James F. Markworth

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.

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

Cold water immersion is a popular strategy to recover from exercise. However, whether regular cold water immersion influences muscle adaptations to strength training is not well understood. We compared the effects of cold water immersion and active recovery on changes in muscle mass and strength after 12 weeks of strength training. We also examined the effects of these two treatments on hypertrophy signalling pathways and satellite cell activity in skeletal muscle after acute strength exercise. Cold water immersion attenuated long term gains in muscle mass and strength. It also blunted the activation of key proteins and satellite cells in skeletal muscle up to 2 days after strength exercise. Individuals who use strength training to improve athletic performance, recover from injury or maintain their health should therefore reconsider whether to use cold water immersion as an adjuvant to their training.

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.

Prostaglandin F2α stimulates PI3K/ERK/mTOR signaling and skeletal myotube hypertrophy

Cyclooxygenase (COX) enzymes mediate the synthesis of proinflammatory prostaglandin (PG) species from cellular arachidonic acid. COX/PGs have been implicated in skeletal muscle growth/regeneration; however, the mechanisms by which PGs influence skeletal muscle adaptation are poorly understood. The present study aimed to investigate PGF(2α) signaling and its role in skeletal myotube hypertrophy. PGF(2α) or the FP receptor agonist fluprostenol increased C2C12 myotube diameter. This effect was abolished by the FP receptor antagonist AL8810 and mammalian target of rapamycin (mTOR) inhibition. PGF(2α) stimulated time- and dose-dependent increases in the phosphorylation of extracellular receptor kinase (ERK)1/2 (Thr202/Tyr204), p70S6 kinase (p70S6K) (Thr389 and Thr421/Ser424), and eukaryotic initiation factor 4G (eIF4G) (Ser1108) without influencing Akt (Ser473). Pretreatment with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and the ERK inhibitor PD98059 blocked F prostanoid receptor signaling responses, whereas rapamycin blocked heightened p70S6K/eIF4G phosphorylation without influencing ERK1/2 phosphorylation. These data suggest that activation of the F prostanoid receptor is coupled to C2C12 myotube growth and intracellular signaling via a PI3K/ERK/mTOR-dependent pathway.

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.

Changes in mitochondrial function and mitochondria associated protein expression in response to 2-weeks of high intensity interval training

Purpose: High-intensity short-duration interval training (HIT) stimulates functional and metabolic adaptation in skeletal muscle, but the influence of HIT on mitochondrial function remains poorly studied in humans. Mitochondrial metabolism as well as mitochondrial-associated protein expression were tested in untrained participants performing HIT over a 2-week period. Methods: Eight males performed a single-leg cycling protocol (12 × 1 min intervals at 120% peak power output, 90 s recovery, 4 days/week). Muscle biopsies (vastus lateralis) were taken pre- and post-HIT. Mitochondrial respiration in permeabilized fibers, citrate synthase (CS) activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC-1α) and respiratory complex components were measured. Results: HIT training improved peak power and time to fatigue. Increases in absolute oxidative phosphorylation (OXPHOS) capacities and CS activity were observed, but not in the ratio of CCO to the electron transport system (CCO/ETS), the respiratory control ratios (RCR-1 and RCR-2) or mitochondrial-associated protein expression. Specific increases in OXPHOS flux were not apparent after normalization to CS, indicating that gross changes mainly resulted from increased mitochondrial mass. Conclusion: Over only 2 weeks HIT significantly increased mitochondrial function in skeletal muscle independently of detectable changes in mitochondrial-associated and mitogenic protein expression.

Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men

The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring 13C6 phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h−1 in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p < 0.001) to 0.057% ± 0.018% and 0.052% ± 0.024% h−1 in the milk and whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

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

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.

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 fromeicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), little is known about themetabolic 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 obtainedfrom humansubjects before andafter 7 d of supplementationwith pure n‐3DPA, 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. FASEB J. 30, 3714–3725 (2016) www.fasebj.org

Impact of resistance exercise on ribosome biogenesis is acutely regulated by post-exercise recovery strategies

Muscle hypertrophy occurs following increased protein synthesis, which requires activation of the ribosomal complex. Additionally, increased translational capacity via elevated ribosomal RNA (rRNA) synthesis has also been implicated in resistance training‐induced skeletal muscle hypertrophy. The time course of ribosome biogenesis following resistance exercise (RE) and the impact exerted by differing recovery strategies remains unknown. In the present study, the activation of transcriptional regulators, the expression levels of pre‐rRNA, and mature rRNA components were measured through 48 h after a single‐bout RE. In addition, the effects of either low‐intensity cycling (active recovery, ACT) or a cold‐water immersion (CWI) recovery strategy were compared. Nine male subjects performed two bouts of high‐load RE randomized to be followed by 10 min of either ACT or CWI. Muscle biopsies were collected before RE and at 2, 24, and 48 h after RE. RE increased the phosphorylation of the p38‐MNK1‐eIF4E axis, an effect only evident with ACT recovery. Downstream, cyclin D1 protein, total eIF4E, upstream binding factor 1 (UBF1), and c‐Myc proteins were all increased only after RE with ACT. This corresponded with elevated abundance of the pre‐rRNAs (45S, ITS‐28S, ITS‐5.8S, and ETS‐18S) from 24 h after RE with ACT. In conclusion, coordinated upstream signaling and activation of transcriptional factors stimulated pre‐rRNA expression after RE. CWI, as a recovery strategy, markedly blunted these events, suggesting that suppressed ribosome biogenesis may be one factor contributing to the impaired hypertrophic response observed when CWI is used regularly after exercise.