Effects of immobilization and aerobic training on proteins related to intramuscular substrate storage and metabolism in young and older men.

PURPOSE: Aging and inactivity lead to skeletal muscle metabolic inflexibility, but the underlying molecular mechanisms are not entirely elucidated. Therefore, we investigated how muscle lipid and glycogen stores and major regulatory proteins were affected by short-term immobilization followed by aerobic training in young and older men. METHODS: 17 young (23 ± 1 years, 24 ± 1 kg m(-2), and 20 ± 2% body fat) and 15 older men (68 ± 1 years; 27 ± 1 kg m(-2), and 29 ± 2% body fat) underwent 2 weeks' one leg immobilization followed by 6 weeks' cycle training. Biopsies were obtained from m. vastus lateralis just before immobilization (at inclusion), after immobilization, and the after 6 weeks' training. The biopsies were analyzed for muscle substrates; muscle perilipin protein (PLIN), glycogen synthase (GS), synaptosomal-associated protein of 23 kDa (SNAP23) protein content, and muscle 3-hydroxyacyl-CoA dehydrogenase (HAD) activity RESULTS: The older men had higher intramuscular triglyceride (IMTG) (73 %) and Glycogen (16%) levels compared to the young men, and IMTG tended to increase with immobilization. PLIN2 and 3 protein content increased with immobilization in the older men only. The young men had higher GS (74%) protein compared to the older men. Immobilization decreased and training restored HAD activity, GS and SNAP23 protein content in young and older men. CONCLUSION: Evidence of age-related metabolic inflexibility is presented, seen as body fat and IMTG accumulation. The question arises as to whether IMTG accumulation in the older men is caused by or leading to the increase in PLIN2 and 3 protein content. Training decreased body fat and IMTG levels in both young and older men; hence, training should be prioritized to reduce the detrimental effect of aging on metabolism.

Skeletal muscle mitochondrial H2 O2 emission increases with immobilization and decreases after aerobic training in young and older men.

Currently, it is not known whether impaired mitochondrial function contributes to human ageing or whether potential impairments in mitochondrial function with age are secondary to physical inactivity. The present study investigated mitochondrial respiratory function and reactive oxygen species emission at a predefined membrane potential in young and older men subjected to 2 weeks of one-leg immobilization followed by 6 weeks of aerobic cycle training. Immobilization increased reactive oxygen species emission and decreased ATP generating respiration. Subsequent aerobic training reversed these effects. By contrast, age had no effect on the measured variables. The results of the present study support the notion that increased mitochondrial reactive oxygen species production mediates the detrimental effects seen after physical inactivity and that ageing per se does not cause mitochondrial dysfunction. Mitochondrial dysfunction, defined as increased oxidative stress and lower capacity for energy production, may be seen with ageing and may cause frailty, or it could be that it is secondary to physical inactivity. We studied the effect of 2 weeks of one-leg immobilization followed by 6 weeks of supervised cycle training on mitochondrial function in 17 young (mean ± SEM: 23 ± 1 years) and 15 older (68 ± 1 years) healthy men. Submaximal H2 O2 emission and respiration were measured simultaneously at a predefined membrane potential in isolated mitochondria from skeletal muscle using two protocols: pyruvate + malate (PM) and succinate + rotenone (SR). This allowed measurement of leak and ATP generating respiration from which the coupling efficiency can be calculated. The protein content of the anti-oxidants manganese superoxide dismuthase (MnSOD), CuZn superoxide dismuthase, catalase and gluthathione peroxidase 1 was measured by western blotting. Immobilization decreased ATP generating respiration using PM and increased H2 O2 emission using both PM and SR similarly in young and older men. Both were restored to baseline after the training period. Furthermore, MnSOD and catalase content increased with endurance training. The young men had a higher leak respiration at inclusion using PM and a higher membrane potential in State 3 using both substrate combinations. Collectively, the findings of the present study support the notion that increased mitochondrial reactive oxygen species mediates the detrimental effects seen after physical inactivity. Age, on the other hand, was not associated with impairments in anti-oxidant protein levels, mitochondrial respiration or H2 O2 emission using either protocol.

The Effect of Reduced Physical Activity and Retraining on Blood Lipids and Body Composition in Young and Older Adult Men.

