Gymnastik- och idrottshögskolan, GIH

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.

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.

Brain-derived neurotrophic factor (BDNF) is essential for neuroplasticity. Exercise caninduce increases in forearm venous plasma and serum BDNF, often assumed to be indicativeof release from the brain. We investigated the effects of exercise on circulating levels of matureBDNF (mBDNF) and its precursor proBDNF. Sixteen healthy, physically fit adults (20–40 years old)cycled for 20 min at 40, 60 and 80% of V˙O2 max, separated by 30 min of rest. BDNF was analysed in blood samples from the brachial artery, internal jugular vein, femoral vein and antecubital vein. Brain/skeletal muscle exchange of BDNF, calculated as arterial-venous differences in BDNF multiplied by blood flow in the middle cerebral artery/common femoral artery, was measured simultaneously with blood sampling. Exercise intensity-dependent increases were observed in blood platelet count, forearm venous serum mBDNF and plasma proBDNF, but not in forearm venous plasma mBDNF. Brain release (or uptake) was not detected for either plasma mBDNF, serum mBDNF or plasma proBDNF. However, muscle uptake of plasma mBDNF and release of plasma proBDNF were observed after high-intensity exercise. Our findings demonstrate that exercise-dependent increases in serum mBDNF are not derived from the brain or the exercised skeletal muscle. Rather, the source of the increase appears to be the increase in platelets that are enriched with mBDNF. Furthermore, in physically fit adults, BDNF is not released from the brain into the bloodstream, after exercise, regardless of exercise intensity. Finally, changes in plasma proBDNF after exercise appear to be dependent on exercised skeletal muscle rather than brain release. KEY POINTS: Previously shown exercise-induced increases in forearm venous brain-derived neurotrophic factor (BDNF) are often assumed to be indicative of release from the brain. We investigated whether exercise-induced changes in forearm venous mature BDNF (mBDNF) and precursor proBDNF are paralleled by concomitant changes in BDNF exchange over the brain and skeletal muscle. We observed exercise intensity-dependent increases in platelet count, forearm venous serum mBDNF and plasma proBDNF, but not in forearm venous plasma mBDNF. We found muscle uptake of plasma mBDNF and release of plasma proBDNF after high-intensity exercise but no exercise intensity-dependent brain exchange of either plasma mBDNF, serum mBDNF or plasma proBDNF. Our findings suggest that acute exercise-induced increases in circulating serum mBDNF may be solely a result of increased platelet count, probably due to splenic platelet release; and that exercised skeletal muscle, and not the brain, responds to high-intensity exercise by releasing plasma proBDNF.

BACKGROUND: Exercising with low muscle glycogen content can improve training adaptation, but the mechanisms underlying the muscular adaptation are still largely unknown. In this study, we measured substrate utilization and cell signaling in different muscle fiber types during exercise and investigated a possible link between these variables.

METHODS: Five subjects performed a single leg cycling exercise in the evening (day 1) with the purpose of reducing glycogen stores. The following morning (day 2), they performed two-legged cycling at ∼70% of VO_2peak for 1 h. Muscle biopsies were taken from both legs pre- and post-exercise for enzymatic analyses of glycogen, metabolite concentrations using LC-MS/MS-based quantification, and protein signaling using Western blot in pools of type I or type II fibers.

RESULTS: Glycogen content was 60-65% lower for both fiber types (P < 0.01) in the leg that exercised on day 1 (low leg) compared to the other leg with normal level of glycogen (normal leg) before the cycling exercise on day 2. Glycogen utilization during exercise was significantly less in both fiber types in the low compared to the normal leg (P < 0.05). In the low leg, there was a 14- and 6-fold increase in long-chain fatty acids conjugated to carnitine in type I and type II fibers, respectively, post-exercise. This increase was 3-4 times larger than in the normal leg (P < 0.05). Post-exercise, mTOR^Ser2448 phosphorylation was increased in both fiber types in the normal leg (P < 0.05) but remained unchanged in both fiber types in the low leg together with an increase in eEF2^Thr56 phosphorylation in type I fibers (P < 0.01). Exercise induced a reduction in the autophagy marker LC3B-II in both fiber types and legs, but the post-exercise level was higher in both fiber types in the low leg (P < 0.05). Accordingly, the LC3B-II/I ratio decreased only in the normal leg (75% for type I and 87% for type II, P < 0.01).

CONCLUSIONS: Starting an endurance exercise session with low glycogen availability leads to profound changes in substrate utilization in both type I and type II fibers. This may reduce the mTORC1 signaling response, primarily in type I muscle fibers, and attenuate the normally observed reduction in autophagy.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays a central role in neuronal health. BDNF exists in two primary isoforms, the mature form (mBDNF) and its precursor (proBDNF), with opposing downstream effects on neuronal function. The positive effect of exercise on plasma levels of the BDNF isoforms has been extensively studied in adults. However, equivalent investigations are lacking in children and adolescents. Twenty healthy children (9-12 years old), 19 adolescents (13-17 years old) and 39 adults (23-49 years old) donated venous blood before and after a 45-minute run. Platelet-poor plasma was analysed for pro- and mBDNF using an enzyme-linked immunosorbent assay. Maximal oxygen uptake and anthropometric data were assessed in all participants, while Tanner stage, circulating sex hormones and accelerometry-based activity level were assessed in children and adolescents only. We found that children, adolescents and adults have similar circulating levels of plasma pro- and mBDNF at rest. For children and adolescents, resting levels of mBDNF correlated with average time spent in vigorous activity. In response to the acute endurance exercise intervention, mBDNF increased in all age groups, but the greatest rise in mBDNF was seen in adults. The acute endurance exercise did not affect proBDNF levels. Our results demonstrate that plasma mBDNF levels, but not proBDNF, increase following endurance exercise in all age groups, with a greater effect in adults. We also show that high-intensity physical activity, but not underlying fitness, is contributing to sustained elevated mBDNF levels. KEY POINTS: We show that in children and adolescents, regular vigorous physical activity is key to increased basal levels of plasma mature brain-derived neurotrophic factor (mBDNF), a factor linked to neuroplasticity and brain health. The ability to elevate mBDNF through exercise is present across all age groups, with the greatest increase in adults. The mBDNF response to physical exercise seems to be independent of underlying physical fitness. Our findings suggest that basal plasma mBDNF levels may reflect the cumulative effects of repeated exercise rather than an individual's overall physical fitness.

