Inflammation and oxidative stress are lower in physically fit and active adults.

The objective of this study was to determine if the inverse relationship between perceived physical fitness (pFIT) and exercise frequency (ExFreq) levels and chronic inflammation and oxidative stress exists after making statistical adjustments for confounders including body mass index (BMI), age, gender, and cigarette smoking. Study participants (60% female and 40% male; n = 998) varied widely in age (18-85 years) and BMI (16.7-52.7 kg/m(2)) completed an extensive medical/health and lifestyle questionnaire, and data were used to establish pFIT and ExFreq tertiles. Biomarkers included serum C-reactive protein (CRP), total blood leukocytes, five plasma cytokines [interleukin (IL)-6, IL-10, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP1), and granulocyte colony-stimulating factor (GCSF)], F2 -isoprostanes, ferric reducing ability of plasma (FRAP), and oxygen radical absorbance capacity (ORAC). A general linear model was used to examine relationships between pFIT and ExFreq with inflammation and oxidative stress while controlling for age, gender, BMI, and smoking. Benjamini-Hochberg method for false discovery rate correction was used for multiple testing corrections. Significant tests (P < 0.05) for trend were found for the effect of pFIT and ExFreq on CRP, white blood cell, IL-6, TNF-α, GCSF, and F2 -isoprostanes, but not MCP1, IL-10, FRAP, and ORAC, after adjustment for confounders. These data indicate that an inverse relationship exists among chronic inflammation, oxidative stress, and pFIT and ExFreq at the community level even after adjustment for important confounders.

Effects of vitamin E and alpha-lipoic acid on skeletal muscle contractile properties.

Initial experiments were conducted using an in situ rat tibialis anterior (TA) muscle preparation to assess the influence of dietary antioxidants on muscle contractile properties. Adult Sprague-Dawley rats were divided into two dietary groups: 1) control diet (Con) and 2) supplemented with vitamin E (VE) and alpha-lipoic acid (alpha-LA) (Antiox). Antiox rats were fed the Con rats' diet (AIN-93M) with an additional 10,000 IU VE/kg diet and 1.65 g/kg alpha-LA. After an 8-wk feeding period, no differences existed (P > 0.05) between the two dietary groups in maximum specific tension before or after a fatigue protocol or in force production during the fatigue protocol. However, in unfatigued muscle, maximal twitch tension and tetanic force production at stimulation frequencies < or = 40 Hz were less (P < 0.05) in Antiox animals compared with Con. To investigate which antioxidant was responsible for the depressed force production, a second experiment was conducted using an in vitro rat diaphragm preparation. Varying concentrations of VE and dihydrolipoic acid, the reduced form of alpha-LA, were added either individually or in combination to baths containing diaphragm muscle strips. The results from these experiments indicate that high levels of VE depress skeletal muscle force production at low stimulation frequencies.

Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

These experiments examined the independent effects of short-term exercise and heat stress on myocardial responses during in vivo ischemia-reperfusion (I/R). Female Sprague-Dawley rats (4 mo old) were randomly assigned to one of four experimental groups: 1) control, 2) 3 consecutive days of treadmill exercise [60 min/day at 60-70% maximal O2 uptake (VO2 max)], 3) 5 consecutive days of treadmill exercise (60 min/day at 60-70% VO2 max), and 4) whole body heat stress (15 min at 42 degrees C). Twenty-four hours after heat stress or exercise, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was maintained for 30-min followed by a 30-min period of reperfusion. Compared with control, both heat-stressed animals and exercised animals (3 and 5 days) maintained higher (P < 0.05) left ventricular developed pressure (LVDP), maximum rate of left ventricular pressure development (+dP/dt), and maximum rate of left ventricular pressure decline (-dP/dt) at all measurement periods during both ischemia and reperfusion. No differences existed between heat-stressed and exercise groups in LVDP, +dP/dt, and -dP/dt at any time during ischemia or reperfusion. Both heat stress and exercise resulted in an increase (P < 0.05) in the relative levels of left ventricular heat shock protein 72 (HSP72). Furthermore, exercise (3 and 5 days) increased (P < 0.05) myocardial glutathione levels and manganese superoxide dismutase activity. These data indicate that 3-5 consecutive days of exercise improves myocardial contractile performance during in vivo I/R and that this exercise-induced myocardial protection is associated with an increase in both myocardial HSP72 and cardiac antioxidant defenses.