We studied the effect of physical inactivity and subsequent retraining on cardiovascular risk factors in 17 young (Y; 23.4 ± 0.5 years) and 15 older adult (O; 68.1 ± 1.1 years) men who underwent 14 days of one leg immobilization followed by six weeks of training. Body weight remained unchanged. Daily physical activity decreased by 31 ± 9% (Y) and 37 ± 9% (O) (p < .001). Maximal oxygen uptake decreased with inactivity (Y) and always increased with training. Visceral fat mass decreased (p < .05) with training. Concentrations of lipids in blood were always highest in the older adults. FFA and glycerol increased with reduced activity (p < .05), but reverted with training. Training resulted in increases in HDL-C (p < .05) and a decrease in LDL-C and TC:HDL-C ratio (p < .05). A minor reduction in daily physical activity for two weeks increased blood lipids in both young and older men. Six weeks of training improved blood lipids along with loss of visceral fat.

Beta-aminoisobutyric acid is released by contracting human skeletal muscle and lowers insulin release from INS-1 832/3 cells by mediating mitochondrial energy metabolism.

AIMS/HYPOTHESIS: This study aimed to examine if beta-aminoisobutyric acid (BAIBA) is (i) secreted by skeletal muscle in humans during exercise, (ii) associated with insulin secretory function in vivo, and (iii) directly linked with acute glucose-mediated insulin release by pancreatic beta cells in vitro. METHODS: Following 2-weeks of single-leg immobilization, plasma BAIBA concentrations were measured in the brachial artery and the femoral veins of each leg in healthy male subjects, at rest and during two-legged dynamic knee-extensor exercise. During a 2-h hyperglycamic clamp, insulin secretory function and levels of plasma BAIBA were assessed in non-diabetic individuals, non-diabetic individuals following 24-h hyperglycemia and patients with type 2 diabetes. Direct effects of BAIBA on acute glucose-mediated insulin release were probed in INS-1832/3 cells under normal and 'diabetes-like' conditions. Finally, the effect of BAIBA on mitochondrial function was assessed in INS-1832/3 cells using extracellular flux analysis. RESULTS: (i) BAIBA is released from skeletal muscle at rest and during exercise under healthy conditions but is suppressed during exercise following leg immobilization, (ii) plasma BAIBA concentrations inversely associate with insulin secretory function in humans, (iii) BAIBA lowers mitochondrial energy metabolism in INS-1 832/3 cells in parallel with decreased insulin secretionConclusion/interpretation: BAIBA is a myokine released by skeletal muscle during exercise and indepedantly alters the triggering pathway of insulin secretion in cultured INS-1832/3 cells.

Simvastatin-Induced Insulin Resistance May Be Linked to Decreased Lipid Uptake and Lipid Synthesis in Human Skeletal Muscle: the LIFESTAT Study.

BACKGROUND: A prevalent side-effect of simvastatin is attenuated glucose homeostasis. The underlying mechanism is unknown, but impaired lipid metabolism may provide the link. The aim of this study was to investigate whether simvastatin-treated patients had a lower capacity to oxidize lipids and reduced expression of the major proteins regulating lipid uptake, synthesis, lipolysis, and storage in skeletal muscle than matched controls. MATERIALS AND METHODS: Ten men were treated with simvastatin (HbA1c: 5.7 ± 0.1%), and 10 healthy men (HbA1c: 5.2 ± 0.1%) underwent an oral glucose tolerance test and a muscle biopsy was obtained. Fat oxidation rates were measured at rest and during exercise. Western blotting was used to assess protein content. RESULTS: Patients treated with simvastatin had impaired glucose tolerance compared with control subjects, but fat oxidation at rest and during exercise was compatible. Skeletal muscle protein content of CD36, lipoprotein lipase (LPL), and diacylglycerol acyltransferase (DGAT) 1 were lower, and DGAT 2 tended to be lower in patients treated with simvastatin. CONCLUSIONS: Patients treated with simvastatin had a reduced capacity to synthesize FA and diacylglycerol (DAG) into triacylglycerol in skeletal muscle compared to matched controls. Decreased lipid synthesis capacity may lead to accumulation of lipotoxic intermediates (FA and DAG) and hence impair glucose tolerance.

Influence of maximal fat oxidation on long-term weight loss maintenance in humans.

