Metabolomics approach to assessing plasma 13- and 9-hydroxy-octadecadienoic acid and linoleic acid metabolite responses to 75-km cycling.

Bioactive oxidized linoleic acid metabolites (OXLAMs) include 13- and 9-hydroxy-octadecadienoic acid (13-HODE + 9-HODE) and have been linked to oxidative stress, inflammation, and numerous pathological and physiological states. The purpose of this study was to measure changes in plasma 13-HODE + 9-HODE following a 75-km cycling bout and identify potential linkages to linoleate metabolism and established biomarkers of oxidative stress (F2-isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists (N = 19, age 38.0 ± 1.6 yr, wattsmax 304 ± 10.5) engaged in a 75-km cycling time trial on their own bicycles using electromagnetically braked cycling ergometers (2.71 ± 0.07 h). Blood samples were collected preexercise, immediately post-, 1.5 h post-, and 21 h postexercise, and analyzed for plasma cytokines (IL-6, IL-8, IL-10, tumor necrosis factor-α, monocyte chemoattractant protein-1, granulocyte colony-stimulating factor), F2-isoprostanes, and shifts in metabolites using global metabolomics procedures with gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS). 13-HODE + 9-HODE increased 3.1-fold and 1.7-fold immediately post- and 1.5 h postexercise (both P < 0.001) and returned to preexercise levels by 21-h postexercise. Post-75-km cycling plasma levels of 13-HODE + 9-HODE were not significantly correlated with increases in plasma cytokines but were positively correlated with postexercise F2-isoprostanes (r = 0.75, P < 0.001), linoleate (r = 0.54, P = 0.016), arachidate (r = 0.77, P < 0.001), 12,13-dihydroxy-9Z-octadecenoate (12,13-DiHOME) (r = 0.60, P = 0.006), dihomo-linolenate (r = 0.57, P = 0.011), and adrenate (r = 0.56, P = 0.013). These findings indicate that prolonged and intensive exercise caused a transient, 3.1-fold increase in the stable linoleic acid oxidation product 13-HODE + 9-HODE and was related to increases in F2-isoprostanes, linoleate, and fatty acids in the linoleate conversion pathway. These data support the use of 13-HODE + 9-HODE as an oxidative stress biomarker in acute exercise investigations.

Evaluation of Rhodiola rosea supplementation on skeletal muscle damage and inflammation in runners following a competitive marathon.

Adaptogens modulate intracellular signaling and increase expression of heat shock protein 72 (HSP72). Rhodiola rosea (RR) is a medicinal plant with demonstrated adaptogenic properties. The purpose of this study was to measure the influence of RR supplementation on exercise-induced muscle damage, delayed onset of muscle soreness (DOMS), plasma cytokines, and extracellular HSP72 (eHSP72) in experienced runners completing a marathon. Experienced marathon runners were randomized to RR (n=24, 6 female, 18 male) or placebo (n=24, 7 female, 17 male) groups and under double-blinded conditions ingested 600mg/day RR extract or placebo for 30days prior to, the day of, and seven days post-marathon. Blood samples were collected, and vertical jump and DOMS assessed the day before, 15min post- and 1.5h post-marathon. DOMS was also assessed for seven days post-marathon. Marathon race performance did not differ between RR and placebo groups (3.87±0.12h and 3.93±0.12h, respectively, p=0.722). Vertical jump decreased post-marathon (time effect, p<0.001) with no difference between groups (interaction effect, p=0.673). Post-marathon DOMS increased significantly (p<0.001) but the pattern of change did not differ between groups (p=0.700). Myoglobin (Mb), creatine phosphokinase (CPK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukin (IL)-6, IL-8, IL-10, monocyte chemotactic protein-1 (MCP-1), granulocyte-colony-stimulating factor (G-CSF), C-reactive protein (CRP), and eHSP72 all increased post-marathon (all p<0.001), with no group differences over time (all p>0.300). In conclusion, RR supplementation (600mg/day) for 30days before running a marathon did not attenuate the post-marathon decrease in muscle function, or increases in muscle damage, DOMS, eHSP72, or plasma cytokines in experienced runners.

Immune and inflammation responses to a 3-day period of intensified running versus cycling.

