Molecular signatures of longissimus dorsi differ between dairy cattle based on prepartum muscle reserves and branched-chain volatile fatty acid supplementation.

Dairy cattle with high (HM) versus low muscle (LM) reserves as determined by longissimus dorsi muscle depth (LDD) in late gestation exhibit differential muscle mobilization related to subsequent milk production. Moreover, branched-chain volatile fatty acid (BCVFA) supplementation increased blood glucose levels. We hypothesized that differences in HM and LM reflect distinct muscle metabolism and that BCVFA supplementation altered metabolic pathways. At 42 days before expected calving (BEC), Holstein dairy cows were enrolled in a 2 × 2 factorial study of diet and muscle reserves, by assignment to control (CON)- or BCVFA-supplemented diets and LDD of HM (>4.6 cm) or LM (≤4.6 cm) groups: HM-CON (n = 13), HM-BCVFA (n = 10), LM-CON (n = 9), and LM-BCVFA (n = 9). Longisumus dorsi muscle was biopsied at 21 days BEC, total RNA was isolated, and protein-coding gene expression was measured with RNA sequencing. Between HM and LM, 713 genes were differentially expressed and 481 between BCVFA and CON (P < 0.05). Transcriptional signatures indicated differential distribution of type II fibers between groups, with MYH1 greater in LM cattle and MYH2 greater in HM cattle (P < 0.05). Signatures of LM cattle relative to HM cattle indicated greater activation of autophagy, ubiquitin-proteasome, and Ca2+-calpain pathways. HM cattle displayed greater expression of genes that encode extracellular matrix proteins and factors that regulate their proteolysis and turnover. BCVFA modified transcriptomes by increasing expression of genes that regulate fatty acid degradation and flux of carbons into the tricarboxylic acid cycle as acetyl CoA. Molecular signatures support distinct metabolic strategies between LM and HM cattle and that BCVFA supplementation increased substrates for energy generation.NEW & NOTEWORTHY Muscle biopsies of the longissimus dorsi of prepartum dairy cattle indicate that molecular signatures support distinct metabolic strategies between low- and high-muscle cattle and that branched-chain volatile fatty acid supplementation increased substrates for energy generation.

Local shifts in inflammatory and resolving lipid mediators in response to tendon overuse.

Tendon inflammation has been implicated in both adaptive connective tissue remodeling and overuse-induced tendinopathy. Lipid mediators control both the initiation and resolution of inflammation, but their roles within tendon are largely unknown. Here, we profiled local shifts in intratendinous lipid mediators via liquid chromatography-tandem mass spectrometry in response to synergist ablation-induced plantaris tendon overuse. Sixty-four individual lipid mediators were detected in homogenates of plantaris tendons from ambulatory control rats. This included many bioactive metabolites of the cyclooxygenase (COX), lipoxygenase (LOX), and epoxygenase (CYP) pathways. Synergist ablation induced a robust inflammatory response at day 3 post-surgery characterized by epitenon infiltration of polymorphonuclear leukocytes and monocytes/macrophages (MΦ), heightened expression of inflammation-related genes, and increased intratendinous concentrations of the pro-inflammatory eicosanoids thromboxane B2 and prostaglandin E2 . By day 7, MΦ became the predominant myeloid cell type in tendon and there were further delayed increases in other COX metabolites including prostaglandins D2 , F2α , and I2 . Specialized pro-resolving mediators including protectin D1, resolvin D2 and D6, as well as related pathway markers of D-resolvins (17-hydroxy-docosahexaenoic acid), E-resolvins (18-hydroxy-eicosapentaenoic acid), and lipoxins (15-hydroxy-eicosatetraenoic acid) were also increased locally in response to tendon overuse, as were anti-inflammatory fatty acid epoxides of the CYP pathway (eg, epoxy-eicosatrienoic acids). Nevertheless, intratendinous prostaglandins remained markedly increased even following 28 days of tendon overuse together with a lingering MΦ presence. These data reveal a delayed and prolonged local inflammatory response to tendon overuse characterized by an overwhelming predominance of pro-inflammatory eicosanoids and a relative lack of specialized pro-resolving lipid mediators.

Daily protein supplementation attenuates immobilization-induced blunting of postabsorptive muscle mTORC1 activation in middle-aged men.

