Effect of α-thalassaemia on exercise-induced oxidative stress in sickle cell trait.

AIM: Alpha-thalassaemia is known to reduce intra-erythrocyte HbS (sickle haemoglobin) concentration in sickle cell trait (SCT) subjects. Because HbS was shown to increase oxidative stress, the purpose of this study was to assess the effects of the coexistence of α-thalassaemia and SCT on oxidative stress markers and nitric oxide (NO) metabolism after an acute physical exercise. METHODS: Forty subjects (age: 23.5 ± 2.21 years), SCT carriers (HbAS) or healthy subjects (HbAA), with (-αT) or without (-NαT) an associated α-thalassaemia took part in the study. Plasma markers of oxidative stress [advanced oxidation protein products (AOPP), protein carbonyl, malondialdehyde (MDA) and nitrotyrosine], anti-oxidant defences and NO metabolism (NOx) were measured at rest (T(rest)), immediately following an incremental maximal exercise test (T(ex)) and during recovery (T(1h), T(2h) and T(24h)). RESULTS: Malondialdehyde expressed as the percentage of changes from baseline was significantly higher in the HbAS-NαT compared with HbAS-αT during recovery (+36.3 ± 14.1% vs. -1.8 ± 13.2% at T(1h), P = 0.02; +36.6 ± 13.4% vs. -11.4 ± 12.5% at T(2h), P = 0.004 and +24.1 ± 12.3% vs. -14.4 ± 11.5% at T(24h), P = 0.02 in HbAS-NαT vs. HbAS-αT). Compared with HbAS-NαT, HbAS-αT had a higher NOx change from baseline at T(ex) (-23.4 ± 20.6% vs. +57.7 ± 19.3%, respectively; P = 0.005) and lower nitrotyrosine change from baseline at T(1h) (+7.2 ± 22.2% vs. +93.5%±29.3%, respectively; P = 0.04). CONCLUSION: All these data suggest that the presence of α-thalassaemia may blunt the higher level of oxidative stress and the impaired bioavailability of NO observed in the SCT carriers.

Liver mitochondrial properties from the obesity-resistant Lou/C rat.

OBJECTIVE: The first objective was to evaluate the influence of caloric intake on liver mitochondrial properties. The second objective was aimed at determining the impact of increasing fat intake on these properties. DESIGN: Lou/C rats, displaying an inborn low caloric intake and resistant to diet-induced obesity, were compared to Wistar rats fed either ad libitum or pair-fed. An additional group of Lou/C rats were allowed to increase their fat intake by adjusting their diet from a standard high carbohydrate low-fat diet to a high-fat carbohydrate-free diet. MEASUREMENTS: Hydrogen peroxide (H(2)O(2)) generation, oxygen consumption rate (J(O(2))), membrane potential (DeltaPsi), activity of respiratory chain complexes, cytochrome contents, oxidative phosphorylation efficiency (OPE) and uncoupling protein 2 (UCP2) expression were determined in liver mitochondria. RESULTS: H(2)O(2) production was higher in Lou/C than Wistar rats with glutamate/malate and/or succinate, octanoyl-carnitine, as substrates. These mitochondrial features cannot be mimicked by pair-feeding Wistar rats and remained unaltered by increasing fat intake. Enhanced H(2)O(2) production by mitochondria from Lou/C rats is due to an increased reverse electron flow through the respiratory-chain complex I and a higher medium-chain acyl-CoA dehydrogenase activity. While J(O(2)) was similar over a large range of DeltaPsi in both strains, Lou/C rats were able to sustain higher membrane potential and respiratory rate. In addition, mitochondria from Lou/C rats displayed a decrease in OPE that cannot be explained by increased expression of UCP2 but rather to a slip in proton pumping by cytochrome oxidase. CONCLUSIONS: Liver mitochondria from Lou/C rats display higher reactive oxygen species (ROS) generation but to deplete upstream electron-rich intermediates responsible for ROS generation, these animals increased intrinsic uncoupling of cytochrome oxidase. It is likely that liver mitochondrial properties allowed this strain of rat to display higher insulin sensitivity and resist diet-induced obesity.