Functional overreaching has been linked to alterations in immunity and host pathogen defense, but little is known as to whether or not running and cycling evoke different responses. This study compared inflammation, muscle damage and soreness, and innate immune function responses to a 3-day period of intensified exercise in trained long distance runners (N=13, age 34.4±2.4year) and cyclists (N=22, age 36.6±1.7year, P=0.452). Upper respiratory tract infection (URTI) symptomatology was monitored for 12weeks using the Wisconsin Upper Respiratory Symptom Survey (WURSS), and subjects from both athletic groups came to the lab during week five and exercised 2.5h/day for 3days in a row at 70% VO2max. Blood samples were collected before and after the 3-day period of exercise, with recovery samples collected 1-, 14-, and 38h-post-exercise. Samples were analyzed for muscle damage [creatine kinase (CK), myoglobin (MYO)], inflammation (CRP, IL-6, IL-8, IL-10, MCP), and innate immunity [granulocyte and monocyte phagocytosis (GR-PHAG and MO-PHAG) and oxidative burst activity (GR-OBA and MO-OBA)]. Runners compared to cyclists experienced significantly more muscle damage (CK 133% and MYO 404% higher post-3days exercise), inflammation (CRP 87%, IL-6 256%, IL 8 61%, IL-10 32%, MCP 29%), and delayed onset of muscle soreness (DOMS, 87%). The 3-day period of exercise caused significant downturns in GR-PHAG, MO-PHAG, GR-OBA, MO-OBA by 14- and 38h-recovery, but the pattern of change did not differ between groups. No group differences were measured for 12-week URTI severity (18.3±5.6 and 16.6±4.0, P=0.803) and symptom scores (33.4±12.6 and 24.7±5.8, P=0.477). These data indicate that a 3-day period of functional overreaching results in substantially more muscle damage and soreness, and systemic inflammation in runners compared to cyclists, but without group differences for 12-week URTI symptomatology and post-exercise decrements in innate immune function.

Influence of vitamin D mushroom powder supplementation on exercise-induced muscle damage in vitamin D insufficient high school athletes.

Incidence of vitamin D deficiency is increasing worldwide. The purpose of this study was to determine if supplementation with vitamin D2 from Portobello mushroom powder would enhance skeletal muscle function and attenuate exercise-induced muscle damage in low vitamin D status high school athletes. Participants were randomised to Portobello mushroom powder (600 IU/d vitamin D2) or placebo for 6 weeks. Participants then completed a 1.5-h exercise session designed to induce skeletal muscle damage. Blood samples and measures of skeletal muscle function were taken pre-supplementation, post-supplementation/pre-exercise and post-exercise. Six weeks supplementation with vitamin D2 increased serum 25(OH)D2 by 9.9-fold and decreased serum 25(OH)D3 by 28%. Changes in skeletal muscle function and circulating markers of skeletal muscle damage did not differ between groups. In conclusion, 600 IU/d vitamin D2 increased 25(OH)D2 with a concomitant decrease in 25(OD)D3, with no effect on muscular function or exercise-induced muscle damage in high school athletes.

Serum metabolic signatures induced by a three-day intensified exercise period persist after 14 h of recovery in runners.

This study investigated changes in the human serum metabolome elicited by a 3-day period of intensified training. Runners (N = 15, mean ± SD age, 35.2 ± 8.7 years) ran for 2.5 h/day on treadmills at ∼70% VO2max for 3 days in a row, with blood samples collected pre-exercise, and immediately and 14 h post-exercise. Samples were analyzed using gas and liquid chromatography/mass spectrometry (GC-MS, LC-MS), with compounds identified based on comparison to more than 2800 purified standards. Repeated measures ANOVA was used to identify metabolites that differed significantly across time, with multiple testing corrected by the false discovery rate (FDR) (q-value). Immediately following the 3-day exercise period, significant 2-fold or higher increases in 75 metabolites were measured, with all but 22 of these metabolites related to lipid/carnitine metabolism, 13 to amino acid/peptide metabolism, 4 to hemoglobin/porphyrin metabolism, and 3 to Krebs cycle intermediates (q-values < 0.001). After a 14 h overnight recovery period, 50 of the 75 metabolites remained elevated, with 8 decreased (primarily amino acid-related metabolites) (q-values < 0.05). Among the top 20 metabolites, the mean fold changes were 12.4 ± 5.3 and 2.9 ± 1.3 immediately and 14-h post-exercise, respectively. Significant decreases (40-70%, q < 0.01) in 22 metabolites (primarily related to lysolipid and bile acid metabolism) were measured post-exercise, with all but 4 of these still decreased after 14 h rest recovery (q < 0.025). Runners experienced a profound systemic shift in blood metabolites related to energy production especially from the lipid super pathway following 3 days of heavy exertion that was not fully restored to pre-exercise levels after 14 h recovery.

Dose-response to 3 months of quercetin-containing supplements on metabolite and quercetin conjugate profile in adults.