Disuse-induced muscle atrophy is accompanied by a blunted postprandial response of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Conflicting observations exist as to whether postabsorptive mTORC1 pathway activation is also blunted by disuse and plays a role in atrophy. It is unknown whether changes in habitual protein intake alter mTORC1 regulatory proteins and how they may contribute to the development of anabolic resistance. The primary objective of this study was to characterize the downstream responsiveness of skeletal muscle mTORC1 activation and its upstream regulatory factors, following 14 days of lower limb disuse in middle-aged men (45-60 yr). The participants were further randomized to receive daily supplementation of 20 g/d of protein (n = 12; milk protein concentrate) or isocaloric carbohydrate placebo (n = 13). Immobilization reduced postabsorptive skeletal muscle phosphorylation of the mTORC1 downstream targets, 4E-BP1, P70S6K, and ribosomal protein S6 (RPS6), with phosphorylation of the latter two decreasing to a greater extent in the placebo, compared with the protein supplementation groups (37% ± 13% vs. 14% ± 11% and 38% ± 20% vs. 25% ± 8%, respectively). Sestrin2 protein was also downregulated following immobilization irrespective of supplement group, despite a corresponding increase in its mRNA content. This decrease in Sestrin2 protein was negatively correlated with the immobilization-induced change in the in silico-predicted regulator miR-23b-3p. No other measured upstream proteins were altered by immobilization or supplementation. Immobilization downregulated postabsorptive mTORC1 pathway activation, and 20 g/day of protein supplementation attenuated the decrease in phosphorylation of targets regulating muscle protein synthesis.

Acute responses of comprehensive gonadosteroids and corticosteroids to resistance exercise before and after 10 weeks of supervised strength training.

NEW FINDINGS: What is the central question of this study? Although acute responses of the principal gonadosteroid and corticosteroid hormones to resistance exercise are well documented, there is no information regarding how the key lower-concentration intermediary hormones respond and potentially influence these hormonal pathways. What is the main finding and its importance? This study provides evidence for cascading conversions of some gonadosteroids, and the data suggest that the testosterone concentration increases independently of these hormones. These findings challenge future studies to determine the exact physiological roles of the lower-concentration gonadosteroids and corticosteroids during and immediately after resistance exercise. ABSTRACT: Resistance training is a potent stimulus for muscle growth, and steroid hormones are known to play a role in this adaptation. However, very little is known about the acute exercise-induced gonadosteroid and corticosteroid hormone responses, including those of key lower-concentration intermediate hormones. The present study determined the acute responses of these steroid hormone families using quantitative ultra-high performance liquid chromatography tandem mass spectrometry after resistance exercise in strength-trained men. Venous and fingertip blood samples were obtained pre-, mid-, 5 min post- and 15 min post-resistance exercise, both before and after 10 weeks of supervised resistance training. The experimental resistance exercise sessions consisted of three sets of 10 repetitions of bilateral leg-press exercise and three sets of 10 repetitions of unilateral knee-extension exercise, with 2 and 1 min recovery between sets, respectively. Statistically significant (P < 0.05) increases in the concentration of hormones in the gonadosteroid [including dehydroepiandrosterone (DHEA), androstenedione, testosterone and estrone] and the corticosteroid (including cortisol, corticosterone and cortisone) families were demonstrated after both experimental resistance exercise sessions, irrespective of training status. Correlation analyses revealed relationships between the following hormones: (i) DHEA and androstenedione; (ii) DHEA and cortisol; (iii) androstenedione and estrone; and (iv) 11-deoxycortisol and cortisol. Testosterone appears to increase acutely and independently of other intermediary hormones after resistance exercise. In conclusion, lower-concentration intermediary gonadosteroids (e.g. estrone) and corticosteroids (e.g. corticosterone) respond robustly to resistance exercise in strength-trained men, although it seems that testosterone concentrations are regulated by factors other than the availability of precursor hormones and changes in plasma volume.

Plasma elemental responses to red meat ingestion in healthy young males and the effect of cooking method.

