Gymnastik- och idrottshögskolan, GIH

Background

Sarcopenia is thought to be underlined by age-associated anabolic resistance and dysregulation of intracellular signalling pathways. However, it is unclear whether these phenomena are driven by ageing per se or other confounding factors.

Methods

Lean and healthy young (n = 10, 22 ± 3 years, BMI; 23.4 ± 0.8 kg/m2) and old men (n = 10, 70 ± 3 years, BMI; 22.7 ± 1.3 kg/m2) performed unilateral resistance exercise followed by intake of essential amino acids (EAA). Muscle biopsies were collected from the rested and the exercised leg before, immediately after and 60 and 180 min after EAA intake. Muscle samples were analysed for amino acid concentrations, muscle protein synthesis (MPS) and associated anabolic signalling.

Results

Following exercise, peak plasma levels of EAA and leucine were similar between groups, but the area under the curve was ~11% and ~28% lower in Young (p < 0.01). Absolute levels of muscle EAA and leucine peaked 60 min after exercise, with ~15 and ~21% higher concentrations in the exercising leg (p < 0.01) but with no difference between groups. MPS increased in both the resting (~0.035%·h−1 to 0.056%·h−1, p < 0.05) and exercising leg (~0.035%·h−1 to 0.083%·h−1, p < 0.05) with no difference between groups. Phosphorylation of S6K1Thr389 increased to a similar extent in the exercising leg in both groups but was 2.8-fold higher in the resting leg of Old at the 60 min timepoint (p < 0.001). Phosphorylation of 4E-BP1Ser65 increased following EAA intake and exercise, but differences between legs were statistically different only at 180 min (p < 0.001). However, phosphorylation of this site was on average 78% greater across all timepoints in Old (p < 0.01). Phosphorylation of eEF2Thr56 was reduced (~66% and 39%) in the exercising leg at both timepoints after EAA intake and exercise, with no group differences (p < 0.05). However, phosphorylation at this site was reduced by ~27% also in the resting leg at 60 min, an effect that was only seen in Old (p < 0.01). Total levels of Rheb (~45%), LAT1 (~31%) and Rag B (~31%) were higher in Old (p < 0.001).

Conclusion

Lean and healthy old men do not manifest AR as evidenced by potent increases in MPS and mTORC1 signalling following EAA intake and exercise. Maintained anabolic sensitivity with age appears to be a function of a compensatory increase in basal levels of proteins involved in anabolic signalling. Therefore, our results suggest that age per se does not appear to cause AR in human skeletal muscle.

Age-related loss of muscle mass and function is underpinned by changes at the myocellular level. However, our understanding of the aged muscle phenotype might be confounded by factors secondary to ageing per se, such as inactivity and adiposity. Here, using healthy, lean, recreationally active, older men, we investigated the impact of ageing on myocellular properties in skeletal muscle. Muscle biopsies were obtained from young men (22 ± 3 years, n = 10) and older men (69 ± 3 years, n = 11) matched for health status, activity level and body mass index. Immunofluorescence was used to assess myofibre composition, morphology (size and shape), capillarization, the content of satellite cells and myonuclei, the spatial relationship between satellite cells and capillaries, denervation and myofibre grouping. Compared with young muscle, aged muscle contained 53% more type I myofibres, in addition to smaller (-32%) and misshapen (3%) type II myofibres (P < 0.05). Aged muscle manifested fewer capillaries (-29%) and satellite cells (-38%) surrounding type II myofibres (P < 0.05); however, the spatial relationship between these two remained intact. The proportion of denervated myofibres was ∼2.6-fold higher in old than young muscle (P < 0.05). Aged muscle had more grouped type I myofibres (∼18-fold), primarily driven by increased size of existing groups rather than increased group frequency (P < 0.05). Aged muscle displayed selective deterioration of type II myofibres alongside increased denervation and myofibre grouping. These data are key to understanding the cellular basis of age-related muscle decline and reveal a pressing need to fine-tune strategies to preserve type II myofibres and innervation status in ageing populations.

Human skeletal muscle fiber type composition varies greatly along the muscle, so one biopsy may not accurately represent the whole muscle. Recommendations on the number of biopsies and fiber counts using immunohistochemistry and whether these findings can be extrapolated to other muscles are lacking. We assessed fiber type composition in the vastus lateralis and gastrocnemius medialis muscles of 40 individuals. Per muscle, we took four biopsy samples from one incision, collecting two samples each from a proximally and distally directed needle. Based on another dataset involving 10 vastus lateralis biopsies per participant (N=7), we calculated 95% limits of agreement for subsets of biopsies and fiber counts compared to the 10-biopsy average. Average absolute differences in type I fiber proportions between proximal and distal, and between within-needle samples were 6.9 and 4.5 percentage points in the vastus lateralis, and 5.5 and 4.4 percentage points in the gastrocnemius medialis, respectively. The 95% limits of agreement narrowed to ±10 percentage points when 200 fibers from at least three biopsies were analyzed, with minimal improvements with greater fiber counts. Type I fiber proportions in the vastus lateralis and gastrocnemius medialis showed a moderate positive association (r²=0.22; p=0.006; at least 200 fibers in each of three to four samples per muscle). In conclusion, three biopsies with a minimum of 200 counted fibers are required to estimate vastus lateralis fiber type composition within ±10 percentage points. Even when using these standards, researchers should be cautious when extrapolating muscle fiber type proportions from one muscle to another.

