Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo.

OBJECTIVES: The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts. MATERIALS AND METHODS: Rat L6 and primary human myoblasts were grown in 21% or 1% O(2) in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT-PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique. RESULTS: After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O(2) conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts. CONCLUSIONS: The data show that exposure to hypoxic conditions (1% O(2)) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.

Erythropoietin induces a shift of muscle phenotype from fast glycolytic to slow oxidative.

Sedentary and trained rat groups were studied. Each of these groups was either erythropoietin or placebo treated. Erythropoietin treatment increased significantly all haematological parameters studied. Training per se failed to modify haematological parameters. In a second time, we studied the specific activity of several oxidative enzymes in three different muscles. In sedentary rats, erythropoietin treatment increased significantly the specific activities of cytochrome c oxidase and L-3-hydroxyacyl CoA dehydrogenase in the soleus and those of L-3-hydroxyacyl CoA dehydrogenase and phosphofructokinase in both locomotor muscles. Training increased the oxidative enzymes activities in all muscles studied. In trained rats, effects of erythropoietin and training on oxidative enzymes activities were additive. In all erythropoietin treated muscles, the expression of slow twitch myosin light chains and oxidative myosin heavy chains increased. A similar phenomenon took place in all trained groups for light chains and in placebo treated trained rats for heavy chains. In trained groups, the effects of the hormone and of training were additive. Our results suggest strongly that two different mechanisms are involved in the response of skeletal muscles to erythropoietin treatment and to endurance training and probably whole body endurance is affected by erythropoietin treatment by an increase of oxygenation of all tissues.

Brain stem NO modulates ventilatory acclimatization to hypoxia in mice.

The objective of our study was to assess the role of neuronal nitric oxide synthase (nNOS) in the ventilatory acclimatization to hypoxia. We measured the ventilation in acclimatized Bl6/CBA mice breathing 21% and 8% oxygen, used a nNOS inhibitor, and assessed the expression of N-methyl-d-aspartate (NMDA) glutamate receptor and nNOS (mRNA and protein). Two groups of Bl6/CBA mice (n = 60) were exposed during 2 wk either to hypoxia [barometric pressure (PB) = 420 mmHg] or normoxia (PB = 760 mmHg). At the end of exposure the medulla was removed to measure the concentration of nitric oxide (NO) metabolites, the expression of NMDA-NR1 receptor, and nNOS by real-time RT-PCR and Western blot. We also measured the ventilatory response [fraction of inspired O(2) (Fi(O(2))) = 0.21 and 0.08] before and after S-methyl-l-thiocitrulline treatment (SMTC, nNOS inhibitor, 10 mg/kg ip). Chronic hypoxia caused an increase in ventilation that was reduced after SMTC treatment mainly through a decrease in tidal volume (Vt) in normoxia and in acute hypoxia. However, the difference observed in the magnitude of acute hypoxic ventilatory response [minute ventilation (Ve) 8% - Ve 21%] in acclimatized mice was not different. Acclimatization to hypoxia induced a rise in NMDA receptor as well as in nNOS and NO production. In conclusion, our study provides evidence that activation of nNOS is involved in the ventilatory acclimatization to hypoxia in mice but not in the hypoxic ventilatory response (HVR) while the increased expression of NMDA receptor expression in the medulla of chronically hypoxic mice plays a role in acute HVR. These results are therefore consistent with central nervous system plasticity, partially involved in ventilatory acclimatization to hypoxia through nNOS.

Moderate exercise in hypoxia induces a greater arterial desaturation in trained than untrained men.

During moderate exercise breathing a low inspired O(2) fraction (F(I)O(2)), arterial O(2) desaturation may depend on the fitness level. Seven trained (TM) and seven untrained men (UTM) cycled in normoxia and in hypoxia (F(I)O(2)=0.187, 0.173, 0.154, 0.13 and 0.117). We compared TM and UTM at submaximal intensities below the ventilatory threshold. Ventilatory variables were monitored and arterial oxygen saturation was measured by pulse oximetry. O(2) saturation was not different between groups at sea level. In hypoxia, O(2) saturation was lower in TM than in UTM at F(I)O(2)=0.154 (87.3 +/- 2.9% vs 90.4 +/- 1.5% at 90 W) and below. Both the ventilatory-equivalent and the end-tidal O(2) pressure were lower in TM at sea level and at every F(I)O(2), with the differences between TM and UTM becoming apparent at lower exercise intensity and increasing in magnitude as the severity of hypoxia increased. O(2) saturation was correlated with the ventilatory parameters at every F(I)O(2) and the correlations were stronger in severe hypoxia. These results demonstrate that a moderate exercise carried out in hypoxia, contrary to normoxic conditions, can lead to a greater arterial desaturation in TM compared with UTM. This phenomenon could be partly attributed to a relative hypoventilation in trained subjects.