PURPOSE: Elemental deficiencies are highly prevalent and have a significant impact on health. However, clinical monitoring of plasma elemental responses to foods remains largely unexplored. Data from in vitro studies show that red meat (beef) is a highly bioavailable source of several key elements, but cooking method may influence this bioavailability. We therefore studied the postprandial responses to beef steak, and the effects of two different cooking methods, in healthy young males. METHODS: In a randomized cross-over controlled trial, healthy males (n = 12, 18-25 years) were fed a breakfast of beef steak (270 ± 20 g) in which the meat was either pan-fried (PF) or sous-vide (SV) cooked. Baseline and postprandial blood samples were collected and the plasma concentrations of 15 elements measured by inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: Concentrations of Fe and Zn changed after meal ingestion, with plasma Fe increasing (p < 0.001) and plasma Zn decreasing (p < 0.05) in response to both cooking methods. The only potential treatment effect was seen for Zn, where the postprandial area under the curve was lower in response to the SV meal (2965 ± 357) compared to the PF meal (3190 ± 310; p < 0.05). CONCLUSIONS: This multi-element approach demonstrated postprandial responsiveness to a steak meal, and an effect of the cooking method used. This suggests the method would provide insight in future elemental metabolic studies to evaluate responses to meat-based meals, including longer-term interventions in more specifically defined cohorts to clearly establish the role of red meat as an important source of elements.

The Effect of Carbohydrate Ingestion Following Eccentric Resistance Exercise on AKT/mTOR and ERK Pathways: A Randomized, Double-Blinded, Crossover Study.

PURPOSE: To determine the acute effects of carbohydrate (CHO) ingestion following a bout of maximal eccentric resistance exercise on key anabolic kinases of mammalian target of rapamycin and extracellular signal-regulated kinase (ERK) pathways. The authors' hypothesis was that the activation of anabolic signaling pathways known to be upregulated by resistance exercise would be further stimulated by the physiological hyperinsulinemia resulting from CHO supplementation. METHODS: Ten resistance-trained men were randomized in a crossover, double-blind, placebo (PLA)-controlled manner to ingest either a noncaloric PLA or 3 g/kg of CHO beverage throughout recovery from resistance exercise. Muscle biopsies were collected at rest, immediately after a single bout of intense lower body resistance exercise, and after 3 hr of recovery. RESULTS: CHO ingestion elevated plasma glucose and insulin concentrations throughout recovery compared with PLA ingestion. The ERK pathway (phosphorylation of ERK1/2 [Thr202/Tyr204], RSK [Ser380], and p70S6K [Thr421/Ser424]) was markedly activated immediately after resistance exercise, without any effect of CHO supplementation. The phosphorylation state of AKT (Thr308) was unchanged postexercise in the PLA trial and increased at 3 hr of recovery above resting with ingestion of CHO compared with PLA. Despite stimulating-marked phosphorylation of AKT, CHO ingestion did not enhance resistance exercise-induced phosphorylation of p70S6K (Thr389) and rpS6 (Ser235/236 and Ser240/244). CONCLUSION: CHO supplementation after resistance exercise and hyperinsulinemia does not influence the ERK pathway nor the mTORC1 target p70S6K and its downstream proteins, despite the increased AKT phosphorylation.

Postnatal tendon growth and remodeling require platelet-derived growth factor receptor signaling.

Platelet-derived growth factor receptor (PDGFR) signaling plays an important role in the fundamental biological activities of many cells that compose musculoskeletal tissues. However, little is known about the role of PDGFR signaling during tendon growth and remodeling in adult animals. Using the hindlimb synergist ablation model of tendon growth, our objectives were to determine the role of PDGFR signaling in the adaptation of tendons subjected to a mechanical growth stimulus, as well as to investigate the biological mechanisms behind this response. We demonstrate that both PDGFRs, PDGFRα and PDGFRß, are expressed in tendon fibroblasts and that the inhibition of PDGFR signaling suppresses the normal growth of tendon tissue in response to mechanical growth cues due to defects in fibroblast proliferation and migration. We also identify membrane type-1 matrix metalloproteinase (MT1-MMP) as an essential proteinase for the migration of tendon fibroblasts through their extracellular matrix. Furthermore, we report that MT1-MMP translation is regulated by phosphoinositide 3-kinase/Akt signaling, while ERK1/2 controls posttranslational trafficking of MT1-MMP to the plasma membrane of tendon fibroblasts. Taken together, these findings demonstrate that PDGFR signaling is necessary for postnatal tendon growth and remodeling and that MT1-MMP is a critical mediator of tendon fibroblast migration and a potential target for the treatment of tendon injuries and diseases.

Divergent effects of cold water immersion versus active recovery on skeletal muscle fiber type and angiogenesis in young men.