Insulin resistance (IR) is a risk factor for the development of several major metabolic diseases. Muscle fiber composition is established early in life and is associated with insulin sensitivity. Hence, muscle fiber composition was used to identify early defects in the development of IR in healthy young individuals in the absence of clinical manifestations. Biopsies were obtained from the thigh muscle, followed by an intravenous glucose tolerance test. Indices of insulin action were calculated and cardiovascular measurements, analyses of blood and muscle were performed. Whole-body insulin sensitivity (SI_galvin) was positively related to expression of type I muscle fibers (r=0.49; P<0.001) and negatively related to resting heart rate (HR, r=-0.39; P<0.001), which was also negatively related to expression of type I muscle fibers (r=-0.41; P<0.001). Muscle protein expression of endothelial nitric oxide synthase (eNOS), whose activation results in vasodilation, was measured in two subsets of subjects expressing a high percentage of type I fibers (59±6%; HR = 57±9 beats/min; SI_galvin = 1.8±0.7 units) or low percentage of type I fibers (30±6%; HR = 71±11; SI_galvin = 0.8±0.3 units; P<0.001 for all variables vs. first group). eNOS expression was: 1. higher in subjects with high type I expression; 2. almost two-fold higher in pools of type I vs. II fibers; 3. only detected in capillaries surrounding muscle fibers; and 4. linearly associated with SI_galvin. These data demonstrate that an altered function of the autonomic nervous system and a compromised capacity for vasodilation in the microvasculature occur early in the development of IR.

OBJECTIVE: Human skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation (OXPHOS). While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic.

METHODS: Here, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within six hours of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity.

RESULTS: Maximal complex I CI+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for CI+II, which was primarily mediated through elevated CII respiration, but not CI or. Furthermore, the abundance of CII protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid- or complex I respiration.

CONCLUSION: Mitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.

Exercise promotes brain plasticity partly by stimulating increases in mature brain-derived neurotrophic factor (mBDNF), but the role of the pro-BDNF isoform in the regulation of BDNF metabolism in humans is unknown. We quantified the expression of pro-BDNF and mBDNF in human skeletal muscle and plasma at rest, after acute exercise (+/- lactate infusion), and after fasting. Pro-BDNF and mBDNF were analyzed with immunoblotting, enzyme-linked immunosorbent assay, immunohistochemistry, and quantitative polymerase chain reaction. Pro-BDNF was consistently and clearly detected in skeletal muscle (40-250 pg mg^-1 dry muscle), whereas mBDNF was not. All methods showed a 4-fold greater pro-BDNF expression in type I muscle fibers compared to type II fibers. Exercise resulted in elevated plasma levels of mBDNF (55%) and pro-BDNF (20%), as well as muscle levels of pro-BDNF (∼10%, all P < 0.05). Lactate infusion during exercise induced a significantly greater increase in plasma mBDNF (115%, P < 0.05) compared to control (saline infusion), with no effect on pro-BDNF levels in plasma or muscle. A 3-day fast resulted in a small increase in plasma pro-BDNF (∼10%, P < 0.05), with no effect on mBDNF. Pro-BDNF is highly expressed in human skeletal muscle, particularly in type I fibers, and is increased after exercise. While exercising with higher lactate augmented levels of plasma mBDNF, exercise-mediated increases in circulating mBDNF likely derive partly from release and cleavage of pro-BDNF from skeletal muscle, and partly from neural and other tissues. These findings have implications for preclinical and clinical work related to a wide range of neurological disorders such as Alzheimer's, clinical depression, and amyotrophic lateral sclerosis.

