Elevated heart rate and decreased muscle endothelial nitric oxide synthase in early development of insulin resistance.

Insulin resistance (IR) is a risk factor for the development of several major metabolic diseases. Muscle fiber composition is established early in life and is associated with insulin sensitivity. Hence, muscle fiber composition was used to identify early defects in the development of IR in healthy young individuals in the absence of clinical manifestations. Biopsies were obtained from the thigh muscle, followed by an intravenous glucose tolerance test. Indices of insulin action were calculated and cardiovascular measurements, analyses of blood and muscle were performed. Whole body insulin sensitivity (SIgalvin) was positively related to expression of type I muscle fibers (r = 0.49; P < 0.001) and negatively related to resting heart rate (HR, r = -0.39; P < 0.001), which was also negatively related to expression of type I muscle fibers (r = -0.41; P < 0.001). Muscle protein expression of endothelial nitric oxide synthase (eNOS), whose activation results in vasodilation, was measured in two subsets of subjects expressing a high percentage of type I fibers (59 ± 6%; HR = 57 ± 9 beats/min; SIgalvin = 1.8 ± 0.7 units) or low percentage of type I fibers (30 ± 6%; HR = 71 ± 11; SIgalvin = 0.8 ± 0.3 units; P < 0.001 for all variables vs. first group). eNOS expression was 1) higher in subjects with high type I expression; 2) almost twofold higher in pools of type I versus II fibers; 3) only detected in capillaries surrounding muscle fibers; and 4) linearly associated with SIgalvin. These data demonstrate that an altered function of the autonomic nervous system and a compromised capacity for vasodilation in the microvasculature occur early in the development of IR.NEW & NOTEWORTHY Insulin resistance (IR) is a risk factor for the development of several metabolic diseases. In healthy young individuals, an elevated heart rate (HR) correlates with low insulin sensitivity and high expression of type II skeletal muscle fibers, which express low levels of endothelial nitric oxide synthase (eNOS) and, hence, a limited capacity to induce vasodilation in response to insulin. Early targeting of the autonomic nervous system and microvasculature may attenuate development of diseases stemming from insulin resistance.

Acute hypoxia attenuates resistance exercise-induced ribosome signaling but does not impact satellite cell pool expansion in human skeletal muscle.

Cumulative evidence supports the hypothesis that hypoxia acts as a regulator of muscle mass. However, the underlying molecular mechanisms remain incompletely understood, particularly in human muscle. Here we examined the effect of hypoxia on signaling pathways related to ribosome biogenesis and myogenic activity following an acute bout of resistance exercise. We also investigated whether hypoxia influenced the satellite cell response to resistance exercise. Employing a randomized, crossover design, eight men performed resistance exercise in normoxia (FiO2 21%) or normobaric hypoxia (FiO2 12%). Muscle biopsies were collected in a time-course manner (before, 0, 90, 180 min and 24 h after exercise) and were analyzed with respect to cell signaling, gene expression and satellite cell content using immunoblotting, RT-qPCR and immunofluorescence, respectively. In normoxia, resistance exercise increased the phosphorylation of RPS6, TIF-1A and UBF above resting levels. Hypoxia reduced the phosphorylation of these targets by ~37%, ~43% and ~ 67% throughout the recovery period, respectively (p < .05 vs. normoxia). Resistance exercise also increased 45 S pre-rRNA expression and mRNA expression of c-Myc, Pol I and TAF-1A above resting levels, but no differences were observed between conditions. Similarly, resistance exercise increased mRNA expression of myogenic regulatory factors throughout the recovery period and Pax7+ cells were elevated 24 h following exercise in mixed and type II muscle fibers, with no differences observed between normoxia and hypoxia. In conclusion, acute hypoxia attenuates ribosome signaling, but does not impact satellite cell pool expansion and myogenic gene expression following a bout of resistance exercise in human skeletal muscle.

The lactate receptor GPR81 is predominantly expressed in type II human skeletal muscle fibers: potential for lactate autocrine signaling.

