Effects of fat loss and low energy availability on the serum cardiometabolic profile of physique athletes.

Low energy availability (LEA) is a health concern for athletes, although it may paradoxically lead to improved cardiometabolic health in the general population. We investigated the associations between LEA, body composition, and serum cardiometabolic profile in 23 physique athletes (DIET) and 21 controls (CONT) during a 5-month pre-competition diet (MID), followed by 1 week of increased energy availability (COMP) and a 5-month weight regain period (POST). Quantification of 250 serum metabolome variables was conducted by NMR spectroscopy, body composition by dual-energy x-ray absorptiometry, dietary intake by food diaries, and exercise levels by training logs. Body fat percentage decreased from 19.5 ± 7.0% to 8.3 ± 5.3% (p < 0.001) in DIET through increased exercise levels and decreased energy intake, while CONT maintained those constant. In MID, DIET had increased (FDR < 0.01) HDL cholesterol, HDL particle size and number, and decreased (FDR < 0.05) VLDL lipids, serum triglycerides, and low-grade inflammation (glycoprotein acetyls) compared to baseline and CONT. The changes were associated with reduced android fat mass (-78 ± 13%) and energy intake (-28 ± 10%). In COMP, most of the metabolic changes found in MID persisted, except for altered triglycerides in all lipoprotein classes. After weight regain in POST, serum metabolome, body composition, energy intake, and exercise levels had reverted to baseline levels. In conclusion, fat loss and LEA may have beneficial yet transient effects on the serum cardiometabolic profile of lean individuals. Especially the HDL lipidome and lipoprotein triglycerides offer potential novel biomarkers for detecting LEA in athletes.

Mimicking exercise in vitro: effects of myotube contractions and mechanical stretch on omics.

The number of studies using skeletal muscle (SkM) cell culture models to study exercise in vitro are rapidly expanding. Progressively, more comprehensive analysis methods, such as different omics approaches including transcriptomics, proteomics, and metabolomics have been used to examine the intra- and extracellular molecular responses to exercise mimicking stimuli in cultured myotubes. Among other techniques, exercise-like electrical pulse stimulation (EL-EPS) and mechanical stretch of SkM cells are the two most commonly used methods to mimic exercise in vitro. In this mini-review, we focus on these two approaches and their effects on the omics of myotubes and/or cell culture media. Furthermore, besides traditional two-dimensional (2-D) methods, the use of three-dimensional (3-D) SkM approaches are increasing in the field of in vitro exercise mimicry. Our aim with this mini-review is to provide the reader with an up-to-date overview of the 2-D and 3-D models and the use of omics approaches to study the molecular response to exercise in vitro.

Vitamin C and E supplementation alters protein signalling after a strength training session, but not muscle growth during 10 weeks of training.

This study investigated the effects of vitamin C and E supplementation on acute responses and adaptations to strength training. Thirty-two recreationally strength-trained men and women were randomly allocated to receive a vitamin C and E supplement (1000 mg day(-1) and 235 mg day(-1), respectively), or a placebo, for 10 weeks. During this period the participants' training involved heavy-load resistance exercise four times per week. Muscle biopsies from m. vastus lateralis were collected, and 1 repetition maximum (1RM) and maximal isometric voluntary contraction force, body composition (dual-energy X-ray absorptiometry), and muscle cross-sectional area (magnetic resonance imaging) were measured before and after the intervention. Furthermore, the cellular responses to a single exercise session were assessed midway in the training period by measurements of muscle protein fractional synthetic rate and phosphorylation of several hypertrophic signalling proteins. Muscle biopsies were obtained from m. vastus lateralis twice before, and 100 and 150 min after, the exercise session (4 × 8RM, leg press and knee-extension). The supplementation did not affect the increase in muscle mass or the acute change in protein synthesis, but it hampered certain strength increases (biceps curl). Moreover, increased phosphorylation of p38 mitogen-activated protein kinase, Extracellular signal-regulated protein kinases 1 and 2 and p70S6 kinase after the exercise session was blunted by vitamin C and E supplementation. The total ubiquitination levels after the exercise session, however, were lower with vitamin C and E than placebo. We concluded that vitamin C and E supplementation interfered with the acute cellular response to heavy-load resistance exercise and demonstrated tentative long-term negative effects on adaptation to strength training.