Lactate transport in rat sarcolemmal vesicles after a single bout of submaximal exercise.

We investigated the effects of a single bout of non-exhaustive exercise (25 m x min(-1), 10% grade, for 30 min) on the initial rates of lactate uptake in rat skeletal muscle sarcolemmal vesicles and the monocarboxylate transporter 1 (MCT1) content in isolated hindlimb muscles in relation to the exercise-induced oxidative stress. The exercise led to a decrease in red gastrocnemius and red vastus lateralis muscle glycogen content by 74% and 83%, respectively, and an increase in blood lactate concentration from 1.67 +/- 0.15 to 3.44 +/- 0.47 mM (p < 0.05). Initial rates of lactate uptake were measured in zero-trans conditions, at pH 7.4, for 1, 10, 30 and 100 mM external lactate concentrations. Lactate transport capacity was significantly decreased at 1 mM in the exercised group (p < 0.05), while a non-significant trend towards an increase was observed at 10, 30 and 100 mM. We failed to obtain any change in soleus, red tibialis anterior and white gastrocnemius muscle MCT1 content (p>0.05), and no evidence of exercise-induced oxidative stress in terms of muscle malondialdehyde content and glutathione peroxidase and superoxide dismutase activities was observed after the 30 min exercise bout. These results indicate that a single bout of submaximal exercise, which did not induce an increase in muscle MCT1 content and apparent oxidative stress, decreased lactate transport capacity at low physiological concentration. Although the changes are small and independent of a MCT1-facilitated lactate transport regulation, we suggest that another MCT isoform with different kinetic properties from MCT1 could be present in the sarcolemma and responsible for lactate exchange alterations.

Training does not protect against exhaustive exercise-induced lactate transport capacity alterations.

The effects of endurance training on lactate transport capacity remain controversial. This study examined whether endurance training 1) alters lactate transport capacity, 2) can protect against exhaustive exercise-induced lactate transport alteration, and 3) can modify heart and oxidative muscle monocarboxylate transporter 1 (MCT1) content. Forty male Wistar rats were divided into control (C), trained (T), exhaustively exercised (E), and trained and exercised (TE) groups. Rats in the T and TE groups ran on a treadmill (1 h/day, 5 days/wk at 25 m/min, 10% incline) for 5 wk; C and E were familiarized with the exercise task for 5 min/day. Before being killed, E and TE rats underwent exhaustive exercise (25 m/min, 10% grade), which lasted 80 and 204 min, respectively (P < 0.05). Although lactate transport measurements (zero-trans) did not differ between groups C and T, both E and TE groups presented an apparent loss of protein saturation properties. In the trained groups, MCT1 content increased in soleus (+28% for T and +26% for TE; P < 0.05) and heart muscle (+36% for T and +33% for TE; P < 0.05). Moreover, despite the metabolic adaptations typically observed after endurance training, we also noted increased lipid peroxidation byproducts after exhaustive exercise. We concluded that 1) endurance training does not alter lactate transport capacity, 2) exhaustive exercise-induced lactate transport alteration is not prevented by training despite increased MCT1 content, and 3) exercise-induced oxidative stress may enhance the passive diffusion responsible for the apparent loss of saturation properties, possibly masking lactate transport regulation.

Endurance training, expression, and physiology of LDH, MCT1, and MCT4 in human skeletal muscle.