Gi-coupled protein receptor 81 (GPR81) was first identified in adipocytes as a receptor for l-lactate, which upon binding inhibits cyclicAMP (cAMP)-protein kinase (PKA)-cAMP-response element binding (CREB) signaling. Moreover, incubation of myotubes with lactate augments expression of GPR81 and genes and proteins involved in lactate- and energy metabolism. However, characterization of GPR81 expression and investigation of related signaling in human skeletal muscle under conditions of elevated circulating lactate levels are lacking. Muscle biopsies were obtained from healthy men and women at rest, after leg extension exercise, with or without venous infusion of sodium lactate, and 90 and 180 min after exercise (8 men and 8 women). Analyses included protein and mRNA levels of GPR81, as well as GPR81-dependent signaling molecules. GPR81 expression was 2.5-fold higher in type II glycolytic compared with type I oxidative muscle fibers, and the expression was inversely related to the percentage of type I muscle fibers. Muscle from women expressed about 25% more GPR81 protein than from men. Global PKA activity increased by 5%-8% after exercise, with no differences between trials. CREBS133 phosphorylation was reduced by 30% after exercise and remained repressed during the entire trials, with no influence of the lactate infusion. The mRNA expression of vascular endothelial growth factor (VEGF) and peroxisome-proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were increased by 2.5-6-fold during recovery, and that of lactate dehydrogenase reduced by 15% with no differences between trials for any gene at any time point. The high expression of GPR81-protein in type II fibers suggests that lactate functions as an autocrine signaling molecule in muscle; however, lactate does not appear to regulate CREB signaling during exercise.

Insulin resistance after a 3-day fast is associated with an increased capacity of skeletal muscle to oxidize lipids.

There is a debate on whether lipid-mediated insulin resistance derives from an increased or decreased capacity of muscle to oxidize fats. Here, we examine the involvement of muscle fiber composition in the metabolic responses to a 3-day fast (starvation, which results in increases in plasma lipids and insulin resistance) in two groups of healthy young subjects: 1), area occupied by type I fibers = 61.0 ± 11.8%; 2), type I area = 36.0 ± 4.9% (P < 0.001). Muscle biopsies and intravenous glucose tolerance tests were performed after an overnight fast and after starvation. Biopsies were analyzed for muscle fiber composition and mitochondrial respiration. Indices of glucose tolerance and insulin sensitivity were determined. Glucose tolerance was similar in both groups after an overnight fast and deteriorated to a similar degree in both groups after starvation. In contrast, whole body insulin sensitivity decreased markedly after starvation in group 1 (P < 0.01), whereas the decrease in group 2 was substantially smaller (P = 0.06). Nonesterified fatty acids and ß-hydroxybutyrate levels in plasma after an overnight fast were similar between groups and increased markedly and comparably in both groups after starvation, demonstrating similar degrees of lipid load. The capacity of permeabilized muscle fibers to oxidize lipids was significantly higher in group 1 versus 2, whereas there was no significant difference in pyruvate oxidation between groups. The data demonstrate that loss of whole body insulin sensitivity after short-term starvation is a function of muscle fiber composition and is associated with an elevated rather than a diminished capacity of muscle to oxidize lipids.NEW & NOTEWORTHY Whether lipid-mediated insulin resistance occurs as a result of an increased or decreased capacity of skeletal muscle to oxidize lipids has been debated. We show that a 3-day fast results in increases in circulating lipids and insulin resistance in subjects expressing a high or low proportion of type I muscle fibers. High expression of type I is associated with a higher capacity to oxidize lipids and a greater loss of insulin sensitivity after starvation.

THRIFTY: a novel high-throughput method for rapid fibre type identification of isolated skeletal muscle fibres.

