Leptin receptors in human skeletal muscle

[EN] Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age = 33.1 +/- 2.0 yr, height = 175.9 +/- 1.7 cm, body mass = 81.2 +/- 3.8 kg, body fat = 22.5 +/- 1.9%; means +/- SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit anti-human leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum.

Strong iron demand during hypoxia-induced erythropoiesis is associated with down-regulation of iron-related proteins and myoglobin in human skeletal muscle

[EN] Iron is essential for oxygen transport because it is incorporated in the heme of the oxygen-binding proteins hemoglobin and myoglobin. An interaction between iron homeostasis and oxygen regulation is further suggested during hypoxia, in which hemoglobin and myoglobin syntheses have been reported to increase. This study gives new insights into the changes in iron content and iron-oxygen interactions during enhanced erythropoiesis by simultaneously analyzing blood and muscle samples in humans exposed to 7 to 9 days of high altitude hypoxia (HA). HA up-regulates iron acquisition by erythroid cells, mobilizes body iron, and increases hemoglobin concentration. However, contrary to our hypothesis that muscle iron proteins and myoglobin would also be up-regulated during HA, this study shows that HA lowers myoglobin expression by 35% and down-regulates iron-related proteins in skeletal muscle, as evidenced by decreases in L-ferritin (43%), transferrin receptor (TfR; 50%), and total iron content (37%). This parallel decrease in L-ferritin and TfR in HA occurs independently of increased hypoxia-inducible factor 1 (HIF-1) mRNA levels and unchanged binding activity of iron regulatory proteins, but concurrently with increased ferroportin mRNA levels, suggesting enhanced iron export. Thus, in HA, the elevated iron requirement associated with enhanced erythropoiesis presumably elicits iron mobilization and myoglobin down-modulation, suggesting an altered muscle oxygen homeostasis.

Leptin receptor 170 kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance

[EN] To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean +/- s.d. age, 31 +/- 5 years; height, 184 +/- 9 cm; weight, 91 +/- 13 kg; and percentage body fat, 24.8 +/- 5.8%) and 10 obese (age, 30 +/- 7 years; height, 184 +/- 8 cm; weight, 115 +/- 8 kg; and percentage body fat, 34.9 +/- 5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P < 0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKalpha and ACCbeta phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKalpha and ACCbeta phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P < 0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles.

Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle

[EN] Increased skeletal muscle capillary density would be a logical adaptive mechanism to chronic hypoxic exposure. However, animal studies have yielded conflicting results, and human studies are sparse. Neoformation of capillaries is dependent on endothelial growth factors such as vascular endothelial growth factor (VEGF), a known target gene for hypoxia inducible factor 1 (HIF-1). We hypothesised that prolonged exposure to high altitude increases muscle capillary density and that this can be explained by an enhanced HIF-1alpha expression inducing an increase in VEGF expression. We measured mRNA levels and capillary density in muscle biopsies from vastus lateralis obtained in sea level residents (SLR; N=8) before and after 2 and 8 weeks of exposure to 4100 m altitude and in Bolivian Aymara high-altitude natives exposed to approximately 4100 m altitude (HAN; N=7). The expression of HIF-1alpha or VEGF mRNA was not changed with prolonged hypoxic exposure in SLR, and both genes were similarly expressed in SLR and HAN. In SLR, whole body mass, mean muscle fibre area and capillary to muscle fibre ratio remained unchanged during acclimatization. The capillary to fibre ratio was lower in HAN than in SLR (2.4+/-0.1 vs 3.6+/-0.2; P<0.05). In conclusion, human muscle VEGF mRNA expression and capillary density are not significantly increased by 8 weeks of exposure to high altitude and are not increased in Aymara high-altitude natives compared with sea level residents.

