The angiotensin-converting enzyme I/D polymorphism does not impact training-induced adaptations in exercise capacity in patients with stable coronary artery disease.

Systematic exercise training effectively improves exercise capacity in patients with coronary artery disease (CAD), but the magnitude of improvements is highly heterogeneous. We investigated whether this heterogeneity in exercise capacity gains is influenced by the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene. Patients with CAD (n = 169) were randomly assigned to 12 weeks of exercise training or standard care, and 142 patients completed the study. The ACE polymorphism was determined for 128 patients (82% males, 67 ± 9 years). Peak oxygen uptake was measured before and after the 12-week intervention. The ACE I/D polymorphism frequency was n = 48 for D/D homozygotes, n = 61 for I/D heterozygotes and n = 19 for I/I homozygotes. Baseline peak oxygen uptake was 23.3 ± 5.0 ml/kg/min in D/D homozygotes, 22.1 ± 5.3 ml/kg/min in I/D heterozygotes and 23.1 ± 6.0 ml/kg/min in I/I homozygotes, with no statistical differences between genotype groups (P = 0.50). The ACE I/D polymorphism frequency in the exercise group was n = 26 for D/D, n = 21 for I/D and n = 12 for I/I. After exercise training, peak oxygen uptake was increased (P < 0.001) in D/D homozygotes by 2.6 ± 1.7 ml/kg/min, in I/D heterozygotes by 2.7 ± 1.9 ml/kg/min, and in I/I homozygotes by 2.1 ± 1.3 ml/kg/min. However, the improvements were similar between genotype groups (time × genotype, P = 0.55). In conclusion, the ACE I/D polymorphism does not affect baseline exercise capacity or exercise capacity gains in response to 12 weeks of high-intensity exercise training in patients with stable CAD.Clinical trial registration: www.clinicaltrials.gov (NCT04268992).

Serum Insulin-like Factor 3, Testosterone, and LH in Experimental and Therapeutic Testicular Suppression.

BACKGROUND: Serum insulin-like factor 3 (INSL3) is a Leydig cell biomarker, but little is known about the circulating concentration of INSL3 during hypothalamus-pituitary-testicular suppression. AIM: To study the concomitant changes in serum concentrations of INSL3, testosterone, and LH during experimental and therapeutic testicular suppression. METHODS: We included serum samples from 3 different cohorts comprising subjects before and after testicular suppression: (1) 6 healthy young men who were treated with androgens (Sustanon, Aspen Pharma, Dublin, Ireland); 2) 10 transgender girls (male sex assigned at birth) who were treated with 3-monthly GnRH agonist injections (Leuprorelinacetat, Abacus Medicine, Copenhagen, Denmark); and (3) 55 patients with prostate cancer who were randomized to surgical castration (bilateral subcapsular orchiectomy) or treatment with GnRH agonist (Triptorelin, Ipsen Pharma, Kista, Sweden). Serum INSL3 and testosterone concentrations were quantified in stored serum samples using validated liquid chromatography-tandem mass spectrometry methodologies, and LH was measured by an ultrasensitive immunoassay. RESULTS: The circulating concentrations of INSL3, testosterone, and LH decreased during experimental testicular suppression in healthy young men by Sustanon injections and subsequently returned to baseline levels after release of suppression. All 3 hormones decreased during therapeutic hormonal hypothalamus-pituitary-testicular suppression in transgender girls and in patients with prostate cancer. CONCLUSION: INSL3 resembles testosterone as a sensitive marker of testicular suppression and reflects Leydig cell function, also during exposure to exogenous testosterone. Serum INSL3 measurements may complement testosterone as a Leydig cell marker in male reproductive disorders, during therapeutic testicular suppression as well as in surveillance of illicit use of androgens.

Oxygen Uptake During Activities of Daily Life in Patients Treated With a Left Ventricular Assist Device.