Fibre type-specific analyses are required for broader understanding of muscle physiology, but such analyses are difficult to conduct due to the extreme time requirements of dissecting and fibre typing individual fibres. Investigations are often confined to a small number of fibres from few participants with low representativeness of the entire fibre population and the participant population. To increase the feasibility of conducting large-scale fibre type-specific studies, a valid and rapid method for high-throughput fibre typing of individually dissected fibres was developed and named THRIFTY (for high-THRoughput Immunofluorescence Fibre TYping). Employing THRIFTY, 400 fibre segments were fixed onto microscope slides with a pre-printed coordinated grid system, probed with antibodies against myosin heavy chain (MyHC)-I and MyHC-II and classified using a fluorescence microscope. The validity and speed of THRIFTY was compared to a previously validated protocol (dot blot) on a fibre-to-fibre basis. Fibre pool purity was evaluated using 'gold standard' SDS-PAGE and silver staining. A modified THRIFTY-protocol using fluorescence western blot equipment was also validated. THRIFTY displayed excellent agreement with the dot blot protocol, κ = 0.955 (95% CI: 0.928, 0.982), P < 0.001. Both the original and modified THRIFTY protocols generated type I and type II fibre pools of absolute purity. Using THRIFTY, 400 fibres were typed just under 11 h, which was approximately 3 times faster than dot blot. THRIFTY is a novel and valid method with high versatility for very rapid fibre typing of individual fibres. THRIFTY can therefore facilitate the generation of large fibre pools for more extensive mechanistic studies into skeletal muscle physiology. KEY POINTS: Skeletal muscle is composed of different fibre types, each with distinct physiological properties. To fully understand how skeletal muscle adapts to external cues such as exercise, nutrition and ageing, fibre type-specific investigations are required. Such investigations are very difficult to conduct due to the extreme time requirements related to classifying individually isolated muscle fibres. To bypass this issue, we have developed a rapid and reliable method named THRIFTY which is cheap as well as versatile and which can easily be implemented in most laboratories. THRIFTY increases the feasibility of conducting larger fibre type-specific studies and enables time-sensitive assays where measurements need to be carried out in close connection with tissue sampling. By using THRIFTY, new insights into fibre type-specific muscle physiology can be gained which may have broad implications in health and disease.

Influence of sex and fiber type on the satellite cell pool in human skeletal muscle.

The repair, remodeling, and regeneration of myofibers are dependent on satellite cells (SCs), although, the distribution of SCs in different fiber types of human muscle remains inconclusive. There is also a paucity of research comparing muscle fiber characteristics in a sex-specific manner. Therefore, the aim of this study was to investigate fiber type-specific SC content in men and women. Muscle biopsies from vastus lateralis were collected from 64 young (mean age 27 ± 5), moderately trained men (n = 34) and women (n = 30). SCs were identified by Pax7-staining together with immunofluorescent analyses of fiber type composition, fiber size, and myonuclei content. In a mixed population, comparable number of SCs was associated to type I and type II fibers (0.07 ± 0.02 vs 0.07 ± 0.02 SCs per fiber, respectively). However, unlike men, women displayed a fiber type-specific distribution, with SC content being lower in type II than type I fibers (P = .041). Sex-based differences were found specifically for type II fibers, where women displayed lower SC content compared to men (P < .001). In addition, positive correlations (r-values between 0.36-0.56) were found between SC content and type I and type II fiber size in men (P = .03 and P < .01, respectively), whereas similar relationships could not be detected in women. Sex-based differences were also noted for fiber type composition and fiber size, but not for myonuclei content. We hereby provide evidence for sex-based differences present at the myocellular level, which may have important implications when studying exercise- and training-induced myogenic responses in skeletal muscle.

Benefits of higher resistance-training volume are related to ribosome biogenesis.

KEY POINTS: For individuals showing suboptimal adaptations to resistance training, manipulation of training volume is a potential measure to facilitate responses. This remains unexplored. Here, 34 untrained individuals performed contralateral resistance training with moderate and low volume for 12 weeks. Moderate volume led to larger increases in muscle cross-sectional area, strength and type II fibre-type transitions. These changes coincided with greater activation of signalling pathways controlling muscle growth and greater induction of ribosome synthesis. Out of 34 participants, thirteen displayed clear benefit of MOD on muscle hypertrophy and sixteen showed clear benefit of MOD on muscle strength gains. This coincided with greater total RNA accumulation in the early phase of the training period, suggesting that ribosomal biogenesis regulates the dose-response relationship between training volume and muscle hypertrophy. These results demonstrate that there is a dose-dependent relationship between training volume and outcomes. On the individual level, benefits of higher training volume were associated with increased ribosomal biogenesis. ABSTRACT: Resistance-exercise volume is a determinant of training outcomes. However not all individuals respond in a dose-dependent fashion. In this study, 34 healthy individuals (males n = 16, 23.6 (4.1) years; females n = 18, 22.0 (1.3) years) performed moderate- (3 sets per exercise, MOD) and low-volume (1 set, LOW) resistance training in a contralateral fashion for 12 weeks (2-3 sessions per week). Muscle cross-sectional area (CSA) and strength were assessed at Weeks 0 and 12, along with biopsy sampling (m. vastus lateralis). Muscle biopsies were also sampled before and 1 h after the fifth session (Week 2). MOD resulted in larger increases in muscle CSA (5.2 (3.8)% versus 3.7 (3.7)%, P < 0.001) and strength (3.4-7.7% difference, all P < 0.05. This coincided with greater reductions in type IIX fibres from Week 0 to Week 12 (MOD, -4.6 percentage points; LOW -3.2 percentage points), greater phosphorylation of S6-kinase 1 (p85 S6K1Thr412 , 19%; p70 S6K1Thr389 , 58%) and ribosomal protein S6Ser235/236 (37%), greater rested-state total RNA (8.8%) and greater exercise-induced c-Myc mRNA expression (25%; Week 2, all P < 0.05). Thirteen and sixteen participants, respectively, displayed clear benefits in response to MOD on muscle hypertrophy and strength. Benefits were associated with greater accumulation of total RNA at Week 2 in the MOD leg, with every 1% difference increasing the odds of MOD benefit by 7.0% (P = 0.005) and 9.8% (P = 0.002). In conclusion, MOD led to greater functional and biological adaptations than LOW. Associations between dose-dependent total RNA accumulation and increases in muscle mass and strength point to ribosome biogenesis as a determinant of dose-dependent training responses.