Resistance training induced increase in muscle fiber size in young and older men.

Muscle strength and mass decline in sedentary individuals with aging. The present study investigated the effects of both age and 21 weeks of progressive hypertrophic resistance training (RT) on skeletal muscle size and strength, and on myostatin and myogenin mRNA expression in 21 previously untrained young men (26.0 ± 4.3 years) and 18 older men (61.2 ± 4.1 years) and age-matched controls. Vastus lateralis muscle biopsies were taken before and after RT. Type I and type II muscle fiber cross-sectional areas increased more in young men than in older men after RT (P < 0.05). Concentric leg extension increased (P < 0.05) more after 10.5 weeks in young men compared to older men, but after 21 weeks no statistical differences existed. The daily energy and protein intake were greater (P < 0.001) in young subjects. Both myostatin and myogenin mRNA expression increased in older when compared with young men after RT (P < 0.05). In conclusion, after RT, muscle fiber size increased less in older compared to young men. This was associated with lower protein and energy intake and increases in myostatin gene expression in older when compared to young men.

Molecular signaling in muscle is affected by the specificity of resistance exercise protocol.

Mammalian target of rapamycin and mitogen-activated protein kinase (MAPK) signaling pathways have been highlighted as important for muscle adaptations and thus, they may distinguish adaptations to different exercises. Typically, resistance exercise designed for muscle hypertrophy has moderate intensity (60-80% of one repetition maximum, 1 RM) while one prioritizing maximal strength with minor hypertrophy has a higher intensity (≥90% of 1 RM). Eight untrained men (28.4 ± 3.7 years) conducted two different bilateral leg press exercise protocols: hypertrophic (5 × 10 RM) and pure maximal strength (15 × 1 RM) in a counterbalanced, cross-over design with 1 week between exercises. Vastus lateralis muscle biopsies were taken before and 0.5 h after resistance exercise, or in six controls (26.5 ± 3.6 years) who rested. The phosphorylation of p70S6K (Thr(421) /Ser(424) ), rpS6 (Ser(240/244) and Ser(235/236) ) and MAPK p38 as increased (∼2-16 fold) after both exercise protocols. However, the phosphorylation of MAPK Erk1/2 and p70S6K at Thr(389) increased only after 5 × 10 RM. The increase in the phosphorylation of p70S6K (Thr(421) /Ser(424) ), rpS6 (Ser(235/236) ) and Erk1/2 were higher after 5 × 10 RM (P<0.05). No changes were seen in controls. In conclusion, MAPK signaling is greater after hypertrophic than maximal strength exercise protocol. This may mediate adaptations specific to these different types of training regimens.

Muscle NAD+ depletion and Serpina3n as molecular determinants of murine cancer cachexia-the effects of blocking myostatin and activins.

OBJECTIVE: Cancer cachexia and muscle loss are associated with increased morbidity and mortality. In preclinical animal models, blocking activin receptor (ACVR) ligands has improved survival and prevented muscle wasting in cancer cachexia without an effect on tumour growth. However, the underlying mechanisms are poorly understood. This study aimed to identify cancer cachexia and soluble ACVR (sACVR) administration-evoked changes in muscle proteome. METHODS: Healthy and C26 tumour-bearing (TB) mice were treated with recombinant sACVR. The sACVR or PBS control were administered either prior to the tumour formation or by continued administration before and after tumour formation. Muscles were analysed by quantitative proteomics with further examination of mitochondria and nicotinamide adenine dinucleotide (NAD+) metabolism. To complement the first prophylactic experiment, sACVR (or PBS) was injected as a treatment after tumour cell inoculation. RESULTS: Muscle proteomics in TB cachectic mice revealed downregulated signatures for mitochondrial oxidative phosphorylation (OXPHOS) and increased acute phase response (APR). These were accompanied by muscle NAD+ deficiency, alterations in NAD+ biosynthesis including downregulation of nicotinamide riboside kinase 2 (Nrk2), and decreased muscle protein synthesis. The disturbances in NAD+ metabolism and protein synthesis were rescued by treatment with sACVR. Across the whole proteome and APR, in particular, Serpina3n represented the most upregulated protein and the strongest predictor of cachexia. However, the increase in Serpina3n expression was associated with increased inflammation rather than decreased muscle mass and/or protein synthesis. CONCLUSIONS: We present evidence implicating disturbed muscle mitochondrial OXPHOS proteome and NAD+ homeostasis in experimental cancer cachexia. Treatment of TB mice with a blocker of activin receptor ligands restores depleted muscle NAD+ and Nrk2, as well as decreased muscle protein synthesis. These results indicate putative new treatment therapies for cachexia and that although acute phase protein Serpina3n may serve as a predictor of cachexia, it more likely reflects a condition of elevated inflammation.

