David Morales-Alamo

What limits performance during whole-body incremental exercise to exhaustion in humans?

At the end of an incremental exercise to exhaustion a large functional reserve remains in the muscles to generate power, even at levels far above the power output at which task failure occurs, regardless of the inspiratory O2 pressure during the incremental exercise. Exhaustion (task failure) is not due to lactate accumulation and the associated muscle acidification; neither the aerobic energy pathways nor the glycolysis are blocked at exhaustion. Muscle lactate accumulation may actually facilitate early recovery after exhaustive exercise even under ischaemic conditions. Although the maximal rate of ATP provision is markedly reduced at task failure, the resynthesis capacity remaining exceeds the rate of ATP consumption, indicating that task failure during an incremental exercise to exhaustion depends more on central than peripheral mechanisms.

Limitations to oxygen transport and utilization during sprint exercise in humans: evidence for a functional reserve in muscle O2 diffusing capacity

Severe acute hypoxia reduces sprint performance. Muscle V̇O2 during sprint exercise in normoxia is not limited by O2 delivery, O2 offloading from haemoglobin or structure‐dependent diffusion constraints in the skeletal muscle of young healthy men. A large functional reserve in muscle O2 diffusing capacity exists and remains available at exhaustion during exercise in normoxia; this functional reserve is recruited during exercise in hypoxia. During whole‐body incremental exercise to exhaustion in severe hypoxia, leg V̇O2 is primarily dependent on convective O2 delivery and less limited by diffusion constraints than previously thought. The kinetics of O2 offloading from haemoglobin does not limit V̇O2 peak in hypoxia. Our results indicate that the limitation to V̇O2 during short sprints resides in mechanisms regulating mitochondrial respiration.

Increased oxidative stress and anaerobic energy release, but blunted Thr172-AMPKα phosphorylation, in response to sprint exercise in severe acute hypoxia in humans

AMP-activated protein kinase (AMPK) is a major mediator of the exercise response and a molecular target to improve insulin sensitivity. To determine if the anaerobic component of the exercise response, which is exaggerated when sprint is performed in severe acute hypoxia, influences sprint exercise-elicited Thr(172)-AMPKα phosphorylation, 10 volunteers performed a single 30-s sprint (Wingate test) in normoxia and in severe acute hypoxia (inspired Po(2): 75 mmHg). Vastus lateralis muscle biopsies were obtained before and immediately after 30 and 120 min postsprint. Mean power output and O(2) consumption were 6% and 37%, respectively, lower in hypoxia than in normoxia. O(2) deficit and muscle lactate accumulation were greater in hypoxia than in normoxia. Carbonylated skeletal muscle and plasma proteins were increased after the sprint in hypoxia. Thr(172)-AMPKα phosphorylation was increased by 3.1-fold 30 min after the sprint in normoxia. This effect was prevented by hypoxia. The NAD(+)-to-NADH.H(+) ratio was reduced (by 24-fold) after the sprints, with a greater reduction in hypoxia than in normoxia (P < 0.05), concomitant with 53% lower sirtuin 1 (SIRT1) protein levels after the sprint in hypoxia (P < 0.05). This could have led to lower liver kinase B1 (LKB1) activation by SIRT1 and, hence, blunted Thr(172)-AMPKα phosphorylation. Ser(485)-AMPKα(1)/Ser(491)-AMPKα(2) phosphorylation, a known negative regulating mechanism of Thr(172)-AMPKα phosphorylation, was increased by 60% immediately after the sprint in hypoxia, coincident with increased Thr(308)-Akt phosphorylation. Collectively, our results indicate that the signaling response to sprint exercise in human skeletal muscle is altered in severe acute hypoxia, which abrogated Thr(172)-AMPKα phosphorylation, likely due to lower LKB1 activation by SIRT1.

Critical role for free radicals on sprint exercise-induced CaMKII and AMPKα phosphorylation in human skeletal muscle.