Elevated plasma lactate levels via exogenous lactate infusion do not alter resistance exercise-induced signaling or protein synthesis in human skeletal muscle.

Lactate has been implicated as a potential signaling molecule. In myotubes, lactate incubation increases mechanistic target of rapamycin complex 1 (mTORC1)- and ERK-signaling and induces hypertrophy, indicating that lactate could be a mediator of muscle adaptations to resistance exercise. However, the potential signaling properties of lactate, at rest or with exercise, have not been explored in human tissue. In a crossover design study, 8 men and 8 women performed one-legged resistance exercise while receiving venous infusion of saline or sodium lactate. Blood was sampled repeatedly, and muscle biopsies were collected at rest and at 0, 90, and 180 min and 24 h after exercise. The primary outcomes examined were intracellular signaling, fractional protein synthesis rate (FSR), and blood/muscle levels of lactate and pH. Postexercise blood lactate concentrations were 130% higher in the Lactate trial (3.0 vs. 7.0 mmol/L, P < 0.001), whereas muscle levels were only marginally higher (27 vs. 32 mmol/kg dry wt, P = 0.003) compared with the Saline trial. Postexercise blood pH was higher in the Lactate trial (7.34 vs. 7.44, P < 0.001), with no differences in intramuscular pH. Exercise increased the phosphorylation of mTORS2448 (∼40%), S6K1T389 (∼3-fold), and p44T202/T204 (∼80%) during recovery, without any differences between trials. FSR over the 24-h recovery period did not differ between the Saline (0.067%/h) and Lactate (0.062%/h) trials. This study does not support the hypothesis that blood lactate levels can modulate anabolic signaling in contracted human muscle. Further in vivo research investigating the impact of exercised versus rested muscle and the role of intramuscular lactate is needed to elucidate its potential signaling properties.

Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise.

Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (Placebo

Intake of branched-chain or essential amino acids attenuates the elevation in muscle levels of PGC-1α4 mRNA caused by resistance exercise.

The transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α is recognized as the master regulator of mitochondrial biogenesis. However, recently a novel isoform, PGC-1α4, that specifically regulates muscle hypertrophy was discovered. Because stimulation of mechanistic target of rapamycin complex 1 (mTORC1) activity is tightly coupled to hypertrophy, we hypothesized that activation of this pathway would upregulate PGC-1α4. Eight male subjects performed heavy resistance exercise (10 × 8-12 repetitions at ∼75% of 1 repetition maximum in leg press) on four different occasions, ingesting in random order a solution containing essential amino acids (EAA), branched-chain amino acids (BCAA), leucine, or flavored water (placebo) during and after the exercise. Biopsies were taken from the vastus lateralis muscle before and immediately after exercise, as well as following 90 and 180 min of recovery. Signaling through mTORC1, as reflected in p70S6 kinase phosphorylation, was stimulated to a greater extent by the EAA and BCAA than the leucine or placebo supplements. Unexpectedly, intake of EAA or BCAA attenuated the stimulatory effect of exercise on PGC-1α4 expression by ∼50% (from a 10- to 5-fold increase with BCAA and EAA, P < 0.05) 3 h after exercise, whereas intake of leucine alone did not reduce this response. The 60% increase (P < 0.05) in the level of PGC-1α1 mRNA 90 min after exercise was uninfluenced by amino acid intake. Muscle glycogen levels were reduced and AMP-activated protein kinase α2 activity and phosphorylation of p38 mitogen-activated protein kinase enhanced to the same extent with all four supplements. In conclusion, induction of PGC-1α4 does not appear to regulate the nutritional (BCAA or EAA)-mediated activation of mTORC1 in human muscle.

