Mazen J. Hamadeh

Aberrant Mitochondrial Homeostasis in the Skeletal Muscle of Sedentary Older Adults

The role of mitochondrial dysfunction and oxidative stress has been extensively characterized in the aetiology of sarcopenia (aging-associated loss of muscle mass) and muscle wasting as a result of muscle disuse. What remains less clear is whether the decline in skeletal muscle mitochondrial oxidative capacity is purely a function of the aging process or if the sedentary lifestyle of older adult subjects has confounded previous reports. The objective of the present study was to investigate if a recreationally active lifestyle in older adults can conserve skeletal muscle strength and functionality, chronic systemic inflammation, mitochondrial biogenesis and oxidative capacity, and cellular antioxidant capacity. To that end, muscle biopsies were taken from the vastus lateralis of young and age-matched recreationally active older and sedentary older men and women (N = 10/group; ♀  =  ♂). We show that a physically active lifestyle is associated with the partial compensatory preservation of mitochondrial biogenesis, and cellular oxidative and antioxidant capacity in skeletal muscle of older adults. Conversely a sedentary lifestyle, associated with osteoarthritis-mediated physical inactivity, is associated with reduced mitochondrial function, dysregulation of cellular redox status and chronic systemic inflammation that renders the skeletal muscle intracellular environment prone to reactive oxygen species-mediated toxicity. We propose that an active lifestyle is an important determinant of quality of life and molecular progression of aging in skeletal muscle of the elderly, and is a viable therapy for attenuating and/or reversing skeletal muscle strength declines and mitochondrial abnormalities associated with aging.

The Effect of Aging on Human Skeletal Muscle Mitochondrial and Intramyocellular Lipid Ultrastructure

The purpose of this study was to determine whether ultrastructural changes in intramyocellular lipid (IMCL) and mitochondria occur with aging. Muscle samples were analyzed from 24 young and 20 old, equally active, individuals for IMCL and mitochondria quantity and size as well as their association. Old men had larger IMCL droplets than all other groups in the total muscle area. Old individuals showed higher IMCL content in the subsarcolemmal area. Young participants had a greater number of mitochondria compared with old participants in both fiber regions and greater enzyme activities of cytochrome c oxidase and citrate synthase. The fraction of IMCL touching mitochondria was lowest in old women in the total area and in old men in the subsarcolemmal region. In summary, older adults have larger IMCL droplets, fewer mitochondria, and a lower proportion of IMCL in contact with mitochondria. These factors likely contribute to age-related reductions in mitochondrial function and lipid metabolism.

Endurance training without weight loss lowers systemic, but not muscle, oxidative stress with no effect on inflammation in lean and obese women.

Obesity is associated with oxidative stress. Endurance training (ET) in healthy individuals increases antioxidant enzyme activity and decreases oxidative stress, whereas its effects on oxidative status in obese humans have yet to be determined. We investigated the effects of obesity and ET on markers of oxidative stress, antioxidant defense, and inflammation. Obese (n=12) and lean (n=12) women underwent 12 weeks of ET with blood, 24-h urine, and muscle biopsies collected prior to and following training for determination of oxidative stress (urinary 8-hydroxy-2-deoxyguanosine and 8-isoprostanes, muscle protein carbonyls, and 4-hydroxynonenal), antioxidant enzyme protein content (muscle CuZnSOD, MnSOD, and catalase), and inflammation (C-reactive protein, leptin, adiponectin, interleukin-6). Obese women had elevated urinary 8-hydroxy-2-deoxyguanosine (P=0.03), muscle protein carbonyls (P=0.03), and 4-hydroxynonenal (P<0.001); serum C-reactive protein (P=0.01); and plasma leptin (P=0.0001) and interleukin-6 (P=0.03). ET decreased urinary 8-hydroxy-2-deoxyguanosine (P=0.006) and 8-isoprostanes (P=0.02) in all subjects and CuZnSOD protein content (P=0.04) in obese women, in the absence of changes in body weight or composition. ET without weight loss decreases systemic oxidative stress, but not markers of inflammation, in obese women.

Effect of Endurance Exercise on Hepatic Lipid Content, Enzymes, and Adiposity in Men and Women

Obesity and physical inactivity are independent risk factors for the development of nonalcoholic fatty liver disease (NAFLD). We determined the effect of endurance exercise training on hepatic lipid content and hepatic enzyme concentration in men and women. Waist circumference (WC), percent body fat (BF), computed tomography (CT) scans for liver attenuation (inverse relationship with hepatic lipid), bilirubin, alanine aminotransferase (ALT), and γ‐glutamyltransferase (GGT) plasma concentrations were measured before and after 12 weeks of endurance training in 41 lean and obese men and women. Exercise training did not change liver attenuation, body weight, percent BF, bilirubin, or ALT concentration, but did lower WC (P < 0.0001), and decreased GGT in men only (P = 0.01). Obese subjects had a lower liver attenuation than lean subjects (P = 0.04). Obese women had lower ALT than obese men (P = 0.03). GGT was lower in women before and after training. WC was positively correlated with GGT (r = 0.32, P = 0.003) and ALT (r = 0.320, P = 0.004) and negatively correlated with liver attenuation (r = −0.340, P = 0.03). Percent BF was negatively correlated with bilirubin (r = −0.374, P = 0.005). Liver attenuation was negatively correlated with ALT (r = −0.405, P = 0.003). Short‐term endurance training without weight loss does not alter hepatic lipid content. There was a strong relationship between GGT/ALT and body composition (percent BF) as well as between ALT and hepatic lipid content.