Vitamin E deficiency fails to affect myocardial performance during in vivo ischemia-reperfusion.

Vitamin E content of cardiac tissue has been proposed to play a major role in the damage caused by myocardial ischemia-reperfusion (I-R). Previous studies using in vitro models have examined vitamin E deficiency and I-R-induced myocardial damage with equivocal results. The purpose of this study was to use an in vivo model of myocardial I-R to determine the effects of vitamin E deficiency on myocardial I-R-induced damage. Female Sprague-Dawley rats (4-mo old) were assigned to either: 1) control diet (CON), or 2) vitamin E deficient diet (VE-DEF). The CON diet was prepared to meet AIN-93M standards, which contains 75 IU vitamin E/kg diet. The VE-DEF diet was the AIN-93M diet prepared with tocopherol stripped corn oil and no vitamin E. Following a 14-week feeding period, significant differences (p < 0.05) existed in mean myocardial VE levels between groups (mean values +/- SEM: CON = 48.2 +/- 3.5; VE-DEF = 12.4 +/- 1.4 micrograms VE/g wet weight). Animals from both experimental groups were subjected to an in vivo I-R protocol consisting of 25 minutes of left coronary artery occlusion followed by 10 minutes of reperfusion. No group differences (p > 0.05) existed in cardiac performance (peak arterial pressure or ventricular work) or the incidence of ventricular arrhythmias during the I-R protocol. VE-DEF animals had significantly higher (p < 0.05) levels of myocardial lipid peroxidation and lower (p < 0.05) protein thiols following I-R compared to the CON animals. These data suggest that although vitamin E deficiency increases oxidative damage resulting from myocardial I-R, it does not affect cardiac performance during the insult.

The homoeobox gene SIX1 alters myosin heavy chain isoform expression in mouse skeletal muscle.

AIM: Six1 is necessary for the genesis of several tissues, but in adults, it is expressed primarily in skeletal muscle where its function is unclear. Overexpression of Six1 with a cofactor in skeletal muscle causes slow-to-fast fibre-type transition. We sought to characterize the effects of a physiologically relevant Six1 knockdown. METHODS: The tibialis anterior (TA) muscles of C57BL/6 mice were electroporated with Six1 knockdown vector (siRNA) or empty vector. Muscles were collected at 2 or 14 days after transfection for Six1 and myosin heavy chain (MHC) expression analysis. C2C12 mouse myoblasts were grown in standard conditions. Cells were cotransfected with MHC promoter vectors and Six1 expression vectors. Cells were harvested after 4 days of differentiation. RESULTS: In vivo, the Six1 siRNA caused a decreased expression of Six1,1.8-fold (±0.1, P < 0.05). With decreased Six1, MHC IIB expression decreased 2.7-fold (±0.7, P = 0.04). Proportion of muscle fibres expressing MHC IIB decreased (45.3 ± 4.8% vs. 65.1 ± 7.3% in control group, P = 0.04), and total area expressing MHC IIB decreased with decreased Six1 (59.6 ± 4.3% vs. 75.2 ± 5.4% in control group, P < 0.05). Decreased Six1 increased MHC IIA expression 1.9-fold (±0.3, P = 0.04). In vitro, Six1 overexpression increased promoter activation of MHC IIB 2.9-fold (±0.3, P < 0.01). Six1 knockdown repressed MHC IIB promoter 2.9-fold (±0.1, P < 0.05) and MHC IIX 3.7-fold (±0.08, P < 0.01). CONCLUSION: Six1 knockdown caused a fast-to-slow shift in MHC isoform, and this was confirmed by promoter activity of MHC genes. Six1 may ultimately control the contractile and metabolic properties that define muscle fibre phenotype.

Ajuga turkestanica increases Notch and Wnt signaling in aged skeletal muscle.

