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Department of Internal Medicine Skeletal muscle responds to physical activity with altered gene and protein expression profiles. Repetitive physical activity ultimately leads to morphological and functional adaptations in skeletal muscle. The aim of this study was to determine the currently unknown effects of training status on skeletal muscle transcriptional responses to a bout of acute endurance exercise. Trained (VO2max ≥ 57 ml/min/kg BW) and untrained (VO2max ≤ 47 ml/min/kg BW) male study participants performed an exhaustive bout of high intensity cycling exercise (60

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minutes at 80% VO2max). Muscle biopsies were taken from the vastus lateralis at rest, 30 minutes and 3h after exercise. Global mRNA expression profiling by microarray analysis (Affymetrix Human Gene 1.0 ST Array) revealed that expression of the nuclear hormone receptor (NR) family NR4A subgroup, Nurr77 (NR4A1) and Nor-1 (NR4A3) was significantly induced by the acute cycling bout in both T and UT.

The NR4A family of orphan nuclear receptors regulates the expression of fiber type specific metabolic and structural genes. Nurr77 expression increased 4.3-fold (p0.01) and 3.5-fold (p0.01) compared to the resting state 30 minutes after exercise in T and UT, respectively. Nurr77 expression continued to increase in UT at 3h (6-fold vs. rest, p0.001), while transcript levels dropped slightly in T (2.1-fold vs.

rest, p0.05). The highest gene induction in this study was measured for Nor-1 at 3h post-exercise (T= 32-fold vs. rest; UT= 52-fold vs.

rest, p0.001). Nor-1 responds to β-adrenergic signaling and reduces myostatin (Mstn) expression. Expression of the β-2 adrenoreceptor (ADRB2) was elevated 2.1-fold vs. rest (p0.01) in both T and UT muscle 30 minutes post exercise. Mstn expression was 2.6-fold (p0.01) higher at rest in UT vs. T and expression decreased only in UT after exercise (3.1-fold vs. rest UT 3h, p0.01). Results suggest that the NR4A family rapidly respond to acute exercise in T and UT skeletal muscle to regulate metabolic and structural genes important for exercise recovery and adaptation. Mstn expression was only perceptive to elevated Nur77 and Nor-1 expression in UT muscle, which suggests that the responsiveness of Mstn to acute exercise is dependent on training status.



Guadalupe Grau, A.1, Plenge, U.2, Belhage, B.2, Dela, F.1, Helge, J.W.1 1University of Copenhagen, 2Bispebjerg Hospital, Denmark Erythropoietin (EPO) is a hypoxia-induced hormone released by the renal cortex, where it promotes proliferation and differentiation of erythroid progenitor cells, regulating its number in the peripheral blood and thus increasing oxygen carrying ability of blood. Therefore, recombinant erythropoietin (rhEPO) treatment is widely used, albeit not legally, to enhance endurance exercise capacity. Parallel to these effects, an increase in resting energy expenditure (REE), as well as whole body fat oxidation (FAO) has been attributed to EPO. Skeletal muscle plays a role in REE, and is one of the main tissues responsible for fatty acid metabolism. This led to our hypothesis that a rhEPOmediated increase in oxygen facilitation to the muscle increases mitochondrial FAO and could explain, at least in part, an increase in systemic REE and FAO. Methods. Human EPO was administered by muscular injection (5000 IU IM once weekly) in 6 healthy volunteers over 8 weeks, titrating the rhEpo dosage to maintain haematocrit level at ~50%. Oral iron (100 mg) supplementation was also given daily.

Body composition was measured by DXA. Subjects performed two graded cycle ergometer exercise tests where VO2max, and maximal fat oxidation (MFO) were measured. ß-hydroxy-acyl-CoA-dehydrogenase (HAD) and citrate synthase (CS) enzyme activity were measured fluorometrically. Mitochondrial respiration was measured in saponin permeabilized muscle using the following substrate-titration protocol: 2 mM Malate + 0.2 mM Octanoyl + 5 mM ADP +10mM Pyruvate + 10 mM Glutamate/ + 10 mM Succinate. Results. As expected, rhEPO significantly increased blood haematocrit to 51%, and VO2max (from 54±3 to 58±3 ml·kg-1·min-1, P0.05) without any exercise intervention. MFO and was not increased after rhEPO treatment, despite the higher intensity and duration of the exercise test. CS activity remained unchanged whereas HAD activity significantly increased after rhEPO (P0.05). Maximal fatty acid mitochondrial capacity (MGPS3) was 1.4 fold higher after rhEPO treatment (from 59.5 ± 10.6 to 82.7 ± 20.8 pmol · s-1· mg-1 P0.01). Conclusion. Skeletal muscle mitochondrial respiratory and fat oxidation capacity is markedly higher after EPO treatment. Whoel body MFO was not increased after rhEPO treatment, despite the higher intensity and duration of the exercise test. Our results suggest that the oxygen delivery capacity was not limiting the magnitude of maximal systemic fat oxidation. These findings may partially account for the enhancement of submaximal endurance performance.



