Vitamin D supplementation associated with physical exercise promotes a tolerogenic immune environment without effect on mammary tumour growth in C57BL/6 mice.

PURPOSE: High plasma vitamin D (VitD) level and regular exercise (Ex) are known to have anti-cancer and immunomodulatory effects. This study aimed to evaluate the impact of VitD supplementation and imposed physical Ex on mammary tumour growth and immune response in ovariectomised mice fed high-fat (HF) diet. METHODS: Ovariectomised 33-week-old mice C57BL/6 (n = 60), housed in enriched environment (EE), were fed HF diet (450 kcal/100 g) supplemented or not with VitD (HF/HF + D: 125/1225 IU/100 g) for 12 weeks and submitted or not to Ex (HF + Ex; HF + D + Ex) on treadmill (45 min/day, 5 days/week). At w8, syngeneic tumour cells EO771 were orthotopically injected into the 4th mammary gland. Spontaneous activity (SPA), maximal speed (MS) and forelimb grip strength (GS) were measured. Tumour immune cells infiltrate was phenotyped by FACS. Data (mean ± SEM) were analysed by two-way ANOVA + Tukey post-test. RESULTS: Ex (p = 0.01) and VitD (p = 0.05) reduced body weight gain. Exercise decreased visceral fat mass [g: 1.5 ± 0.8 (HF); 1.2 ± 0.65 (HF + Ex); 0.9 ± 0.6 (HF + D + Ex); p = 0.03]. SPA (p < 0.0001) and GS (p = 0.01) were higher in HF + D + Ex mice vs others. No effect of Ex or VitD on tumour growth was detected. In tumour, VitD decreased the proportion of NK (p = 0.03), while Ex increased it (p = 0.03). The Th1/Th2 ratio is lowered by VitD (p = 0.05), while Tc/Treg ratio was not affected either by Exercise or VitD. CONCLUSION: In our experimental conditions, VitD supplementation and physical exercise have synergetic effects reducing the weight gain under HF diet and improving the physical capacities of mice. VitD coupled with exercise induces an immunosuppressive response without effect on tumour growth.

Lung Stereotactic Arc Therapy in Mice: Development of Radiation Pneumopathy and Influence of HIF-1α Endothelial Deletion.

PURPOSE: Stereotactic body radiation therapy offers good lung local tumor control by the administration of a high dose per fraction in small volumes. Stereotactic body radiation therapy preclinical modeling is now possible, and our aim was to develop a model of focal irradiation of the mouse lung and to investigate the impact of conditional hypoxia-inducible factor 1α (HIF-1α) deletion in the endothelium on radiation-induced tissue damage. METHODS AND MATERIALS: The Small Animal Radiation Research Platform was used to create a mouse model of focal irradiation of the lung using arc therapy. HIF-1α conditional deletion was obtained by crossing mice expressing Cre recombinase under the endothelial promoter VE-cadherin (VECad-Cre+/+ mice) with HIF-1α floxed mice. RESULTS: Lung stereotactic arc therapy allows thoracic wall sparing and long-term studies. However, isodose curves showed that neighboring organs received significant doses of radiation, as revealed by ipsilateral lung acute red hepatization and major gene expression level modifications. Conditional HIF-1α deletion reduced acute lung edema and tended to diminish neutrophil infiltrate, but it had no impact on long-term global tissue damage. CONCLUSIONS: Arc therapy for focal high-dose irradiation of mouse lung is an efficient model for long-term studies. However, irradiation may have a strong impact on the structure and function of neighboring organs, which must be considered. HIF-1α conditional deletion has no beneficial impact on lung damage in this irradiation schedule.

Case Studies in Physiology: Maximal oxygen consumption and performance in a centenarian cyclist.

The purpose of this study was to examine the physiological characteristics of an elite centenarian cyclist who, at 101 yr old, established the 1-h cycling record for individuals ≥100 yr old (24.25 km) and to determine the physiological factors associated with his performance improvement 2 yr later at 103 yr old (26.92 km; +11%). Before each record, he performed an incremental test on a cycling ergometer. For 2 yr, he trained 5,000 km/yr with a polarized training that involved cycling 80% of mileage at "light" rate of perceived exertion (RPE) ≤12 and 20% at "hard" RPE ≥15 at a cadence between 50 and 70 rpm. His body weight and lean body mass did not change, while his maximal oxygen consumption (V̇o2max) increased (31-35 ml·kg-1·min-1; +13%). Peak power output increased from 90 to 125 W (+39%), mainly because of increasing the maximal pedaling frequency (69-90 rpm; +30%). Maximal heart rate did not change (134-137 beats/min) in contrast to the maximal ventilation (57-70 l/min, +23%), increasing with both the respiratory frequency (38-41 cycles/min; +8%) and the tidal volume (1.5-1.7 liters; +13%). Respiratory exchange ratio increased (1.03-1.14) to the same extent as tolerance to V̇co2 In conclusion, it is possible to increase performance and V̇o2max with polarized training focusing on a high pedaling cadence even after turning 100 yr old.NEW & NOTEWORTHY This study shows, for the first time, that maximal oxygen consumption (+13%) and performance (+11%) can still be increased between 101 and 103 yr old with 2 yr of training and that a centenarian is able, at 103 yr old, to cover 26.9 km/h in 1 h.