Cumulative evidence supports the hypothesis that hypoxia acts as a regulator of muscle mass. However, the underlying molecular mechanisms remain incompletely understood, particularly in human muscle. Here we examined the effect of hypoxia on signaling pathways related to ribosome biogenesis and myogenic activity following an acute bout of resistance exercise. We also investigated whether hypoxia influenced the satellite cell response to resistance exercise. Employing a randomized, crossover design, eight men performed resistance exercise in normoxia (FiO₂ 21%) or normobaric hypoxia (FiO₂ 12%). Muscle biopsies were collected in a time-course manner (before, 0, 90, 180 min and 24 h after exercise) and were analyzed with respect to cell signaling, gene expression and satellite cell content using immunoblotting, RT-qPCR and immunofluorescence, respectively. In normoxia, resistance exercise increased the phosphorylation of RPS6, TIF-1A and UBF above resting levels. Hypoxia reduced the phosphorylation of these targets by ~37%, ~43% and ~ 67% throughout the recovery period, respectively (p < .05 vs. normoxia). Resistance exercise also increased 45 S pre-rRNA expression and mRNA expression of c-Myc, Pol I and TAF-1A above resting levels, but no differences were observed between conditions. Similarly, resistance exercise increased mRNA expression of myogenic regulatory factors throughout the recovery period and Pax7⁺ cells were elevated 24 h following exercise in mixed and type II muscle fibers, with no differences observed between normoxia and hypoxia. In conclusion, acute hypoxia attenuates ribosome signaling, but does not impact satellite cell pool expansion and myogenic gene expression following a bout of resistance exercise in human skeletal muscle.

Oxidative stress plays a vital role for the adaptive responses to physical training. However, excessive oxidative stress can precipitate cellular damage, necessitating protective mechanisms to mitigate this effect. Glucosinolates, found predominantly in cruciferous vegetables, can be converted into isothiocyanates, known for their antioxidative properties. These compounds activate crucial antioxidant defence pathways and support mitochondrial function and protein integrity under oxidative stress, in both Nrf2-dependent and independent manners. We here administered glucosinolate-rich broccoli sprouts (GRS), in a randomized double-blinded cross-over fashion to 9 healthy subjects in combination with daily intense exercise training for 7 days. We found that exercise in combination with GRS significantly decreased the levels of carbonylated proteins in skeletal muscle and the release of myeloperoxidase into blood. Moreover, it lowered lactate accumulation during submaximal exercise, and attenuated the severe nocturnal hypoglycaemic episodes seen during the placebo condition. Furthermore, GRS in combination with exercise improved physical performance, which was unchanged in the placebo condition.

There is a debate on whether lipid-mediated insulin resistance derives from an increased or decreased capacity of muscle to oxidize fats. Here we examine the involvement of muscle fiber composition in the metabolic responses to a 3-day fast (starvation, which results in increases in plasma lipids and insulin resistance) in two groups of healthy young subjects: 1, area occupied by type I fibers = 61.0 ± 11.8%; 2, type I area = 36.0 ± 4.9% (P<0.001). Muscle biopsies and intravenous glucose tolerance tests were performed after an overnight fast and after starvation. Biopsies were analyzed for muscle fiber composition and mitochondrial respiration. Indices of glucose tolerance and insulin sensitivity were determined. Glucose tolerance was similar in both groups after an overnight fast and deteriorated to a similar degree in both groups after starvation. In contrast, whole-body insulin sensitivity decreased markedly after starvation in group 1 (P<0.01), whereas the decrease in group 2 was substantially smaller (P=0.06). Non-esterified fatty acids and β-hydroxybutyrate levels in plasma after an overnight fast were similar between groups and increased markedly and comparably in both groups after starvation, demonstrating similar degrees of lipid load. The capacity of permeabilized muscle fibers to oxidize lipids was significantly higher in group 1 vs. 2, whereas there was no significant difference in pyruvate oxidation between groups. The data demonstrate that loss of whole-body insulin sensitivity after short-term starvation is a function of muscle fiber composition and is associated with an elevated rather than a diminished capacity of muscle to oxidize lipids.

We have previously shown that maximal over-the-counter doses of ibuprofen, compared with low doses of acetylsalicylic acid, reduce muscle hypertrophy in young individuals after 8 weeks of resistance training. Because the mechanism behind this effect has not been fully elucidated, we here investigated skeletal muscle molecular responses and myofiber adaptations in response to acute and chronic resistance training with concomitant drug intake. Thirty-one young (aged 18-35 years) healthy men (n=17) and women (n=14) were randomized to receive either ibuprofen (IBU;1200mg daily; n=15) or acetylsalicylic acid (ASA; 75mg daily; n=16) while undergoing 8 weeks of knee extension training. Muscle biopsies from the vastus lateralis were obtained before, at week 4 after an acute exercise session, and after 8 weeks of resistance training and analyzed for mRNA markers and mTOR signaling, as well as quantification of total RNA content (marker of ribosome biogenesis) and immunohistochemical analyzes of muscle fiber size, satellite cell content, myonuclear accretion, and capillarization. There were only two treatment ´ time interaction in selected molecular markers after acute exercise (atrogin-1 and MuRF1 mRNA), but several exercise effects. Muscle fiber size, satellite cell and myonuclear accretion, and capillarization were not affected by chronic training or drug intake. RNA content increased comparably (~14%) in both groups. Collectively, these data suggest that established acute and chronic hypertrophy regulators (including mTOR signaling, ribosome biogenesis, satellite cell content, myonuclear accretion, and angiogenesis) were not differentially affected between groups and therefore do not explain the deleterious effects of ibuprofen on muscle hypertrophy in young adults.