Caloric restriction transiently improves motor performance but hastens clinical onset of disease in the Cu/Zn-superoxide dismutase mutant G93A mouse

Caloric restriction (CR) prolongs lifespan in insects, rodents, and nonhuman primates, a process attributed to a reduction in oxidative stress. Transgenic mice that overexpress the mutant human Cu/Zn‐superoxide dismutase (SOD1) gene (G93A mice) are an animal model of amyotrophic lateral sclerosis showing progressively lower motor neuron weakness and increased oxidative stress. We investigated the effect of CR on motor performance, clinical onset, disease progression, and lifespan in G93A mice. Starting at 40 days of age, 14 separately caged G93A mice were randomly divided into two groups: ad libitum (AL; n = 6) and calorie‐restricted (CR; n = 8) with a diet equal to 60% of AL. The CR mice (mean ± SEM: 14.0 ± 0.7 g) weighed 31% less than the AL mice (20.3 ± 1.0 g) (P = 0.0002). From 74 to 93 days of age, the CR mice performed better on the rotarod than the AL mice: fall time, P = 0.039; fall speed, P = 0.009. The CR mice had a faster rate of reaching clinical onset than the AL mice (hazard ratio = 4.3, P = 0.0006). The CR and AL mice reached clinical onset of disease at age 99 ± 1 and 110 ± 2 days, respectively (P = 0.0003), with no significant difference in disease progression. The CR mice tended to reach endpoint sooner than the AL mice (age‐specific death: 125 ± 3 vs. 133 ± 3 days, respectively, P = 0.09). We conclude that CR diet transiently improves motor performance but hastens clinical onset of disease in G93A mice. These results suggest that CR diet is not a protective strategy for patients with amyotrophic lateral sclerosis (ALS) and hence is contraindicated. Muscle Nerve, 2005

Caloric restriction shortens lifespan through an increase in lipid peroxidation, inflammation and apoptosis in the G93A mouse, an animal model of ALS.

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G93A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G93A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS – citrate synthase content and activity, cytochrome c oxidase - COX activity and protein content of COX subunit- I and IV and UCP3- uncoupling protein 3), oxidative damage (MDA – malondialdehyde and PC – protein carbonyls), antioxidant enzyme capacity (Mn-SOD, Cu/Zn-SOD and catalase), inflammation (TNF-α), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G93A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 13; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (35%), despite a 2.3-fold higher COX-IV/CS protein content. UCP3 was higher in CR vs. AL females only. MnSOD and Cu/Zn-SOD were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (83%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (30%) gastrocnemius. TNF-α was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G93A mice.

Age at menarche in Canada: results from the National Longitudinal Survey of Children & Youth

BackgroundGiven the downward trend in age at menarche and its implications for the reproductive health and wellbeing of women, little is known about menarcheal age in Canada. Most Canadian studies are only representative of specific populations. The present study, therefore, aims to assess the distribution of age at menarche for Canadian girls and explore its variation across socio-economic and demographic factors.MethodsThe analysis of the study was based on all female respondents aged 14 to 17 years during Cycle 4 (2000/2001) of the National Longitudinal Survey of Children & Youth (NLSCY). The main outcome was age at menarche assessed as the month and year of the occurrence of the first menstrual cycle. Kaplan Meier was used to estimate the mean and median of age at menarche. Chi-square test was used to assess the differences in early, average and later maturers across the different levels of socio-economic and demographic variables. Bootstrapping was performed to account for the complex sampling design.ResultsThe total number of girls analyzed in this study was 1,403 weighted to represent 601,911 Canadian girls. The estimated mean and median of age at menarche was 12.72 years (standard deviation = 1.05) and 12.67 years, respectively. The proportions of early (< 11.53 years), average (≥11.53 years and ≤13.91 years) and late maturers (> 13.91 years) were 14.6% (95% confidence interval (CI): 11.92-17.35), 68.0% (95% CI: 63.82-72.17) and 17.4% (95% CI: 14.10-20.63), respectively. Variations across the menarcheal groups were statistically significant for the province of residence, household income and family type.ConclusionThe findings of the study pave the way for future Canadian research. More studies are warranted to understand menarcheal age in terms of its variation across the provinces, the secular trend over time and its potential predictors.