Validation of a Ramp Running Protocol for Determination of the True VO2max in Mice.

In the field of comparative physiology, it remains to be established whether the concept of VO2max is valid in the mouse and, if so, how this value can be accurately determined. In humans, VO2max is generally considered to correspond to the plateau observed when VO2 no longer rises with an increase in workload. In contrast, the concept of VO2peak tends to be used in murine studies. The objectives of the present study were to determine whether (i) a continuous ramp protocol yielded a higher VO2peak than a stepwise, incremental protocol, and (ii) the VO2peak measured in the ramp protocol corresponded to VO2max. The three protocols (based on intensity-controlled treadmill running until exhaustion with eight female FVB/N mice) were performed in random order: (a) an incremental protocol that begins at 10 m.min(-1) speed and increases by 3 m.min(-1) every 3 min. (b) a ramp protocol with slow acceleration (3 m.min(-2)), and (c) a ramp protocol with fast acceleration (12 m.min(-2)). Each protocol was performed with two slopes (0 and 25°). Hence, each mouse performed six exercise tests. We found that the value of VO2peak was protocol-dependent (p < 0.05) and was highest (59.0 ml.kg (0.75).min(-1)) for the 3 m.min(-2) 0° ramp protocol. In the latter, the presence of a VO2max plateau was associated with the fulfillment of two secondary criteria (a blood lactate concentration >8 mmol.l(-1) and a respiratory exchange ratio >1). The total duration of the 3 m.min(-2) 0° ramp protocol was shorter than that of the incremental protocol. Taken as a whole, our results suggest that VO2max in the mouse is best determined by applying a ramp exercise protocol with slow acceleration and no treadmill slope.

Myostatin is a key mediator between energy metabolism and endurance capacity of skeletal muscle.

Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn(-/-) mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn(-/-) mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn(-/-) mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn(-/-) mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.

The sustainability of VO2max: effect of decreasing the workload.

The study examined the maintenance of VO(2max) using VO(2max) as the controlling variable instead of power. Therefore, ten subjects performed three exhaustive cycling exercise bouts: (1) an incremental test to determine VO(2max) and the minimal power at VO(2max) (PVO(max)), (2) a constant-power test at PVO(max) and (3) a variable-power test (VPT) during which power was varied to control VO(2) at VO(2max). Stroke volume (SV) was measured by impedance in each test and the stroke volume reserve was calculated as the difference between the maximal and the average 5-s SV. Average power during VPT was significantly lower than PVO(max) (238 ± 79 vs. 305 ± 86 W; p < 0.0001). All subjects, regardless of their VO(2max) values and/or their ability to achieve a VO(2max) plateau during incremental test, were able to sustain VO(2max) for a significantly longer time during VPT compared to constant-power test (CPT) (958 ± 368 s vs. 136 ± 81 s; p < 0.0001). Time to exhaustion at VO(2max) during VPT was correlated with the power drop in the first quarter of the time to exhaustion at VO(2max) (r = 0.71; p < 0.02) and with the stroke volume reserve (r = 0.70, p = 0.02) but was not correlated with VO(2max). This protocol, using VO(2max) rather than power as the controlling variable, demonstrates that the maintenance of exercise at VO(2max) can exceed 15 min independent of the VO(2max) value, suggesting that the ability to sustain exercise at VO(2max) has different limiting factors than those related to the VO(2max) value.

Skeletal muscle alterations and exercise performance decrease in erythropoietin-deficient mice: a comparative study.