Strength, [corrected] endurance or combined training elicit diverse skeletal muscle myosin heavy chain isoform proportion but unaltered androgen receptor concentration in older men.

We investigated whether the myosin heavy chain (MyHC) proportion and androgen receptor (AR) concentration in skeletal muscle differ following 21 weeks of strength, endurance and combined training in untrained older men. Strength (S) and endurance (E) groups trained twice per week and combined (S+E) group trained four times per week (two strength and two endurance). Muscle biopsies were obtained before and after the training period from m. vastus lateralis (VL) and AR mRNA and protein concentration and MyHC proportion were determined. 1RM increased during the training period in S, S+E and E but the changes were greater in S and S+E than in E. Statistically significant increases were observed only in S and S+E in maximal isometric force as well as in VL thickness. VO (2max) increased significantly only in E. MyHCIIa proportion increased in S, while MyHCIIa proportion decreased and MyHCI increased (p<0.05) in E. No statistically significant changes were observed in serum testosterone and in AR mRNA or protein concentrations. The present results indicate that 21 weeks of strength, endurance or combined training changed MyHC proportion according to the training method but did not have an effect on AR mRNA or protein expression in skeletal muscle at rest.

Beneficial effects of running and milk protein supplements on Sirtuins and risk factors of metabolic disorders in rats with low aerobic capacity.

BACKGROUND: Physical activity and dietary intake of dairy products are associated with improved metabolic health. Dairy products are rich with branched chain amino acids that are essential for energy production. To gain insight into the mechanisms underlying the benefit of the sub-chronic effects of running and intake of milk protein supplements, we studied Low Capacity Runner rats (LCR), a rodent exercise model with risk for metabolic disorders. We especially focused on the role of Sirtuins, energy level dependent proteins that affect many cellular metabolic processes. METHODS: Forty-seven adult LCR female rats sedentary or running voluntarily in wheels were fed normal chow and given supplements of either whey or milk protein drink (PD)-supplemented water, or water only for 21 weeks. Physiological responses were measured in vivo. Blood lipids were determined from serum. Mitochondrial markers and Sirtuins (Sirt1-7) including downstream targets were measured in plantaris muscle by western blotting. RESULTS: For the first 10 weeks whey-drinking rats ran about 50% less compared to other groups; still, in all runners glucose tolerance improved and triglycerides decreased. Generally, running induced a ∼six-fold increase in running capacity and a ∼8% decrease in % body fat. Together with running, protein supplements increased the relative lean mass of the total body weight by ∼11%. In comparison with sedentary controls, running and whey increased HDL (21%) and whey, with or without running, lowered LDL (-34%). Running increased mitochondrial biogenesis and Sirtuins 3 and 4. When combined with exercise, both whey and milk protein drink induced about a 4-fold increase in Sirt3, compared to runners drinking water only, and about a 2-fold increase compared to the respective sedentary group. Protein supplements, with or without running, enhanced the phosphorylation level of the acetyl-coA-carboxylase, suggesting increased fat oxidation. Both supplemented diets increased Sirt5 and Sirt7 without an additional effect from exercise. Running diminished and PD supplement increased Sirt6. CONCLUSION: We demonstrate in rats new sub-chronic effects of milk proteins on metabolism that involve Sirtuins and their downstream targets in skeletal muscle. The results show that running and milk proteins act on reducing the risk factors of metabolic disorders and suggest that the underlying mechanisms may involve Sirtuins. Notably, we found that milk protein supplements have some favorable effects on metabolism even without running.