The extremely high energy demand elicited by sprint exercise is satisfied by an increase in O2 consumption combined with a high glycolytic rate, leading to a marked lactate accumulation, increased AMP-to-ATP ratio, and reduced NAD(+)/NADH.H(+) and muscle pH, which are accompanied by marked Thr(172) AMP-activated protein kinase (AMPK)-α phosphorylation during the recovery period by a mechanism not fully understood. To determine the role played by reactive nitrogen and oxygen species (RNOS) on Thr(172)-AMPKα phosphorylation in response to cycling sprint exercise, nine voluntary participants performed a single 30-s sprint (Wingate test) on two occasions: one 2 h after the ingestion of placebo and another after the intake of antioxidants (α-lipoic acid, vitamin C, and vitamin E) in a double-blind design. Vastus lateralis muscle biopsies were obtained before, immediately postsprint, and 30 and 120 min postsprint. Performance and muscle metabolism were similar during both sprints. The NAD(+)-to-NADH.H(+) ratio was similarly reduced (84%) and the AMP-to-ATP ratio was similarly increased (×21-fold) immediately after the sprints. Thr(286) Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and Thr(172)-AMPKα phosphorylations were increased after the control sprint (with placebo) but not when the sprints were preceded by the ingestion of antioxidants. Ser(485)-AMPKα1/Ser(491)-AMPKα2 phosphorylation, a known inhibitory mechanism of Thr(172)-AMPKα phosphorylation, was increased only with antioxidant ingestion. In conclusion, RNOS play a crucial role in AMPK-mediated signaling after sprint exercise in human skeletal muscle. Antioxidant ingestion 2 h before sprint exercise abrogates the Thr(172)-AMPKα phosphorylation response observed after the ingestion of placebo by reducing CaMKII and increasing Ser(485)-AMPKα1/Ser(491)-AMPKα2 phosphorylation. Sprint performance, muscle metabolism, and AMP-to-ATP and NAD(+)-to-NADH.H(+) ratios are not affected by the acute ingestion of antioxidants.

High-intensity sprint training inhibits mitochondrial respiration through aconitase inactivation

Intense exercise training is a powerful stimulus that activates mitochondrial biogenesis pathways and thus increases mitochondrial density and oxidative capacity. Moderate levels of reactive oxygen species (ROS) during exercise are considered vital in the adaptive response, but high ROS production is a serious threat to cellular homeostasis. Although biochemical markers of the transition from adaptive to maladaptive ROS stress are lacking, it is likely mediated by redox sensitive enzymes involved in oxidative metabolism. One potential enzyme mediating such redox sensitivity is the citric acid cycle enzyme aconitase. In this study, we examined biopsy specimens of vastus lateralis and triceps brachii in healthy volunteers, together with primary human myotubes. An intense exercise regimen inactivated aconitase by 55‐72%, resulting in inhibition of mitochondrial respiration by 50‐65%. In the vastus, the mitochondrial dysfunction was compensated for by a 15‐72% increase in mitochondrial proteins, whereas H2O2 emission was unchanged. In parallel with the inactivation of aconitase, the intermediary metabolite citrate accumulated and played an integral part in cellular protection against oxidative stress. In contrast, the triceps failed to increase mitochondrial density, and citrate did not accumulate. Instead, mitochondrial H2O2 emission was decreased to 40% of the pretraining levels, together with a 6‐fold increase in protein abundance of catalase. In this study, a novel mitochondrial stress response was highlighted where accumulation of citrate acted to preserve the redox status of the cell during periods of intense exercise.—Larsen, F. J., Schiffer, T. A., Ørtenblad, N., Zinner, C., Morales‐Alamo, D., Willis, S. J., Calbet, J. A., Holmberg, H.‐C., Boushel, R. High‐intensity sprint training inhibits mitochondrial respiration through aconitase inactivation. FASEB J. 30, 417‐427 (2016). www.fasebj.org

Is sprint exercise a leptin signaling mimetic in human skeletal muscle

This study was designed to determine whether sprint exercise activates signaling cascades linked to leptin actions in human skeletal muscle and how this pattern of activation may be interfered by glucose ingestion. Muscle biopsies were obtained in 15 young healthy men in response to a 30-s sprint exercise (Wingate test) randomly distributed into two groups: the fasting (n = 7, C) and the glucose group (n = 8, G), who ingested 75 g of glucose 1 h before the Wingate test. Exercise elicited different patterns of JAK2, STAT3, STAT5, ERK1/2, p38 MAPK phosphorylation, and SOCS3 protein expression during the recovery period after glucose ingestion. Thirty minutes after the control sprint, STAT3 and ERK1/2 phosphorylation levels were augmented (both, P < 0.05). SOCS3 protein expression was increased 120 min after the control sprint but PTP1B protein expression was unaffected. Thirty and 120 min after the control sprint, STAT5 phosphorylation was augmented (P < 0.05). Glucose abolished the 30 min STAT3 and ERK1/2 phosphorylation and the 120 min SOCS3 protein expression increase while retarding the STAT5 phosphorylation response to sprint. Activation of these signaling cascades occurred despite a reduction of circulating leptin concentration after the sprint. Basal JAK2 and p38 MAPK phosphorylation levels were reduced and increased (both P < 0.05), respectively, by glucose ingestion prior to exercise. During recovery, JAK2 phosphorylation was unchanged and p38 MAPK phosphorylation was transiently reduced when the exercise was preceded by glucose ingestion. In conclusion, sprint exercise performed under fasting conditions is a leptin signaling mimetic in human skeletal muscle.

