Carsten Lundby

Proteins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia.

High altitude hypoxia is a paraphysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. In man, skeletal muscle, after prolonged exposure to hypoxia, undergoes mass reduction and alterations at the cellular level featuring a reduction of mitochondrial volume density, accumulation of lipofuscin, a product of lipid peroxidation, and dysregulation of enzymes whose time course is unknown. The effects of 7–9u2005days exposure to 4559u2005m (Margherita Hut, Monte Rosa, Italy) on the muscle proteins pattern were investigated, pre‐ and post‐exposure, in ten young subjects, by 2‐D DIGE and MS. Ten milligram biopsies were obtained from the mid part of the vastus lateralis muscle at sea level (control) and at altitude, after 7–9u2005days hypoxia. Differential analysis indicates that proteins involved in iron transport, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and oxidative stress responses were significantly (p<0.05) decreased in hypoxia. Parenthetically, hypoxia markers such as hypoxia inducible factor 1 α (HIF‐1α) and pyruvate dehydrogenase kinase 1 (PDK1) were still at the pre‐hypoxia levels, whereas the mammalian target of rapamycin (mTOR), a marker of protein synthesis, was reduced.

Prolonged administration of recombinant human erythropoietin increases submaximal performance more than maximal aerobic capacity.

The effects of recombinant human erythropoietin (rHuEpo) treatment on aerobic power (VO2max) are well documented, but little is known about the effects of rHuEpo on submaximal exercise performance. The present study investigated the effect on performance (ergometer cycling, 20–30xa0min at 80% of maximal attainable workload), and for this purpose eight subjects received either 5,000xa0IU rHuEpo or placebo every second day for 14xa0days, and subsequently a single dose of 5,000xa0IU/placebo weekly/10xa0weeks. Exercise performance was evaluated before treatment and after 4 and 11xa0weeks of treatment. With rHuEpo treatment VO2max increased (Pxa0<xa00.05) by 12.6 and 11.6% in week 4 and 11, respectively, and time-to-exhaustion (80% VO2max) was increased by 54.0 and 54.3% (Pxa0<xa00.05) after 4 and 11xa0weeks of treatment, respectively. However, when normalizing the workload to the same relative intensity (only done at time point week 11), TTE was decreased by 26.8% as compared to pre rHuEpo administration. In conclusion, in healthy non-athlete subjects rHuEpo administration prolongs submaximal exercise performance by about 54% independently of the approximately 12% increase in VO2max.

Alterations of systemic and muscle iron metabolism in human subjects treated with low-dose recombinant erythropoietin

The high iron demand associated with enhanced erythropoiesis during high-altitude hypoxia leads to skeletal muscle iron mobilization and decrease in myoglobin protein levels. To investigate the effect of enhanced erythropoiesis on systemic and muscle iron metabolism under nonhypoxic conditions, 8 healthy volunteers were treated with recombinant erythropoietin (rhEpo) for 1 month. As expected, the treatment efficiently increased erythropoiesis and stimulated bone marrow iron use. It was also associated with a prompt and considerable decrease in urinary hepcidin and a slight transient increase in GDF-15. The increased iron use and reduced hepcidin levels suggested increased iron mobilization, but the treatment was associated with increased muscle iron and L ferritin levels. The muscle expression of transferrin receptor and ferroportin was up-regulated by rhEpo administration, whereas no appreciable change in myoglobin levels was observed, which suggests unaltered muscle oxygen homeostasis. In conclusion, under rhEpo stimulation, the changes in the expression of muscle iron proteins indicate the occurrence of skeletal muscle iron accumulation despite the remarkable hepcidin suppression that may be mediated by several factors, such as rhEpo or decreased transferrin saturation or both.

Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness?