Exercise, sex, menstrual cycle phase, and 17β-estradiol influence metabolism-related genes in human skeletal muscle

Higher fat and lower carbohydrate and amino acid oxidation are observed in women compared with men during endurance exercise. We hypothesized that the observed sex difference is due to estrogen and that menstrual cycle phase or supplementation of men with 17beta-estradiol (E(2)) would coordinately influence the mRNA content of genes involved in lipid and/or carbohydrate metabolism in skeletal muscle. Twelve men and twelve women had muscle biopsies taken before and immediately after 90 min of cycling at 65% peak oxygen consumption (Vo(2peak)). Women were studied in the midfollicular (Fol) and midluteal (Lut) phases, and men were studied after 8 days of E(2) or placebo supplementation. Targeted RT-PCR was used to compare mRNA content for genes involved in transcriptional regulation and lipid, carbohydrate, and amino acid metabolism. Sex was the greatest predictor of substrate metabolism gene content. Sex affected the mRNA content of FATm, FABPc, SREBP-1c, mtGPAT, PPARdelta, PPARalpha, CPTI, TFP-alpha, GLUT4, HKII, PFK, and BCOADK (P < 0.05). E(2) administration significantly (P < 0.05) affected the mRNA content of PGC-1alpha, PPARalpha, PPARdelta, TFP-alpha, CPTI, SREBP-1c, mtGPAT, GLUT4, GS-1, and AST. Acute exercise increased the mRNA abundance for PGC-1alpha, HSL, FABPc, CPTI, GLUT4, HKII, and AST (P < 0.05). Menstrual cycle had a small effect on PPARdelta, GP, and glycogenin mRNA content. Overall, women have greater mRNA content for several genes involved in lipid metabolism, which is partially due to an effect of E(2).

The effect of endurance exercise on both skeletal muscle and systemic oxidative stress in previously sedentary obese men

Background:Obesity is associated with low-grade systemic inflammation, in part because of secretion of proinflammatory cytokines, resulting into peripheral insulin resistance (IR). Increased oxidative stress is proposed to link adiposity and chronic inflammation. The effects of endurance exercise in modulating these outcomes in insulin-resistant obese adults remain unclear. We investigated the effect of endurance exercise on markers of oxidative damage (4-hydroxy-2-nonenal (4-HNE), protein carbonyls (PCs)) and antioxidant enzymes (superoxide dismutase (SOD), catalase) in skeletal muscle; urinary markers of oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG), 8-isoprostane); and plasma cytokines (C-reactive protein (CRP), interleukin-6 (IL-6), leptin, adiponectin).Methods:Age- and fitness-matched sedentary obese and lean men (n=9 per group) underwent 3 months of moderate-intensity endurance cycling training with a vastus lateralis biopsy, 24-h urine sample and venous blood samples taken before and after the intervention.Results:Obese subjects had increased levels of oxidative damage: 4-HNE (+37%; P⩽0.03) and PC (+63%; P⩽0.02); evidence of increased adaptive response to oxidative stress because of elevated levels of copper/zinc SOD (Cu/ZnSOD) protein content (+84%; P⩽0.01); increased markers of inflammation: CRP (+737%; P⩽0.0001) and IL-6 (+85%; P⩽0.03), and these correlated with increased markers of obesity; and increased leptin (+262%; P⩽0.0001) with lower adiponectin (−27%; P⩽0.01) levels vs lean controls. Training reduced 4-HNE (−10%; P⩽0.04), PC (−21%; P⩽0.05), 8-isoprostane (−26%; P⩽0.02) and leptin levels (−33%; P⩽0.01); had a tendency to decrease IL-6 levels (−21%; P=0.07) and IR (−17%; P=0.10); and increased manganese SOD (MnSOD) levels (+47%; P⩽0.01).Conclusion:Endurance exercise reduced skeletal muscle-specific and systemic oxidative damage while improving IR and cytokine profile associated with obesity, independent of weight loss. Hence, exercise is a useful therapeutic modality to reduce risk factors associated with the pathogenesis of IR in obesity.

Comparison of Total Protein Concentration in Skeletal Muscle as Measured by the Bradford and Lowry Assays

The Lowry and Bradford assays are the most commonly used methods of total protein quantification, yet vary in several aspects. To date, no comparisons have been made in skeletal muscle. We compared total protein concentrations of mouse red and white gastrocnemius, reagent stability, protein stability and range of linearity using both assays. The Lowry averaged protein concentrations 15% higher than the Bradford with a moderate correlation (r = 0.36, P = 0.01). However, Bland-Altman analysis revealed considerable bias (15.8 +/- 29.7%). Both Lowry reagents and its protein-reagent interactions were less stable over time than the Bradford. The linear range of concentration was smaller for the Lowry (0.05-0.50 mg/ml) than the Bradford (0-2.0 mg/ml). We conclude that the Bradford and Lowry measures of total protein concentration in skeletal muscle are not interchangeable. The Bradford and Lowry assays have various strengths and weaknesses in terms of substance interference and protein size. However, the Bradford provides greater reagent stability, protein-reagent stability and range of linearity, and requires less time to analyse compared to the Lowry assay.