Leucine does not affect mechanistic target of rapamycin complex 1 assembly but is required for maximal ribosomal protein s6 kinase 1 activity in human skeletal muscle following resistance exercise.

We examined how the stimulatory effect of leucine on the mechanistic target of rapamycin complex 1 (mTORC1) pathway is affected by the presence of the remaining essential amino acids (EAAs). Nine male subjects performed resistance exercise on 4 occasions and were randomly supplied EAAs with leucine, EAAs without leucine (EAA-Leu), leucine alone, or flavored water (placebo; control). Muscle biopsies were taken from the vastus lateralis before and 60 and 90 min after exercise. Biopsies were analyzed for protein phosphorylation, kinase activity, protein-protein interactions, amino acid concentrations, and tracer incorporation. Leucine alone stimulated ribosomal protein s6 kinase 1 (S6K1) phosphorylation ∼280% more than placebo and EAA-Leu after exercise. Moreover, this response was enhanced by 60-75% after intake of EAAs compared with that of leucine alone (P < 0.05). Kinase activity of S6K1 reflected that of S6K1 phosphorylation; 60 min after exercise, the activity was elevated 3.3- and 4.2-fold with intake of leucine alone and with EAAs, respectively (P < 0.05). The interaction between mammalian target of rapamycin and regulatory-associated protein of mammalian target of rapamycin was unaltered in response to both resistance exercise and amino acid provision. Leucine alone stimulates mTORC1 signaling, although this response is enhanced by other EAAs and does not appear to be caused by alterations in mTORC1 assembly.

Resistance exercise-induced S6K1 kinase activity is not inhibited in human skeletal muscle despite prior activation of AMPK by high-intensity interval cycling.

Combining endurance and strength training in the same session has been reported to reduce the anabolic response to the latter form of exercise. The underlying mechanism, based primarily on results from rodent muscle, is proposed to involve AMPK-dependent inhibition of mTORC1 signaling. This hypothesis was tested in eight trained male subjects who in randomized order performed either resistance exercise only (R) or interval cycling followed by resistance exercise (ER). Biopsies taken from the vastus lateralis before and after endurance exercise and repeatedly after resistance exercise were assessed for glycogen content, kinase activity, protein phosphorylation, and gene expression. Mixed muscle fractional synthetic rate was measured at rest and during 3 h of recovery using the stable isotope technique. In ER, AMPK activity was elevated immediately after both endurance and resistance exercise (∼90%, P < 0.05) but was unchanged in R. Thr(389) phosphorylation of S6K1 was increased severalfold immediately after exercise (P < 0.05) in both trials and increased further throughout recovery. After 90 and 180 min recovery, S6K1 activity was elevated (∼55 and ∼110%, respectively, P < 0.05) and eukaryotic elongation factor 2 phosphorylation was reduced (∼55%, P < 0.05) with no difference between trials. In contrast, markers for protein catabolism were differently influenced by the two modes of exercise; ER induced a significant increase in gene and protein expression of MuRF1 (P < 0.05), which was not observed following R exercise only. In conclusion, cycling-induced elevation in AMPK activity does not inhibit mTOR complex 1 signaling after subsequent resistance exercise but may instead interfere with the hypertrophic response by influencing key components in protein breakdown.

Need for speed: Human fast-twitch mitochondria favor power over efficiency.

OBJECTIVE: Human skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation. While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic. METHODS: Here, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity. RESULTS: Maximal complex I+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per mitochondrial volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for complex I+II, which was primarily mediated through elevated complex II respiration. Furthermore, the abundance of complex II protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid-or complex I respiration. CONCLUSION: Mitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.

Pro-Brain-Derived Neurotrophic Factor (BDNF), but Not Mature BDNF, Is Expressed in Human Skeletal Muscle: Implications for Exercise-Induced Neuroplasticity.