BACKGROUND: Erythropoietin (EPO) is known to improve exercise performance by increasing oxygen blood transport and thus inducing a higher maximum oxygen uptake (VO2max). Furthermore, treatment with (or overexpression of) EPO induces protective effects in several tissues, including the myocardium. However, it is not known whether EPO exerts this protective effect when present at physiological levels. Given that EPO receptors have been identified in skeletal muscle, we hypothesized that EPO may have a direct, protective effect on this tissue. Thus, the objectives of the present study were to confirm a decrease in exercise performance and highlight muscle transcriptome alterations in a murine EPO functional knock-out model (the EPO-d mouse). METHODS: We determined VO2max peak velocity and critical speed in exhaustive runs in 17 mice (9 EPO-d animals and 8 inbred controls), using treadmill enclosed in a metabolic chamber. Mice were sacrificed 24h after a last exhaustive treadmill exercise at critical speed. The tibialis anterior and soleus muscles were removed and total RNA was extracted for microarray gene expression analysis. RESULTS: The EPO-d mice's hematocrit was about 50% lower than that of controls (p<0.05) and their performance level was about 25% lower (p<0.001). A total of 1583 genes exhibited significant changes in their expression levels. However, 68 genes were strongly up-regulated (normalized ratio>1.4) and 115 were strongly down-regulated (normalized ratio<0.80). The transcriptome data mining analysis showed that the exercise in the EPO-d mice induced muscle hypoxia, oxidative stress and proteolysis associated with energy pathway disruptions in glycolysis and mitochondrial oxidative phosphorylation. CONCLUSIONS: Our results showed that the lack of functional EPO induced a decrease in the aerobic exercise capacity. This decrease was correlated with the hematocrit and reflecting poor oxygen supply to the muscles. The observed alterations in the muscle transcriptome suggest that physiological concentrations of EPO exert both direct and indirect muscle-protecting effects during exercise. However, the signaling pathway involved in these protective effects remains to be described in detail.

NMR metabolomics for assessment of exercise effects with mouse biofluids.

Exercise modulates the metabolome in urine or blood as demonstrated previously for humans and animal models. Using nuclear magnetic resonance (NMR) metabolomics, the present study compares the metabolic consequences of an exhaustive exercise at peak velocity (Vp) and at critical velocity (Vc) on mice. Since small-volume samples (blood and urine) were collected, dilution was necessary to acquire NMR spectra. Consequently, specific processing methods were applied before statistical analysis. According to the type of exercise (control group, Vp group and Vc group), 26 male mice were divided into three groups. Mice were sacrificed 2 h after the end of exercise, and urine and blood samples were drawn from each mouse. Proton NMR spectra were acquired with urine and deproteinized blood. The NMR data were aligned with the icoshift method and normalised using the probabilistic quotient method. Finally, data were analysed with the orthogonal projection of latent-structure analysis. The spectra obtained with deproteinized blood can neither discriminate the control mice from exercised mice nor discriminate according to the duration of the exercise. With urine samples, a significant statistical model can be estimated when comparing the control mice to both groups, Vc and Vp. The best model is obtained according to the exercise duration with all mice. Taking into account the spectral regions having the highest correlations, the discriminant metabolites are allantoin, inosine and branched-chain amino acids. In conclusion, metabolomic profiles assessed with NMR are highly dependent on the exercise. These results show that urine samples are more informative than blood samples and that the duration of the exercise is a more important parameter to influence the metabolomic status than the exercise velocity.

Cardiac output and performance during a marathon race in middle-aged recreational runners.

PURPOSE: Despite the increasing popularity of marathon running, there are no data on the responses of stroke volume (SV) and cardiac output (CO) to exercise in this context. We sought to establish whether marathon performance is associated with the ability to sustain high fractional use of maximal SV and CO (i.e, cardiac endurance) and/or CO, per meter (i.e., cardiac cost). METHODS: We measured the SV, heart rate (HR), CO, and running speed of 14 recreational runners in an incremental, maximal laboratory test and then during a real marathon race (mean performance: 3 hr 30 min ± 45 min). RESULTS: Our data revealed that HR, SV and CO were all in a high but submaximal steady state during the marathon (87.0 ± 1.6%, 77.2 ± 2.6%, and 68.7 ± 2.8% of maximal values, respectively). Marathon performance was inversely correlated with an upward drift in the CO/speed ratio (mL of CO × m(-1)) (r = -0.65, P < 0.01) and positively correlated with the runner's ability to complete the race at a high percentage of the speed at maximal SV (r = 0.83, P < 0.0002). CONCLUSION: Our results showed that marathon performance is inversely correlated with cardiac cost and positively correlated with cardiac endurance. The CO response could be a benchmark for race performance in recreational marathon runners.

A new incremental test for VO2max accurate measurement by increasing VO2max plateau duration, allowing the investigation of its limiting factors.