Oxidative stress in hemodialyzed patients during exhausting exercise.

BACKGROUND: This study was designed to study the effect of an exhausting exercise test on plasma lipid peroxidation marker (malondialdehyde, MDA) and on vitamin E levels in hemodialyzed patients (HD) compared to healthy control subjects (HC). METHODS: Eight sedentary chronically HD, (39.0+/-10.8 years) and eight sedentary HC (38.4+/-12.4 years) were studied. Before exercise, the activity of scavenger enzymes [plasma and erythrocyte glutathione peroxidase (GPX), and erythrocyte superoxide dismutase (SOD)] was determined. All subjects performed a symptom-limited exercise test; respiratory gas exchanges were collected on-line and blood was sampled five min before exercise, at exhaustion and after 30 min of recovery. RESULTS: At rest, the activity of plasma and erythrocyte GPX and erythrocyte SOD was significantly lower in HD than in HC. Exercise provoked an increase of plasma MDA concentration after recovery in HD patients, whereas our HC group show a decrease in MDA plasma level immediately after exercise, returning to the rest values during recovery. Exercise induced no change of vitamin E concentration in HD whereas in HC it increased during recovery. CONCLUSIONS: Our results indicate that: 1) at rest: although hemodialyzed patients showed lower scavenger enzymes activity than HC subjects, there was no difference in MDA plasma level between groups; 2) there is some evidence that physical exercise contribute to oxidative stress during recovery in HD patients, and 3) exercise induced an increase in plasma vitamin E concentration in healthy subjects.

Exercise does not induce oxidative stress in trained heart transplant recipients.

PURPOSE: The objectives of this study were twofold: 1) to determine the effect of incremental exercise to volitional fatigue on plasma levels of lipid peroxidation (malondialdehyde) in heart transplant recipients (HRT) and 2) to examine blood antioxidant capacity in HTR by assessment of antioxidant enzyme activities and vitamin E levels. METHODS: Seven endurance-trained HTR (mean +/- SD; age 39.7 +/- 12.8 yr) and seven endurance-trained healthy, age-matched control subjects (HC) (mean age 40.6 +/- 10.7 yr) performed a symptom-limited incremental exercise test on a cycle ergometer. Venous blood samples were obtained at rest, exercise, and during recovery and analyzed for plasma levels of malondialdehyde (MDA) as well as markers of blood antioxidant capacity. After exercise and during recovery, all dependent measures were corrected for plasma volume changes induced by exercise. Significance was established at (P < 0.05). RESULTS: No group differences existed in plasma levels of MDA at rest. Further, graded exercise did not alter plasma levels of MDA in either group. Resting erythrocyte glutathione peroxidase (GPX) activity was significantly lower and erythrocyte superoxide dismutase (SOD) activity was higher in HTR compared with HC. Finally, at rest, no group differences existed in plasma GPX activity or vitamin E levels. CONCLUSIONS: Graded exercise to fatigue does not promote an increase in oxidative stress in blood of exercise trained HTR. Therefore, physical exercise does not appear to pose an oxidative-stress risk for these patients.

[Screening for exogenous erythropoietin]. / Erythropoïétine: dépistage d'apport exogène.

r-HuEPO: Ever since it was first produced, illicit use recombinant human erythropoietin (r-HuEPO), a human polypeptide which accelerates production and maturation of red cells and consequently improves aerobic potential, has been observed in athletic competitions. IMPORTANCE OF SCREENING: Unfortunately, abuse of r-HuEPO can have severe adverse effects, particularly on the cardiovascular system. Screening methods capable of detecting exogenous erythropoietin were thus developed not only to detect illicit drug use in sports competition, but also to preserve athletesí health. THEORETICAL POSSIBILITIES: Several methods have been explored. Direct methods using electrophoresis or indirect methods measuring different biological parameters (fibrin degradation products, number of soluble transferrin receptors) as well specific hematology tests (red cell morphology, hematocrit...) can theoretically detect exogenous erythropoietin. METHODS TO VALIDATE: It would be advisable to examine all of the available work on the proposed methods to develop a reliable diagnostic method for detecting r-HuEPO. Soluble transferrin receptor counts appear to be the most promising method, but specificity is not totally satisfactory.

Chronotropic competence in endurance trained heart transplant recipients: heart rate is not a limiting factor for exercise capacity.