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

[EN] Chronic hypoxia is accompanied by changes in blood and skeletal muscle acid-base control. We hypothesized that the underlying mechanisms include altered protein expression of transport systems and the enzymes involved in lactate, HCO3- and H+ fluxes in skeletal muscle and erythrocytes. Immunoblotting was used to quantify densities of the transport systems and enzymes. Muscle and erythrocyte samples were obtained from eight Danish lowlanders at sea level and after 2 and 8 weeks at 4100 m (Bolivia). For comparison, samples were obtained from eight Bolivian natives. In muscle membranes there were no changes in fibre-type distribution, lactate dehydrogenase isoforms, Na+,K+-pump subunits or in the lactate-H+ co-transporters MCT1 and MCT4. The Na+-H+ exchanger protein NHE1 was elevated by 39 % in natives compared to lowlanders. The Na+-HCO3- co-transporter density in muscle was elevated by 47-69 % after 2 and 8 weeks at altitude. The membrane-bound carbonic anhydrase (CA) IV in muscle increased in the lowlanders by 39 %, whereas CA XIV decreased by 23-47 %. Levels of cytosolic CA II and III in muscle and CA I and II in erythrocytes were unchanged. The erythrocyte lactate-H+ co-transporter MCT1 increased by 230-405 % in lowlanders and was 324 % higher in natives. The erythrocyte inorganic anion exchanger (Cl--HCO3- exchanger AE1) was increased by 149-228 %. In conclusion, chronic hypoxia induces dramatic changes in erythrocyte proteins, but only moderate changes in muscle proteins involved in acid-base control. Together, these changes suggest a hypoxia-induced increase in the capacity for lactate, HCO3- and H+ fluxes from muscle to blood and from blood to erythrocytes.

Skeletal muscle mitochondrial DNA content in exercising humans

[EN] Several weeks of intense endurance training enhances mitochondrial biogenesis in humans. Whether a single bout of exercise alters skeletal muscle mitochondrial DNA (mtDNA) content remains unexplored. Double-stranded mtDNA, estimated by slot-blot hybridization and real time PCR and expressed as mtDNA-to-nuclear DNA ratio (mtDNA/nDNA) was obtained from the vastus lateralis muscle of healthy human subjects to investigate whether skeletal muscle mtDNA changes during fatiguing and nonfatiguing prolonged moderate intensity [2.0-2.5 h; approximately 60% maximal oxygen consumption (Vo(2 max))] and short repeated high-intensity exercise (5-8 min; approximately 110% Vo(2 max)). In control resting and light exercise (2 h; approximately 25% Vo(2 max)) studies, mtDNA/nDNA did not change. Conversely, mtDNA/nDNA declined after prolonged fatiguing exercise (0.863 +/- 0.061 vs. 1.101 +/- 0.067 at baseline; n = 14; P = 0.005), remained lower after 24 h of recovery, and was restored after 1 wk. After nonfatiguing prolonged exercise, mtDNA/nDNA tended to decline (n = 10; P = 0.083) but was reduced after three repeated high-intensity exercise bouts (0.900 +/- 0.049 vs. 1.067 +/- 0.071 at baseline; n = 7; P = 0.013). Our findings indicate that prolonged and short repeated intense exercise can lead to significant reductions in human skeletal muscle mtDNA content, which might function as a signal stimulating mitochondrial biogenesis with exercise training.

Gender dimorphism in skeletal muscle leptin receptors, serum leptin and insulin sensitivity

[EN] To determine if there is a gender dimorphism in the expression of leptin receptors (OB-R170, OB-R128 and OB-R98) and the protein suppressor of cytokine signaling 3 (SOCS3) in human skeletal muscle, the protein expression of OB-R, perilipin A, SOCS3 and alpha-tubulin was assessed by Western blot in muscle biopsies obtained from the m. vastus lateralis in thirty-four men (age = 27.1+/-6.8 yr) and thirty-three women (age = 26.7+/-6.7 yr). Basal serum insulin concentration and HOMA were similar in both genders. Serum leptin concentration was 3.4 times higher in women compared to men (P<0.05) and this difference remained significant after accounting for the differences in percentage of body fat or soluble leptin receptor. OB-R protein was 41% (OB-R170, P<0.05) and 163% (OB-R128, P<0.05) greater in women than men. There was no relationship between OB-R expression and the serum concentrations of leptin or 17beta-estradiol. In men, muscle OB-R128 protein was inversely related to serum free testosterone. In women, OB-R98 and OB-R128 were inversely related to total serum testosterone concentration, and OB-R128 to serum free testosterone concentration. SOCS3 protein expression was similar in men and women and was not related to OB-R. In women, there was an inverse relationship between the logarithm of free testosterone and SCOS3 protein content in skeletal muscle (r = -0.46, P<0.05). In summary, there is a gender dimorphism in skeletal muscle leptin receptors expression, which can be partly explained by the influence of testosterone. SOCS3 expression in skeletal muscle is not up-regulated in women, despite very high serum leptin concentrations compared to men. The circulating form of the leptin receptor can not be used as a surrogate measure of the amount of leptin receptors expressed in skeletal muscles.

Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

[EN] As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O(2). With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-(13)C-leucine in nine healthy male subjects at sea level and subsequently at high-altitude (4559 m) after 7-9 days of acclimatization. Physical activity levels and food and energy intake were controlled prior to the two experimental conditions with the aim to standardize these confounding factors. Blood samples and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041+/-0.018 at sea-level to 0.080+/-0.018%hr(-1) (p<0.05) when acclimatized to high altitude. The sarcoplasmic protein synthesis rate was in contrast unaffected by altitude exposure; 0.052+/-0.019 at sea-level to 0.059+/-0.010%hr(-1) (p>0.05). Trends to increments in whole body protein kinetics were seen: Degradation rate elevated from 2.51+/-0.21 at sea level to 2.73+/-0.13 micromolkg(-1)min(-1) (p = 0.05) at high altitude and synthesis rate similar; 2.24+/-0.20 at sea level and 2.43+/-0.13 micromolkg(-1)min(-1) (p>0.05) at altitude. We conclude that whole body amino acid flux is increased due to an elevated protein turnover rate. Resting skeletal muscle myocontractile protein synthesis rate was concomitantly elevated by high-altitude induced hypoxia, whereas the sarcoplasmic protein synthesis rate was unaffected by hypoxia. These changed responses may lead to divergent adaptation over the course of prolonged exposure.

Reductions in systemic and skeletal muscle blood flow and oxygen delivery limit maximal aerobic capacity in humans

[EN] BACKGROUND: A classic, unresolved physiological question is whether central cardiorespiratory and/or local skeletal muscle circulatory factors limit maximal aerobic capacity (VO2max) in humans. Severe heat stress drastically reduces VO2max, but the mechanisms have never been studied. METHODS AND RESULTS: To determine the main contributing factor that limits VO2max with and without heat stress, we measured hemodynamics in 8 healthy males performing intense upright cycling exercise until exhaustion starting with either high or normal skin and core temperatures (+10 degrees C and +1 degrees C). Heat stress reduced VO2max, 2-legged VO2, and time to fatigue by 0.4+/-0.1 L/min (8%), 0.5+/-0.2 L/min (11%), and 2.2+/-0.4 minutes (28%), respectively (all P<0.05), despite heart rate and core temperature reaching similar peak values. However, before exhaustion in both heat stress and normal conditions, cardiac output, leg blood flow, mean arterial pressure, and systemic and leg O2 delivery declined significantly (all 5% to 11%, P<0.05), yet arterial O2 content and leg vascular conductance remained unchanged. Despite increasing leg O2 extraction, leg VO2 declined 5% to 6% before exhaustion in both heat stress and normal conditions, accompanied by enhanced muscle lactate accumulation and ATP and creatine phosphate hydrolysis. CONCLUSIONS: These results demonstrate that in trained humans, severe heat stress reduces VO2max by accelerating the declines in cardiac output and mean arterial pressure that lead to decrements in exercising muscle blood flow, O2 delivery, and O2 uptake. Furthermore, the impaired systemic and skeletal muscle aerobic capacity that precedes fatigue with or without heat stress is largely related to the failure of the heart to maintain cardiac output and O2 delivery to locomotive muscle.

The lactate paradox revisited in lowlanders during acclimatization to 4100 m and in high-altitude natives