Impaired maximal fat oxidation has been linked to obesity and weight regain after weight loss. The aim was to investigate the relationship between maximal fat oxidation (MFO) and long-term weight loss maintenance. Eighty subjects [means (SD): age, 36(13) yrs; BMI, 38(1) kg/m2] were recruited from a total of 2,420 former participants of an 11- to 12-wk lifestyle intervention. Three groups were established based on percent weight loss at follow-up [5.3(3.3) yr]: clinical weight loss maintenance (CWL), >10% weight loss; moderate weight loss (MWL), 1-10% weight loss; and weight regain (WR). Body composition (dual X-ray absorptiometry) and fat oxidation (indirect calorimetry) during incremental exercise were measured at follow-up. Blood and a muscle biopsy were sampled. At follow-up, a U-shaped parabolic relationship between MFO and percent weight loss was observed (r = 0.448; P < 0.001). Overall differences between CWL, MWL, and WR were observed in MFO (mean [95% confidence interval], in g/min, respectively: 0.46 [0.41-0.52]; 0.32 [0.27-0.38]; 0.45 [0.38-0.51]; P = 0.002), maximal oxygen uptake (V̇o2max, in ml·min-1·FFM-1, respectively; 49 [46-51]; 43 [40-47]; 41 [39-44]; P = 0.007), HAD-activity (in µmol·g-1·min-1, respectively: 123 [113-133]; 104 [91-118]; 97 [88-105]; P < 0.001), muscle protein content of CD36 (in AU, respectively: 1.1 [1.0-1.2]; 0.9 [0.8-1.0]; 0.9 [0.8-0.9]; P = 0.008) and FABPpm (in AU, respectively, 1.0 [0.8-1.2]; 0.7 [0.5-0.8]; 0.7 [0.5-0.9]; P = 0.008), body fat (in %, respectively: 33 [29-38]; 42 [38-46]; 52 [49-55]; P < 0.001), and plasma triglycerides (in mM, respectively: 0.8 [0.7-1.0]; 1.3 [0.9-1.7]; 1.6 [1.0-2.1]; P = 0.013). CWL and WR both had higher MFO compared with MWL, but based on different mechanisms. CWL displayed higher V̇o2max and intramuscular capacity for fat oxidation, whereas abundance of lipids at whole-body level and in plasma was higher in WR.NEW & NOTEWORTHY Impaired maximal fat oxidation has been linked to obesity and weight regain after weight loss. Noteworthy, maximal fat oxidation was equally high after clinical weight loss maintenance and weight regain compared with moderate weight loss. A high maximal fat oxidation after clinical weight loss maintenance was related to higher maximal oxygen updake, content of key proteins involved in transport of lipids across the plasma membrane and ß-oxidation. In contrast, a high maximal fat oxidation after weight regain was related to higher availability of lipids, i.e., general adiposity and plasma concentration of triglycerides.

miRNAs in human subcutaneous adipose tissue: Effects of weight loss induced by hypocaloric diet and exercise.

OBJECTIVE: Obesity is central in the development of insulin resistance. However, the underlying mechanisms still need elucidation. Dysregulated microRNAs (miRNAs; post-transcriptional regulators) in adipose tissue may present an important link. METHODS: The miRNA expression in subcutaneous adipose tissue from 19 individuals with severe obesity (10 women and 9 men) before and after a 15-week weight loss intervention was studied using genome-wide microarray analysis. The microarray results were validated with RT-qPCR, and pathway enrichment analysis of in silico predicted targets was performed to elucidate the biological consequences of the miRNA dysregulation. Lastly, the messenger RNA (mRNA) and/or protein expression of multiple predicted targets as well as several proteins involved in lipolysis were investigated. RESULTS: The intervention led to upregulation of miR-29a-3p and miR-29a-5p and downregulation of miR-20b-5p. The mRNA and protein expression of predicted targets was not significantly affected by the intervention. However, negative correlations between miR-20b-5p and the protein levels of its predicted target, acyl-CoA synthetase long-chain family member 1, were observed. Several other miRNA-target relationships correlated negatively, indicating possible miRNA regulation, including miR-29a-3p and lipoprotein lipase mRNA levels. Proteins involved in lipolysis were not affected by the intervention. CONCLUSIONS: Weight loss influenced several miRNAs, some of which were negatively correlated with predicted targets. These dysregulated miRNAs may affect adipocytokine signaling and forkhead box protein O signaling.

Maintaining a clinical weight loss after intensive lifestyle intervention is the key to cardiometabolic health.

