A role for ß-catenin in diet-induced skeletal muscle insulin resistance.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. ß-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle ß-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated ß-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific ß-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower ß-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated ß-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, ß-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total ß-catenin expression was unchanged. These findings suggest that ß-catenin dysfunction is associated with the development of insulin resistance.

Wool keratin - A novel dietary protein source: Nutritional value and toxicological assessment.

Keratin derived protein (KDP) was extracted from sheep wool using high pressure microwave technology and food acids and investigated for its potential as a novel dietary protein. The proximate composition, amino acid profile, element profile, in vitro cytotoxicity and digestibility of KDP were evaluated. Nutritive effects of KDP at 50% dietary supplementation were compared with a casein-based diet in a growing rat model for 95 days. Results indicate KDP to be rich in protein (86%), amino acid cysteine (8.8 g/100 g) and element selenium (0.29 µg/g). KDP was non-cytotoxic in vitro at ≤ 2 mg/mL concentration. There were no differences in the rat's weight gain compared to the control group (P > 0.05). Overall, the inclusion of the KDP in the diet was an effective substitute for casein protein at 50% and KDP has the potential to be used in the food industry as a novel dietary protein, free of fat and carbohydrate.

Six weeks of high intensity cycle training reduces H2O2 emission and increases antioxidant protein levels in obese adults with risk factors for type 2 diabetes.

Obesity has been associated with increased production of reactive oxygen species (ROS), which may be involved in the development of cardiovascular disease and type 2 diabetes (T2D). Endurance exercise lowers ROS production and increases antioxidant capacity in muscle cells, but it is currently unknown whether high intensity interval training (HIT) elicits the same effects. Twelve sedentary obese subjects at risk of developing T2D took part in a six-week intervention, performing three HIT sessions per week (five 1-min sets of high-intensity cycling (125% of VO2peak), with 90 s recovery in between sets). Muscle biopsies were obtained for assessment of ROS production (H2O2 emission), mitochondrial respiratory capacity, and antioxidant protein levels before and after the intervention. H2O2 emission decreased 60.4% after the intervention (Succinate 3 mmolï½¥l-1), concurrent with a 35.1% increase in protein levels of the antioxidant manganese superoxide dismutase (MnSOD) and a trend towards increased levels of the antioxidant catalase (p = 0.06, 72.9%). These findings were accompanied by a 19% increased mitochondrial respiratory capacity (CI + II), a 6.9% increased VO2peak and a 1.7% lower body fat percentage. These effects were achieved after just 15 min of high-intensity work and 40 min of total time spent per week. Overall, this suggests that a relatively small amount of HIT is sufficient to induce beneficial effects on ROS production and antioxidant status in muscle cells, which may lower oxidative stress and potentially protect against the development of cardiovascular disease.

Beta-aminoisobutyric acid is released by contracting human skeletal muscle and lowers insulin release from INS-1 832/3 cells by mediating mitochondrial energy metabolism.

AIMS/HYPOTHESIS: This study aimed to examine if beta-aminoisobutyric acid (BAIBA) is (i) secreted by skeletal muscle in humans during exercise, (ii) associated with insulin secretory function in vivo, and (iii) directly linked with acute glucose-mediated insulin release by pancreatic beta cells in vitro. METHODS: Following 2-weeks of single-leg immobilization, plasma BAIBA concentrations were measured in the brachial artery and the femoral veins of each leg in healthy male subjects, at rest and during two-legged dynamic knee-extensor exercise. During a 2-h hyperglycamic clamp, insulin secretory function and levels of plasma BAIBA were assessed in non-diabetic individuals, non-diabetic individuals following 24-h hyperglycemia and patients with type 2 diabetes. Direct effects of BAIBA on acute glucose-mediated insulin release were probed in INS-1832/3 cells under normal and 'diabetes-like' conditions. Finally, the effect of BAIBA on mitochondrial function was assessed in INS-1832/3 cells using extracellular flux analysis. RESULTS: (i) BAIBA is released from skeletal muscle at rest and during exercise under healthy conditions but is suppressed during exercise following leg immobilization, (ii) plasma BAIBA concentrations inversely associate with insulin secretory function in humans, (iii) BAIBA lowers mitochondrial energy metabolism in INS-1 832/3 cells in parallel with decreased insulin secretionConclusion/interpretation: BAIBA is a myokine released by skeletal muscle during exercise and indepedantly alters the triggering pathway of insulin secretion in cultured INS-1832/3 cells.