INTRODUCTION: Oxygen consumption during activities of daily life (ADL) is not described in recipients of left ventricular assist device (LVAD). We aimed to investigate the relation between oxygen consumption during predefined ADLs and measures of functional capacity (FC) in stable-phase LVAD recipients. METHODS: LVADs and controls were matched on gender, age, BMI, smoking status, and ethnicity. VO2 was measured using mobile equipment (K5, Cosmed, Rome, Italy) while putting on vest and LVAD equipment(1), folding towels(2), putting on socks and shoes(3), putting bottles in a cupboard(4), making a bed(5), walking on stairs without(6) and with extra weight(7), and sweeping the floor(8). Submaximal FC was tested by means of 6 minute walk test (6MWT) and peak oxygen uptake (pVO2) to test maximal FC. RESULTS: Fifteen LVAD patients and 16 controls were included; Patients were 61 ± 10years, all males with BMI 28 ± 5kg/m2 and implanted with Heartmate 3 (DT: 60%). PVO2 was 14.9 ± 2.2 ml/kg/min in patients and 39.6 ± 7.7 in controls (p < 0.001). Oxygen consumption expressed as percent of pVO2 for each task in patients vs controls was (%): ADL1: 41 ± 5 vs 21 ± 4, ADL2: 41 ± 6 vs 22 ± 5 %, ADL3: 50 ± 16 vs 24 ± 5%, ADL4: 45 ± 12 vs 22 ± 4, ADL5: 50 ± 8 vs 23 ± 4, ADL6: 66 ± 10 vs 30 ± 4, ADL7: 65 ± 10 vs 31 ± 5, ADL8: 75 ± 10 vs 39 ± 12, (p < 0.001 for all). During 6MWT LVAD patients used 96% ± 8 % of their pVO2. CONCLUSION: Recipients of durable LVADs perform daily life activities at oxygen uptake levels much closer to their peak cardiopulmonary reserve than matched healthy controls.

Beta2 -adrenergic agonist clenbuterol increases energy expenditure and fat oxidation, and induces mTOR phosphorylation in skeletal muscle of young healthy men.

Clenbuterol is a beta2 -adrenoceptor agonist marketed as an asthma reliever but is not approved for human use in most countries due to concerns of adverse cardiac effects. Given its demonstrated hypertrophic and lipolytic actions in rodents, clenbuterol is one of the most widely abused doping substances amongt athletes and recreational body-builders seeking leanness. Herein, we examined the effect of clenbuterol ingestion on metabolic rate as well as skeletal muscle mammalian target of rapamycin (mTOR) phosphorylation and protein kinase A (PKA)-signaling in six young men. Before and 140 min after ingestion of 80 µg clenbuterol, resting metabolic rate and contractile function of the quadriceps muscle were measured, and blood samples as well as vastus lateralis muscle biopsies were collected. Clenbuterol increased resting energy expenditure by 21% (P < 0.001), and fat oxidation by 39% (P = 0.006), whereas carbohydrate oxidation was unchanged. Phosphorylation of mTORSer2448 and PKA substrates increased by 121% (P = 0.004) and 35% (P = 0.006), respectively, with clenbuterol. Maximal voluntary contraction torque decreased by 4% (P = 0.026) and the half-relaxation time shortened by 9% (P = 0.046), while voluntary activation, time to peak twitch, and peak twitch torque did not change significantly with clenbuterol. Glycogen content of the vastus lateralis muscle did not change with clenbuterol. Clenbuterol increased circulating levels of glucose (+30%; P < 0.001), lactate (+90%; P = 0.004), insulin (+130%; P = 0.009), and fatty acids (+180%; P = 0.001). Collectively, these findings indicate that clenbuterol is an efficient thermogenic substance that possibly also exerts muscle hypertrophic actions in humans. For these reasons, the restrictions imposed against clenbuterol in competitive sports seem warranted.

Purinergic effects on Na,K-ATPase activity differ in rat and human skeletal muscle.