Quercetin, a flavonol in fruits and vegetables, has been demonstrated to have antioxidant, anti-inflammatory and immunomodulating influences. The purpose of the present study was to determine if quercetin, vitamin C and niacin supplements (Q-500 = 500 mg/d of quercetin, 125 mg/d of vitamin C and 5 mg/d of niacin; Q-1000 = 1000 mg/d of quercetin, 250 mg/d of vitamin C and 10 mg/d of niacin) would alter small-molecule metabolite profiles and serum quercetin conjugate levels in adults. Healthy adults (fifty-eight women and forty-two men; aged 40-83 years) were assigned using a randomised double-blinded placebo-controlled trial to one of three supplement groups (Q-1000, Q-500 or placebo). Overnight fasted blood samples were collected at 0, 1 and 3 months. Quercetin conjugate concentrations were measured using ultra-performance liquid chromatography (UPLC)-MS/MS, and metabolite profiles were measured using two MS platforms (UPLC-quadrupole time-of-flight MS (TOFMS) and GC-TOFMS). Statistical procedures included partial least square discriminant analysis (PLS-DA) and linear mixed model analysis with repeated measures. After accounting for age, sex and BMI, quercetin supplementation was associated with significant shifts in 163 metabolites/quercetin conjugates (false discovery rate, P<0·05). The top five metabolite shifts were an increase in serum guaiacol, 2-oxo-4-methylthiobutanoic acid, allocystathionine and two bile acids. Inflammatory and oxidative stress metabolites were not affected. PLS-DA revealed a clear separation only between the 1000 mg/d and placebo groups (Q(2)Y = 0·763). The quercetin conjugate, isorhamnetin-3-glucuronide, had the highest concentration at 3 months followed by quercetin-3-glucuronide, quercetin-3-sulphate and quercetin diglucuronide. In human subjects, long-term quercetin supplementation exerts disparate and wide-ranging metabolic effects and changes in quercetin conjugate concentrations. Metabolic shifts were apparent at the 1000 mg/d dose; further research is required to understand the health implications of these shifts.

A commercialized dietary supplement alleviates joint pain in community adults: a double-blind, placebo-controlled community trial.

BACKGROUND: The purpose of this study was to assess the effect of 8-weeks ingestion of a commercialized joint pain dietary supplement (Instaflex™ Joint Support, Direct Digital, Charlotte, NC) compared to placebo on joint pain, stiffness, and function in adults with self-reported joint pain. Instaflex™ is a joint pain supplement containing glucosamine sulfate, methylsufonlylmethane (MSM), white willow bark extract (15% salicin), ginger root concentrate, boswella serrata extract (65% boswellic acid), turmeric root extract, cayenne, and hyaluronic acid. METHODS: Subjects included 100 men and women, ages 50-75 years, with a history (>3 months) of joint pain, and were randomized to Instaflex™ or placebo (3 colored gel capsules per day for 8 weeks, double-blind administration). Subjects agreed to avoid the use of non-steroidal anti-inflammatory drugs (NSAID) and all other medications and supplements targeted for joint pain. Primary outcome measures were obtained pre- and post-study and included joint pain severity, stiffness, and function (Western Ontario and McMaster Universities [WOMAC]), and secondary outcome measures included health-related quality of life (Short Form 36 or SF-36), systemic inflammation (serum C-reactive protein and 9 plasma cytokines), and physical function (6-minute walk test). Joint pain symptom severity was assessed bi-weekly using a 12-point Likert visual scale (12-VS). RESULTS: Joint pain severity was significantly reduced in Instaflex™ compared to placebo (8-week WOMAC, ↓37% versus ↓16%, respectively, interaction effect P = 0.025), with group differences using the 12-VS emerging by week 4 of the study (interaction effect, P = 0.0125). Improvements in ability to perform daily activities and stiffness scores in Instaflex™ compared to placebo were most evident for the 74% of subjects reporting knee pain (8-week WOMAC function score, ↓39% versus ↓14%, respectively, interaction effect P = 0.027; stiffness score, ↓30% versus ↓12%, respectively, interaction effect P = 0.081). Patterns of change in SF-36, systemic inflammation biomarkers, and the 6-minute walk test did not differ significantly between groups during the 8-week study CONCLUSIONS: Results from this randomized, double blind, placebo-controlled community trial support the use of the Instaflex™ dietary supplement in alleviating joint pain severity in middle-aged and older adults, with mitigation of difficulty performing daily activities most apparent in subjects with knee pain.

Latent cytomegalovirus infection and innate immune function following a 75 km cycling time trial.