To evaluate the effects of endurance training on the expression of monocarboxylate transporters (MCT) in human vastus lateralis muscle, we compared the amounts of MCT1 and MCT4 in total muscle preparations (MU) and sarcolemma-enriched (SL) and mitochondria-enriched (MI) fractions before and after training. To determine if changes in muscle lactate release and oxidation were associated with training-induced changes in MCT expression, we correlated band densities in Western blots to lactate kinetics determined in vivo. Nine weeks of leg cycle endurance training [75% peak oxygen consumption (VO(2 peak))] increased muscle citrate synthase activity (+75%, P < 0.05) and percentage of type I myosin heavy chain (+50%, P < 0.05); percentage of MU lactate dehydrogenase-5 (M4) isozyme decreased (-12%, P < 0.05). MCT1 was detected in SL and MI fractions, and MCT4 was localized to the SL. Muscle MCT1 contents were consistent among subjects both before and after training; in contrast, MCT4 contents showed large interindividual variations. MCT1 amounts significantly increased in MU, SL, and MI after training (+90%, +60%, and +78%, respectively), whereas SL but not MU MCT4 content increased after training (+47%, P < 0.05). Mitochondrial MCT1 content was negatively correlated to net leg lactate release at rest (r = -0.85, P < 0.02). Sarcolemmal MCT1 and MCT4 contents correlated positively to net leg lactate release at 5 min of exercise at 65% VO(2 peak) (r = 0.76, P < 0.03 and r = 0. 86, P < 0.01, respectively). Results support the conclusions that 1) endurance training increases expression of MCT1 in muscle because of insertion of MCT1 into both sarcolemmal and mitochondrial membranes, 2) training has variable effects on sarcolemmal MCT4, and 3) both MCT1 and MCT4 participate in the cell-cell lactate shuttle, whereas MCT1 facilitates operation of the intracellular lactate shuttle.

Cardiac and skeletal muscle mitochondria have a monocarboxylate transporter MCT1.

To evaluate the potential role of monocarboxylate transporter-1 (MCT1) in tissue lactate oxidation, isolated rat subsarcolemmal and interfibrillar cardiac and skeletal muscle mitochondria were probed with an antibody to MCT1. Western blots indicated presence of MCT1 in sarcolemmal membranes and in subsarcolemmal and interfibrillar mitochondria. Minimal cross-contamination of mitochondria by cell membrane fragments was verified by probing for the sarcolemmal protein GLUT-1. In agreement, immunolabeling and electron microscopy showed mitochondrial MCT1 in situ. Along with lactic dehydrogenase, the presence of MCT1 in striated muscle mitochondria permits mitochondrial lactate oxidation and facilitates function of the "intracellular lactate shuttle."

Lactate transport activity in rat skeletal muscle sarcolemmal vesicles after acute exhaustive exercise.

The effect of a single bout of exhaustive exercise on muscle lactate transport capacity was studied in rat skeletal muscle sarcolemmal (SL) vesicles. Rats were assigned to a control (C) group (n = 14) or an acutely exercised (E) group (n = 20). Exercise consisted of treadmill running (25 m/min, 10% grade) to exhaustion. SL vesicles purified from C and E rats were sealed because of sensitivity to osmotic forces. The time course of 1 mM lactate uptake in zero-trans conditions showed that the equilibrium level in the E group was significantly lower than in the C group (P < 0.05). The initial rate of 1 mM lactate uptake decreased significantly from 2.44 +/- 0.22 to 1.03 +/- 0.08 nmol. min(-1). mg protein(-1) (P < 0.05) after exercise, whereas that of 50 mM lactate uptake did not differ significantly between the two groups. For 100 mM external lactate concentration ([lactate]), exhaustive exercise increased initial rates of lactate uptake (219.6 +/- 36.3 to 465.4 +/- 80.2 nmol. min(-1). mg protein(-1), P < 0.05). Although saturation kinetics were observed in the C group with a maximal transport velocity of 233 nmol. min(-1). mg protein(-1) and a Michealis-Menten constant of 24.5 mM, saturation properties were not seen after exhaustive exercise in the E group, because initial rates of lactate uptake increased linearly with external [lactate]. We conclude that a single bout of exhaustive exercise significantly modified SL lactate transport activity, resulting in a decrease in 1 mM lactate uptake and was associated with alterations in the saturable properties at [lactate] above 50 mM. These results suggest that changes in sarcolemmal lactate transport activity may alter lactate and proton exchanges after exhaustive exercise.

Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle.

