, and vastus lateralis muscles of six male subjects. The fiber-type compositions of the muscles were evaluated from the occurrence of the myosin heavy chain isoforms, and the fibers were classified as type I, IIA, or IIX. The total content of MCT1 and MCT4 was determined in muscle homogenates by Western blotting, and MCT1 and MCT4 were visualized on cross-sectional muscle sections by immunofluorescence microscopy. The Western blotting revealed a positive, linear relationship between the MCT1 content and the occurrence of type I fibers in the muscle, but no significant relation was found between MCT4 content and fiber type. Moreover, the interindividual variation in MCT4 content was much larger than the interindividual variation in MCT1 content in homogenate samples. The immunofluorescence microscopy showed that within a given muscle section, the MCT4 isoform was clearly more abundant in type II fibers than in type I fibers, whereas only minor differences existed in the occurrence of the MCT1 isoform between type I and II fibers. Together the present results indicate that the content of MCT1 in a muscle varies between different muscles, whereas fiber-type differences in MCT1 content are minor within a given muscle section. In contrast, the content of MCT4 is clearly fiber-type specific but apparently quite similar in various muscles.

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

Chronic hypoxia is accompanied by changes in blood and skeletal muscle acid‐base control. We hypothesized that the underlying mechanisms include altered protein expression of transport systems and the enzymes involved in lactate, HCO3− and H+ fluxes in skeletal muscle and erythrocytes. Immunoblotting was used to quantify densities of the transport systems and enzymes. Muscle and erythrocyte samples were obtained from eight Danish lowlanders at sea level and after 2 and 8 weeks at 4100 m (Bolivia). For comparison, samples were obtained from eight Bolivian natives. In muscle membranes there were no changes in fibre‐type distribution, lactate dehydrogenase isoforms, Na+,K+‐pump subunits or in the lactate‐H+ co‐transporters MCT1 and MCT4. The Na+–H+ exchanger protein NHE1 was elevated by 39 % in natives compared to lowlanders. The Na+‐HCO3− co‐transporter density in muscle was elevated by 47–69 % after 2 and 8 weeks at altitude. The membrane‐bound carbonic anhydrase (CA) IV in muscle increased in the lowlanders by 39 %, whereas CA XIV decreased by 23–47 %. Levels of cytosolic CA II and III in muscle and CA I and II in erythrocytes were unchanged. The erythrocyte lactate‐H+ co‐transporter MCT1 increased by 230–405 % in lowlanders and was 324 % higher in natives. The erythrocyte inorganic anion exchanger (Cl−‐HCO3− exchanger AE1) was increased by 149–228 %. In conclusion, chronic hypoxia induces dramatic changes in erythrocyte proteins, but only moderate changes in muscle proteins involved in acid‐base control. Together, these changes suggest a hypoxia‐induced increase in the capacity for lactate, HCO3− and H+ fluxes from muscle to blood and from blood to erythrocytes.

Metabolic alkalosis reduces exercise‐induced acidosis and potassium accumulation in human skeletal muscle interstitium

Skeletal muscle releases potassium during activity. Interstitial potassium accumulation is important for muscle function and the development of fatigue resulting from exercise. In the present study we used sodium citrate ingestion as a tool to investigate the relationship between interstitial H+ concentration and K+ accumulation during exercise. Seven healthy subjects performed one‐legged knee‐extensor exercise on two separate days with and without sodium citrate ingestion. Interstitial H+ and K+ concentrations were measured with the microdialysis technique. Citrate ingestion reduced the plasma H+ concentration and increased the plasma HCO3− concentration. Citrate had no effect on interstitial H+ at rest. The increase in interstitial H+ concentration during intense exercise was significantly lower (P < 0.05) with citrate ingestion compared to control (peak interstitial H+ concentration 79 versus 131 nm). After 3 min of exercise interstitial K+ concentration was reduced (P < 0.05) in the citrate (alkalosis) compared to the control experiment (8.0 ± 0.9 versus 11.0 ± 2 mm) and interstitial K+ concentration remained lower during the rest of the exercise period. The present study demonstrated a link between interstitial H+ and K+ accumulation, which may be through the ATP‐sensitive K+ channels (KATP channels), which are sensitive to changes in H+.