Task Failure during Exercise to Exhaustion in Normoxia and Hypoxia Is Due to Reduced Muscle Activation Caused by Central Mechanisms While Muscle Metaboreflex Does Not Limit Performance

To determine whether task failure during incremental exercise to exhaustion (IE) is principally due to reduced neural drive and increased metaboreflex activation eleven men (22 ± 2 years) performed a 10 s control isokinetic sprint (IS; 80 rpm) after a short warm-up. This was immediately followed by an IE in normoxia (Nx, PIO2:143 mmHg) and hypoxia (Hyp, PIO2:73 mmHg) in random order, separated by a 120 min resting period. At exhaustion, the circulation of both legs was occluded instantaneously (300 mmHg) during 10 or 60 s to impede recovery and increase metaboreflex activation. This was immediately followed by an IS with open circulation. Electromyographic recordings were obtained from the vastus medialis and lateralis. Muscle biopsies and blood gases were obtained in separate experiments. During the last 10 s of the IE, pulmonary ventilation, VO2, power output and muscle activation were lower in hypoxia than in normoxia, while pedaling rate was similar. Compared to the control sprint, performance (IS-Wpeak) was reduced to a greater extent after the IE-Nx (11% lower P < 0.05) than IE-Hyp. The root mean square (EMGRMS) was reduced by 38 and 27% during IS performed after IE-Nx and IE-Hyp, respectively (Nx vs. Hyp: P < 0.05). Post-ischemia IS-EMGRMS values were higher than during the last 10 s of IE. Sprint exercise mean (IS-MPF) and median (IS-MdPF) power frequencies, and burst duration, were more reduced after IE-Nx than IE-Hyp (P < 0.05). Despite increased muscle lactate accumulation, acidification, and metaboreflex activation from 10 to 60 s of ischemia, IS-Wmean (+23%) and burst duration (+10%) increased, while IS-EMGRMS decreased (−24%, P < 0.05), with IS-MPF and IS-MdPF remaining unchanged. In conclusion, close to task failure, muscle activation is lower in hypoxia than in normoxia. Task failure is predominantly caused by central mechanisms, which recover to great extent within 1 min even when the legs remain ischemic. There is dissociation between the recovery of EMGRMS and performance. The reduction of surface electromyogram MPF, MdPF and burst duration due to fatigue is associated but not caused by muscle acidification and lactate accumulation. Despite metaboreflex stimulation, muscle activation and power output recovers partly in ischemia indicating that metaboreflex activation has a minor impact on sprint performance.

The Physiological Mechanisms of Performance Enhancement with Sprint Interval Training Differ between the Upper and Lower Extremities in Humans

To elucidate the mechanisms underlying the differences in adaptation of arm and leg muscles to sprint training, over a period of 11 days 16 untrained men performed six sessions of 4–6 × 30-s all-out sprints (SIT) with the legs and arms, separately, with a 1-h interval of recovery. Limb-specific VO2peak, sprint performance (two 30-s Wingate tests with 4-min recovery), muscle efficiency and time-trial performance (TT, 5-min all-out) were assessed and biopsies from the m. vastus lateralis and m. triceps brachii taken before and after training. VO2peak and Wmax increased 3–11% after training, with a more pronounced change in the arms (P < 0.05). Gross efficiency improved for the arms (+8.8%, P < 0.05), but not the legs (−0.6%). Wingate peak and mean power outputs improved similarly for the arms and legs, as did TT performance. After training, VO2 during the two Wingate tests was increased by 52 and 6% for the arms and legs, respectively (P < 0.001). In the case of the arms, VO2 was higher during the first than second Wingate test (64 vs. 44%, P < 0.05). During the TT, relative exercise intensity, HR, VO2, VCO2, VE, and Vt were all lower during arm-cranking than leg-pedaling, and oxidation of fat was minimal, remaining so after training. Despite the higher relative intensity, fat oxidation was 70% greater during leg-pedaling (P = 0.017). The aerobic energy contribution in the legs was larger than for the arms during the Wingate tests, although VO2 for the arms was enhanced more by training, reducing the O2 deficit after SIT. The levels of muscle glycogen, as well as the myosin heavy chain composition were unchanged in both cases, while the activities of 3-hydroxyacyl-CoA-dehydrogenase and citrate synthase were elevated only in the legs and capillarization enhanced in both limbs. Multiple regression analysis demonstrated that the variables that predict TT performance differ for the arms and legs. The primary mechanism of adaptation to SIT by both the arms and legs is enhancement of aerobic energy production. However, with their higher proportion of fast muscle fibers, the arms exhibit greater plasticity.