PURPOSE: This study compared the acute immune response, inflammation, and lipid peroxidation to a 75 km cycling time trial in male athletes testing positive or negative for latent cytomegalovirus (CMV) infection. DESIGN: Trained cyclists (N = 20) were tested for CMV serostatus, and cycled 75 km on a mountainous course using indoor trainers with continuous workload monitoring. Pre-, post-, and 1 h post-exercise blood samples were analyzed for total blood leukocyte counts, blood granulocyte (GR) and monocyte (MO) phagocytosis (PHAG) and oxidative burst activity (OBA), four plasma cytokines, and plasma F2-isoprostanes. RESULTS: Forty percent of the subjects tested positive for CMV. No differences in subject characteristics were found between CMVpos and CMVneg groups. Mean power (57.3 ± 1.6, 59.4 ± 1.8 % maximal Watts, p = 0.803), heart rate (87.0 ± 1.0, 86.5 ± 1.3 % maximal heart rate, p = 0.376), and total time (2.56 ± 0.08, 2.60 ± 0.08 h, p = 0.744) to complete the 75 km cycling time trial did not differ between CMVpos and CMVneg groups. Whereas exercise induced significant changes in total blood leukocyte counts, GR and MO-PHAG, four plasma cytokines, and plasma F2-isoprostanes (p < 0.05, ω(2) > 0.03), these exercise-induced changes did not differ between CMVpos and CMVneg groups (p > 0.05, ω(2) < 0.01). CONCLUSIONS: CMV serostatus does not appear to influence these innate immune responses or markers of inflammation and lipid peroxidation in response to a single bout of heavy exertion.

Effects of a flavonoid-rich juice on inflammation, oxidative stress, and immunity in elite swimmers: a metabolomics-based approach.

The effects of a flavonoid-rich fresh fruit and vegetable juice (JUICE) on chronic resting and postexercise inflammation, oxidative stress, immune function, and metabolic profiles (metabolomics analysis, gas-chromatography mass-spectrometry platform) in elite sprint and middle-distance swimmers were studied. In a randomized, crossover design with a 3-wk washout period, swimmers (n = 9) completed 10-d training with or without 16 fl oz of JUICE (230 mg flavonoids) ingested pre- and postworkout. Blood samples were taken presupplementation, post-10-d supplementation, and immediately postexercise, with data analyzed using a 2 × 3 repeated-measures ANOVA. Prestudy blood samples were also acquired from nonathletic controls (n = 7, age- and weight-matched) and revealed higher levels of oxidative stress in the swimmers, no differences in inflammation or immune function, and a distinct separation in global metabolic scores (R2Y [cum] = .971). Swim workouts consisted of high-intensity intervals (1:1, 1:2 swim-to-rest ratio) and induced little inflammation, oxidative stress, or immune changes. A distinct separation in global metabolic scores was found pre- to postexercise (R2Y [cum] = .976), with shifts detected in a small number of metabolites related to substrate utilization. No effect of 10-d JUICE was found on chronic resting levels or postexercise inflammation, oxidative stress, immune function, and shifts in metabolites. In conclusion, sprint and middle-distance swimmers had a slight chronic elevation in oxidative stress compared with nonathletic controls, experienced a low magnitude of postworkout perturbations in the biomarkers included in this study, and received no apparent benefit other than added nutrient intake from ingesting JUICE pre- and postworkout for 10 days.

Influence of red pepper spice and turmeric on inflammation and oxidative stress biomarkers in overweight females: a metabolomics approach.

Red pepper spice (RP) and turmeric (TM) are used as flavorings in foods and for medicinal purposes. Utilizing a randomized, doubled-blinded, placebo-controlled, crossover design (2-week washout), 4-week supplementation with RP (1 g/d) or TM (2.8 g/d) was tested for influences on inflammation and oxidative stress in 62 overweight/obese (body mass index ≥ 27 kg/m²) females (40-75 years) with systemic inflammation (C-reactive protein, CRP ≥ 2 mg/l). Overnight, fasted blood samples were collected pre- and post-supplementation, and analyzed for oxidative stress (F2-isoprostanes, oxidized low density lipoprotein), inflammation (CRP and seven inflammatory cytokines), and metabolic profiles using gas chromatography-mass spectrometry with multivariate partial least square discriminant analysis (PLS-DA). Pre- to post-supplementation measures of inflammation and oxidative stress for both RP and TM did not differ when compared to placebo (all interaction effects, P > 0.05), and global metabolic difference scores calculated through PLS-DA were non-significant (both spices, Q²Y < 0.40). These data indicate that 4-week supplementation with RP or TM at culinary levels does not alter oxidative stress or inflammation in overweight/obese females with systemic inflammation, or cause a significant shift in the global metabolic profile.