The purpose of this study was to (1) validate a new exercise protocol for accurate measurement of VO(2max) by obtention of a VO(2max) plateau for all subjects fit and unfit (2) test the hypothesis that VO(2max) plateau duration is not correlated with VO(2max) and (3) verify that limiting factors of VO(2max) plateau duration are different from those of VO(2max) amplitude. Therefore, 14 subjects performed two incremental cycling tests: (1) a classical incremental test (CIT) to determine VO(2max), the power at VO(2max) (PVO(2max)) and at the lactate threshold (PLT) (2) a new incremental test (NIT) in which the power was decreased just after the subject reached VO(2max). During both protocols, heart rate, stroke volume, cardiac output, the arterio-venous difference and the oxygen blood saturation were recorded. The results showed that, with the NIT, subject could maintain a long VO(2max) plateau (6 ± 3 min), even those who could not reach VO(2max) plateau at the end of CIT (n = 5). The VO(2max) plateau duration was not correlated with VO(2max) amplitude which was correlated with the power at SV(max) (r = 0.888, p < 0.001). The VO(2max) plateau duration was correlated with the power decrease (W/s) during the VO(2max) plateau (r = -0.72, p = 0.003) but not with cardiac-related factors nor with PVO(2max). In conclusion, these experiments showed that it was possible to get a long VO(2max) plateau at the end of NIT whatever the individual VO(2max) amplitude was. The limiting factor of VO(2max) duration was the power output.

Athletes' dietary intake was closer to French RDA's than those of young sedentary counterparts.

It has been demonstrated that athletes' dietary intake was relatively well-balanced according to the recommended dietary allowances (RDAs). In contrast, other studies have shown that athletes may have low energy intake or imbalance of protein and fat and insufficient minerals and vitamins. Nonetheless, we hypothesized that practicing a sport may allow young adults to have a nutritional status closer to recommended values. The purpose of this experiment was to study the nutritional status of young French adults, particularly to compare the nutritional status of trained young male and female athletes to those of young sedentary control subjects, and to national RDAs. A total of 85 young adults were recruited and filled a 4-day food and physical activity record. Dietary intake, energy expenditure, energy balance, carbohydrate, protein, fat, water, vitamins, and minerals were recorded. Data were analyzed with a software Nutrilog and statistics with Sigma Stat. Energy intake values were 9874 +/- 3050 kJ for the athletes and 7506 +/- 1845 kJ for control subjects. Athletes' nutritional status was closer to French RDAs than those of sedentary subjects who present a lower energy intake, a greater percentage of the energy intake from fat and lower values for minerals and vitamins. In conclusion, practicing a sport may allow athletes to balance their energy intake and expenditure and could be a good way to have a nutritional status closer to RDAs. Educational programs for students on proper food selection, eating habits and physical activity are needed to improve the nutritional status of these young French adults, particularly in sedentary students.

Objective and subjective analysis of the training content in young cyclists.

The purpose of this study was to analyse the objective and subjective training for young cyclists that is prescribed by their coaches. Seven cyclists performed an incremental exercise to exhaustion before and after 14 weeks of training using an incremental test to determine their maximal oxygen uptake (VO(2) (max)), the velocity associated with VO(2) (max) (vVO(2) (max)), and the velocity associated with the ventilatory threshold (vVT). Cyclists completed a training record with the actual content and the perceived exertion of each training session during these 14 weeks. We have focused on the actual content of the training prescribed by the coaches. Analysis of the content of each session allowed us to calculate the objective training load (volume at different intensities) and to determine the subjective training load from perceived exertion ratings (training load, monotony, strain, and fitness-fatigue). The results showed that cyclists were training at a relatively low intensity and that training rating of perceived exhaustion was weak. Moreover, after 14 weeks of training, VO2 max did not change whereas vVO(2) (max) and vVT increased significantly. Therefore, a discrepancy may exist between what is perceived during training and the effects of training. Consequently, objective and subjective indices collected from training books provided useful information supplementary to that recorded from the physiological indices alone.

1H NMR urine analysis as an effective tool to detect creatine supplementation.

Creatine is one of the main compounds in muscular energetic metabolism leading to phosphocreatine to maintain high ATP levels. Creatine is found in blood and excreted in small amounts in urine. Creatine supplementation and athletic performances are supposed to be correlated, particularly in intensive and intermittent efforts. After oral creatine supplementation, a 1H nuclear magnetic resonance (NMR) spectroscopy method was developed for its direct analysis, without any pretreament of urine samples. This method can be used to detect any supplementation of creatine, a substance prohibited in France. The detection limit is 10 micromol/L (1.31 mg/L) and analysis is performed in 10 min. After a single oral supplementation of 2.1 g to three subjects, a kinetic investigation reveals a maximum concentration of 20 mmol/L (2.62 g/L), observed between 1 and 6 h after ingestion. This procedure was used to test 13 urine specimens obtained from bodybuilders. From the concentrations measured (range: 0.41 to 10.30 mmol/L, 54 to 1350 mg/L), the doping practices of at least nine athletes could be observed. Creatine is not often analyzed in hospital laboratories. This paper documents how easily creatine can be determined and quantitated by 1H NMR spectroscopy.