To evaluate the potential role of mitochondrial lactate dehydrogenase (LDH) in tissue lactate clearance and oxidation in vivo, isolated rat liver, cardiac, and skeletal muscle mitochondria were incubated with lactate, pyruvate, glutamate, and succinate. As well, alpha-cyano-4-hydroxycinnamate (CINN), a known monocarboxylate transport inhibitor, and oxamate, a known LDH inhibitor were used. Mitochondria readily oxidized pyruvate and lactate, with similar state 3 and 4 respiratory rates, respiratory control (state 3/state 4), and ADP/O ratios. With lactate or pyruvate as substrates, alpha-cyano-4-hydroxycinnamate blocked the respiratory response to added ADP, but the block was bypassed by addition of glutamate (complex I-linked) and succinate (complex II-linked) substrates. Oxamate increased pyruvate (approximately 10-40%), but blocked lactate oxidation. Gel electrophoresis and electron microscopy indicated LDH isoenzyme distribution patterns to display tissue specificity, but the LDH isoenzyme patterns in isolated mitochondria were distinct from those in surrounding cell compartments. In heart, LDH-1 (H4) was concentrated in mitochondria whereas LDH-5 (M4) was present in both mitochondria and surrounding cytosol and organelles. LDH-5 predominated in liver but was more abundant in mitochondria than elsewhere. Because lactate exceeds cytosolic pyruvate concentration by an order of magnitude, we conclude that lactate is the predominant monocarboxylate oxidized by mitochondria in vivo. Mammalian liver and striated muscle mitochondria can oxidize exogenous lactate because of an internal LDH pool that facilitates lactate oxidation.

Effects of 4 wk of hindlimb suspension on skeletal muscle mitochondrial respiration in rats.

We investigated in rats the effect of 4 wk of hypodynamia on the respiration of mitochondria isolated from four distinct muscles [soleus, extensor digitorum longus, tibial anterior, and gastrocnemius (Gas)] and from subsarcolemmal (SS) and intermyofibrillar (IMF) regions of mixed hindlimb muscles that mainly contained the four cited muscles. With pyruvate plus malate as respiratory substrate, 4 wk of hindlimb suspension produced an 18% decrease in state 3 respiration for IMF mitochondria compared with those in the control group (P < 0.05). The SS mitochondria state 3 were not significantly changed. Concerning the four single muscles, the mitochondrial respiration was significantly decreased in the Gas muscle, which showed a 59% decrease in state 3 with pyruvate + malate (P < 0.05). The other muscles presented no significant decrease in respiratory rate in comparison with the control group. With succinate + rotenone, there was no significant difference in the respiratory rate compared with the respective control group, whatever the mitochondrial origin (SS, or IMF, or from single muscle). We conclude that 4 wk of hindlimb suspension alters the respiration of IMF mitochondria in hindlimb skeletal muscles and seems to act negatively on complex I of the electron-transport chain or prior sites. The muscle mitochondria most affected are those isolated from the Gas muscle.

Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles.

2-Chloropropionate (2-CP) is a halogenated monocarboxylic acid generally used to decrease blood lactate concentration in various metabolic states. To investigate whether it has an inhibitory effect on sarcolemmal lactate transport, we compared the initial rate of lactate transport in sarcolemmal membrane vesicles purified from 20 male Wistar rats with and without 2-CP. Transport by these vesicles was measured as uptake of L-(+)-[U-14C]lactate under pH gradient-stimulated cis inhibition. The time courses of 1 mM L-(+)-lactate uptake into vesicles both with and without 10 mM 2-CP (L- or D-) displayed saturation kinetics. Lactate uptake values were lower with 10 mM L-2-CP and 10 mM D-2-CP in comparison to the control values. Both 10 mM L-2-CP and 10 mM D-2-CP significantly inhibited 1 mM L-(+)-lactate uptake (55.8 +/- 9.1 and 53.5 +/- 12.1%, respectively; P < 0.001), whereas a smaller inhibition was observed with a higher lactate concentration of 50 mM (40.2 +/- 11.2 and 38.7 +/- 12.4%; P < 0.001 and P < 0.05, respectively). However, a higher D-2-CP concentration (50 mM) increased the inhibition of pH-stimulated 1 mM L-(+)-lactate uptake (77.0 +/- 9.4%; P < 0.001). D-2-CP had a trans-stimulation effect on the initial rate of lactate efflux of 1 mM L-(+)-lactate compared with baseline efflux (9.5 +/- 0.8 vs. 5.1 +/- 0.4 nmol.min-1.mg protein-1; P < 0.05). 2-CP significantly inhibited the initial rate of lactate uptake in skeletal muscle sarcolemmal membrane vesicles. This result suggests that 2-CP is a nonstereoselective substrate of the lactate muscle carrier that impairs lactate transport.