Research Toolbox for Peripheral Arterial Disease　- Minimally Invasive Assessment of the Vasculature and Skeletal Muscle.

In 2010, more than 200 million people were afflicted with peripheral arterial disease (PAD). Because it is atherosclerotic in etiology, it is not surprising that PAD is a leading cause of cardiovascular morbidity. Cardiovascular disease (CVD) risk can be decreased if ambulatory physical function is improved. However, physical function is limited by a mismatch between oxygen supply and demand in the legs, which results in exertional pain, leg weakness, and balance problems. Therefore, a key factor for improving physical function, and decreasing CVD outcomes, is ensuring oxygen supply meets the oxygen demand. The purpose of this review is to highlight and evaluate practical and minimally invasive tools for assessing PAD etiology, with a specific focus on tools suited to studies focusing on improving physical function and CVD outcomes. Specifically, the macrovascular, microvascular, and skeletal muscle pathology of PAD is briefly outlined. Subsequently, the tools for assessing each of these components is discussed, including, where available, the evidence to contextualize these tools to PAD pathology as well as physical function and CVD outcomes. The goal of this review is to guide researchers to the appropriate tools with respect to their methodological design.

Exercise promotes IL-6 release from legs in older men with minor response to unilateral immobilization.

Physical inactivity is a major contributor to low-grade systemic inflammation. Most of the studies characterizing interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) release from exercising legs have been done in young, healthy men, but studies on inactivity in older people are lacking. The impact of 14 days of one-leg immobilization (IM) on IL-6 and TNF-α release during exercise in comparison to the contralateral control (CON) leg was investigated. Fifteen healthy men (age 68.1 ± 1.1 year (mean ± SEM); BMI 27.0 ± 0.4 kg·m(2); VO2max 33.3 ± 1.6 ml·kg(‒1)·min(‒1)) performed 45 min of two-leg dynamic knee extensor exercise at 19.5 ± 0.9 W. Arterial and femoral venous blood samples from the CON and the IM legs were collected every 15 min during exercise, and thigh blood flow was measured with ultrasound Doppler. Arterial plasma IL-6 concentration increased with exercise (rest vs. 45 min, main effect p < .05). IL-6 release increased with exercise (rest vs. 30 min, main effect p < .05). Furthermore, IL-6 release was borderline (main effect, p = .085, effect size 0.28) higher in the IM leg compared to the CON leg (288 (95% CI: 213-373) vs. 220 (95% CI: 152-299) pg·min(‒1), respectively). There was no release of TNF-α in either leg and arterial concentrations remained unchanged during exercise (p > .05). In conclusion, exercise induces more pronounced IL-6 secretion in healthy older men. Two weeks of unilateral immobilization on the other hand had only a minor influence on IL-6 release. Neither immobilization nor exercise had an effect on TNF-α release across the working legs in older men.

Reliability of Central Adiposity Assessments Using B-Mode Ultrasound: A Comparison of Linear and Curved Array Transducers.

Recently, it was reported that intra-abdominal thickness (IAT) assessments using ultrasound are most reliable if measured from the linea alba to the anterior vertebral column. These 2 anatomical sites can be simultaneously visualized using a linear array transducer. Linear array transducers have different operational characteristics when compared with conventional curved array transducers and are more reliable for some ultrasound-derived measures such as abdominal subcutaneous fat thickness. However, it is unknown whether linear array transducers facilitate more reliable IAT measurements than curved array transducers. The purpose of the current study was to (1) compare the reliability of linear and curved array transducer assessments of IAT and maximal abdominal ratio (MAR) and (2) use the findings to update central adiposity measurement guidelines. Fifteen healthy adults (mean [SD], 27 [10] years; 60% female) with a range of somatotypes (body mass index: mean [SD], 24 [4]; range, 19-33 kg/m; waist circumference: mean [SD], 75 [11]; range, 61-96 cm) were tested on 3 mornings under standardized conditions. Intra-abdominal thickness was assessed 2 cm above the umbilicus (transverse plane), measuring from linea alba to the anterior vertebral column. Maximal abdominal ratio was defined as the ratio of IAT to abdominal subcutaneous fat thickness. The IAT range was 25 to 87 mm, and the MAR range was 0.15 to 0.77. Between-day intraclass correlation coefficient values for IAT measurements made were comparable (0.96-0.97) for both transducers, as were MAR values (0.95). In conclusion, while both transducers provided equally reliable measurement of IAT, the use of a single linear array transducer simplifies the assessment of central adiposity.

Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men.

PURPOSE: Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed by aerobic training in young and older men. METHODS: 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity RESULTS: The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. CONCLUSION: Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises as to whether IMTG accumulation in the older men is caused by or leading to the increase in PLIN2 and 3 protein content. Training decreased body fat and IMTG levels in both young and older men; hence, training should be prioritized to reduce the detrimental effect of aging on metabolism.

A novel method for determining human ex vivo submaximal skeletal muscle mitochondrial function.

The present study utilized a novel method aiming to investigate mitochondrial function in human skeletal muscle at submaximal levels and at a predefined membrane potential. The effect of age and training status was investigated using a cross-sectional design. Ageing was found to be related to decreased leak regardless of training status. Increased training status was associated with increased mitochondrial hydrogen peroxide emission. Despite numerous studies, there is no consensus about whether mitochondrial function is altered with increased age. The novelty of the present study is the determination of mitochondrial function at submaximal activity rates, which is more physiologically relevant than the ex vivo functionality protocols used previously. Muscle biopsies were taken from 64 old or young male subjects (aged 60-70 or 20-30 years). Aged subjects were recruited as trained or untrained. Muscle biopsies were used for the isolation of mitochondria and subsequent measurements of DNA repair, anti-oxidant capacity and mitochondrial protein levels (complexes I-V). Mitochondrial function was determined by simultaneous measurement of oxygen consumption, membrane potential and hydrogen peroxide emission using pyruvate + malate (PM) or succinate + rotenone (SR) as substrates. Proton leak was lower in aged subjects when determined at the same membrane potential and was unaffected by training status. State 3 respiration was lower in aged untrained subjects. This effect, however, was alleviated in aged trained subjects. H2 O2 emission with PM was higher in aged subjects, and was exacerbated by training, although it was not changed when using SR. However, with a higher manganese superoxide dismuthase content, the trained aged subjects may actually have lower or similar mitochondrial superoxide emission compared to the untrained subjects. We conclude that ageing and the physical activity level in aged subjects are both related to changes in the intrinsic functionality of the mitochondrion in skeletal muscle. Both of these changes could be important factors in determining the metabolic health of the aged skeletal muscle cell.

Skeletal muscle mitochondrial H2 O2 emission increases with immobilization and decreases after aerobic training in young and older men.

Currently, it is not known whether impaired mitochondrial function contributes to human ageing or whether potential impairments in mitochondrial function with age are secondary to physical inactivity. The present study investigated mitochondrial respiratory function and reactive oxygen species emission at a predefined membrane potential in young and older men subjected to 2 weeks of one-leg immobilization followed by 6 weeks of aerobic cycle training. Immobilization increased reactive oxygen species emission and decreased ATP generating respiration. Subsequent aerobic training reversed these effects. By contrast, age had no effect on the measured variables. The results of the present study support the notion that increased mitochondrial reactive oxygen species production mediates the detrimental effects seen after physical inactivity and that ageing per se does not cause mitochondrial dysfunction. Mitochondrial dysfunction, defined as increased oxidative stress and lower capacity for energy production, may be seen with ageing and may cause frailty, or it could be that it is secondary to physical inactivity. We studied the effect of 2 weeks of one-leg immobilization followed by 6 weeks of supervised cycle training on mitochondrial function in 17 young (mean ± SEM: 23 ± 1 years) and 15 older (68 ± 1 years) healthy men. Submaximal H2 O2 emission and respiration were measured simultaneously at a predefined membrane potential in isolated mitochondria from skeletal muscle using two protocols: pyruvate + malate (PM) and succinate + rotenone (SR). This allowed measurement of leak and ATP generating respiration from which the coupling efficiency can be calculated. The protein content of the anti-oxidants manganese superoxide dismuthase (MnSOD), CuZn superoxide dismuthase, catalase and gluthathione peroxidase 1 was measured by western blotting. Immobilization decreased ATP generating respiration using PM and increased H2 O2 emission using both PM and SR similarly in young and older men. Both were restored to baseline after the training period. Furthermore, MnSOD and catalase content increased with endurance training. The young men had a higher leak respiration at inclusion using PM and a higher membrane potential in State 3 using both substrate combinations. Collectively, the findings of the present study support the notion that increased mitochondrial reactive oxygen species mediates the detrimental effects seen after physical inactivity. Age, on the other hand, was not associated with impairments in anti-oxidant protein levels, mitochondrial respiration or H2 O2 emission using either protocol.