Resistance training (RT) increases muscle fiber size and induces angiogenesis to maintain capillary density. Cold water immersion (CWI), a common postexercise recovery modality, may improve acute recovery, but it attenuates muscle hypertrophy compared with active recovery (ACT). It is unknown if CWI following RT alters muscle fiber type expression or angiogenesis. Twenty-one men strength trained for 12 wk, with either 10 min of CWI ( n = 11) or ACT ( n = 10) performed following each session. Vastus lateralis biopsies were collected at rest before and after training. Type IIx myofiber percent decreased ( P = 0.013) and type IIa myofiber percent increased with training ( P = 0.012), with no difference between groups. The number of capillaries per fiber increased from pretraining in the CWI group ( P = 0.004) but not the ACT group ( P = 0.955). Expression of myosin heavy chain genes ( MYH1 and MYH2), encoding type IIx and IIa fibers, respectively, decreased in the ACT group, whereas MYH7 (encoding type I fibers) increased in the ACT group versus CWI ( P = 0.004). Myosin heavy chain IIa protein increased with training ( P = 0.012) with no difference between groups. The proangiogenic vascular endothelial growth factor protein decreased posttraining in the ACT group versus CWI ( P < 0.001), whereas antiangiogenic Sprouty-related, EVH1 domain-containing protein 1 protein increased with training in both groups ( P = 0.015). Expression of microRNAs that regulate muscle fiber type (miR-208b and -499a) and angiogenesis (miR-15a, -16, and -126) increased only in the ACT group ( P < 0.05). CWI recovery after each training session altered the angiogenic and fiber type-specific response to RT through regulation at the levels of microRNA, gene, and protein expression.

Considerations on mTOR regulation at serine 2448: implications for muscle metabolism studies.

The mammalian target of rapamycin (mTOR) complex exerts a pivotal role in protein anabolism and cell growth. Despite its importance, few studies adequately address the complexity of phosphorylation of the mTOR protein itself to enable conclusions to be drawn on the extent of kinase activation following this event. In particular, a large number of studies in the skeletal muscle biology field have measured Serine 2448 (Ser2448) phosphorylation as a proxy of mTOR kinase activity. However, the evidence to be described is that Ser2448 is not a measure of mTOR kinase activity nor is a target of AKT activity and instead has inhibitory effects on the kinase that is targeted by the downstream effector p70S6K in a negative feedback loop mechanism, which is evident when revisiting muscle research studies. It is proposed that this residue modification acts as a fine-tuning mechanism that has been gained during vertebrate evolution. In conclusion, it is recommended that Ser2448 is an inadequate measure and that preferential analysis of mTORC1 activation should focus on the downstream and effector proteins, including p70S6K and 4E-BP1, along mTOR protein partners that bind to mTOR protein to form the active complexes 1 and 2.

The effects of cold water immersion and active recovery on inflammation and cell stress responses in human skeletal muscle after resistance exercise.

KEY POINTS: Cold water immersion and active recovery are common post-exercise recovery treatments. A key assumption about the benefits of cold water immersion is that it reduces inflammation in skeletal muscle. However, no data are available from humans to support this notion. We compared the effects of cold water immersion and active recovery on inflammatory and cellular stress responses in skeletal muscle from exercise-trained men 2, 24 and 48 h during recovery after acute resistance exercise. Exercise led to the infiltration of inflammatory cells, with increased mRNA expression of pro-inflammatory cytokines and neurotrophins, and the subcellular translocation of heat shock proteins in muscle. These responses did not differ significantly between cold water immersion and active recovery. Our results suggest that cold water immersion is no more effective than active recovery for minimizing the inflammatory and stress responses in muscle after resistance exercise. ABSTRACT: Cold water immersion and active recovery are common post-exercise recovery treatments. However, little is known about whether these treatments influence inflammation and cellular stress in human skeletal muscle after exercise. We compared the effects of cold water immersion versus active recovery on inflammatory cells, pro-inflammatory cytokines, neurotrophins and heat shock proteins (HSPs) in skeletal muscle after intense resistance exercise. Nine active men performed unilateral lower-body resistance exercise on separate days, at least 1 week apart. On one day, they immersed their lower body in cold water (10°C) for 10 min after exercise. On the other day, they cycled at a low intensity for 10 min after exercise. Muscle biopsies were collected from the exercised leg before, 2, 24 and 48 h after exercise in both trials. Exercise increased intramuscular neutrophil and macrophage counts, MAC1 and CD163 mRNA expression (P < 0.05). Exercise also increased IL1ß, TNF, IL6, CCL2, CCL4, CXCL2, IL8 and LIF mRNA expression (P < 0.05). As evidence of hyperalgesia, the expression of NGF and GDNF mRNA increased after exercise (P < 0.05). The cytosolic protein content of αB-crystallin and HSP70 decreased after exercise (P < 0.05). This response was accompanied by increases in the cytoskeletal protein content of αB-crystallin and the percentage of type II fibres stained for αB-crystallin. Changes in inflammatory cells, cytokines, neurotrophins and HSPs did not differ significantly between the recovery treatments. These findings indicate that cold water immersion is no more effective than active recovery for reducing inflammation or cellular stress in muscle after a bout of resistance exercise.

Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid.

In contrast to the well-characterized effects of specialized proresolving lipid mediators (SPMs) derived from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), little is known about the metabolic fate of the intermediary long-chain (LC) n-3 polyunsaturated fatty acid (PUFA) docosapentaenoic acid (DPA). In this double blind crossover study, shifts in circulating levels of n-3 and n-6 PUFA-derived bioactive lipid mediators were quantified by an unbiased liquid chromatography-tandem mass spectrometry lipidomic approach. Plasma was obtained from human subjects before and after 7 d of supplementation with pure n-3 DPA, n-3 EPA or placebo (olive oil). DPA supplementation increased the SPM resolvin D5n-3DPA (RvD5n-3DPA) and maresin (MaR)-1, the DHA vicinal diol 19,20-dihydroxy-DPA and n-6 PUFA derived 15-keto-PG E2 (15-keto-PGE2). EPA supplementation had no effect on any plasma DPA or DHA derived mediators, but markedly elevated monohydroxy-eicosapentaenoic acids (HEPEs), including the e-series resolvin (RvE) precursor 18-HEPE; effects not observed with DPA supplementation. These data show that dietary n-3 DPA and EPA have highly divergent effects on human lipid mediator profile, with no overlap in PUFA metabolites formed. The recently uncovered biologic activity of n-3 DPA docosanoids and their marked modulation by dietary DPA intake reveals a unique and specific role of n-3 DPA in human physiology.-Markworth, J. F., Kaur, G., Miller, E. G., Larsen, A. E., Sinclair, A. J., Maddipati, K. R., Cameron-Smith, D. Divergent shifts in lipid mediator profile following supplementation with n-3 docosapentaenoic acid and eicosapentaenoic acid.

Emerging roles of pro-resolving lipid mediators in immunological and adaptive responses to exercise-induced muscle injury.

Lipid mediators are bioactive metabolites of the essential polyunsaturated fatty acids (PUFA) that play diverse roles inthe initiation, self-limitation, and active resolution of inflammation. Prostaglandins, classical pro-inflammatory lipid metabolites of arachidonic acid, have long been implicated in immunological and adaptive muscle responses to acute injury and exercise-induced stress. More recently, PUFA metabolites have been discovered during the resolution phase of inflammation which collectively function as endogenous 'stop signals' to control inflammation whilst actively promoting the return to a non-inflamed state. The apparent self-resolving nature of inflammatory responses holds important implications for contexts of musculoskeletal injury, exercise recovery, and chronic inflammatory diseases originati ng in or impacting upon muscle. 'Anti-inflammatory' interventions that strive to control inflammation via antagonism of pro-inflammatory signals are currently commonplace in efforts to hasten muscle recovery from damaging or exhaustive exercise, as well as to relieve the pain associated with musculoskeletal injury. However, the scientific literature does not clearly support a benefit of this anti-inflammatory approach. Additionally, recent evidence suggests that strategies to block pro-inflammatory lipid mediator pathways (e.g. NSAIDs) may be counterintuitive and inadvertently derange or impair timely resolution of inflammation; with potentially deleterious implications on skeletal muscle remodelling. The current review will provide an overview of the current understanding of diverse roles of bioactive lipid mediators in the initiation, control, and active resolution of acute inflammation. The established and putative roles of lipid mediators in mediating immunological and adapt ive skeletal muscle responses to acute muscle injury and exercise-induced muscle load/stress will be discussed.

Older adults have an altered chylomicron response to a high-fat meal.

