Jens Hudemann

Short-Term Treadmill Running as a Model for Studying Cell-Free DNA Kinetics In Vivo

BACKGROUND Increased plasma concentrations of cell-free DNA (cf-DNA) are considered a hallmark of various clinical conditions. Despite intensive research in this field, limited data are available concerning the time course of release and clearance of cf-DNA in vivo. METHODS We extracted cf-DNA from plasma samples taken before and immediately after a 10-km cross-country run, and from samples taken before, immediately after, and 30 min after exhaustive short-term treadmill exercise. The contribution of nuclear (nDNA) and mitochondrial DNA (mtDNA) was measured by quantitative real-time PCR. The incremental treadmill exercise setup was exploited to delineate the precise sequencing and timing of cf-nDNA, lactate, and high-mobility group box 1 protein (HMGB1) release during the exercise and recovery phases. RESULTS Postexercise plasma cf-nDNA concentrations in cross-country and treadmill runners were significantly increased, by 7.6-fold and 9.9-fold, respectively (P < 0.001). cf-nDNA concentrations were not correlated with age, sex, or body mass index. Plasma concentrations of cf-nDNA and HMGB1 in postexercise samples of treadmill runners were significantly correlated (r = 0.84; P = 0.004). cf-mtDNA concentrations were not affected by treadmill exercise. Time-course analyses demonstrated that cf-nDNA is released within minutes after the onset of exercise and is rapidly cleared from the circulation after the cessation of exercise. Nearly congruent kinetics for cf-nDNA, lactate, and HMGB1 were observed during the exercise phase. CONCLUSIONS A single bout of exhaustive short-term treadmill exercise constitutes a versatile model system suitable for addressing basic questions about cf-DNA biology.

Neutrophils release extracellular DNA traps in response to exercise

Intense exercise evokes a rapid and transient increase in circulating cell-free DNA (cf-DNA), a phenomenon that is commonly observed in a variety of acute and chronic inflammatory conditions. In this study, we aimed to shed new light on the release and clearance mechanisms of cf-DNA in response to exercise. We hypothesized that activated neutrophils may primarily contribute to exercise-evoked cf-DNA levels by releasing neutrophil extracellular traps (NETs). Analysis of plasma and/or serum samples from male athletes at rest and in response to exhaustive treadmill exercise revealed an immediate and transient increase in cf-DNA that was concomitantly counterbalanced by an increase in serum DNase activity. Consistently, rapid release and clearance kinetics for cf-DNA could also be observed in response to intensive cycling exercise, with no significant differences between endurance-trained (V̇o2max >57 ml·min(-1)·kg(-1)) and healthy (V̇o2max <49 ml·min(-1)·kg(-1)) sedentary individuals. In postexercise blood smear samples, we detected seemingly intact neutrophils displaying morphological signs of NET release, as indicated by abnormal swollen nuclei and emanating DNA fibers. In support, we observed a striking correlation of postexercise cf-DNA concentrations with plasma levels of the granule-derived enzyme myeloperoxidase. Our study indicates that intense exercise induces liberation of NETs, which is sufficiently counterbalanced in healthy individuals by a concomitant rise in serum DNase activity. As aberrant release of NETs has been linked to diverse disease states, monitoring of cf-DNA/DNase levels or activities in response to standardized exercise testing could provide a valuable tool to identify people who are at increased risk for cardiac ischemia, thrombosis, autoimmunity, or chronic fatigue.

Basal and exercise induced label-free quantitative protein profiling of m. vastus lateralis in trained and untrained individuals.

UNLABELLED Morphological and metabolic adaptations of the human skeletal muscle to exercise are crucial to improve performance and prevent chronic diseases and metabolic disorders. In this study we investigated human skeletal muscle protein composition in endurance trained (ET) versus untrained individuals (UT) and its modulation by an acute bout of endurance exercise. Participants were recruited based on their VO2max and subjected to a bicycle exercise test. M. vastus lateralis biopsies were taken before and three hours after exercise. Muscle lysates were analyzed using off-gel LC-MS/MS. Relative protein abundances were compared between ET and UT at rest and after exercise. Comparing UT and ET, we identified 92 significantly changed proteins under resting conditions. Specifically, fiber-type-specific and proteins of the oxidative phosphorylation and tricarboxylic acid cycle were increased in ET. In response to acute exercise, 71 proteins in ET and 44 in UT were altered. Here, a decrease of proteins involved in energy metabolism accompanied with alterations of heat shock and proteasomal proteins could be observed. In summary, long-term endurance training increased the basal level of structural and mitochondrial proteins in skeletal muscle. In contrast, acute exercise resulted in a depletion of proteins related to substrate utilization, especially in trained athletes. BIOLOGICAL SIGNIFICANCE The investigation of the human skeletal muscle proteome in response to exercise may provide novel insights into the process of muscular plasticity. It is of importance in the development of exercise-based strategies in the prevention and therapy of many chronic inflammatory and degenerative diseases which are often accompanied by muscular deconditioning. Up to date, proteomic investigations of the human muscle proteome in adaptation to exercise are mainly focused on untrained individuals and often restricted to animal studies. In the present study we compare the protein composition in endurance trained athletes and untrained individuals in the resting muscle and its modulation in response to acute exercise. To our knowledge, we present the first comprehensive analysis of skeletal muscle proteome alterations in response to acute and long-term exercise intervention.