Six weeks' aerobic retraining after two weeks' immobilization restores leg lean mass and aerobic capacity but does not fully rehabilitate leg strength in young and older men.

OBJECTIVE: To determine the effect of aerobic retraining as rehabilitation after short-term leg immobilization on leg strength, leg work capacity, leg lean mass, leg muscle fibre type composition and leg capillary supply, in young and older men.Subjects and design: Seventeen young (23 ± 1 years) and 15 older (68 ± 1 [standard error of the mean; SEM] years) men had one leg immobilized for 2 weeks, followed by 6 weeks' bicycle endurance retraining. METHODS: Maximal voluntary contraction, leg work capacity (Wmax), and leg lean mass by dual energy X-ray absorptiometry were measured at inclusion, after immobilization and after 3 and 6 weeks' retraining. Muscle biopsies were evaluated for fibre type, fibre area, and capillarization. RESULTS: Immobilization decreased maximal voluntary contraction (-28 ± 6% and -23 ± 3%); Wmax (-13 ± 5% and -9 ± 4%) and leg lean mass (only in young, -485 ± 105g) in young and older men, respectively. Six weeks' retraining increased maximal voluntary contraction (34 ± 8% and 17 ± 6%), Wmax (33 ± 5% and 20 ± 5%) and leg lean mass (only in young 669 ± 69 g) in young and older men, respectively, compared with the immobilized value. CONCLUSION: Short-term leg immobilization had marked effects on leg strength, and work capacity and 6 weeks' retraining was sufficient to increase, but not completely rehabilitate, muscle strength, and to rehabilitate aerobic work capacity and leg lean mass (in the young men).

The Effect of Reduced Physical Activity and Retraining on Blood Lipids and Body Composition in Young and Older Adult Men.

We studied the effect of physical inactivity and subsequent retraining on cardiovascular risk factors in 17 young (Y; 23.4 ± 0.5 years) and 15 older adult (O; 68.1 ± 1.1 years) men who underwent 14 days of one leg immobilization followed by six weeks of training. Body weight remained unchanged. Daily physical activity decreased by 31 ± 9% (Y) and 37 ± 9% (O) (p < .001). Maximal oxygen uptake decreased with inactivity (Y) and always increased with training. Visceral fat mass decreased (p < .05) with training. Concentrations of lipids in blood were always highest in the older adults. FFA and glycerol increased with reduced activity (p < .05), but reverted with training. Training resulted in increases in HDL-C (p < .05) and a decrease in LDL-C and TC:HDL-C ratio (p < .05). A minor reduction in daily physical activity for two weeks increased blood lipids in both young and older men. Six weeks of training improved blood lipids along with loss of visceral fat.

Two weeks of one-leg immobilization decreases skeletal muscle respiratory capacity equally in young and elderly men.

Physical inactivity affects human skeletal muscle mitochondrial oxidative capacity but the influence of aging combined with physical inactivity is not known. This study investigates the effect of two weeks of immobilization followed by six weeks of supervised cycle training on muscle oxidative capacity in 17 young (23±1years) and 15 elderly (68±1years) healthy men. We applied high-resolution respirometry in permeabilized fibers from muscle biopsies at inclusion after immobilization and training. Furthermore, protein content of mitochondrial complexes I-V, mitochondrial heat shock protein 70 (mtHSP70) and voltage dependent anion channel (VDAC) were measured in skeletal muscle by Western blotting. The elderly men had lower content of complexes I-V and mtHSP70 but similar respiratory capacity and content of VDAC compared to the young. In both groups the respiratory capacity and protein content of VDAC, mtHSP70 and complexes I, II, IV and V decreased with immobilization and increased with retraining. Moreover, there was no overall difference in the response between the groups. When the intrinsic mitochondrial capacity was evaluated by normalizing respiration to citrate synthase activity, the respiratory differences with immobilization and training disappeared. In conclusion, aging is not associated with a decrease in muscle respiratory capacity in spite of lower complexes I-V and mtHSP70 protein content. Furthermore, immobilization decreased and aerobic training increased the respiratory capacity and protein contents of complexes I-V, mtHSP70 and VDAC similarly in the two groups. This suggests that inactivity and training alter mitochondrial biogenesis equally in young and elderly men.

The best approach: homogenization or manual permeabilization of human skeletal muscle fibers for respirometry?