BACKGROUND: The declining myogenic potential of aged skeletal muscle is multifactorial. Insufficient satellite cell activity is one factor in this process. Notch and Wnt signaling are involved in various biological processes including orchestrating satellite cell activity within skeletal muscle. These pathways become dysfunctional during the aging process and may contribute to the poor skeletal muscle competency. Phytoecdysteroids are natural adaptogenic compounds with demonstrated benefit on skeletal muscle. AIM: To determine the extent to which a phytoecdysteroid enriched extract from Ajuga turkestanica (ATE) affects Notch and Wnt signaling in aged skeletal muscle. MATERIALS AND METHODS: Male C57BL/6 mice (20 months) were randomly assigned to Control (CT) or ATE treatment groups. Chow was supplemented with either vehicle (CT) or ATE (50 mg/kg/day) for 28 days. Following supplementation, the triceps brachii muscles were harvested and immunohistochemical analyses performed. Components of Notch or Wnt signaling were co-labelled with Pax7, a quiescent satellite cell marker. RESULTS: ATE supplementation significantly increased the percent of active Notch/Pax7+ nuclei (p = 0.005), Hes1/Pax7+ nuclei (p = 0.038), active B-catenin/Pax7+ nuclei (p = 0.011), and Lef1/Pax7+ nuclei (p = 0.022), compared to CT. ATE supplementation did not change the resting satellite cell number. CONCLUSIONS: ATE supplementation in aged mice increases Notch and Wnt signaling in triceps brachii muscle. If Notch and Wnt benefit skeletal muscle, then phytoecdysteroids may provide a protective effect and maintain the integrity of aged skeletal muscle.

The variable plasma quercetin response to 12-week quercetin supplementation in humans.

BACKGROUND/OBJECTIVES: Quercetin supplementation results in a variable plasma quercetin response in humans. The purpose of this study was to determine whether this variance is related to gender, age, body mass index (BMI), and other demographic and lifestyle factors. SUBJECTS/METHODS: Subjects (N=1002, ages 18-85 years, 60% female and 40% male) were recruited from the community and randomized to one of three groups, with supplements administered using double-blinded procedures: Q-500 (500 mg/day), Q-1000 (1000 mg/day), or placebo. Subjects ingested two soft chew supplements twice daily during the 12-week study. Fasting blood samples were obtained pre- and post-study, analyzed for plasma quercetin, and then compared between and within groups by gender, age group (<40, 40-59, and >or=60 years), BMI (<25, 25-29.9, and >or=30 kg/m(2)), self-reported physical fitness level, and diet intake (food group servings). RESULTS: Quercetin supplementation over 12 weeks caused a significant increase in overnight-fasted plasma quercetin, with a net increase of 332+/-21.0 and 516+/-30.8 microg/l for Q-500 and Q-1000 compared with 53.6+/-6.4 microg/l for placebo (interaction effect, P<0.001). The increase in plasma quercetin was highly variable within each quercetin supplementation group, but was unrelated to age, gender, BMI, fitness levels, or diet intake. CONCLUSIONS: In summary, quercetin supplementation in doses of 500 and 1000 mg/day caused large but highly variable increases in plasma quercetin that were unrelated to demographic or lifestyle factors.

Lactic acidosis and diaphragmatic function in vitro.

Diaphragm fatigue occurs during heavy exercise. Acidosis leads to skeletal muscle fatigue, yet the diaphragm is not a net producer of either lactic acid or hydrogen ions. We tested the hypothesis that hydrogen ion and lactic acid concentrations similar to those seen in arterial blood at maximal exercise decrease contractility of the in vitro isolated rat diaphragm. Diaphragm strips were exposed to a control solution for 15 min and then to one of the following treatment solutions: control (C, pH = 7.4) or 10 mM lactic acid buffered to pH 7.4 (L74), pH 7.2 (L72), pH 7.1 (L71), or pH 6.8 (L68). After 15 min, the force-frequency relationship of the strip was measured. The strips were then stressed with 75 contractions at 25 Hz (250-ms train duration) at the rate of one per second and the force-frequency curve was measured after 15 min of recovery. The L74, L72, and L71 strips responded similarly to the C strips at all times and frequencies. Decrements in force associated with acidosis were only seen in L68. Within L68, we found decreases in force at stimulation frequencies < 100 Hz. These data suggest that physiologic levels of exogenous hydrogen ions are not a primary cause of in vitro diaphragm fatigue.