BACKGROUND: P2Y receptor activation may link the effect of purines to increased maximal in vitro activity of the Na,K-ATPase in rat muscle. The hypothesis that a similar mechanism is present in human skeletal muscle was investigated with membranes from rat and human skeletal muscle. RESULTS: Membranes purified from rat and human muscles were used in the Na,K-ATPase assay. Incubation with ADP, the stable ADP analogue MeS-ADP and UDP increased the Na+ dependent Na,K-ATPase activity in rat muscle membranes, whereas similar treatments of human muscle membranes lowered the Na,K-ATPase activity. UTP incubation resulted in unchanged Na,K-ATPase activity in humans, but pre-incubation with the antagonist suramin resulted in inhibition with UTP, suggesting that P2Y receptors are involved. The Na,K-ATPase in membranes from both rat and human could be stimulated by protein kinase A and C activation. Thus, protein kinase A and C activation can increase Na,K-ATPase activity in human muscle but not via P2Y receptor stimulation. CONCLUSION: The inhibitory effects of most purines (with the exception of UTP) in human muscle membranes are probably due to mass law inhibition of ATP hydrolysis. This inhibition could be blurred in rat due to receptor mediated activation of the Na,K-ATPase. The different effects could be related to a high density of ADP sensitive P2Y1 and P2Y13 receptors in rat, whereas the UTP sensitive P2Y11 could be more abundant in human. Alternatively, rat could possesses a mechanism for protein-protein interaction between P2Y receptors and the Na,K-ATPase, and this mechanism could be absent in human skeletal muscle (perhaps with the exception of the UTP sensitive P2Y11 receptor). PERSPECTIVE: Rat muscle is not a reliable model for purinergic effects on Na,K-ATPase in human skeletal muscle.

Exercise-induced increase in maximal in vitro Na-K-ATPase activity in human skeletal muscle.

The present study investigated whether maximal in vitro Na-K-ATPase activity in human skeletal muscle is changed with exercise and whether it was altered by acute hypoxia. Needle biopsies from 14 subjects were obtained from vastus lateralis before and after 4 min of intense muscle activity. In addition, six subjects exercised also in hypoxia (12.5% oxygen). The Na-K-ATPase assay revealed a 19% increase (P < 0.05) in maximal velocity (Vmax) for Na⁺-dependent Na-K-ATPase activity after exercise and a tendency (P < 0.1) toward a decrease in Km for Na⁺ (increased Na⁺ affinity) in both normoxia and hypoxia. In contrast, the in vitro Na-K-ATPase activity determined with the 3-O-MFPase technique was 11-32% lower after exercise in normoxia (P < 0.05) and hypoxia (P < 0.1). Based on the different results obtained with the Na-K-ATPase assay and the 3-O-MFPase technique, it was suggested that the 3-O-MFPase method is insensitive to changes in Na-K-ATPase activity. To test this possibility, changes in Na-K-ATPase activity was induced by protein kinase C activation. The changes quantified with the Na-K-ATPase assay could not be detected with the 3-O-MFPase method. In addition, purines stimulated Na-K-ATPase activity in rat muscle membranes; these changes could not be detected with the 3-O-MFPase method. Therefore, the 3-O-MFPase technique is not sensitive to changes in Na⁺ sensitivity, and the method is not suited to detecting changes in Na-K-ATPase activity with exercise. In conclusion, muscle activity in humans induces an increased in vitro Na⁺-dependent Na-K-ATPase activity, which contributes to the upregulation of the Na-K-ATPase in association with exercise both in normoxia and hypoxia.

Lactate oxidation in human skeletal muscle mitochondria.

Lactate is an important intermediate metabolite in human bioenergetics and is oxidized in many different tissues including the heart, brain, kidney, adipose tissue, liver, and skeletal muscle. The mechanism(s) explaining the metabolism of lactate in these tissues, however, remains unclear. Here, we analyze the ability of skeletal muscle to respire lactate by using an in situ mitochondrial preparation that leaves the native tubular reticulum and subcellular interactions of the organelle unaltered. Skeletal muscle biopsies were obtained from vastus lateralis muscle in 16 human subjects. Samples were chemically permeabilized with saponin, which selectively perforates the sarcolemma and facilitates the loss of cytosolic content without altering mitochondrial membranes, structure, and subcellular interactions. High-resolution respirometry was performed on permeabilized muscle biopsy preparations. By use of four separate and specific substrate titration protocols, the respirometric analysis revealed that mitochondria were capable of oxidizing lactate in the absence of exogenous LDH. The titration of lactate and NAD(+) into the respiration medium stimulated respiration (P ≤ 0.003). The addition of exogenous LDH failed to increase lactate-stimulated respiration (P = 1.0). The results further demonstrate that human skeletal muscle mitochondria cannot directly oxidize lactate within the mitochondrial matrix. Alternately, these data support previous claims that lactate is converted to pyruvate within the mitochondrial intermembrane space with the pyruvate subsequently taken into the mitochondrial matrix where it enters the TCA cycle and is ultimately oxidized.