OBJECTIVES: The purpose of this study was to show that the chronotropic potential of the well trained heart transplant recipient (HTR) does not limit exercise capacity. BACKGROUND: Chronotropic incompetence is considered to be the main limiting factor of the functional capacity of heart transplant recipients. However, no systematic study had been published on patients who had spontaneously undergone heavy endurance training for several years. METHODS: Heart rate (HR) and respiratory gas exchanges (VO2, VCO2, VE) were measured in 14 trained HTRs (T-HTRs) during exercise tests on a bicycle, on a treadmill and by Holter electrocardiography during a race. RESULTS: Peak values observed in T-HTRs during the treadmill test were higher than those reached during the bicycle test (VO2peak: 39.8+/-6.9 vs. 32.5+/-7.8 ml x kg(-1) x min(-1), p < 0.001; HRpeak: 169+/-14 vs. 159+/-16 bpm, p < 0.01). During treadmill exercise VO2peak and HRpeak values observed were very close to the mean predicted VO2pmax and HRpmax. The maximum heart rate during the race (HRrace) was greater than HRpeak values during the treadmill test (179+/-14 vs 169+/-14 bpm, p < 0.01) and slightly above the mean predicted values (HRrace/HRpmax X 100 = 101+/-10%). The treadmill exercise test yields more reliable data than does the bicycle test. CONCLUSIONS: Extensive endurance training enables heart transplant recipients to reach physical fitness levels similar to those of normal sedentary subjects; heart rate does not limit their exercise capacity.

Mobilization of visceral adipose tissue related to the improvement in insulin sensitivity in response to physical training in NIDDM. Effects of branched-chain amino acid supplements.

OBJECTIVE: To evaluate the effects of an intense physical training program on abdominal fat distribution, glycemic control, and insulin sensitivity in patients with NIDDM and to determine whether branched-chain amino acid (BCAA) supplements influence these effects. RESEARCH DESIGN AND METHODS: Twenty-four patients (ages 45 +/- 2 [mean +/- SE] years, BMI 30.2 +/- 0.9 kg/m2, HbA1c 7.9 +/- 0.3%) were randomly assigned to four groups: training plus BCAA supplement (n = 6), training plus placebo (n = 6), sedentary plus BCAA supplement (n = 6), and sedentary plus placebo (n = 6). Physical training consisted of a supervised 45-min cycling exercise at 75% of their oxygen uptake peak (VO2 peak) two times per week and an intermittent exercise one time per week for 2 months. RESULTS: Patients who exercised increased their VO2 peak by 41% and their insulin sensitivity by 46%. Physical training significantly decreased abdominal fat evaluated by magnetic resonance imaging (umbilicus), with a greater loss of visceral adipose tissue (VAT) (48%) in comparison with the loss of subcutaneous adipose tissue (18%), but did not significantly affect body weight. The change in visceral abdominal fat was associated with the improvement in insulin sensitivity (r = 0.84, P = 0.001). BCAA supplementation had no effect on abdominal fat and glucose metabolism. CONCLUSIONS: Physical training resulted in an improvement in insulin sensitivity with concomitant loss of VAT and should be included in the treatment program for patients with NIDDM.

Influence of simulated altitude on the performance of five blood glucose meters.

OBJECTIVE: To determine the reliability of five blood glucose meters (BGMs) at various simulated altitudes using a hypobaric chamber. RESEARCH DESIGN AND METHODS: Blood glucose levels (ranged from 1.5 to 26.3 mmol/l, according to the reference method) were measured in 18 venous blood samples by each BGM at 200, 1,000, and every 500 m up to 4,000 m in a hypobaric chamber, where temperature and humidity were held constant. RESULTS: Four BGMs underestimated and one overestimated blood glucose concentration while barometric pressure decreased. The average percent error varied in relation to simulated altitude from 0.26 +/- 4.8% (SD) at 200 m to -28.9 +/- 4.5% at 4,000 m (Glucometer 3; P < 0.05), from 28.4 +/- 5.7 to 49.3 +/- 5.9% (Accu-Chek Easy; P < 0.05), from -10.5 +/- 2.6 to 19.8 +/- 4.3% (Tracer, P < 0.05), from -5.5 +/- 2.6 to -11.2 +/- 3.0% (Reflolux; NS), and from 17.8 +/- 4.3 to 14.8 +/- 3.6% (One Touch; NS). The most accurate seemed to be the Reflolux, except for high blood glucose levels at simulated high altitudes. The One Touch II showed a good agreement, whatever the barometric pressure and the range of blood glucose concentrations. The highest underestimation was seen with the Glucometer 3. CONCLUSIONS: Except for the Accu-Chek Easy, low barometric pressure underestimated the BGM results in comparison with measurements taken at simulated low altitudes. The lack of accuracy and consistency of performance > 2,000 m should be known by diabetic patients practicing sports activities, such as trekking or skiing at high altitudes.

Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin in athletes. Blood sampling and doping control.

1. The pharmacokinetics of recombinant human erythropoietin (rHuEpo) were initially determined in two healthy volunteers after a single subcutaneous dose (50 u kg-1). Twenty subjects then received repeated subcutaneous administrations of high dose (200 u kg-1) rHuEpo and 10 subjects received placebo. An immunoradiometric assay was used to measure the concentrations of erythropoietin (Epo) in serum and urine. 2. Serum Epo concentration-time profiles were best described by a one-compartment open model with zero-order input. The mean elimination half-life (+/- s.d.) was 42.0 +/- 34.2 h. Clearance, uncorrected for bioavailability, was 0.05 +/- 0.011 h-1 kg-1. Erythropoietin concentrations returned to normal values in serum and urine, 7 and 4 days after the last administration, respectively. 3. The recombinant hormone was well tolerated. Significant changes in reticulocytes and red blood cells, haemoglobin concentrations and haematocrit were observed after administration of rHuEpo. In the control group, these parameters remained unchanged. 4. The change in reticulocytes was used as an index of the therapeutic effect of rHuEpo. The concentration-effect relationship was best described by an exponential model. 5. These data show the limitations of the measurement of Epo concentrations in blood and urine samples, collected in athletes during competition, for antidoping control. Epo doping can be detected only during or within 4 to 7 days of ending a course of rHuEpo.

[Cardiovascular risks during isokinetic tests for peripheral functional studies]. / Risques cardio-vasculaires lors des tests d'explorations fonctionnelles périphériques par isocinétisme.

OBJECTIVES: Isokinetic tests used to assess both muscle function and joint disorders stimulate the entire organism and in particular the cardiovascular system. We evaluated the risk of cardiovascular or vascular impairment during isokinetic knee extension and flexion tests. METHODS: Cardiac electrical activity, heart rate and systolic and diastolic blood pressures were measured in 251 patients during 50-sec isokinetic tests. RESULTS: Abnormal findings were recorded in 15 patients (5.9%) including 6 with altered blood pressure, 4 with altered cardiac electrical activity and 5 with heart rate abnormalities. Maximal heart rate for a given subject recorded during the isokinetic test was 199 beats per minute and heart rate exceeded 190 bpm in 92 subjects (36.6%). Maximal efforts with cardiac barometric charge produced dramatic, occasionally pathological, changes in heart rate or blood pressure. CONCLUSION: These findings emphasize the need to carefully assess patients before isokinetic tests and carefully monitor cardiovascular parameters. A preliminary physical examination and medical surveillance during testing are required.

Lactate transport during differentiation of skeletal muscle cells: evidence for a specific carrier in L6 myotubes.

Cellular uptake of lactate involves a carrier in numerous types of mammalian cells. In cultured rat L6 myoblasts, lactate diffuses freely. We show that during myogenesis a carrier appears at the myotube stage. The degree of cellular differentiation was determined by microscopic observation and by measurement of a marker of biochemical differentiation: the increase in specific activity of creatine kinases.

Blood mononuclear cells energy metabolism response to muscular exercise.

In a study of 4 enzymatic activities in human blood mononuclear cells before and immediately after standard maximal exercise test (25 min) on treadmill we noted: (1) a significant decrease in the activity of the pyruvate dehydrogenase complex (PDHc); (2) a significant increase in the activity of the citrate synthase (CS); and (3) no significant changes in the activities of cytochrome c oxydase and succinate cytochrome reductase. Although non-specifically stimulated (antigen or even mitogen), the blood mononuclear cells responded metabolically to muscular exercise. Pyruvate dehydrogenase (PDHc) activity of blood mononuclear cells appeared to decrease via enzyme interconversion regulation of PDHc. It is not known if these changes can be linked to the studies indicated altered immune function after a single bout of exercise.

Lactate transport in skeletal muscle cells: uptake in L6 myoblasts.

During exercise, lactate is produced by degradation of glucose-6-phosphate during glycolysis in the contracting muscles. This lactate is metabolized during and after exercise in the muscle itself and also in the liver and other muscles, which can use it as an energy metabolite or can resynthetize glycogen. Lactate is transported in the blood, and the rate of muscular utilization may be limited by two factors: the rate of metabolic utilization by the muscle cell; and the rate of transport across the membrane regulating lactate transfer from the blood to the cell. We have studied lactate uptake in L6 muscle cells by incorporation of 14C-lactate. The uptake rates were linear for 20 seconds with 5 mM lactate and 10 seconds with 20 mM. The uptake during 10 seconds for physiological lactate concentrations (1-20 mM) gave a straight line passing through the origin. Lactate uptake was not altered by specific inhibitors of lactate transport (2.5 mM alpha cyano-4-hydroxycinnamic acid. 5 microM 4,4'-diisothiocyanostilbene-2,2'disulphonic acid) or by the stereospecific D-lactate inhibitor. The results suggest that L-lactate uptake in L6 cells occurs by passive diffusion.