Obesity is associated with increased myocardial oxidative stress.

OBJECTIVE: To determine: 1) whether obesity predisposes the myocardium to oxidative stress as evidenced by higher tissue levels of myocardial lipid peroxidation, and 2) what cellular mechanisms are responsible for this predisposition. DESIGN: Comparative, descriptive study of the myocardial tissue of lean and obese Fatty Zucker animals. ANIMALS: 12 month old lean (-/fa; n = 6; mean body weight = 590 g) and obese (fa/fa; na = 7; mean body weight= 882 g) male Fatty Zucker rats. MEASUREMENTS: Basal lipid peroxidation (assessed using thiobarbituric reactive acid substances (TBARS) and cumene hydroperoxide equivalents), oxidative and antioxidant enzyme activities (citrate synthase (CS), superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT), thiol content, heat shock protein expression (HSP72/73) and TBARS concentrations following an iron-mediated challenge in vitro. RESULTS: Compared to lean, lipid peroxidation was greater (P < 0.05) in the left ventricle (LV) from obese rats as indicated by higher levels of lipid hydroperoxides (mean = 11.48 vs 13.7 cumene hydroperoxide equivalents (CHPE)/mg lipid) and TBARS (mean = 11.1 vs 13.9 nMol/mg lipid.). The activity of the manganese isoform of superoxide dismutase in the LV was higher (P < 0.05) in obese animals, compared to controls (mean = 135 vs 117 U/mg protein). In contrast, LV catalase and glutathione peroxidase activities did not differ (P > 0.05) between groups. Also, LV levels of HSP 72 (inducible) and 73 (constitutive) did not differ (P > 0.05)( between lean and obese animals. Following an iron-stimulated oxidative challenge in vitro, TBARS concentration was significantly greater (P < 0.05) in LV of obese rats compared to the lean (mean = 12.7 vs 16.7 nMol/mg lipid). CONCLUSIONS: These results support the notion that obesity predisposes the myocardium to oxidative stress. However, the postulate that obesity is associated with elevated myocardial antioxidant enzyme activities and HSPs was only partially supported by these findings.

Short-term exercise training improves diaphragm antioxidant capacity and endurance.

These experiments tested the hypothesis that short-term endurance exercise training would rapidly improve (within 5 days) the diaphragm oxidative/antioxidant capacity and protect the diaphragm against contraction-induced oxidative stress. To test this postulate, male Sprague-Dawley rats (6 weeks old) ran on a motorized treadmill for 5 consecutive days (40-60 min x day(-1)) at approximately 65% maximal oxygen uptake. Costal diaphragm strips were excised from both sedentary control (CON, n=14) and trained (TR, n=13) animals 24 h after the last exercise session, for measurement of in vitro contraction properties and selected biochemical parameters of oxidative/antioxidant capacity. Training did not alter diaphragm force-frequency characteristics over a full range of submaximal and maximal stimulation frequencies (P > 0.05). In contrast, training improved diaphragm resistance to fatigue as contraction forces were better-maintained by the diaphragms of the TR animals during a submaximal 60-min fatigue protocol (P < 0.05). Following the fatigue protocol, diaphragm strips from the TR animals contained 30% lower concentrations of lipid hydroperoxides compared to CON (P < 0.05). Biochemical analysis revealed that exercise training increased diaphragm oxidative and antioxidant capacity (citrate synthase activity +18%, catalase activity +24%, total superoxide dismutase activity +20%, glutathione concentration +10%) (P < 0.05). These data indicate that short-term exercise training can rapidly elevate oxidative capacity as well as enzymatic and non-enzymatic antioxidant defenses in the diaphragm. Furthermore, this up-regulation in antioxidant defenses would be accompanied by a reduction in contraction-induced lipid peroxidation and an increased fatigue resistance.

