Quantitation of Mitochondrial DNA Deletions Via Restriction Digestion/Long-Range Single-Molecule PCR.

Quantification of deletions in mtDNA is a long-standing problem in mutational analysis. We describe here an approach that combines the power of single-molecule PCR of the entire mitochondrial genome with the enrichment of the deletions by restriction digestion. This approach is indispensable if information about wide range of deletion types in a sample is critical, such as in studies concerning distribution of deletion breakpoints (as opposed to approaches where fraction of a single deletion or a limited set of deletions is used as a proxy for total deletion load). Because deletions in a sample are quantified almost exhaustively, the other important application of this approach involves studies where only small amounts of tissue, such as biopsies, are available.

Efficacy of whey protein supplementation on resistance exercise-induced changes in lean mass, muscle strength, and physical function in mobility-limited older adults.

BACKGROUND: Whey protein supplementation may augment resistance exercise-induced increases in muscle strength and mass. Further studies are required to determine whether this effect extends to mobility-limited older adults. The objectives of the study were to compare the effects of whey protein concentrate (WPC) supplementation to an isocaloric control on changes in whole-body lean mass, mid-thigh muscle cross-sectional area, muscle strength, and stair-climbing performance in older mobility-limited adults in response to 6 months of resistance training (RT). METHODS: Eighty mobility-limited adults aged 70-85 years were randomized to receive WPC (40g/day) or an isocaloric control for 6 months. All participants also completed a progressive high-intensity RT intervention. Sample sizes were calculated based on the primary outcome of change in whole-body lean mass to give 80% power for a 0.05-level, two-sided test. RESULTS: Lean mass increased 1.3% and 0.6% in the WPC and control groups, respectively. Muscle cross-sectional area was increased 4.6% and 2.9% in the WPC and control groups, respectively, and muscle strength increased 16%-50% in WPC and control groups. Stair-climbing performance also improved in both groups. However, there were no statistically significant differences in the change in any of these variables between groups. CONCLUSIONS: These data suggest that WPC supplementation at this dose does not offer additional benefit to the effects of RT in mobility-limited older adults.

Systemic vascular function is associated with muscular power in older adults.

Age-associated loss of muscular strength and muscular power is a critical determinant of loss of physical function and progression to disability in older adults. In this study, we examined the association of systemic vascular function and measures of muscle strength and power in older adults. Measures of vascular endothelial function included brachial artery flow-mediated dilation (FMD) and the pulse wave amplitude reactive hyperemia index (PWA-RHI). Augmentation index (AIx) was taken as a measure of systemic vascular function related to arterial stiffness and wave reflection. Measures of muscular strength included one repetition maximum (1RM) for a bilateral leg press. Peak muscular power was measured during 5 repetitions performed as fast as possible for bilateral leg press at 40% 1RM. Muscular power was associated with brachial FMD (r = 0.43, P < 0.05), PWA-RHI (r = 0.42, P < 0.05), and AIx (r = -0.54, P < 0.05). Muscular strength was not associated with any measure of vascular function. In conclusion, systemic vascular function is associated with lower-limb muscular power but not muscular strength in older adults. Whether loss of muscular power with aging contributes to systemic vascular deconditioning or vascular dysfunction contributes to decrements in muscular power remains to be determined.

Muscle power failure in mobility-limited older adults: preserved single fiber function despite lower whole muscle size, quality and rate of neuromuscular activation.

This study investigated the physiological and gender determinants of the age-related loss of muscle power in 31 healthy middle-aged adults (aged 40-55 years), 28 healthy older adults (70-85 years) and 34 mobility-limited older adults (70-85 years). We hypothesized that leg extensor muscle power would be significantly lower in mobility-limited elders relative to both healthy groups and sought to characterize the physiological mechanisms associated with the reduction of muscle power with aging. Computed tomography was utilized to assess mid-thigh body composition and calculate specific muscle power and strength. Surface electromyography was used to assess rate of neuromuscular activation and muscle biopsies were taken to evaluate single muscle fiber contractile properties. Peak muscle power, strength, muscle cross-sectional area, specific muscle power and rate of neuromuscular activation were significantly lower among mobility-limited elders compared to both healthy groups (P ≤ 0.05). Mobility-limited older participants had greater deposits of intermuscular adipose tissue (P < 0.001). Single fiber contractile properties of type I and type IIA muscle fibers were preserved in mobility-limited elders relative to both healthy groups. Male gender was associated with greater decrements in peak and specific muscle power among mobility-limited participants. Impairments in the rate of neuromuscular activation and concomitant reductions in muscle quality are important physiological mechanisms contributing to muscle power deficits and mobility limitations. The dissociation between age-related changes at the whole muscle and single fiber level suggest that, even among older adults with overt mobility problems, contractile properties of surviving muscle fibers are preserved in an attempt to maintain overall muscle function.

Leucine-enriched essential amino acid supplementation during moderate steady state exercise enhances postexercise muscle protein synthesis.