Ageing is associated with a prolonged and exaggerated postprandial lipaemia. This study aimed to examine the contribution of alterations in chylomicron synthesis, size and lipid composition to increased lipaemia. Healthy older (60-75 years; n 15) and younger (20-25 years; n 15) subjects consumed a high-fat breakfast. Chylomicron dynamics and fatty acid composition were analysed for 5 h in the postprandial state. Plasma TAG levels were elevated following the meal in the older subjects, relative to younger subjects (P<0·01). For older subjects compared with younger subjects, circulating chylomicron particle size was smaller (P<0·05), with greater apoB content (P<0·05) at all postprandial time points. However, total chylomicron TAG concentration between the groups was unaltered post-meal. Compared with younger subjects, the older subjects exhibited a greater proportion of oleic acid in the TAG and phospholipid (PL) fraction (P<0·05), plus lower proportions of linoleic acid in the TAG fraction of the chylomicrons (P<0·01). Thus, following the ingestion of a high-fat meal, older individuals demonstrate both smaller, more numerous chylomicrons, with a greater total MUFA and lower PUFA contents. These data suggest that the increased postprandial lipaemia of ageing cannot be attributed to increased chylomicron TAG. Rather, ageing is associated with changes in chylomicron particle size, apoB content and fatty acid composition of the chylomicron TAG and PL fractions.

Post-exercise cold water immersion attenuates acute anabolic signalling and long-term adaptations in muscle to strength training.

We investigated functional, morphological and molecular adaptations to strength training exercise and cold water immersion (CWI) through two separate studies. In one study, 21 physically active men strength trained for 12 weeks (2 days per week), with either 10 min of CWI or active recovery (ACT) after each training session. Strength and muscle mass increased more in the ACT group than in the CWI group (P < 0.05). Isokinetic work (19%), type II muscle fibre cross-sectional area (17%) and the number of myonuclei per fibre (26%) increased in the ACT group (all P < 0.05), but not the CWI group. In another study, nine active men performed a bout of single-leg strength exercises on separate days, followed by CWI or ACT. Muscle biopsies were collected before and 2, 24 and 48 h after exercise. The number of satellite cells expressing neural cell adhesion molecule (NCAM) (10-30%) and paired box protein (Pax7) (20-50%) increased 24-48 h after exercise with ACT. The number of NCAM(+) satellite cells increased 48 h after exercise with CWI. NCAM(+) - and Pax7(+) -positive satellite cell numbers were greater after ACT than after CWI (P < 0.05). Phosphorylation of p70S6 kinase(Thr421/Ser424) increased after exercise in both conditions but was greater after ACT (P < 0.05). These data suggest that CWI attenuates the acute changes in satellite cell numbers and activity of kinases that regulate muscle hypertrophy, which may translate to smaller long-term training gains in muscle strength and hypertrophy. The use of CWI as a regular post-exercise recovery strategy should be reconsidered.

Ribosome biogenesis adaptation in resistance training-induced human skeletal muscle hypertrophy.

Resistance training (RT) has the capacity to increase skeletal muscle mass, which is due in part to transient increases in the rate of muscle protein synthesis during postexercise recovery. The role of ribosome biogenesis in supporting the increased muscle protein synthetic demands is not known. This study examined the effect of both a single acute bout of resistance exercise (RE) and a chronic RT program on the muscle ribosome biogenesis response. Fourteen healthy young men performed a single bout of RE both before and after 8 wk of chronic RT. Muscle cross-sectional area was increased by 6 ± 4.5% in response to 8 wk of RT. Acute RE-induced activation of the ERK and mTOR pathways were similar before and after RT, as assessed by phosphorylation of ERK, MNK1, p70S6K, and S6 ribosomal protein 1 h postexercise. Phosphorylation of TIF-IA was also similarly elevated following both RE sessions. Cyclin D1 protein levels, which appeared to be regulated at the translational rather than transcriptional level, were acutely increased after RE. UBF was the only protein found to be highly phosphorylated at rest after 8 wk of training. Also, muscle levels of the rRNAs, including the precursor 45S and the mature transcripts (28S, 18S, and 5.8S), were increased in response to RT. We propose that ribosome biogenesis is an important yet overlooked event in RE-induced muscle hypertrophy that warrants further investigation.

Regular postexercise cooling enhances mitochondrial biogenesis through AMPK and p38 MAPK in human skeletal muscle.