Substantial fat mass loss reduces low-grade inflammation and induces positive alteration in cardiometabolic factors in normal-weight individuals.

The accumulation of fat, especially in visceral sites, is a significant risk factor for several chronic diseases with altered cardiometabolic homeostasis. We studied how intensive long-term weight loss and subsequent weight regain affect physiological changes, by longitudinally interrogating the lipid metabolism and white blood cell transcriptomic markers in healthy, normal-weight individuals. The current study examined 42 healthy, young (age: 27.5 ± 4.0 years), normal-weight (body mass index, BMI: 23.4 ± 1.7 kg/m2) female athletes, of which 25 belong to the weight loss and regain group (diet group), and 17 to the control group. Participants were evaluated, and fasting blood samples were drawn at three time points: at baseline (PRE); at the end of the weight loss period (MID: 21.1 ± 3.1 weeks after PRE); and at the end of the weight regain period (POST: 18.4 ± 2.9 weeks after MID). Following the weight loss period, the diet group experienced a ~73% reduction (~0.69 kg) in visceral fat mass (false discovery rate, FDR < 2.0 × 10-16), accompanied by anti-atherogenic effects on transcriptomic markers, decreased low-grade inflammation (e.g., as α1-acid glycoprotein (FDR = 3.08 × 10-13) and hs-CRP (FDR = 2.44 × 10-3)), and an increase in functionally important anti-atherogenic high-density lipoprotein -associated metabolites (FDR < 0.05). This occurred even though these values were already at favorable levels in these participants, who follow a fitness-lifestyle compared to age- and BMI-matched females from the general population (n = 58). Following the weight regain period, most of the observed beneficial changes in visceral fat mass, and metabolomic and transcriptomic profiles dissipated. Overall, the beneficial anti-atherogenic effects of weight loss can be observed even in previously healthy, normal-weight individuals.

Effect of strength training session on plasma amino acid concentration following oral ingestion of arginine or taurine in men.

This study examined the acute effects of a one-hour hypertrophic strength training session (STS) on plasma amino acid concentration following oral ingestion of arginine or taurine in nine physically active men participating in a double-blind and randomised experiment. The subjects took placebo, arginine or taurine capsules (50 mg/kg) in either rest (REST) or STS condition. Blood samples were taken before and at 30, 60, 90, and 120 min after the beginning of the treatment and assayed for plasma amino acids with HPLC. There was a significant interaction effect with STS and sample time for both arginine and taurine in the raw data (p < 0.05). The modelled polynomial data for the arginine treatment showed that the peak concentration of arginine occurred at 69 min at rest and at 104 min in STS, and for the taurine treatment, the peak concentration of taurine occurred at 89 min at rest and at 112 min in STS. In conclusion, one hour of hypertrophic STS slows the increase in the peak concentration of plasma arginine and taurine after oral ingestion of the respective amino acids.

Autophagy is induced by resistance exercise in young men, but unfolded protein response is induced regardless of age.

AIM: Autophagy and unfolded protein response (UPR) appear to be important for skeletal muscle homoeostasis and may be altered by exercise. Our aim was to investigate the effects of resistance exercise and training on indicators of UPR and autophagy in healthy untrained young men (n = 12, 27 ± 4 years) and older men (n = 8, 61 ± 6 years) as well as in resistance-trained individuals (n = 15, 25 ± 5 years). METHODS: Indicators of autophagy and UPR were investigated from the muscle biopsies after a single resistance exercise bout and after 21 weeks of resistance training. RESULTS: Lipidated LC3II as an indicator of autophagosome content increased at 48 hours post-resistance exercise (P < .05) and after a resistance training period (P < .01) in untrained young men but not in older men. Several UPRER markers, typically induced by protein misfolding in endoplasmic reticulum, were increased at 48 hours post-resistance exercise in untrained young and older men (P < .05) but were unaltered after the 21-week resistance training period regardless of age. UPR was unchanged within the first few hours after the resistance exercise bout regardless of the training status. Changes in autophagy and UPRER indicators did not correlate with a resistance training-induced increase in muscle strength and size. CONCLUSION: Autophagosome content is increased by resistance training in young previously untrained men, but this response may be blunted by ageing. However, unfolded protein response is induced by an unaccustomed resistance exercise bout in a delayed manner regardless of age.