BACKGROUND: The effects of essential amino acid (EAA) supplementation during moderate steady state (ie, endurance) exercise on postexercise skeletal muscle metabolism are not well described, and the potential role of supplemental leucine on muscle protein synthesis (MPS) and associated molecular responses remains to be elucidated. OBJECTIVE: This randomized crossover study examined whether EAA supplementation with 2 different concentrations of leucine affected post-steady state exercise MPS, whole-body protein turnover, and mammalian target of rapamycin 1 (mTORC1) intracellular signaling. DESIGN: Eight adults completed 2 separate bouts of cycle ergometry [60 min, 60% VO(2)peak (peak oxygen uptake)]. Isonitrogenous (10 g EAA) drinks with different leucine contents [leucine-enriched (l)-EAA, 3.5 g leucine; EAA, 1.87 g leucine] were consumed during exercise. MPS and whole-body protein turnover were determined by using primed continuous infusions of [(2)H(5)]phenylalanine and [1-(13)C]leucine. Multiplex and immunoblot analyses were used to quantify mTORC1 signaling. RESULTS: MPS was 33% greater (P < 0.05) after consumption of L-EAA (0.08 ± 0.01%/h) than after consumption of EAA (0.06 ± 0.01%/h). Whole-body protein breakdown and synthesis were lower (P < 0.05) and oxidation was greater (P < 0.05) after consumption of L-EAA than after consumption of EAA. Regardless of dietary treatment, multiplex analysis indicated that Akt and mammalian target of rapamycin phosphorylation were increased (P < 0.05) 30 min after exercise. Immunoblot analysis indicated that phosphorylation of ribosomal protein S6 and extracellular-signal regulated protein kinase increased (P < 0.05) and phosphorylation of eukaryotic elongation factor 2 decreased (P < 0.05) after exercise but was not affected by dietary treatment. CONCLUSION: These findings suggest that increasing the concentration of leucine in an EAA supplement consumed during steady state exercise elicits a greater MPS response during recovery. This trial is registered at clinicaltrials.gov as NCT01366924.

Molecular responses to moderate endurance exercise in skeletal muscle.

This study examined alterations in skeletal-muscle growth and atrophy-related molecular events after a single bout of moderate-intensity endurance exercise. Muscle biopsies were obtained from 10 men (23 ± 1 yr, body mass 80 ± 2 kg, and VO(2peak) 45 ± 1 ml x kg⁻¹ x min⁻¹) immediately (0 hr) and 3 hr after a 60-min bout of cycle exercise (60% +/- 5% VO(2peak)). Corresponding muscle biopsies were also obtained under resting conditions. The phosphorylation status of insulin/IGF-PI3K molecular-signaling proteins, ubiquitin-proteasome-related gene expression, FOXO transcription factors, and myogenic regulatory factors in muscle samples was analyzed using multiplex analysis, Western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR). A condition-time interaction was observed for Akt phosphorylation (p < .05) with multiplexing. Regardless of endurance exercise, Akt phosphorylation decreased and ERK phosphorylation increased at 3 hr compared with 0 hr (p < .05). Levels of p70(S6K) phosphorylation were 110% greater (p < .05) at 3 hr than at 0 hr using Western blots. MuRF mRNA expression postexercise increased; levels were 4.7- and 5.7-fold greater (p < .05) at 0 hr and 3 hr, respectively, than at rest with qRT-PCR. Atrogin mRNA expression was up-regulated 3.2-fold 3 hr postexercise compared with rest. These findings demonstrate modest changes in the molecular responses to moderate endurance exercise in the absence of nutrition. This study provides the groundwork for future investigations designed to optimize the metabolic conditions necessary to positively influence the cellular mechanisms specific to skeletal-muscle protein turnover during recovery from endurance exercise.

Resistance training and timed essential amino acids protect against the loss of muscle mass and strength during 28 days of bed rest and energy deficit.

Spaceflight and bed rest (BR) result in losses of muscle mass and strength. Resistance training (RT) and amino acid (AA) supplementation are potential countermeasures to minimize these losses. However, it is unknown if timing of supplementation with exercise can optimize benefits, particularly with energy deficit. We examined the effect of these countermeasures on body composition, strength, and insulin levels in 31 men (ages 31-55 yr) during BR (28 days) followed by active recovery (14 days). Subjects were randomly assigned to essential AA supplementation (AA group, n = 7); RT with AA given 3 h after training (RT group, n = 12); or RT with AA given 5 min before training (AART group, n = 12). Energy intake was reduced by 8 +/- 6%. Midthigh muscle area declined with BR for the AA > RT > AART groups: -11%, -3%, -4% (P = 0.05). Similarly, greatest losses in lower body muscle strength were seen in the AA group (-22%). These were attenuated in the exercising groups [RT (-8%) and AART (-6%; P < 0.05)]. Fat mass and midthigh intramuscular fat increased after BR in the AA group (+3% and +14%, respectively), and decreased in the RT (-5% and -4%) and AART groups (-1 and -5%; P = 0.05). Muscle mass and strength returned toward baseline after recovery, but the AA group showed the lowest regains. Combined resistance training with AA supplementation pre- or postexercise attenuated the losses in muscle mass and strength by approximately two-thirds compared with AA supplement alone during BR and energy deficit. These data support the efficacy of combined AA and RT as a countermeasure against muscle wasting due to low gravity.

Reduction of abdominal obesity in lipodystrophy associated with human immunodeficiency virus infection by means of diet and exercise: case report and proof of principle.

Lipodystrophy associated with human immunodeficiency virus infection causes abdominal fat gain, peripheral subcutaneous fat atrophy, insulin resistance, low levels of high-density lipoprotein cholesterol, and hypertriglyceridemia. An exercise program combined with a moderate-fat, low-glycemic-index, high-fiber diet can reverse several aspects of lipodystrophy, and, until specific treatment is available, should be considered for treatment of lipodystrophy.