Exercise training improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

Experimental studies examining the effects of regular exercise on cardiac responses to ischemia and reperfusion (I/R) are limited. Therefore, these experiments examined the effects of endurance exercise training on myocardial biochemical and physiological responses during in vivo I/R. Female Sprague-Dawley rats (4 mo old) were randomly assigned to either a sedentary control group or to an exercise training group. After a 10-wk endurance exercise training program, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was achieved by a ligature around the left coronary artery; occlusion was maintained for 20 min, followed by a 10-min period of reperfusion. Compared with untrained, exercise-trained animals maintained higher (P < 0.05) peak systolic blood pressure throughout I/R. Training resulted in a significant (P < 0.05) increase in ventricular nonprotein thiols, heat shock protein (HSP) 72, and the activities of superoxide dismutase (SOD), phosphofructokinase (PFK), and lactate dehydrogenase. Furthermore, compared with untrained controls, left ventricles from trained animals exhibited lower levels (P < 0. 05) of lipid peroxidation after I/R. These data demonstrate that endurance exercise training improves myocardial contractile performance and reduces lipid peroxidation during I/R in the rat in vivo. It appears likely that the improvement in the myocardial responses to I/R was related to training-induced increases in nonprotein thiols, HSP72, and the activities of SOD and PFK in the myocardium.

Effect of combined supplementation with vitamin E and alpha-lipoic acid on myocardial performance during in vivo ischaemia-reperfusion.

Reactive oxygen species (ROS) contribute significantly to myocardial ischaemia-reperfusion (I-R) injury. Recently the combination of the antioxidants vitamin E (VE) and alpha-lipoic acid (alpha-LA) has been reported to improve cardiac performance and reduce myocardial lipid peroxidation during in vitro I-R. The purpose of these experiments was to investigate the effects of VE and alpha-LA supplementation on cardiac performance, incidence of dysrhythmias and biochemical alterations during an in vivo myocardial I-R insult. Female Sprague-Dawley rats (4-months old) were assigned to one of the two dietary treatments: (1) control diet (CON) or (2) VE and alpha-LA supplementation (ANTIOXID). The CON diet was prepared to meet AIN-93M standards, which contains 75 IU VE kg-1 diet. The ANTIOXID diet contained 10 000 IU VE kg(-1) diet and 1.65 g alpha-LA kg(-1) diet. After the 14-week feeding period, significant differences (P<0.05) existed in mean myocardial VE levels between dietary groups. Animals in each experimental group were subjected to an in vivo I-R protocol which included 25 min of left anterior coronary artery occlusion followed by 10 min of reperfusion. No group differences (P>0.05) existed in cardiac performance (e.g. peak arterial pressure or ventricular work) or the incidence of ventricular dysrhythmias during the I-R protocol. Following I-R, two markers of lipid peroxidation were lower (P<0.05) in the ANTIOXID animals compared with CON. These data indicate that dietary supplementation of the antioxidants, VE and alpha-LA do not influence cardiac performance or the incidence of dysrhythmias but do decrease lipid peroxidation during in vivo I-R in young adult rats.

Improved cardiac performance after ischemia in aged rats supplemented with vitamin E and alpha-lipoic acid.

The purpose of these experiments was to examine the effects of dietary antioxidant supplementation with vitamin E (VE) and alpha-lipoic acid (alpha-LA) on biochemical and physiological responses to in vivo myocardial ischemia-reperfusion (I-R) in aged rats. Male Fischer-334 rats (18 mo old) were assigned to either 1) a control diet (CON) or 2) a VE and alpha-LA supplemented diet (ANTIOX). After a 14-wk feeding period, animals in each group underwent an in vivo I-R protocol (25 min of myocardial ischemia and 15 min of reperfusion). During reperfusion, peak arterial pressure was significantly higher (P < 0.05) in ANTIOX animals compared with CON diet animals. I-R resulted in a significant increase (P < 0.05) in myocardial lipid peroxidation in CON diet animals but not in ANTIOX animals. Compared with ANTIOX animals, heart homogenates from CON animals experienced significantly less (P < 0.05) oxidative damage when exposed to five different in vitro radical producing systems. These data indicate that dietary supplementation with VE and alpha-LA protects the aged rat heart from I-R-induced lipid peroxidation by scavenging numerous reactive oxygen species. Importantly, this protection is associated with improved cardiac performance during reperfusion.