Phytoecdysteroids Accelerate Recovery of Skeletal Muscle Function Following in vivo Eccentric Contraction-Induced Injury in Adult and Old Mice.

Background: Eccentric muscle contractions are commonly used in exercise regimens, as well as in rehabilitation as a treatment against muscle atrophy and weakness. If repeated multiple times, eccentric contractions may result in skeletal muscle injury and loss of function. Skeletal muscle possesses the remarkable ability to repair and regenerate after an injury or damage; however, this ability is impaired with aging. Phytoecdysteroids are natural plant steroids that possess medicinal, pharmacological, and biological properties, with no adverse side effects in mammals. Previous research has demonstrated that administration of phytoecdysteroids, such as 20-hydroxyecdysone (20E), leads to an increase in protein synthesis signaling and skeletal muscle strength. Methods: To investigate whether 20E enhances skeletal muscle recovery from eccentric contraction-induced damage, adult (7-8 mo) and old (26-27 mo) mice were subjected to injurious eccentric contractions (EC), followed by 20E or placebo (PLA) supplementation for 7 days. Contractile function via torque-frequency relationships (TF) was measured three times in each mouse: pre- and post-EC, as well as after the 7-day recovery period. Mice were anesthetized with isoflurane and then electrically-stimulated isometric contractions were performed to obtain in vivo muscle function of the anterior crural muscle group before injury (pre), followed by 150 EC, and then again post-injury (post). Following recovery from anesthesia, mice received either 20E (50 mg•kg-1 BW) or PLA by oral gavage. Mice were gavaged daily for 6 days and on day 7, the TF relationship was reassessed (7-day). Results: EC resulted in significant reductions of muscle function post-injury, regardless of age or treatment condition (p < 0.001). 20E supplementation completely recovered muscle function after 7 days in both adult and old mice (pre vs. 7-day; p > 0.05), while PLA muscle function remained reduced (pre vs. 7-day; p < 0.01). In addition, histological markers of muscle damage appear lower in damaged muscle from 20E-treated mice after the 7-day recovery period, compared to PLA. Conclusions: Taken together, these findings demonstrate that 20E fully recovers skeletal muscle function in both adult and old mice just 7 days after eccentric contraction-induced damage. However, the underlying mechanics by which 20E contributes to the accelerated recovery from muscle damage warrant further investigation.

Phytoecdysteroids Do Not Have Anabolic Effects in Skeletal Muscle in Sedentary Aging Mice.

Skeletal muscle mass and strength are lost with aging. Phytoecdysteroids, in particular 20-hydroxyecdysone (20E), increase protein synthesis in C2C12 skeletal muscle cells and muscle strength in young rats. The objective of this study was to determine whether an extract from Ajuga turkestanica (ATE), enriched in phytoecdysteroids, and 20E affect skeletal muscle mass and fiber size, fiber type, activation of the PI3K-Akt signaling pathway, and the mRNA levels of MAFbx, MuRF-1, and myostatin in sedentary aging mice. Aging male C57BL/6 mice (20 months old) received ATE, 20E, or vehicle (CT) once per day for 28 days or a single acute dose. Treatment did not alter body, muscle, or organ mass; fiber cross-sectional area; or fiber type in the triceps brachii or plantaris muscles. Likewise, protein synthesis signaling markers (i.e., phosphorylation of AktSer473 and p70S6kThr389) measured after either 28 days or acutely were unchanged. Neither ATE nor 20E treatment for 28 days affected the mRNA levels of MAFbx, MuRF-1, and myostatin. In conclusion, these data indicate that phytoecdysteroid treatment does not alter muscle mass or fiber type, nor does it activate protein synthesis signaling in the skeletal muscle of sedentary aging mice.

Daily watermelon consumption decreases plasma sVCAM-1 levels in overweight and obese postmenopausal women.

Postmenopausal status is associated with an increase in total and abdominal body fat as well as increased incidence of insulin resistance and cardiovascular disease. The purpose of this study was to determine if watermelon supplementation affects select systemic markers of atherosclerosis and measures of insulin resistance in overweight and obese postmenopausal women. We hypothesized that overweight and obese postmenopausal women consuming 100% watermelon puree daily for 6 weeks would have improved levels of select systemic markers connected with cardiovascular disease without changing markers of insulin resistance. To test this hypothesis, overweight and obese postmenopausal women were recruited to participate in this study. Participants were randomly assigned to either the control group (no intervention) or the watermelon puree group (WM) for 6 weeks. Plasma concentration of markers connected with atherosclerosis and glycemic control were measured pre- and poststudy. A significant 6% decrease in soluble vascular cell adhesion molecule-1 occurred pre- to poststudy in WM, P = .003. The pattern of change in fasting blood glucose (P = .633), insulin (P = .158), and homeostatic model assessment-estimated insulin resistance (P = .174) did not differ between groups. Pre- to poststudy increases were measured in the fasting plasma concentration of l-arginine (8%, P = .005), cis-lycopene (32%, P = .003), and trans-lycopene (42%, P = .003) in WM. We conclude that 6 weeks of watermelon supplementation improved soluble vascular cell adhesion molecule-1 levels, a marker connected to atherogenesis, independent of changes in body composition or glycemic control.