This study examined whether hypoxia causes free radical-mediated disruption of the blood-brain barrier (BBB) and impaired cerebral oxidative metabolism and whether this has any bearing on neurological symptoms ascribed to acute mountain sickness (AMS). Ten men provided internal jugular vein and radial artery blood samples during normoxia and 9-h passive exposure to hypoxia (12.9% O(2)). Cerebral blood flow was determined by the Kety-Schmidt technique with net exchange calculated by the Fick principle. AMS and headache were determined with clinically validated questionnaires. Electron paramagnetic resonance spectroscopy and ozone-based chemiluminescence were employed for direct detection of spin-trapped free radicals and nitric oxide metabolites. Neuron-specific enolase (NSE), S100beta, and 3-nitrotyrosine (3-NT) were determined by ELISA. Hypoxia increased the arterio-jugular venous concentration difference (a-v(D)) and net cerebral output of lipid-derived alkoxyl-alkyl free radicals and lipid hydroperoxides (P < 0.05 vs. normoxia) that correlated with the increase in AMS/headache scores (r = -0.50 to -0.90, P < 0.05). This was associated with a reduction in a-v(D) and hence net cerebral uptake of plasma nitrite and increased cerebral output of 3-NT (P < 0.05 vs. normoxia) that also correlated against AMS/headache scores (r = 0.74-0.87, P < 0.05). In contrast, hypoxia did not alter the cerebral exchange of S100beta and both global cerebral oxidative metabolism (cerebral metabolic rate of oxygen) and neuronal integrity (NSE) were preserved (P > 0.05 vs. normoxia). These findings indicate that hypoxia stimulates cerebral oxidative-nitrative stress, which has broader implications for other clinical models of human disease characterized by hypoxemia. This may prove a risk factor for AMS by a mechanism that appears independent of impaired BBB function and cerebral oxidative metabolism.

Interleukin-6 response to exercise during acute and chronic hypoxia

Prolonged exercise is associated with increased plasma levels of the cytokine interleukin-6 (IL-6). Both circulating catecholamine levels and exercise intensity have been related to the exercise-derived IL-6. During hypoxia and acclimatization, changes in sympathetic activity is seen, and also a given workload becomes more intense in hypoxia. Therefore, hypoxia offers a unique opportunity to study the effect of catecholamines and intensity on exercise-derived IL-6. In the present study, eight Danish sea-level residents performed 60xa0min of cycle ergometer exercise at sea level (SL) (154xa0W, 45% maximal O2 consumption, V̇O2max), in acute (AH) and chronic hypoxia (CH), at the same absolute (abs) (AHabs=154xa0W, 54% V̇O2max; CHabs=154xa0W, 59% V̇O2max) and same relative (rel) (AHrel=130xa0W, 46% V̇O2max; CHrel=120xa0W, 44% V̇O2max) workload. We hypothesized that the IL-6 response to exercise at the same absolute workload would be augmented during hypoxia compared with sea level, and that these changes would not correlate with changes in catecholamines. In AHabs (2.35xa0pg·ml−1) and CHabs (3.34xa0pg·ml−1) the IL-6 response to exercise was augmented (p<0.05) compared with that at sea level (0.78·ml−1). In addition, after 60xa0min of bicycling at sea level, AHrel (1.02xa0pg·ml−1) and CHrel (1.31xa0pg·ml−1) resulted in similar IL-6 responses. The augmented IL-6 response during AHabs and CHabs did not match changes in circulating catecholamine levels when comparing all trials. We conclude that the plasma IL-6 concentration during exercise in hypoxia is intensity dependent, and that factors other than catecholamine levels are more important for its regulation.

Erythropoietin augments the cytokine response to acute endotoxin-induced inflammation in humans.