Effect of NaHCO3 on lactate kinetics in forearm muscles during leg exercise in man.

We investigated NaHCO3 infusion effects on plasma lactate removal by forearm muscles and performance during intensive leg exercise. Seven subjects performed the force-velocity (FV) test with placebo and NaHCO3 (2 mEq.min-1) with a double-blind crossover protocol. Blood samples for arterial ([LA]A) and venous ([LA]V) lactate determinations were taken 1) at rest before infusion, and 2, 6, 10, 14, 18, and 22 min following its start; and 2) at the end of each exercise bout. The arteriovenous difference ([LA]A-V) was determined for each sampling. NaHCO3 significantly increased arterial bicarbonate concentration and pH during rest (P < 0.001; P < 0.001) and the FV test (P < 0.001; P < 0.05). During the test, [LA]A and [LA]V were significantly higher with NaHCO3 (P < 0.05, P < 0.001). At test onset, [LA]A-V became positive and increased until the braking force of 6 kg, with NaHCO3 and placebo, with values significantly lower for NaHCO3 (P < 0.001). Peak anaerobic power (Wanae, peak) and the corresponding braking force (Fmax) were also determined. Fmax was significantly increased with NaHCO3 (P < 0.001). In conclusion, the increasing rise in [LA]A and [LA]V induced by NaHCO3 may be partly explained by a decreased rate of lactate uptake by forearm skeletal muscles. NaHCO3 did not improve Wanae, peak, but improved Fmax, thus increasing FV duration.

Effects of active recovery on plasma lactate and anaerobic power following repeated intensive exercise.

The purpose of this study was to investigate the effects of active recovery (AR) on plasma lactate concentration [La] and anaerobic power output as measured during repeated bouts of intense exercise (6 s) against increasing braking forces. Ten male subjects performed two randomly assigned exercise trials: one with a 5-min passive recovery (PR) after each exercise bout and one with a 5-min active recovery (AR) at a workload corresponding to 32% of maximal aerobic power. Blood samples were taken at rest, at the end of each exercise bout (S1) and at the 5th minute between bout-recovery (S2) for plasma lactate assay. During the tests, [La]S1 was not significantly different after AR and PR, but [La]S2 was significantly lower after AR for power outputs obtained at braking forces 6 kg (5.66 +/- 0.38 vs 7.56 +/- 0.51 mmol.l-1) and peak anaerobic power (PAnP) (6.73 +/- 0.61 vs 8.54 +/- 0.89 mmol.l-1). Power outputs obtained at 2 and 4 kg did not differ after AR and PR. However, when compared with PR, AR induced a significant increase in both power outputs at 6 kg (842 +/- 35 vs 798 +/- 33 W) and PAnP (945 +/- 56 vs 883 +/- 58 W). These results showed that AR between bouts of intensive exercise decreased blood lactate concentration at high braking forces. This decrease was accompanied by higher anaerobic power outputs at these forces.

Lactate uptake by skeletal muscle sarcolemmal vesicles decreases after 4 wk of hindlimb unweighting in rats.