The number of studies on mitochondrial function is growing as a result of the recognition of the pivotal role of an intact mitochondrial function in numerous diseases. Measurements of oxygen consumption by the mitochondria in human skeletal muscle are used in many studies. There are several advantages of studying mitochondrial respiration in permeabilized fibers (Pfi), but the method requires a manual procedure of mechanical separation of the fiber bundles in the biopsy and chemical permeabilization of the cell membrane. This is time-consuming and subject to interpersonal variability. An alternative is to use a semiautomatic tool for preparation of a homogenate of the muscle biopsy. We investigated whether the PBI shredder is useful in preparing a muscle homogenate for measurements of mitochondrial respiratory capacity. The homogenate is compared with the Pfi preparation. Maximal respiratory capacity was significantly reduced in the homogenate compared with the Pfi from human skeletal muscle. A marked cytochrome c response was observed in the homogenate, which was not the case with the Pfi, indicating that the outer mitochondrial membrane was not intact. The mitochondria in the homogenate were more uncoupled compared with the Pfi. Manual permeabilization is an advantageous technique for preparing human skeletal muscle biopsies for respirometry.

Physical inactivity and muscle oxidative capacity in humans.

Physical inactivity is associated with a high prevalence of type 2 diabetes and is an independent predictor of mortality. It is possible that the detrimental effects of physical inactivity are mediated through a lack of adequate muscle oxidative capacity. This short review will cover the present literature on the effects of different models of inactivity on muscle oxidative capacity in humans. Effects of physical inactivity include decreased mitochondrial content, decreased activity of oxidative enzymes, changes in markers of oxidative stress and a decreased expression of genes and contents of proteins related to oxidative phosphorylation. With such a substantial down-regulation, it is likely that a range of adenosine triphosphate (ATP)-dependent pathways such as calcium signalling, respiratory capacity and apoptosis are affected by physical inactivity. However, this has not been investigated in humans, and further studies are required to substantiate this hypothesis, which could expand our knowledge of the potential link between lifestyle-related diseases and muscle oxidative capacity. Furthermore, even though a large body of literature reports the effect of physical training on muscle oxidative capacity, the adaptations that occur with physical inactivity may not always be opposite to that of physical training. Thus, it is concluded that studies on the effect of physical inactivity per se on muscle oxidative capacity in functional human skeletal muscle are warranted.

Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells.

Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality. Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters. However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects of mitochondrial oxidative phosphorylation capacity in PBMCs, but may have other underlying cellular dysfunctions that contribute to dNTP imbalance and altered ROS production.

Immobilization increases interleukin-6, but not tumour necrosis factor-α, release from the leg during exercise in humans.

Data on interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) release during acute exercise are not conclusive, and information is lacking about the impact of physical inactivity. Some studies have shown an increase, but others report no changes in IL-6 and TNF-α release during exercise. We have now studied the temporal relationship of leg IL-6 and TNF-α release before and during isolated two-legged exercise after 14 days of one-leg immobilization (IM) while the other leg served as the control (CON) leg. Fifteen healthy male subjects (mean ± SEM age, 23 ± 1 years; body mass index, 23.6 ± 0.7 kg m(-2); and maximal oxygen uptake, 46.8 ± 1.4 ml kg(-1) min(-1)) performed 45 min of two-legged dynamic knee-extensor exercise at 19.6 ± 0.8 W. Arterial and femoral venous blood samples from the CON and the IM leg were collected every 15 min during exercise, and leg blood flow was measured with Doppler ultrasound. The arterial plasma IL-6 concentration increased (P < 0.05) with exercise (rest, 1.3 ± 0.1 pg ml(-1); 15 min, 1.9 ± 0.2 pg ml(-1); 30 min, 2.4 ± 0.2 pg ml(-1); and 45 min, 3.1 ± 0.3 pg ml(-1)). Interleukin-6 release occurred after 15 min of exercise, and the release from the IM leg was significantly greater compared with the CON leg after 45 min (1114 ± 152 versus 606 ± 14 pg min(-1), respectively, P < 0.05). Tumour necrosis factor-α release did not differ between the CON and the IM leg, and arterial concentrations remained unchanged during exercise (P > 0.05). In conclusion, prior immobilization enhances release of IL-6 from the leg during exercise at a moderate workload, and the release is already present in the early phase of exercise. Neither immobilization nor exercise had an effect on TNF-α release in the working legs.