AMPK signaling in skeletal muscle during exercise: Role of reactive oxygen and nitrogen species.

Reactive oxygen and nitrogen species (RONS) are generated during exercise depending on intensity, duration and training status. A greater amount of RONS is released during repeated high-intensity sprint exercise and when the exercise is performed in hypoxia. By activating adenosine monophosphate-activated kinase (AMPK), RONS play a critical role in the regulation of muscle metabolism but also in the adaptive responses to exercise training. RONS may activate AMPK by direct an indirect mechanisms. Directly, RONS may activate or deactivate AMPK by modifying RONS-sensitive residues of the AMPK-α subunit. Indirectly, RONS may activate AMPK by reducing mitochondrial ATP synthesis, leading to an increased AMP:ATP ratio and subsequent Thr(172)-AMPK phosphorylation by the two main AMPK kinases: LKB1 and CaMKKβ. In presence of RONS the rate of Thr(172)-AMPK dephosphorylation is reduced. RONS may activate LKB1 through Sestrin2 and SIRT1 (NAD(+)/NADH.H(+)-dependent deacetylase). RONS may also activate CaMKKβ by direct modification of RONS sensitive motifs and, indirectly, by activating the ryanodine receptor (Ryr) to release Ca(2+). Both too high (hypoxia) and too low (ingestion of antioxidants) RONS levels may lead to Ser(485)-AMPKα1/Ser(491)-AMPKα2 phosphorylation causing inhibition of Thr(172)-AMPKα phosphorylation. Exercise training increases muscle antioxidant capacity. When the same high-intensity training is applied to arm and leg muscles, arm muscles show signs of increased oxidative stress and reduced mitochondrial biogenesis, which may be explained by differences in RONS-sensing mechanisms and basal antioxidant capacities between arm and leg muscles. Efficient adaptation to exercise training requires optimal exposure to pulses of RONS. Inappropriate training stimulus may lead to excessive RONS formation, oxidative inactivation of AMPK and reduced adaptation or even maladaptation. Theoretically, exercise programs should be designed taking into account the intrinsic properties of different skeletal muscles, the specific RONS induction and the subsequent signaling responses.

Cerebral blood flow, frontal lobe oxygenation and intra-arterial blood pressure during sprint exercise in normoxia and severe acute hypoxia in humans

Cerebral blood flow (CBF) is regulated to secure brain O2 delivery while simultaneously avoiding hyperperfusion; however, both requisites may conflict during sprint exercise. To determine whether brain O2 delivery or CBF is prioritized, young men performed sprint exercise in normoxia and hypoxia (PIO2 = 73 mmHg). During the sprints, cardiac output increased to ∼22 L min−1, mean arterial pressure to ∼131 mmHg and peak systolic blood pressure ranged between 200 and 304 mmHg. Middle-cerebral artery velocity (MCAv) increased to peak values (∼16%) after 7.5 s and decreased to pre-exercise values towards the end of the sprint. When the sprints in normoxia were preceded by a reduced PETCO2, CBF and frontal lobe oxygenation decreased in parallel (r = 0.93, P < 0.01). In hypoxia, MCAv was increased by 25%, due to a 26% greater vascular conductance, despite 4–6 mmHg lower PaCO2 in hypoxia than normoxia. This vasodilation fully accounted for the 22 % lower CaO2 in hypoxia, leading to a similar brain O2 delivery during the sprints regardless of PIO2. In conclusion, when a conflict exists between preserving brain O2 delivery or restraining CBF to avoid potential damage by an elevated perfusion pressure, the priority is given to brain O2 delivery.