Exercise promotes brain plasticity partly by stimulating increases in mature brain-derived neurotrophic factor (mBDNF), but the role of the pro-BDNF isoform in the regulation of BDNF metabolism in humans is unknown. We quantified the expression of pro-BDNF and mBDNF in human skeletal muscle and plasma at rest, after acute exercise (+/- lactate infusion), and after fasting. Pro-BDNF and mBDNF were analyzed with immunoblotting, enzyme-linked immunosorbent assay, immunohistochemistry, and quantitative polymerase chain reaction. Pro-BDNF was consistently and clearly detected in skeletal muscle (40-250 pg mg-1 dry muscle), whereas mBDNF was not. All methods showed a 4-fold greater pro-BDNF expression in type I muscle fibers compared to type II fibers. Exercise resulted in elevated plasma levels of mBDNF (55%) and pro-BDNF (20%), as well as muscle levels of pro-BDNF (∼10%, all P < 0.05). Lactate infusion during exercise induced a significantly greater increase in plasma mBDNF (115%, P < 0.05) compared to control (saline infusion), with no effect on pro-BDNF levels in plasma or muscle. A 3-day fast resulted in a small increase in plasma pro-BDNF (∼10%, P < 0.05), with no effect on mBDNF. Pro-BDNF is highly expressed in human skeletal muscle, particularly in type I fibers, and is increased after exercise. While exercising with higher lactate augmented levels of plasma mBDNF, exercise-mediated increases in circulating mBDNF likely derive partly from release and cleavage of pro-BDNF from skeletal muscle, and partly from neural and other tissues. These findings have implications for preclinical and clinical work related to a wide range of neurological disorders such as Alzheimer's, clinical depression, and amyotrophic lateral sclerosis.

Resistance exercise induced mTORC1 signaling is not impaired by subsequent endurance exercise in human skeletal muscle.

The current dogma is that the muscle adaptation to resistance exercise is blunted when combined with endurance exercise. The suggested mechanism (based on rodent experiments) is that activation of adenosine monophosphate-activated protein kinase (AMPK) during endurance exercise impairs muscle growth through inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). The purpose of this study was to investigate potential interference of endurance training on the signaling pathway of resistance training [mTORC1 phosphorylation of ribosomal protein S6 kinase 1 (S6K1)] in human muscle. Ten healthy and moderately trained male subjects performed on two separate occasions either acute high-intensity and high-volume resistance exercise (leg press, R) or R followed by 30 min of cycling (RE). Muscle biopsies were collected before and 1 and 3 h post resistance exercise. Phosphorylation of mTOR (Ser²448) increased 2-fold (P < 0.05) and that of S6K1 (Thr³89) 14-fold (P < 0.05), with no difference between R and RE. Phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) was reduced ~70% during recovery in both trials (P < 0.05). An interesting finding was that phosphorylation of AMPK (Thr¹7²) and acetyl-CoA carboxylase (ACC, Ser79) decreased ~30% and ~50%, respectively, 3 h postexercise (P < 0.05). Proliferator-activated receptor-γ coactivator-1 (PGC-1α) mRNA increased more after RE (6.5-fold) than after R (4-fold) (RE vs. R: P < 0.01) and was the only gene expressed differently between trials. These data show that the signaling of muscle growth through the mTORC1-S6K1 axis after heavy resistance exercise is not inhibited by subsequent endurance exercise. It is also suggested that prior activation of mTORC1 signaling may repress subsequent phosphorylation of AMPK.

Acute low-load resistance exercise with and without blood flow restriction increased protein signalling and number of satellite cells in human skeletal muscle.

PURPOSE: To investigate hypertrophic signalling after a single bout of low-load resistance exercise with and without blood flow restriction (BFR). METHODS: Seven subjects performed unilateral knee extensions at 30 % of their one repetition maximum. The subjects performed five sets to failure with BFR on one leg, and then repeated the same amount of work with the other leg without BFR. Biopsies were obtained from m. vastus lateralis before and 1, 24 and 48 h after exercise. RESULTS: At 1-h post-exercise, phosphorylation of p70S6KThr389 and p38MAPKThr180/Tyr182 was elevated in the BFR leg, but not in the free-flow leg. Phospho-p70S6KThr389 was elevated three- to fourfold in both legs at 24-h post-exercise, but back to baseline at 48 h. The number of visible satellite cells (SCs) per muscle fibre was increased for all post-exercise time points and in both legs (33­53 %). The proportion of SCs with cytoplasmic extensions was elevated at 1-h post in the BFR leg and the number of SCs positive for myogenin and/or MyoD was increased at 1- and 24-h post-exercise for both legs combined. CONCLUSION: Acute low-load resistance exercise with BFR resulted in early (1 h) and late (24 h) enhancement of phospho-p70S6KThr389, an early response of p38MAPK, and an increased number of SCs per muscle fibre. Enhanced phospho-p70S6KThr389 at 24-h post-exercise and increases in SC numbers were seen also in the free-flow leg. Implications of these findings for the hypertrophic effects of fatiguing low-load resistance exercise with and without BFR are discussed.