IGF-I activates the mouse type IIb myosin heavy chain gene.

IGF-I increases skeletal muscle mass, but whether IGF-I increases type IIb myosin heavy chain (MyHC) transcriptional activity is not known. C2C12 myotubes were cultured with or without IGF-I to determine whether IGF-I increases type IIb MyHC promoter activity, and if so, what region of the promoter might IGF-I signaling regulate. At differentiation days 3 and 4, IGF-I increased type IIb MyHC mRNA and mouse 3.0-kb type IIb MyHC promoter activity. Deletion construct studies identified a potential IGF-I-responsive region between 1.25 and 1.2 kb of the type IIb MyHC promoter, which contained an exact 6-bp T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding site at position -1206 to -1201. Site-specific mutation of the putative Tcf/Lef binding site reduced IGF-I-induced 1.3-kb type IIb MyHC promoter activity. To identify potential IGF-I signaling molecules, the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY-294002 were both found to markedly attenuate IGF-I activation of the 1.3-kb type IIb MyHC promoter. Downstream signaling of IGF-I can phosphorylate and inactivate GSK-3beta, thereby enhancing beta-catenin protein. The GSK-3beta inhibitor, LiCl, dramatically enhanced IGF-I induction of the 1.3-kb type IIb MyHC promoter, and constitutively active GSK-3beta attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity. Finally, IGF-I increased nuclear beta-catenin protein, and small interfering RNA knockdown of beta-catenin attenuated IGF-I-induced 1.3-kb type IIb MyHC promoter activity and type IIb MyHC mRNA. In summary, IGF-I stimulation of C2C12 myotubes increases mouse type IIb MyHC promoter activity, likely through signaling of PI3K, GSK-3beta, beta-catenin, and a Tcf/Lef binding site at -1,206 to -1,201 bp in the promoter.

Quercetin and Green Tea Extract Supplementation Downregulates Genes Related to Tissue Inflammatory Responses to a 12-Week High Fat-Diet in Mice.

Quercetin (Q) and green tea extract (E) are reported to counter insulin resistance and inflammation and favorably alter fat metabolism. We investigated whether a mixture of E + Q (EQ) could synergistically influence metabolic and inflammation endpoints in a high-fat diet (HFD) fed to mice. Male C57BL/6 mice (n = 40) were put on HFD (fat = 60%kcal) for 12 weeks and randomly assigned to Q (25 mg/kg of body weight (BW)/day), E (3 mg of epigallocatechin gallate/kg BW/day), EQ, or control groups for four weeks. At 16 weeks, insulin sensitivity was measured via the glucose tolerance test (GTT), followed by area-under-the-curve (AUC) estimations. Plasma cytokines and quercetin were also measured, along with whole genome transcriptome analysis and real-time polymerase chain reaction (qPCR) on adipose, liver, and skeletal muscle tissues. Univariate analyses were conducted via analysis of variance (ANOVA), and whole-genome expression profiles were examined via gene set enrichment. At 16 weeks, plasma quercetin levels were higher in Q and EQ groups vs. the control and E groups (p < 0.05). Plasma cytokines were similar among groups (p > 0.05). AUC estimations for GTT was 14% lower for Q vs. E (p = 0.0311), but non-significant from control (p = 0.0809). Genes for cholesterol metabolism and immune and inflammatory response were downregulated in Q and EQ groups vs. control in adipose tissue and soleus muscle tissue. These data support an anti-inflammatory role for Q and EQ, a result best captured when measured with tissue gene downregulation in comparison to changes in plasma cytokine levels.

A Mixed Flavonoid-Fish Oil Supplement Induces Immune-Enhancing and Anti-Inflammatory Transcriptomic Changes in Adult Obese and Overweight Women-A Randomized Controlled Trial.