OBJECTIVE: Intensive lifestyle interventions (ILI) are criticised for ineffective obesity treatment because weight loss over time is modest and thus of limited clinical relevance. However, a subgroup (5-30%) maintains a clinical weight loss >10%, but it is not clear if cardiometabolic health follows this pattern. The aim was to study the effect of different magnitudes of weight loss maintenance after ILI on cardiometabolic health. METHODS: Eighty out of 2420 former participants (age: 36±1, BMI: 38±1, (means ±SE)) in an 11-12-week ILI were recruited into 3 groups; clinical weight loss maintenance (>10% weight loss), moderate maintenance (1-10%), and weight regain based on weight loss at follow-up (5.3±0.4years). Weight loss during the ILI was achieved by increased physical activity and hypo-caloric diet. Dual X-ray Absorptiometry, blood sample, skeletal muscle biopsy and VO2max test were used to determine cardiometabolic health at follow-up. RESULTS: At follow-up, the clinical weight loss maintenance group scored better in the following variables compared to the other groups: BMI (31±1, 33±2, 43±2kg/m2), composition (34±2, 40±1, 49±1% fat), visceral adipose tissue (0.8±0.2, 1.7±0.5, 2.4±0.4kg), plasma triglycerides (0.8±0.2, 1.3±0.4, 1.6±0.3mmol/L), plasma glucose (4.9±0.1, 5.9±0.4, 5.9±0.1mmol/L), Hb1Ac (5.1±0.0, 5.6±0.2, 5.8±0.2%), protein content in skeletal muscle of GLUT4 (1.5±0.2, 0.9±0.1, 1.0±0.1 AU) and hexokinase II (1.6±0.2, 1.0±0.2, 0.7±0.1 AU), citrate synthase activity (155±6, 130±5, 113±5µmol/g/min) and VO2max (49±1, 43±1, 41±1mL/min/FFM) (p<0.05). CONCLUSION: Cardiometabolic health is better in participants who have maintained >10% weight loss compared to moderate weight loss and weight regain.

Exercise promotes IL-6 release from legs in older men with minor response to unilateral immobilization.

Physical inactivity is a major contributor to low-grade systemic inflammation. Most of the studies characterizing interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) release from exercising legs have been done in young, healthy men, but studies on inactivity in older people are lacking. The impact of 14 days of one-leg immobilization (IM) on IL-6 and TNF-α release during exercise in comparison to the contralateral control (CON) leg was investigated. Fifteen healthy men (age 68.1 ± 1.1 year (mean ± SEM); BMI 27.0 ± 0.4 kg·m(2); VO2max 33.3 ± 1.6 ml·kg(‒1)·min(‒1)) performed 45 min of two-leg dynamic knee extensor exercise at 19.5 ± 0.9 W. Arterial and femoral venous blood samples from the CON and the IM legs were collected every 15 min during exercise, and thigh blood flow was measured with ultrasound Doppler. Arterial plasma IL-6 concentration increased with exercise (rest vs. 45 min, main effect p < .05). IL-6 release increased with exercise (rest vs. 30 min, main effect p < .05). Furthermore, IL-6 release was borderline (main effect, p = .085, effect size 0.28) higher in the IM leg compared to the CON leg (288 (95% CI: 213-373) vs. 220 (95% CI: 152-299) pg·min(‒1), respectively). There was no release of TNF-α in either leg and arterial concentrations remained unchanged during exercise (p > .05). In conclusion, exercise induces more pronounced IL-6 secretion in healthy older men. Two weeks of unilateral immobilization on the other hand had only a minor influence on IL-6 release. Neither immobilization nor exercise had an effect on TNF-α release across the working legs in older men.

Training Does Not Alter Muscle Ceramide and Diacylglycerol in Offsprings of Type 2 Diabetic Patients Despite Improved Insulin Sensitivity.

Ceramide and diacylglycerol (DAG) may be involved in the early phase of insulin resistance but data are inconsistent in man. We evaluated if an increase in insulin sensitivity after endurance training was accompanied by changes in these lipids in skeletal muscle. Nineteen first-degree type 2 diabetes Offsprings (Offsprings) (age: 33.1 ± 1.4 yrs; BMI: 26.4 ± 0.4 kg/m2) and sixteen matched Controls (age: 31.3 ± 1.5 yrs; BMI: 25.3 ± 0.7 kg/m2) performed 10 weeks of endurance training three times a week at 70% of VO2max on a bicycle ergometer. Before and after the intervention a hyperinsulinemic-euglycemic clamp and VO2max test were performed and muscle biopsies obtained. Insulin sensitivity was significantly lower in Offsprings compared to control subjects (p < 0.01) but improved in both groups after 10 weeks of endurance training (Off: 17 ± 6%; Con: 12 ± 9%, p < 0.01). The content of muscle ceramide, DAG, and their subspecies were similar between groups and did not change in response to the endurance training except for an overall reduction in C22:0-Cer (p < 0.05). Finally, the intervention induced an increase in AKT protein expression (Off: 27 ± 11%; Con: 20 ± 24%, p < 0.05). This study showed no relation between insulin sensitivity and ceramide or DAG content suggesting that ceramide and DAG are not major players in the early phase of insulin resistance in human muscle.

GAPDH and ß-actin protein decreases with aging, making Stain-Free technology a superior loading control in Western blotting of human skeletal muscle.

Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [ß-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and ß-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of ß-actin and GAPDH was lower in older men compared with young men. In conclusion, ß-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology.

Six weeks' aerobic retraining after two weeks' immobilization restores leg lean mass and aerobic capacity but does not fully rehabilitate leg strength in young and older men.

OBJECTIVE: To determine the effect of aerobic retraining as rehabilitation after short-term leg immobilization on leg strength, leg work capacity, leg lean mass, leg muscle fibre type composition and leg capillary supply, in young and older men.Subjects and design: Seventeen young (23 ± 1 years) and 15 older (68 ± 1 [standard error of the mean; SEM] years) men had one leg immobilized for 2 weeks, followed by 6 weeks' bicycle endurance retraining. METHODS: Maximal voluntary contraction, leg work capacity (Wmax), and leg lean mass by dual energy X-ray absorptiometry were measured at inclusion, after immobilization and after 3 and 6 weeks' retraining. Muscle biopsies were evaluated for fibre type, fibre area, and capillarization. RESULTS: Immobilization decreased maximal voluntary contraction (-28 ± 6% and -23 ± 3%); Wmax (-13 ± 5% and -9 ± 4%) and leg lean mass (only in young, -485 ± 105g) in young and older men, respectively. Six weeks' retraining increased maximal voluntary contraction (34 ± 8% and 17 ± 6%), Wmax (33 ± 5% and 20 ± 5%) and leg lean mass (only in young 669 ± 69 g) in young and older men, respectively, compared with the immobilized value. CONCLUSION: Short-term leg immobilization had marked effects on leg strength, and work capacity and 6 weeks' retraining was sufficient to increase, but not completely rehabilitate, muscle strength, and to rehabilitate aerobic work capacity and leg lean mass (in the young men).

The relationship between skeletal muscle mitochondrial citrate synthase activity and whole body oxygen uptake adaptations in response to exercise training.

Citrate synthase (CS) activity is a validated biomarker for mitochondrial density in skeletal muscle. CS activity is also used as a biochemical marker of the skeletal muscle oxidative adaptation to a training intervention, and a relationship between changes in whole body aerobic capacity and changes in CS activity is often assumed. However, this relationship and absolute values of CS and maximal oxygen uptake (V.O2max) has never been assessed across different studies. A systematic PubMed search on literature published from 1983 to 2013 was performed. The search profile included: citrate, synthase, human, skeletal, muscle, training, not electrical stimulation, not in-vitro, not rats. Studies that reported changes in CS activity and V.O2max were included. Different training types and subject populations were analyzed independently to assess correlation between relative changes in V.O2max and CS activity. 70 publications with 97 intervention groups were included. There was a positive (r = 0.45) correlation (P < 0.001) between the relative change in V.O2max and the relative change in CS activity. All reported absolute values of CS and V.O2max did not correlate (r =- 0.07, n = 148, P = 0.4). Training induced changes in whole body oxidative capacity is matched by changes in muscle CS activity in a nearly 1:1 relationship. Absolute values of CS across different studies cannot be compared unless a standardized analytical method is used by all laboratories.

Two weeks of one-leg immobilization decreases skeletal muscle respiratory capacity equally in young and elderly men.

Physical inactivity affects human skeletal muscle mitochondrial oxidative capacity but the influence of aging combined with physical inactivity is not known. This study investigates the effect of two weeks of immobilization followed by six weeks of supervised cycle training on muscle oxidative capacity in 17 young (23±1years) and 15 elderly (68±1years) healthy men. We applied high-resolution respirometry in permeabilized fibers from muscle biopsies at inclusion after immobilization and training. Furthermore, protein content of mitochondrial complexes I-V, mitochondrial heat shock protein 70 (mtHSP70) and voltage dependent anion channel (VDAC) were measured in skeletal muscle by Western blotting. The elderly men had lower content of complexes I-V and mtHSP70 but similar respiratory capacity and content of VDAC compared to the young. In both groups the respiratory capacity and protein content of VDAC, mtHSP70 and complexes I, II, IV and V decreased with immobilization and increased with retraining. Moreover, there was no overall difference in the response between the groups. When the intrinsic mitochondrial capacity was evaluated by normalizing respiration to citrate synthase activity, the respiratory differences with immobilization and training disappeared. In conclusion, aging is not associated with a decrease in muscle respiratory capacity in spite of lower complexes I-V and mtHSP70 protein content. Furthermore, immobilization decreased and aerobic training increased the respiratory capacity and protein contents of complexes I-V, mtHSP70 and VDAC similarly in the two groups. This suggests that inactivity and training alter mitochondrial biogenesis equally in young and elderly men.