THRIFTY-A High-throughput Single Muscle Fiber Typing Method Based on Immunofluorescence Detection.

Skeletal muscle consists of a mixture of fiber types with different functional and metabolic characteristics. The relative composition of these muscle fiber types has implications for muscle performance, whole-body metabolism, and health. However, analyses of muscle samples in a fiber type-dependent manner are very time consuming. Therefore, these are often neglected in favor of more time-efficient analyses on mixed muscle samples. Methods such as western blot and myosin heavy chain separation by SDS-PAGE have previously been utilized to fiber type-isolated muscle fibers. More recently, the introduction of the dot blot method significantly increased the speed of fiber typing. However, despite recent advancements, none of the current methodologies are feasible for large-scale investigations because of their time requirements. Here, we present the protocol for a new method, which we have named THRIFTY (high-THRoughput Immunofluorescence Fiber TYping), that enables rapid fiber type identification using antibodies towards the different myosin heavy chain (MyHC) isoforms of fast and slow twitch muscle fibers. First, a short segment (<1 mm) is cut off from isolated muscle fibers and mounted on a customized gridded microscope slide holding up to 200 fiber segments. Second, the fiber segments attached to the microscope slide are stained with MyHC-specific antibodies and then visualized using a fluorescence microscope. Lastly, the remaining pieces of the fibers can either be collected individually or pooled together with fibers of the same type for subsequent analyses. The THRIFTY protocol is approximately three times as fast as the dot blot method, which enables not only time-sensitive assays to be performed but also increases the feasibility to conduct large-scale investigations into fiber type specific physiology. Graphical Overview Graphical overview of the THRIFTY workflow. Cut off a small segment (0.5 mm) of an individually dissected muscle fiber and mount it onto the customized microscope slide containing a printed grid system. Using a Hamilton syringe, fixate the fiber segment by applying a small droplet of distilled water on the segment and let it fully dry (1A). The remaining large segment of the fiber should be placed in the corresponding square on a black A4 paper (1B). Once the microscope slide has been fully mounted with fiber segments, submerge the slide in a polypropylene slide mailer (illustrated as a Coplin jar in the figure) containing acetone to permeabilize the fiber segments. Thereafter, incubate the slide with primary antibodies targeting MyHC-I and MyHC-II. Following washes in PBS solution, incubate the slides with fluorescently labeled secondary antibodies, wash again, and mount with a cover glass and antifade reagent (2). Identification of fiber type can be performed using a digital fluorescence microscope (3), whereafter the remaining pieces of the fiber segments (large) are pooled together according to their fiber type or individually collected for experiments on single fibers (4). Image modified from Horwath et al. (2022).

Reduced glucose tolerance and insulin sensitivity after prolonged exercise in endurance athletes.

AIM: The purpose of this study was to 1. investigate if glucose tolerance is affected after one acute bout of different types of exercise; 2. assess if potential differences between two exercise paradigms are related to changes in mitochondrial function; and 3. determine if endurance athletes differ from nonendurance-trained controls in their metabolic responses to the exercise paradigms. METHODS: Nine endurance athletes (END) and eight healthy nonendurance-trained controls (CON) were studied. Oral glucose tolerance tests (OGTT) and mitochondrial function were assessed on three occasions: in the morning, 14 h after an overnight fast without prior exercise (RE), as well as after 3 h of prolonged continuous exercise at 65% of VO2 max (PE) or 5 × 4 min at ~95% of VO2 max (HIIT) on a cycle ergometer. RESULTS: Glucose tolerance was markedly reduced in END after PE compared with RE. END also exhibited elevated fasting serum FFA and ketones levels, reduced insulin sensitivity and glucose oxidation, and increased fat oxidation during the OGTT. CON showed insignificant changes in glucose tolerance and the aforementioned measurements compared with RE. HIIT did not alter glucose tolerance in either group. Neither PE nor HIIT affected mitochondrial function in either group. END also exhibited increased activity of 3-hydroxyacyl-CoA dehydrogenase activity in muscle extracts vs. CON. CONCLUSION: Prolonged exercise reduces glucose tolerance and increases insulin resistance in endurance athletes the following day. These findings are associated with an increased lipid load, a high capacity to oxidize lipids, and increased fat oxidation.