Flavonoids and fish oils have anti-inflammatory and immune-modulating influences. The purpose of this study was to determine if a mixed flavonoid-fish oil supplement (Q-Mix; 1000 mg quercetin, 400 mg isoquercetin, 120 mg epigallocatechin (EGCG) from green tea extract, 400 mg n3-PUFAs (omega-3 polyunsaturated fatty acid) (220 mg eicosapentaenoic acid (EPA) and 180 mg docosahexaenoic acid (DHA)) from fish oil, 1000 mg vitamin C, 40 mg niacinamide, and 800 µg folic acid) would reduce complications associated with obesity; that is, reduce inflammatory and oxidative stress markers and alter genomic profiles in overweight women. Overweight and obese women (n = 48; age = 40-70 years) were assigned to Q-Mix or placebo groups using randomized double-blinded placebo-controlled procedures. Overnight fasted blood samples were collected at 0 and 10 weeks and analyzed for cytokines, C-reactive protein (CRP), F2-isoprostanes, and whole-blood-derived mRNA, which was assessed using Affymetrix HuGene-1_1 ST arrays. Statistical analysis included two-way ANOVA models for blood analytes and gene expression and pathway and network enrichment methods for gene expression. Plasma levels increased with Q-Mix supplementation by 388% for quercetin, 95% for EPA, 18% for DHA, and 20% for docosapentaenoic acid (DPA). Q-Mix did not alter plasma levels for CRP (p = 0.268), F2-isoprostanes (p = 0.273), and cytokines (p > 0.05). Gene set enrichment analysis revealed upregulation of pathways in Q-Mix vs. placebo related to interferon-induced antiviral mechanism (false discovery rate, FDR < 0.001). Overrepresentation analysis further disclosed an inhibition of phagocytosis-related inflammatory pathways in Q-Mix vs. placebo. Thus, a 10-week Q-Mix supplementation elicited a significant rise in plasma quercetin, EPA, DHA, and DPA, as well as stimulated an antiviral and inflammation whole-blood transcriptomic response in overweight women.

Comparison of Watermelon and Carbohydrate Beverage on Exercise-Induced Alterations in Systemic Inflammation, Immune Dysfunction, and Plasma Antioxidant Capacity.

Consuming carbohydrate- and antioxidant-rich fruits during exercise as a means of supporting and enhancing both performance and health is of interest to endurance athletes. Watermelon (WM) contains carbohydrate, lycopene, l-citrulline, and l-arginine. WM may support exercise performance, augment antioxidant capacity, and act as a countermeasure to exercise-induced inflammation and innate immune changes. Trained cyclists (n = 20, 48 ± 2 years) participated in a randomized, placebo controlled, crossover study. Subjects completed two 75 km cycling time trials after either 2 weeks ingestion of 980 mL/day WM puree or no treatment. Subjects drank either WM puree containing 0.2 gm/kg carbohydrate or a 6% carbohydrate beverage every 15 min during the time trials. Blood samples were taken pre-study and pre-, post-, 1 h post-exercise. WM ingestion versus no treatment for 2-weeks increased plasma l-citrulline and l-arginine concentrations (p < 0.0125). Exercise performance did not differ between WM puree or carbohydrate beverage trials (p > 0.05), however, the rating of perceived exertion was greater during the WM trial (p > 0.05). WM puree versus carbohydrate beverage resulted in a similar pattern of increase in blood glucose, and greater increases in post-exercise plasma antioxidant capacity, l-citrulline, l-arginine, and total nitrate (all p < 0.05), but without differences in systemic markers of inflammation or innate immune function. Daily WM puree consumption fully supported the energy demands of exercise, and increased post-exercise blood levels of WM nutritional components (l-citrulline and l-arginine), antioxidant capacity, and total nitrate, but without an influence on post-exercise inflammation and changes in innate immune function.

Long-term quercetin dietary enrichment decreases muscle injury in mdx mice.

BACKGROUND & AIMS: Duchenne muscular dystrophy results from a mutation in the dystrophin gene, which leads to a dystrophin-deficiency. Dystrophic muscle is marked by progressive muscle injury and loss of muscle fibers. Activation of the PGC-1α pathway has been previously shown to decrease disease-related muscle damage. Oral administration of the flavonol, quercetin, appears to be an effective and safe method to activate the PGC-1α pathway. The aim of this investigation was to determine the extent to which long term dietary quercetin enrichment would decrease muscle injury in dystrophic skeletal muscle. We hypothesized that a quercetin enriched diet would rescue dystrophic muscle from further decline and increase utrophin abundance. METHODS: Beginning at three-months of age and continuing to nine-months of age mdx mice (n = 10/group) were assigned to either to mdx-control receiving standard chow or to mdx-quercetin receiving a 0.2% quercetin-enriched diet. At nine-months of age mice were sacrificed and costal diaphragms collected. One hemidiaphragm was used for histological analysis and the second hemidiaphragm was used to determine gene expression via RT-qPCR. RESULTS: The diaphragm from the mdx-quercetin group had 24% (p ≤ 0.05) more muscle fibers/area and 34% (p ≤ 0.05) fewer centrally nucleated fibers compared to the mdx-control group. Further, there were 44% (p ≤ 0.05) fewer infiltrating immune cells/area, a corresponding 31% (p ≤ 0.05) reduction in TNF gene expression, and a near 50% reduction in fibrosis. The quercetin-enriched diet increased expression of genes associated with oxidative metabolism but did not increase utrophin protein abundance. CONCLUSIONS: Long-term quercetin supplementation decreased disease-related muscle injury in dystrophic skeletal muscle; however the role of PGC-1α pathway activation as a mediator of this response is unclear.