Guéritat, J., Lefeuvre-Orfila, L., Vincent, S., Delamarche, A., Rannou-Bekono, F., Rebillard, A.

EA 1274, Introduction Prostate cancer (PC) is the most common form of cancer affecting men in the Western world. Various studies suggest a decrease of PC evolution with exercise or antioxidants but the association of these two strategies has never been investigated. We hypothesize that oxidative stress is a key regulator factor of PC. In this context, we suppose that the potential decrease of oxidative stress (OS) induced by pomegranate juice (PJ) consumption alone, running training alone or the associated strategies could delay the development of PC. The objectives of this project are (1) to identify the effect of PJ and/or physical activity on PC progression in a rodent model; (2) to determine the molecular mechanisms involved in this prevention. Methods Forty male Copenhagen rats with subcutaneous prostate tumor are divided into four groups: (1) control, (2) daily consumption of pomegranate juice PJ, (3) daily running on a treadmill (training), (4) PJ + training. Each week, tumor growth is evaluated using a sliding caliper. Four weeks after the various treatments, the rodents are sacrificed. The tumors are resected and frozen. Muscles (EDL, soleus, gastrocnemus and tibialis) are removed as well as blood and urine.

The levels of antioxidant vitamins, antioxidant enzyme activities, lipid peroxidation, carbonylated proteins and DNA oxidation are investigated and the signaling pathways are identified. Data are compared by analysis of variance (ANOVA) and the degree of significance is set at p0.05. Results PJ consumption alone or training alone decreases prostate tumor growth in the Copenhagen rat (p0.05) but the association of the two strategies is not also effective. Indeed, tumor volume doubling time is respectively of about six days for control and PJ+ training, whereas it is of eight days for PJ alone or training alone. We observe a significant difference of reduced GSH and vitamin A between PJ and control groups (p0.05). Moreover, training increases significantly GPx activity vs control and PJ groups (p0.05). The combination of PJ and training inhibits antioxidant adaptations induced by each treatment. Discussion Although none study has demonstrated the benefits of both PJ and training, urologists recommend consumption of food antioxidants associated with physical exercise as chemoprevention for active monitoring PC patients. However our innovative study shows for the first time that combined effects of PJ and training may inhibit antioxidant adaptations and could prevent the modulation of redox-dependent signaling pathways involved in the prostate tumorigenesis.

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Wessner, B., Berkowitsch, B., Laaber, O., Bachl, N., Tschan, H.

University of Vienna Introduction: MicroRNAs (miRs) have been shown to mediate adaptation to exercise by influencing many physiological processes [1,2].

Besides their specific abundance within different tissues, significant levels of miRs were detected in serum where they are remarkably stable [3]. It is assumed that muscle-specific miRs could serve as diagnostic markers for cardiac damage but the influence of mechanical stress due to exercise is questioned [4]. Therefore, the aims of this study were to investigate whether circulating levels of skeletal and cardiac muscle-specific miRs are detectable after extreme exercise and to correlate them with biochemical markers of skeletal and cardiac muscle damage. Methods: 18 male triathletes (age: 40 ± 9 y, BMI: 23 ± 2 kg/m²) participating at the Ironman Austria agreed to donate blood 1d before, immediately as well as 1 and 7d after the race (finishing time: 11.8 ± 1.1h). Total RNA was isolated from serum using the miRNeasy Serum Kit (Qiagen). After reverse transcription, miR-133a, -206, -208b, and -499-5p levels were quantified by qRTPCR. Creatine kinase (CK), myoglobin (MYO), high sensitive troponin (hs-TNT), and NT-proBNP were determined on a COBAS 8000 system.