Limited effect of over-the-counter doses of ibuprofen on mechanisms regulating muscle hypertrophy during resistance training in young adults.

We have previously shown that maximal over-the-counter doses of ibuprofen, compared with low doses of acetylsalicylic acid, reduce muscle hypertrophy in young individuals after 8 wk of resistance training. Because the mechanism behind this effect has not been fully elucidated, we here investigated skeletal muscle molecular responses and myofiber adaptations in response to acute and chronic resistance training with concomitant drug intake. Thirty-one young (aged 18-35 yr) healthy men (n = 17) and women (n = 14) were randomized to receive either ibuprofen (IBU; 1,200 mg daily; n = 15) or acetylsalicylic acid (ASA; 75 mg daily; n = 16) while undergoing 8 wk of knee extension training. Muscle biopsies from the vastus lateralis were obtained before, at week 4 after an acute exercise session, and after 8 wk of resistance training and analyzed for mRNA markers and mTOR signaling, as well as quantification of total RNA content (marker of ribosome biogenesis) and immunohistochemical analysis of muscle fiber size, satellite cell content, myonuclear accretion, and capillarization. There were only two treatment × time interaction in selected molecular markers after acute exercise (atrogin-1 and MuRF1 mRNA), but several exercise effects. Muscle fiber size, satellite cell and myonuclear accretion, and capillarization were not affected by chronic training or drug intake. RNA content increased comparably (∼14%) in both groups. Collectively, these data suggest that established acute and chronic hypertrophy regulators (including mTOR signaling, ribosome biogenesis, satellite cell content, myonuclear accretion, and angiogenesis) were not differentially affected between groups and therefore do not explain the deleterious effects of ibuprofen on muscle hypertrophy in young adults.NEW & NOTEWORTHY Here we show that mTOR signaling, fiber size, ribosome biogenesis, satellite cell content, myonuclear accretion, and angiogenesis were not differentially affected between groups undergoing 8 wk of resistance training with concomitant anti-inflammatory medication (ibuprofen versus low-dose aspirin). Atrogin-1 and MuRF-1 mRNA were more downregulated after acute exercise in the low-dose aspirin group than in the ibuprofen group. Taken together it appears that these established hypertrophy regulators do not explain the previously reported deleterious effects of high doses of ibuprofen on muscle hypertrophy in young adults.

Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle.

Resistance exercise and amino acids are two major factors that influence muscle protein turnover. Here, we examined the effects of resistance exercise and branched-chain amino acids (BCAA), individually and in combination, on the expression of anabolic and catabolic genes in human skeletal muscle. Seven subjects performed two sessions of unilateral leg press exercise with randomized supplementation with BCAA or flavored water. Biopsies were collected from the vastus lateralis muscle of both the resting and exercising legs before and repeatedly after exercise to determine levels of mRNA, protein phosphorylation, and amino acid concentrations. Intake of BCAA reduced (P < 0.05) MAFbx mRNA by 30 and 50% in the resting and exercising legs, respectively. The level of MuRF-1 mRNA was elevated (P < 0.05) in the exercising leg two- and threefold under the placebo and BCAA conditions, respectively, whereas MuRF-1 total protein increased by 20% (P < 0.05) only in the placebo condition. Phosphorylation of p70(S6k) increased to a larger extent (∼2-fold; P < 0.05) in the early recovery period with BCAA supplementation, whereas the expression of genes regulating mTOR activity was not influenced by BCAA. Muscle levels of phenylalanine and tyrosine were reduced (13-17%) throughout recovery (P < 0.05) in the placebo condition and to a greater extent (32-43%; P < 0.05) following BCAA supplementation in both resting and exercising muscle. In conclusion, BCAA ingestion reduced MAFbx mRNA and prevented the exercise-induced increase in MuRF-1 total protein in both resting and exercising leg. Further-more, resistance exercise differently influenced MAFbx and MuRF-1 mRNA expression, suggesting both common and divergent regulation of these two ubiquitin ligases.