Brain-derived neurotrophic factor (BDNF) is a key mediator of neuroplasticity and responsive to acute physical exercise, providing a link between exercise and brain health. Lactate, a metabolite related to exercise, has been proposed as a potential mediator of the BDNF exercise response; however, lactate's role in isolation has not yet been determined. To investigate this, 18 young, healthy volunteers (50% female) were recruited to donate blood and muscle before, during, and after a 1-h venous infusion of sodium lactate (125 μmol × kg FFM^-1 × min^-1) or isotonic saline. Muscle and blood samples were collected during 120 min of recovery from the infusion. Samples were analyzed for pro-BDNF and mBDNF using enzyme-linked immunosorbent assay and immunoblotting. The participants reached a peak plasma lactate level of 5.9 ± 0.37 mmol × L^-1 in the lactate trial (p = 0.0002 vs. Pre). Plasma pro-BDNF levels increased 15 min post lactate infusion and stayed elevated throughout the recovery (55%-68%, p < 0.0286 vs. Saline) while plasma and serum levels of mBDNF showed no significant change (p > 0.05 vs. Saline). Muscle pro-BDNF levels were also unaltered by the lactate infusion (p > 0.05 vs. Saline); however, the expression of pro-BDNF correlated with the proportion of type I muscle fiber area (fCSA%) of the participants (n = 18, r = 0.6746, p = 0.0021). Muscle levels of the mBDNF isoform were non-detectable. In conclusion, these results suggest that lactate in isolation affects circulatory pro-BDNF, but not mBDNF levels. This implies that lactate may partly mediate the exercise response of pro-BDNF in humans.

CONTEXT: Insulin resistance (IR) is a major risk factor for the development of several diseases that have reached epidemic proportions worldwide, including hypertension, obesity and type 2 diabetes. In many diseased states, IR is associated with fasting hyperinsulinemia/excessive glucose-stimulated insulin secretion. However, it is not known whether hyperinsulinemia precedes/leads to the natural development of IR or vice versa.

OBJECTIVE: Here, we assess the relationship between hyperinsulinemia and insulin sensitivity in a cohort of healthy young lean men and women, where IR is observed in those who exhibit a low expression of type I skeletal muscle fibers and a high resting heart rate.

METHODS: Biopsies were obtained from the vastus lateralis muscle, followed by an intravenous glucose tolerance test. Insulin secretion and whole-body insulin sensitivity were calculated.

RESULTS: In this young population of normoglycemic, glucose-tolerant individuals, insulin sensitivity was significantly and negatively associated with fasting levels of plasma insulin, as well as insulin secretion in response to glucose infusion. Surprisingly, however, all the correlations became stronger when calculated in women, but became insignificant when calculated in men. In contrast, insulin sensitivity was significantly correlated with expression of type I skeletal muscle fibers and resting heart rate to similar extents in both sexes.

CONCLUSIONS: In the natural development of IR in men, it appears that hyperinsulinemia is a compensatory adaptation to peripheral IR rather than its cause.

OBJECTIVE: Here we use skeletal muscle fiber composition to investigate whether defects in amino acid metabolism are involved in the early development of IR in healthy young individuals before onset of clinical manifestations.

DESIGN: Two groups consisting of healthy young men and women, insulin-sensitive and insulin resistant, were studied using a cross-sectional design.

METHODS: Biopsies were obtained from the vastus lateralis muscle and an intravenous glucose tolerance test was performed. Plasma and muscle tissue were analyzed by metabolomics.

RESULTS: Subjects in group 1 (n=20; age 28±5 yrs; body mass index 22.3±2.7 kg/m2) had an expression of type I muscle fibers and whole-body insulin sensitivity, respectively, of 58.8±5.7% and 1.8±0.7 units. Subjects in group 2 (n=16; age 25±6 yrs; body mass index 22.6±3.0 kg/m2) had an expression of type I muscle fibers and whole-body insulin sensitivity, respectively, of 29.8±6.6% and 0.8±0.3 units (P<0.001 vs. group 1 for both). Anserine and β-alanine contents in muscle were significantly higher and taurine lower in group 2 vs. 1, consistent with the differences in muscle fiber composition between groups. Taurine correlated well with insulin sensitivity and expression of type I muscle fibers (r=0.63; P<0.001 for both). In contrast, there were no significant differences in plasma or tissue contents of glutamine, arginine, or branch-chain amino acids between groups.

CONCLUSIONS: These data demonstrate that the early development of IR is not a consequence of defects in amino acid metabolism. Rather, defects in amino acid metabolism in diseased states are more likely a consequence of IR.