Systemic blockade of ACVR2B ligands prevents chemotherapy-induced muscle wasting by restoring muscle protein synthesis without affecting oxidative capacity or atrogenes.

Doxorubicin is a widely used and effective chemotherapy drug. However, cardiac and skeletal muscle toxicity of doxorubicin limits its use. Inhibiting myostatin/activin signalling can prevent muscle atrophy, but its effects in chemotherapy-induced muscle wasting are unknown. In the present study we investigated the effects of doxorubicin administration alone or combined with activin receptor ligand pathway blockade by soluble activin receptor IIB (sACVR2B-Fc). Doxorubicin administration decreased body mass, muscle size and bone mineral density/content in mice. However, these effects were prevented by sACVR2B-Fc administration. Unlike in many other wasting situations, doxorubicin induced muscle atrophy without markedly increasing typical atrogenes or protein degradation pathways. Instead, doxorubicin decreased muscle protein synthesis which was completely restored by sACVR2B-Fc. Doxorubicin administration also resulted in impaired running performance without effects on skeletal muscle mitochondrial capacity/function or capillary density. Running performance and mitochondrial function were unaltered by sACVR2B-Fc administration. Tumour experiment using Lewis lung carcinoma cells demonstrated that sACVR2B-Fc decreased the cachectic effects of chemotherapy without affecting tumour growth. These results demonstrate that blocking ACVR2B signalling may be a promising strategy to counteract chemotherapy-induced muscle wasting without damage to skeletal muscle oxidative capacity or cancer treatment.

Effect of diet composition on acid-base balance in adolescents, young adults and elderly at rest and during exercise.

BACKGROUND: Diets rich in animal protein and cereal grains and deficient in vegetables and fruits may cause low-grade metabolic acidosis, which may impact exercise and health. We hypothesized that (1) a normal-protein diet with high amount of vegetables and fruits (HV) induces more alkaline acid-base balance compared with a high-protein diet with no vegetables and fruits (HP) and (2) diet composition has a greater impact on acid-base balance in the elderly (ELD). SUBJECTS/METHODS: In all, 12-15 (adolescents (ADO)), 25-35 (young adults (YAD)) and 60-75 (ELD)-year-old male and female subjects (n=88) followed a 7-day HV and a 7-day HP in a randomized order and at the end performed incremental cycle ergometer tests. We investigated the effect of diet composition and age on capillary (c-pH) and urine pH (u-pH), strong ion difference (SID), partial pressure of carbon dioxide (pCO2) and total concentration of weak acids (Atot). Linear regression analysis was used to examine the contribution of SID, pCO2 and Atot to c-pH. RESULTS: In YAD and ELD, c-pH (P⩽0.038) and u-pH (P<0.001) were higher at rest after HV compared with HP. During cycling, c-pH was higher (P⩽0.034) after HV compared with HP at submaximal workloads in YAD and at 75% of VO2max (maximal oxygen consumption) in ELD. The contribution of SID, pCO2 and Atot to c-pH varied widely. Gender effects or changes in acid-base balance of ADO were not detected. CONCLUSIONS: A high intake of vegetables and fruits increases blood and u-pH in YAD and ELD. ELD compared with younger persons may be more sensitive for the diet-induced acid-base changes.

Cellular and tissue expression of DAPIT, a phylogenetically conserved peptide.

DAPIT (Diabetes Associated Protein in Insulin-sensitive Tissues) is a small, phylogenetically conserved, 58 amino acid peptide that was previously shown to be down-regulated at mRNA level in insulin-sensitive tissues of type 1 diabetes rats. In this study we characterize a custom made antibody against DAPIT and confirm the mitochondrial presence of DAPIT on cellular level. We also show that DAPIT is localized in lysosomes of HUVEC and HEK 293T cells. In addition, we describe the histological expression of DAPIT in several tissues of rat and man and show that it is highly expressed especially in cells with high aerobic metabolism and epithelial cells related to active transport of nutrients and ions. We propose that DAPIT, in addition to indicated subunit of mitochondrial F-ATPase, is also a subunit of lysosomal V-ATPase suggesting that it is a common component in different proton pumps.