We investigated the effects of 4 wk of hypodynamia on the rate of lactate transport in skeletal muscle sarcolemmal vesicles from control and hindlimb-suspended rats. Characterization of the sarcolemmal preparations was achieved with a marker enzyme (K+-p-nitrophenylphosphatase) and measurement of 1 mM [U-14C]lactate transport activity under zero-trans conditions with or without a pH gradient or the transport inhibitor alpha-hydroxycinnamate. Preparations from the two groups were not significantly different concerning yield and purification. Based on these results, we used this model to analyze the lactate transport activity after hypodynamia by tail suspension. Hindlimb suspension caused a shift from slow to fast myosin heavy chain isoforms in soleus muscles with a 40% decrease in the citrate synthase activity (from 35.3 +/- 3.7 to 21.4 +/- 2.1 mu mol x g-1 x min-1; P < 0.05). Lactate (1 mM) uptake in vesicles from the two groups was a function of time, and the rate after hindlimb suspension was significantly decreased in the suspended compared with the control group (2.25 +/- 0.44 and 3.50 +/- 0.26 nmol x min-1 x mg protein-1, respectively; P < 0.05). These differences were not observed for a higher lactate concentration (50 mM). These results suggest that the level of physical activity plays a role in the regulation of sarcolemmal lactate transport activity implicated in the exchanges of lactate between producing and utilizing cells, organs, and tissues, which are major ways of carbohydrate energy distribution in humans and others species.

Lactate uptake by forearm skeletal muscles during repeated periods of short-term intense leg exercise in humans.

We investigated the role of the forearm skeletal muscles in the removal of lactate during repeated periods of short-term intensive leg exercise, i.e. a force-velocity (FV) test known to induce a marked accumulation of lactate in the blood. The leg FV test was performed by seven untrained male subjects. Arterial and venous blood samples for determination of arterial ([la-]a) and venous ([la-]v) plasma lactate concentrations were concomitantly taken at rest before the test, during the FV test at the end of each period of intensive exercise just before the 5-min between-sprint recovery period, and after the completion of the test at 2, 4, 6, 8, 10, 15, and 20 min of the final recovery. The arteriovenous difference in concentration for plasma lactate ([la-]a-v) was determined for each blood sample. During the test, [la-]a and [la-]v increased significantly (P < 0.001; P < 0.001) with significantly higher values for [la-]a (P < 0.001). At the onset of the test, [la-]a-v became positive and increased up to a braking force of 6 kg, correlating significantly with [la-]a (r = 0.61, P < 0.001) with power (r = 0.58, P < 0.001) during the test. At the end of the test, [la-]a, [la-]v and [la-]a-v decreased (P < 0.001; P < 0.001; P < 0.001 respectively) but were still higher than the basal values after 20-min of passive recovery. In conclusion, forearm skeletal muscles would seem to have been involved in the removal of lactate from the blood during the leg FV test, with an increase in lactate uptake proportional to the increase in plasma lactate concentration and power.

[Effect of hypodynamia on initial speed of lactate transport in skeletal muscle sarcolemmal vesicles in rats]. / Effet de l'hypodynamie sur la vitesse initiale du transport du lactate au niveau des vésicules de sarcolemme de muscles squelettiques de rat.

Skeletal muscle sarcolemmal vesicles from control (C) and hindlimb suspended (S) rats were used to investigate the effect of unweighting on the lactate transporter activity. Sarcolemmal preparations were not different between the two groups. The efficiency of 4 weeks of hindlimb suspension was confirmed by a 40% decrease of citrate synthetase activity and a shift towards faster myosin isoforms in soleus muscle. The time course of 1 mM lactate uptake showed that the equilibrium was reached faster in group C (20 s) than in group S (40 s). The initial rate of 1 mM of lactate uptake decreased significantly (p < 0.05) after 4 weeks of hindlimb suspension. The initial rate of 50 mM lactate uptake did not differ significantly between the two groups. We conclude that 4 weeks of unweighting decreases significantly the skeletal muscle sarcolemmal lactate transport activity in rats. This result suggests that the level of physical activity probably plays a role on lactate transport regulation in muscle.