This study investigated the effect of regular postexercise cold water immersion (CWI) on muscle aerobic adaptations to endurance training. Eight males performed 3 sessions/wk of endurance training for 4 wk. Following each session, subjects immersed one leg in a cold water bath (10°C; COLD) for 15 min, while the contralateral leg served as a control (CON). Muscle biopsies were obtained from vastus lateralis of both CON and COLD legs prior to training and 48 h following the last training session. Samples were analyzed for signaling kinases: p38 MAPK and AMPK, peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), enzyme activities indicative of mitochondrial biogenesis, and protein subunits representative of respiratory chain complexes I-V. Following training, subjects' peak oxygen uptake and running velocity were improved by 5.9% and 6.2%, respectively (P < 0.05). Repeated CWI resulted in higher total AMPK, phosphorylated AMPK, phosphorylated acetyl-CoA carboxylase, ß-3-hydroxyacyl-CoA-dehydrogenase and the protein subunits representative of complex I and III (P < 0.05). Moreover, large effect sizes (Cohen's d > 0.8) were noted with changes in protein content of p38 (d = 1.02, P = 0.064), PGC-1α (d = 0.99, P = 0.079), and peroxisome proliferator-activated receptor α (d = 0.93, P = 0.10) in COLD compared with CON. No differences between conditions were observed in the representative protein subunits of respiratory complexes II, IV, and V and in the activities of several mitochondrial enzymes (P > 0.05). These findings indicate that regular CWI enhances p38, AMPK, and possibly mitochondrial biogenesis.

Metabolic and hormonal responses to isoenergetic high-intensity interval exercise and continuous moderate-intensity exercise.

This study investigated the effects of high-intensity interval training (HIIT) vs. work-matched moderate-intensity continuous exercise (MOD) on metabolism and counterregulatory stress hormones. In a randomized and counterbalanced order, 10 well-trained male cyclists and triathletes completed a HIIT session [81.6 ± 3.7% maximum oxygen consumption (V̇o2 max); 72.0 ± 3.2% peak power output; 792 ± 95 kJ] and a MOD session (66.7 ± 3.5% V̇o2 max; 48.5 ± 3.1% peak power output; 797 ± 95 kJ). Blood samples were collected before, immediately after, and 1 and 2 h postexercise. Carbohydrate oxidation was higher (P = 0.037; 20%), whereas fat oxidation was lower (P = 0.037; -47%) during HIIT vs. MOD. Immediately after exercise, plasma glucose (P = 0.024; 20%) and lactate (P < 0.01; 5.4×) were higher in HIIT vs. MOD, whereas total serum free fatty acid concentration was not significantly different (P = 0.33). Targeted gas chromatography-mass spectromtery metabolomics analysis identified and quantified 49 metabolites in plasma, among which 11 changed after both HIIT and MOD, 13 changed only after HIIT, and 5 changed only after MOD. Notable changes included substantial increases in tricarboxylic acid intermediates and monounsaturated fatty acids after HIIT and marked decreases in amino acids during recovery from both trials. Plasma adrenocorticotrophic hormone (P = 0.019), cortisol (P < 0.01), and growth hormone (P < 0.01) were all higher immediately after HIIT. Plasma norepinephrine (P = 0.11) and interleukin-6 (P = 0.20) immediately after exercise were not significantly different between trials. Plasma insulin decreased during recovery from both HIIT and MOD (P < 0.01). These data indicate distinct differences in specific metabolites and counterregulatory hormones following HIIT vs. MOD and highlight the value of targeted metabolomic analysis to provide more detailed insights into the metabolic demands of exercise.

Arachidonic acid supplementation enhances in vitro skeletal muscle cell growth via a COX-2-dependent pathway.

Arachidonic acid (AA) is the metabolic precursor to a diverse range of downstream bioactive lipid mediators. A positive or negative influence of individual eicosanoid species [e.g., prostaglandins (PGs), leukotrienes, and hydroxyeicosatetraenoic acids] has been implicated in skeletal muscle cell growth and development. The collective role of AA-derived metabolites in physiological states of skeletal muscle growth/atrophy remains unclear. The present study aimed to determine the direct effect of free AA supplementation and subsequent eicosanoid biosynthesis on skeletal myocyte growth in vitro. C2C12 (mouse) skeletal myocytes induced to differentiate with supplemental AA exhibited dose-dependent increases in the size, myonuclear content, and protein accretion of developing myotubes, independent of changes in cell density or the rate/extent of myogenic differentiation. Nonselective (indomethacin) or cyclooxygenase 2 (COX-2)-selective (NS-398) nonsteroidal anti-inflammatory drugs blunted basal myogenesis, an effect that was amplified in the presence of supplemental free AA substrate. The stimulatory effects of AA persisted in preexisting myotubes via a COX-2-dependent (NS-389-sensitive) pathway, specifically implying dependency on downstream PG biosynthesis. AA-stimulated growth was associated with markedly increased secretion of PGF(2α) and PGE(2); however, incubation of myocytes with PG-rich conditioned medium failed to mimic the effects of direct AA supplementation. In vitro AA supplementation stimulates PG release and skeletal muscle cell hypertrophy via a COX-2-dependent pathway.