Repeated measures ANOVA was used to detect differences between pre and post values and Pearson’s correlation analyses served to describe correlations between single markers. Results: Muscle-specific miRs were highly enhanced directly after completing the race (miR-133a: 32-fold, miR-206: 61-fold, miR208b: 41-fold, miR-499-5p: 13-fold; p0.001 for all comparisons). In general, levels decreased 24h thereafter but still were significantly higher than pre-race levels (miR-133a: 10-fold, miR-206: 10-fold, miR208b: 28-fold, miR-499-5p 12-fold; p0.001 for all comparisons). 7d post-race all miRs returned to baseline levels. Markers for skeletal muscle damage peaked immediately (MYO: 35-fold, p0.001) or 1d (CK: 21-fold, p0.001) post-race. Cardiac stress markers (NT-proBNP, hs-TNT) were significantly enhanced immediately after the Ironman. Correlation analyses revealed high and significant correlations between muscle-damage markers and miRNAs. Discussion: This is one of the first studies revealing that intense exercise such as an Ironman triathlon causes increases in circulating muscle-specific miRs and that these increases are highly correlated to common markers of muscle-damage. It has to be proven whether they could serve as new diagnostic markers to clearly discriminate between skeletal (miR-206) and cardiac muscle damage (miR-208b). References [1] McCarthy J et al (2011). Exerc Sport Sci Rev 39(3):150-4. [2] Wessner B et al. (2010). Exerc Immunol Rev 16:22-39. [3] Chen X et al. (2008). Cell Res 18(10):997-1006. [4] Tijsen AJ et al (2012). Am J Physiol Heart Circ Physiol 303(9):H1085-95.


Mannion, S.1,2, Mtintsilana, A.1, van der Merwe, W.4, O’Cuinneagain, D.4, Collins, M.1,3, September, A.V.1 1 UCT Human Biology (Cape Town, South Africa), 2 UCT Human Genetics (Cape Town, South Africa), 3 MRC (Cape Town, South Africa), 4 SSOC (Cape Town, South Africa) Introduction Anterior cruciate ligament (ACL) ruptures are one of the most detrimental injuries sustained in sports. Multiple risk factors, including genetic factors, are associated with the injury (September et al., 2007; 2012). Proteoglycan content is lowered in ruptured ACL tissue in comparison to non-ruptured controls (Young el al., 2011). Genes encoding proteoglycans thus form candidate genes to be investigated for an association with ACL injury risk. The aim of this study was to investigate whether sequence variants within the ACAN, BGN, DCN, FMOD and LUM proteoglycan genes are associated with risk of ACL injury. Aggrecan is a major structural constituent of cartilage, while the small leucine rich proteoglycans are important for collagen fibrillogenesis. Methods A case-control genetic association study was conducted. In total, 210 participants with surgically diagnosed ACL ruptures and 234 control participants without any history of ACL injury were genotyped for the following single nucleotide polymorphisms using standard PCR based methods: ACAN rs2351491, rs1042631, rs1516797; BGN rs1126499, rs1042103; DCN rs13312816, rs516115; FMOD rs7543148, rs10800912; and LUM rs2268578. Statistical analysis determined whether genotype or allele frequencies differed significantly between cases and controls. Results The GG genotype of ACAN rs1516797 was significantly overrepresented in ACL participants (p=0.042, OR=2.018, 95% CI 1.015-4.015) when compared to controls. For BGN rs1126499 the TT genotype was overrepresented in ACL participants (p=0.032, OR=1.515, 95% CI 1.037-2.215) while the CT genotype was overrepresented in controls (p=0.004, OR=0.470, 95% CI 0.277-0.796). The AA genotype of DCN rs516115 was significantly overrepresented in female ACL participants (p=0.022, OR=2.279, 95% CI 1.120-4.636) and interestingly the GG genotype was absent in female ACL participants (p=0.008, OR=0.063, 95% CI 0.004-1.096). No other significant genotype or allele associations were noted. Discussion Preliminary evidence suggests that ACAN rs1516797, BGN rs1126499 and DCN rs516115 are associated with ACL injury risk. This novel study highlights the potential role of proteoglycans in ACL ruptures. References September AV, Schwellnus MP, Collins M.

Br J Sports Med 2007;41:241-6. September AV, Posthumus M, Collins M. Recent Pat DNA Gene Seq. 2012;6(3):216-23. Young K, Samiric T, Feller J, et al. Knee 2011;18:242-6.



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