Relationship between performance at different exercise intensities and skeletal muscle characteristics.

The hypothesis investigated whether exercise performance over a broad range of intensities is determined by specific skeletal muscle characteristics. Seven subjects performed 8-10 exhaustive cycle trials at different workloads, ranging from 150 to 700 W (150 min to 20 s). No relationships between the performance times at high and low workloads were observed. A relationship (P < 0.05) was noticed between the percentage of fast-twitch x fibers and the exercise time at 579 ± 21 W (∼30 s; r(2) = 0.88). Capillary-to-fiber-ratio (r(2): 0.58-0.85) was related (P < 0.05) to exercise time at work intensities ranging from 395 to 270 W (2.5-21 min). Capillary density was correlated (r(2) = 0.68; P < 0.05) with the net rate of plasma K(+) accumulation during an ∼3-min bout and was estimated to explain 50-80% (P < 0.05) of the total variance observed in exercise performances lasting ∼30 s to 3 min. The Na(+)-K(+) pump ß(1)-subunit expression was found to account for 13-34% (P < 0.05) during exhaustive exercise of ∼1-4 min. In conclusion, exercise performance at different intensities is related to specific physiological variables. A large distribution of fast-twitch x fibers may play a role during very intense efforts, i.e., ∼30 s. Muscle capillaries and the Na(+)-K(+) pump ß(1)-subunit seem to be important determinants for performance during exhaustive high-intensity exercises lasting between 30 s and 4 min.

Exercise-induced regulation of muscular Na+-K+ pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type.

It is investigated if exercise-induced mRNA changes cause similar protein expression changes of Na(+)-K(+) pump isoforms (α(1), α(2), ß(1), ß(2)), FXYD1, and Na(+)/K(+) exchanger (NHE1) in rat skeletal muscle. Expression was evaluated (n = 8 per group) in soleus and extensor digutorum longus after 1 day, 3 days, and 3 wk (5 sessions/wk) of either sprint (4 × 3-min sprint + 1-min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of soleus α(2) (4.9 ± 0.8-fold; P < 0.05), ß(2) (13.2 ± 4.4-fold; P < 0.001), and NHE1 (12.0 ± 3.1-fold; P < 0.01). Two hours after sprint exercise, protein expression normalized to control samples was higher on day 3 than day 1 for soleus α(1) (41 ± 18% increase vs. 15 ± 8% reduction; P < 0.05), α(2) (64 ± 35% increase vs. 37 ± 12% reduction; P < 0.05), ß(1) (17 ± 21% increase vs. 14 ± 29% reduction; P < 0.05), and FXYD1 (35 ± 16% increase vs. 13 ± 10% reduction; P < 0.05). In contrast, on day 3, soleus α(1) (0.1 ± 0.1-fold; P < 0.001), α(2) (0.2 ± 0.1-fold; P < 0.001), ß(1) (0.4 ± 0.1-fold; P < 0.05), and ß(2)-mRNA (2.9 ± 1.7-fold; P < 0.001) expression was lower than after exercise on day 1. After 3 wk of training, no change in protein expression relative to control existed. In conclusion, increased expression of Na(+)-K(+) pump subunits, FXYD1 and NHE1 after 3 days exercise training does not appear to be an effect of increased constitutive mRNA levels. Importantly, sprint exercise can reduce mRNA expression concomitant with increased protein expression.