Recent studies have shown that erythropoietin (EPO) offers protection against ischemia, hemorrhagic shock and systemic inflammation in many tissues and it has been suggested that EPO has anti-inflammatory effects. With the aim of investigating the potential acute anti-inflammatory effects of EPO in a human in vivo model of acute systemic low-grade inflammation, we measured circulating inflammatory mediators after intravenous administration of Escherichia coli endotoxin (LPS) bolus injection (0.1 ng/kg of body weight) in young healthy male subjects. The subjects were divided into three groups receiving either (1) LPS alone, (2) EPO alone (15,000 IE of rHuEPO) or (3) EPO and LPS. Endotoxin administration alone induced a 3-, 12- and 5-fold increase in plasma concentrations of TNF-alpha, IL-6 and IL-10, respectively, 3h after LPS challenge. When EPO was given prior to a bolus injection with endotoxin, the levels of TNF-alpha and IL-6 were enhanced by 5- and 40-fold, respectively, whereas the endotoxin-induced increase in IL-10 response was not influenced by EPO. In contrast to our hypothesis, we find that EPO augments the acute inflammatory effect.

Cerebral net exchange of large neutral amino acids after lipopolysaccharide infusion in healthy humans

IntroductionAlterations in circulating large neutral amino acids (LNAAs), leading to a decrease in the plasma ratio between branched-chain and aromatic amino acids (BCAA/AAA ratio), may be involved in sepsis-associated encephalopathy. We hypothesised that a decrease in the BCAA/AAA ratio occurs along with a net cerebral influx of the neurotoxic AAA phenylalanine in a human experimental model of systemic inflammation.MethodsThe BCAA/AAA ratio, the cerebral delivery, and net exchange of LNAAs and ammonia were measured before and 1 hour after a 4-hour intravenous infusion of Escherichia coli lipopolysaccharide (LPS) in 12 healthy young men.ResultsLPS induced systemic inflammation, reduced the BCAA/AAA ratio, increased the cerebral delivery and unidirectional influx of phenylalanine, and abolished the net cerebral influx of the BCAAs leucine and isoleucine. Furthermore, a net cerebral efflux of glutamine, which was independent of the cerebral net exchange of ammonia, was present after LPS infusion.ConclusionsSystemic inflammation may affect brain function by reducing the BCAA/AAA ratio, thereby changing the cerebral net exchange of LNAAs.

Calcitonin gene-related peptide and adrenomedullin release in humans: Effects of exercise and hypoxia

Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are potent vasorelaxant peptides. This study examined exercise-induced changes in CGRP and AM levels in 12 healthy sea level natives at sea level (SL) and subsequently after 24 h (HA1) and 5 days (HA5) in high altitude hypoxia (4559 m). Plasma values of CGRP, AM, calcitonin, noradrenaline, adrenaline, lactate and heart rate were measured at rest and during maximal exercise (W(max)). On each study day, the dopamine D(2)-receptor antagonist, domperidone (30 mg; n=6), or no medication (n=6) was given 1 h before exercise. W(max) at SL, HA1 and HA5 increased CGRP and AM along with heart rate, lactate and catecholamines, whereas, calcitonin remained unchanged. The maximal CGRP levels at W(max) were significantly decreased at HA1 (74.3+/-6.1 pmol/l; p=0.002) and HA5 (69.6+/-6.0 pmol/l; p<0.001) compared to maximal CGRP at SL (85.1+/-4.9 pmol/l). A similar pattern was observed for lactate and the relation between CGRP and lactate release showed a close linear correlation (r(2)=0.63, P<0.0001). Domperidone produced a marked increase in noradrenaline at W(max), but had no affect on CGRP or AM. In conclusion, CGRP release during hypoxic exercise does not respond to domperidone-induced changes in circulating levels of noradrenaline, rather the release may be directly related to the production of lactate.

Contraction‐induced changes in skeletal muscle Na+,K+ pump mRNA expression – importance of exercise intensity and Ca2+‐mediated signalling

Aim:u2002 To investigate if exercise intensity and Ca2+ signalling regulate Na+,K+ pump mRNA expression in skeletal muscle.

Setup for human sera MALDI profiling: The case of rhEPO treatment

The implementation of high‐throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α‐cyano‐4‐hydroxycinnamic acid and 2′,4′‐dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2‐D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2′,4′‐dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.