Human inflammatory and resolving lipid mediator responses to resistance exercise and ibuprofen treatment.

Classical proinflammatory eicosanoids, and more recently discovered lipid mediators with anti-inflammatory and proresolving bioactivity, exert a complex role in the initiation, control, and resolution of inflammation. Using a targeted lipidomics approach, we investigated circulating lipid mediator responses to resistance exercise and treatment with the NSAID ibuprofen. Human subjects undertook a single bout of unaccustomed resistance exercise (80% of one repetition maximum) following oral ingestion of ibuprofen (400 mg) or placebo control. Venous blood was collected during early recovery (0-3 h and 24 h postexercise), and serum lipid mediator composition was analyzed by LC-MS-based targeted lipidomics. Postexercise recovery was characterized by elevated levels of cyclooxygenase (COX)-1 and 2-derived prostanoids (TXB2, PGE2, PGD2, PGF2α, and PGI2), lipooxygenase (5-LOX, 12-LOX, and 15-LOX)-derived hydroxyeicosatetraenoic acids (HETEs), and leukotrienes (e.g., LTB4), and epoxygenase (CYP)-derived epoxy/dihydroxy eicosatrienoic acids (EpETrEs/DiHETrEs). Additionally, we detected elevated levels of bioactive lipid mediators with anti-inflammatory and proresolving properties, including arachidonic acid-derived lipoxins (LXA4 and LXB4), and the EPA (E-series) and DHA (D-series)-derived resolvins (RvD1 and RvE1), and protectins (PD1 isomer 10S, 17S-diHDoHE). Ibuprofen treatment blocked exercise-induced increases in COX-1 and COX-2-derived prostanoids but also resulted in off-target reductions in leukotriene biosynthesis, and a diminished proresolving lipid mediator response. CYP pathway product metabolism was also altered by ibuprofen treatment, as indicated by elevated postexercise serum 5,6-DiHETrE and 8,9-DiHETrE only in those receiving ibuprofen. These findings characterize the blood inflammatory lipid mediator response to unaccustomed resistance exercise in humans and show that acute proinflammatory signals are mechanistically linked to the induction of a biological active inflammatory resolution program, regulated by proresolving lipid mediators during postexercise recovery.

Cold water immersion in recovery following a single bout resistance exercise suppresses mechanisms of miRNA nuclear export and maturation.

Cold water immersion (CWI) following intense exercise is a common athletic recovery practice. However, CWI impacts muscle adaptations to exercise training, with attenuated muscle hypertrophy and increased angiogenesis. Tissue temperature modulates the abundance of specific miRNA species and thus CWI may affect muscle adaptations via modulating miRNA expression following a bout of exercise. The current study focused on the regulatory mechanisms involved in cleavage and nuclear export of mature miRNA, including DROSHA, EXPORTIN-5, and DICER. Muscle biopsies were obtained from the vastus lateralis of young males (n = 9) at rest and at 2, 4, and 48 h of recovery from an acute bout of resistance exercise, followed by either 10 min of active recovery (ACT) at ambient temperature or CWI at 10°C. The abundance of key miRNA species in the regulation of intracellular anabolic signaling (miR-1 and miR-133a) and angiogenesis (miR-15a and miR-126) were measured, along with several gene targets implicated in satellite cell dynamics (NCAM and PAX7) and angiogenesis (VEGF and SPRED-1). When compared to ACT, CWI suppressed mRNA expression of DROSHA (24 h p = 0.025 and 48 h p = 0.017), EXPORTIN-5 (24 h p = 0.008), and DICER (24 h p = 0.0034). Of the analyzed miRNA species, miR-133a (24 h p < 0.001 and 48 h p = 0.007) and miR-126 (24 h p < 0.001 and 48 h p < 0.001) remained elevated at 24 h post-exercise in the CWI trial only. Potential gene targets of these miRNA, however, did not differ between trials. CWI may therefore impact miRNA abundance in skeletal muscle, although the precise physiological relevance needs further investigation.