[EN] Chronic hypoxia has been proposed to induce a closer coupling in human skeletal muscle between ATP utilization and production in both lowlanders (LN) acclimatizing to high altitude and high-altitude natives (HAN), linked with an improved match between pyruvate availability and its use in mitochondrial respiration. This should result in less lactate being formed during exercise in spite of the hypoxaemia. To test this hypothesis six LN (22-31 years old) were studied during 15 min warm up followed by an incremental bicycle exercise to exhaustion at sea level, during acute hypoxia and after 2 and 8 weeks at 4100 m above sea level (El Alto, Bolivia). In addition, eight HAN (26-37 years old) were studied with a similar exercise protocol at altitude. The leg net lactate release, and the arterial and muscle lactate concentrations were elevated during the exercise in LN in acute hypoxia and remained at this higher level during the acclimatization period. HAN had similar high values; however, at the moment of exhaustion their muscle lactate, ADP and IMP content and Cr/PCr ratio were higher than in LN. In conclusion, sea-level residents in the course of acclimatization to high altitude did not exhibit a reduced capacity for the active muscle to produce lactate. Thus, the lactate paradox concept could not be demonstrated. High-altitude natives from the Andes actually exhibit a higher anaerobic energy production than lowlanders after 8 weeks of acclimatization reflected by an increased muscle lactate accumulation and enhanced adenine nucleotide breakdown.

Arterial O2 content and tension in regulation of cardiac output and leg blood flow during exercise in humans

[EN] A universal O2 sensor presumes that compensation for impaired O2 delivery is triggered by low O2 tension, but in humans, comparisons of compensatory responses to altered arterial O2 content (CaO2) or tension (PaO2) have not been reported. To directly compare cardiac output (QTOT) and leg blood flow (LBF) responses to a range of CaO2 and PaO2, seven healthy young men were studied during two-legged knee extension exercise with control hemoglobin concentration ([Hb] = 144.4 +/- 4 g/l) and at least 1 wk later after isovolemic hemodilution ([Hb] = 115 +/- 2 g/l). On each study day, subjects exercised twice at 30 W and on to voluntary exhaustion with an FIO2 of 0.21 or 0.11. The interventions resulted in two conditions with matched CaO2 but markedly different PaO2 (hypoxia and anemia) and two conditions with matched PaO2 and different CaO2 (hypoxia and anemia + hypoxia). PaO2 varied from 46 +/- 3 Torr in hypoxia to 95 +/- 3 Torr (range 37 to >100) in anemia (P < 0.001), yet LBF at exercise was nearly identical. However, as CaO2 dropped from 190 +/- 5 ml/l in control to 132 +/- 2 ml/l in anemia + hypoxia (P < 0.001), QTOT and LBF at 30 W rose to 12.8 +/- 0.8 and 7.2 +/- 0.3 l/min, respectively, values 23 and 47% above control (P < 0.01). Thus regulation of QTOT, LBF, and arterial O2 delivery to contracting intact human skeletal muscle is dependent for signaling primarily on CaO2, not PaO2. This finding suggests that factors related to CaO2 or [Hb] may play an important role in the regulation of blood flow during exercise in humans.

Human muscle net K(+) release during exercise is unaffected by elevated anaerobic metabolism, but reduced after prolonged acclimatization to 4,100 m

[EN] It was investigated whether skeletal muscle K(+) release is linked to the degree of anaerobic energy production. Six subjects performed an incremental bicycle exercise test in normoxic and hypoxic conditions prior to and after 2 and 8 wk of acclimatization to 4,100 m. The highest workload completed by all subjects in all trials was 260 W. With acute hypoxic exposure prior to acclimatization, venous plasma [K(+)] was lower (P < 0.05) in normoxia (4.9 +/- 0.1 mM) than hypoxia (5.2 +/- 0.2 mM) at 260 W, but similar at exhaustion, which occurred at 400 +/- 9 W and 307 +/- 7 W (P < 0.05), respectively. At the same absolute exercise intensity, leg net K(+) release was unaffected by hypoxic exposure independent of acclimatization. After 8 wk of acclimatization, no difference existed in venous plasma [K(+)] between the normoxic and hypoxic trial, either at submaximal intensities or at exhaustion (360 +/- 14 W vs. 313 +/- 8 W; P < 0.05). At the same absolute exercise intensity, leg net K(+) release was less (P < 0.001) than prior to acclimatization and reached negative values in both hypoxic and normoxic conditions after acclimatization. Moreover, the reduction in plasma volume during exercise relative to rest was less (P < 0.01) in normoxic than hypoxic conditions, irrespective of the degree of acclimatization (at 260 W prior to acclimatization: -4.9 +/- 0.8% in normoxia and -10.0 +/- 0.4% in hypoxia). It is concluded that leg net K(+) release is unrelated to anaerobic energy production and that acclimatization reduces leg net K(+) release during exercise.