Exhaustive exercise modifies different gene expression profiles and pathways in LPS-stimulated and un-stimulated whole blood cultures

Exhaustive exercise can interfere with immunity, causing transient immunosuppression and infections/inflammation in athletes. We used microarray technology to analyze the gene expression profiles of whole blood in short time (1h) LPS-stimulated and un-stimulated cultures drawn before, 30min after, 3h after and 24h after a half-marathon run. Four male and 4 female athletes participated. Exercise induced differential expression of genes known to be involved in innate immunity/inflammatory response, metabolic response, DNA methylation, apoptosis and regulation of brain function. Several genes with prominent anti-inflammatory function were up-regulated in un-stimulated cultures, including ARG-1, SOCS3, DUSP-1, ORMs, IRAK3, and GJB6. Some of these genes were also strongly up-regulated in LPS-stimulated cultures (ARG-1, ORM2, and GJB6). Some genes were strongly up-regulated through exercise in LPS-stimulated cultures, but not in un-stimulated cultures (TNIP3, PLAU, and HIVEP1). There was also a row of genes, which were strongly down-regulated by exercise in LPS-stimulated cultures, notably IFN-β1 and CXCL10. Exercise also significantly changed the expression of genes (OLIG2, TMEM106B) which are known to be related to brain function and expression of which has never been documented in peripheral blood. In summary, exhaustive exercise, in addition to modifying gene expression in un-stimulated cells, could also interfere with the early gene expression response to endotoxin. There was an anti-inflammatory bias of gene regulation by exercise, including genes involved in the negative regulation of TLRs signalling. The results of the present study demonstrate that some potentially important effects of exercise can only be detected in relation to pathogen stimulation.

TGF-β Contributes to Impaired Exercise Response by Suppression of Mitochondrial Key Regulators in Skeletal Muscle.

A substantial number of people at risk of developing type 2 diabetes could not improve insulin sensitivity by physical training intervention. We studied the mechanisms of this impaired exercise response in 20 middle-aged individuals at high risk of developing type 2 diabetes who performed 8 weeks of controlled cycling and walking training at 80% individual Vo2 peak. Participants identified as nonresponders in insulin sensitivity (based on the Matsuda index) did not differ in preintervention parameters compared with high responders. The failure to increase insulin sensitivity after training correlates with impaired upregulation of mitochondrial fuel oxidation genes in skeletal muscle, and with the suppression of the upstream regulators PGC1α and AMPKα2. The muscle transcriptomes of the nonresponders are further characterized by the activation of transforming growth factor (TGF)-β and TGF-β target genes, which is associated with increases in inflammatory and macrophage markers. TGF-β1 as inhibitor of mitochondrial regulators and insulin signaling is validated in human skeletal muscle cells. Activated TGF-β1 signaling downregulates the abundance of PGC1α, AMPKα2, the mitochondrial transcription factor TFAM, and mitochondrial enzymes. Thus, the data suggest that increased TGF-β activity in skeletal muscle can attenuate the improvement of mitochondrial fuel oxidation after training and contribute to the failure to increase insulin sensitivity.

Effects of Acute Endurance Exercise on Plasma Protein Profiles of Endurance-Trained and Untrained Individuals over Time

Acute physical exercise and repeated exercise stimuli affect whole-body metabolic and immunologic homeostasis. The aim of this study was to determine plasma protein profiles of trained (EET, n = 19) and untrained (SED, n = 17) individuals at rest and in response to an acute bout of endurance exercise. Participants completed a bicycle exercise test at an intensity corresponding to 80% of their VO2max. Plasma samples were taken before, directly after, and three hours after exercise and analyzed using multiplex immunoassays. Seventy-eight plasma variables were included in the final analysis. Twenty-nine variables displayed significant acute exercise effects in both groups. Seven proteins differed between groups, without being affected by acute exercise. Among these A2Macro and IL-5 were higher in EET individuals while leptin showed elevated levels in SED individuals. Fifteen variables revealed group and time differences with elevated levels for IL-3, IL-7, IL-10, and TNFR2 in EET individuals. An interaction effect could be observed for nine variables including IL-6, MMP-2, MMP-3, and muscle damage markers. The proteins that differ between groups indicate a long-term exercise effect on plasma protein concentrations. These findings might be of importance in the development of exercise-based strategies in the prevention and therapy of chronic metabolic and inflammatory diseases and for training monitoring.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Quality Control of Serum and Plasma by Quantification of (4E,14Z)-Sphingadienine-C18-1-Phosphate Uncovers Common Preanalytical Errors During Handling of Whole Blood

BACKGROUND Nonadherence to standard operating procedures (SOPs) during handling and processing of whole blood is one of the most frequent causes affecting the quality of serum and plasma. Yet, the quality of blood samples is of the utmost importance for reliable, conclusive research findings, valid diagnostics, and appropriate therapeutic decisions. METHODS UHPLC-MS-driven nontargeted metabolomics was applied to identify biomarkers that reflected time to processing of blood samples, and a targeted UHPLC-MS analysis was used to quantify and validate these biomarkers. RESULTS We found that (4E,14Z)-sphingadienine-C18-1-phosphate (S1P-d18:2) was suitable for the reliable assessment of the pronounced changes in the quality of serum and plasma caused by errors in the phase between collection and centrifugation of whole blood samples. We rigorously validated S1P-d18:2, which included the use of practicality tests on >1400 randomly selected serum and plasma samples that were originally collected during single- and multicenter trials and then stored in 11 biobanks in 3 countries. Neither life-threatening disease states nor strenuous metabolic challenges (i.e., high-intensity exercise) affected the concentration of S1P-d18:2. Cutoff values for sample assessment were defined (plasma, ≤0.085 μg/mL; serum, ≤0.154 μg/mL). CONCLUSIONS Unbiased valid monitoring to check for adherence to SOP-dictated time for processing to plasma or serum and/or time to storage of whole blood at 4 °C is now feasible. This novel quality assessment step could enable scientists to uncover common preanalytical errors, allowing for identification of serum and plasma samples that should be excluded from certain investigations. It should also allow control of samples before long-term storage in biobanks.