The energy cost of running increases with the distance covered.

The net energy cost of running per unit of body mass and distance (Cr, ml O2.kg-1.km-1) was determined on ten amateur runners before and immediately after running 15, 32 or 42 km on an indoor track at a constant speed. The Cr was determined on a treadmill at the same speed and each run was performed twice. The average value of Cr, as determined before the runs, amounted to 174.9 ml O2.kg-1.km-1, SD 13.7. After 15 km, Cr was not significantly different, whereas it had increased significantly after 32 or 42 km, the increase ranging from 0.20 to 0.31 ml O2.kg-1.km-1 per km of distance (D). However, Cr before the runs decreased, albeit at a progressively smaller rate, with the number of trials (N), indicating an habituation effect (H) to treadmill running. The effects of D alone were determined assuming that Cr increased linearly with D, whereas H decreased exponentially with increasing N, i.e. Cr = Cr0 + a D + He-bN. The Cr0, the "true" energy cost of running in nonfatigued subjects accustomed to treadmill running, was assumed to be equal to the average value of Cr before the run for N equal to or greater than 7 (171.1 ml O2.kg-1.km-1, SD 12.7; n = 30).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of previous supramaximal work on lacticaemia during supra-anaerobic threshold exercise.

Twelve male and female subjects (eight trained, four untrained) exercised for 30 min on a treadmill at an intensity of maximal O2 consumption (% VO2max) 90.0%, SD 4.7 greater than the anaerobic threshold of 4 mmol.l-1 (Than = 83.6% VO2max, SD 8.9). Time-dependent changes in blood lactate concentration [( lab]) during exercise occurred in two phases: the oxygen uptake (VO2) transient phase (from 0 to 4 min) and the VO2 steady-state phase (4-30 min). During the transient phase, [lab] increased markedly (1.30 mmol.l-1.min-1, SD (0.13). During the steady-state phase, [lab] increased slightly (0.02 mmol.l-1.min-1, SD 0.06) and when individual values were considered, it was seen that there were no time-dependent increases in [lab] in half of the subjects. Following hyperlacticaemia (8.8 mmol.l-1, SD 2.0) induced by a previous 2 min of supramaximal exercise (120% VO2max), [lab] decreased during the VO2 transient (-0.118 mmol.l-1.min-1, SD 0.209) and steady-state (-0.088 mmol.l-1.min-1, SD 0.103) phases of 30 min exercise (91.4% VO2max, SD 4.8). In conclusion, it was not possible from the Than to determine the maximal [lab] steady state for each subject. In addition, lactate accumulated during previous supramaximal exercise was eliminated during the VO2 transient phase of exercise performed at an intensity above the Than. This effect is probably largely explained by the reduction in oxygen deficit during the transient phase. Under these conditions, the time-course of changes in [lab] during the VO2 steady state was also affected.

Changes in blood leucocyte populations induced by acute maximal and chronic submaximal exercise.

Absolute (x 10(3).mm-3) or relative (%) numbers of blood leucocyte types (monocytes, lymphocytes, neutrophils) and lymphocyte subsets (T11+, T4+, T8+, B1+, and NKH1+) reacting with specific monoclonal antibodies were determined at rest, immediately after maximal exercise on a treadmill, in six controls (C), and in six young cyclists before training (BT) and after 5 months of training (AT). Maximal exercise significantly increased the absolute number (mobilization) of virtually all the types of leucocytes and subsets of lymphocytes in C, BT and AT subjects. In these subjects mobilization of natural killer cells (NKH1+) and cytotoxic/suppressor T lymphocytes (T8+) was greater than mobilization of the other leucocyte types and lymphocyte subsets; however, maximal exercise induced no significant changes in the relative numbers of any leucocyte types and lymphocyte subsets, except in the case of T4+ lymphocytes in At cyclists. Chronic submaximal exercise induced increased mobilization of neutrophils and decreased mobilization of lymphocytes during maximal exercise, except in the case of B lymphocytes (B1+) and NKH1+ cells, and decreases in the absolute and relative number of neutrophils at rest. It remains to be seen how these results can explain the modifications of leucocyte activities noted in vitro after isolated or chronic exercise.