Ultrasonic assessment of exercise-induced change in skeletal muscle glycogen content.

BACKGROUND: Ultrasound imaging is a valuable tool in exercise and sport science research, and has been used to visualize and track real-time movement of muscles and tendons, estimate hydration status in body tissues, and most recently, quantify skeletal muscle glycogen content. In this validation study, direct glycogen quantification from pre-and post-exercise muscle biopsy samples was compared with glycogen content estimates made through a portable, diagnostic high-frequency ultrasound and cloud-based software system (MuscleSound®, Denver, CO). METHODS: Well-trained cyclists (N = 20, age 38.4 ± 6.0 y, 351 ± 57.6 wattsmax) participated in a 75-km cycling time trial on their own bicycles using CompuTrainer Pro Model 8001 trainers (RacerMate, Seattle, WA). Muscle biopsy samples and ultrasound measurements were acquired pre- and post-exercise. Specific locations on the vastus lateralis were marked, and a trained technician used a 12 MHz linear transducer and a standard diagnostic high resolution GE LOGIQ-e ultrasound machine (GE Healthcare, Milwaukee, WI) to make three ultrasound measurements. Ultrasound images were pre-processed to isolate the muscle area under analysis, with the mean pixel intensity averaged from the three scans and scaled (0 to 100 scale) to create the glycogen score. Pre- and post-exercise muscle biopsy samples were acquired at the vastus lateralis location (2 cm apart) using the suction-modified percutaneous needle biopsy procedure, and analyzed for glycogen content. RESULTS: The 20 cyclists completed the 75-km cycling time trial in 168 ± 26.0 minutes at a power output of 193 ± 57.8 watts (54.2 ± 9.6% wattsmax). Muscle glycogen decreased 77.2 ± 17.4%, with an absolute change of 71.4 ± 23.1 mmol glycogen per kilogram of muscle. The MuscleSound® change score at the vastus lateralis site correlated highly with change in measured muscle glycogen content (R = 0.92, P < 0.001). CONCLUSIONS: MuscleSound® change scores acquired from an average of three ultrasound scans at the vastus lateralis site correlated significantly with change in vastus lateralis muscle glycogen content. These data support the use of the MuscleSound® system for accurately and non-invasively estimating exercise-induced decreases in vastus lateralis skeletal muscle glycogen content.

Rhodiola rosea Exerts Antiviral Activity in Athletes Following a Competitive Marathon Race.

Rhodiola rosea, a medicinal plant with demonstrated adaptogenic properties, has recently been reported to contain active compounds with antimicrobial activity. The goal of this study was to measure the antiviral and antibacterial properties of the bioactive metabolites of Rhodiola rosea in the serum of experienced marathon runners following supplementation. Marathon runners, randomly divided into two groups, ingested 600 mg/day of Rhodiola rosea (n = 24, 6 female, 18 male) or placebo (n = 24, 7 females, 17 males) for 30 days prior to, the day of, and 7 days post-marathon. Blood serum samples were collected the day before, 15 min post-, and 1.5 h post-marathon. Serum from Rhodiola rosea-supplemented runners collected after marathon running did not attenuate the marathon-induced susceptibility of HeLa cells to killing by vesicular stomatitis virus. However, the use of Rhodiola rosea induced antiviral activity at early times post-infection by delaying an exercise-dependent increase in virus replication (P = 0.013 compared to placebo). Serum from both groups collected 15 min post-marathon significantly promoted the growth of Escherichia coli in culture as compared to serum collected the day before the marathon (P = 0.003, all subjects). Furthermore, the serum from subjects ingesting Rhodiola rosea did not display antibacterial properties at any time point as indicated by a lack of group differences immediately (P = 0.785) or 1.5 h (P = 0.633) post-marathon. These results indicate that bioactive compounds in the serum of subjects ingesting Rhodiola rosea may exert protective effects against virus replication following intense and prolonged exercise by inducing antiviral activity.