A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

Nampt controls skeletal muscle development by maintaining Ca2+ homeostasis and mitochondrial integrity.

OBJECTIVE: NAD+ is a co-factor and substrate for enzymes maintaining energy homeostasis. Nicotinamide phosphoribosyltransferase (NAMPT) controls NAD+ synthesis, and in skeletal muscle, NAD+ is essential for muscle integrity. However, the underlying molecular mechanisms by which NAD+ synthesis affects muscle health remain poorly understood. Thus, the objective of the current study was to delineate the role of NAMPT-mediated NAD+ biosynthesis in skeletal muscle development and function. METHODS: To determine the role of Nampt in muscle development and function, we generated skeletal muscle-specific Nampt KO (SMNKO) mice. We performed a comprehensive phenotypic characterization of the SMNKO mice, including metabolic measurements, histological examinations, and RNA sequencing analyses of skeletal muscle from SMNKO mice and WT littermates. RESULTS: SMNKO mice were smaller, with phenotypic changes in skeletal muscle, including reduced fiber area and increased number of centralized nuclei. The majority of SMNKO mice died prematurely. Transcriptomic analysis identified that the gene encoding the mitochondrial permeability transition pore (mPTP) regulator Cyclophilin D (Ppif) was upregulated in skeletal muscle of SMNKO mice from 2 weeks of age, with associated increased sensitivity of mitochondria to the Ca2+-stimulated mPTP opening. Treatment of SMNKO mice with the Cyclophilin D inhibitor, Cyclosporine A, increased membrane integrity, decreased the number of centralized nuclei, and increased survival. CONCLUSIONS: Our study demonstrates that NAMPT is crucial for maintaining cellular Ca2+ homeostasis and skeletal muscle development, which is vital for juvenile survival.

Isolation and characterization of muscle stem cells, fibro-adipogenic progenitors, and macrophages from human skeletal muscle biopsies.

Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11c+/-) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.

Exercise Training Promotes Sex-Specific Adaptations in Mouse Inguinal White Adipose Tissue.

Recent studies demonstrate that adaptations to white adipose tissue (WAT) are important components of the beneficial effects of exercise training on metabolic health. Exercise training favorably alters the phenotype of subcutaneous inguinal WAT (iWAT) in male mice, including decreasing fat mass, improving mitochondrial function, inducing beiging, and stimulating the secretion of adipokines. In this study, we find that despite performing more voluntary wheel running compared with males, these adaptations do not occur in the iWAT of female mice. Consistent with sex-specific adaptations, we report that mRNA expression of androgen receptor coactivators is upregulated in iWAT from trained male mice and that testosterone treatment of primary adipocytes derived from the iWAT of male, but not female mice, phenocopies exercise-induced metabolic adaptations. Sex specificity also occurs in the secretome profile, as we identify cysteine-rich secretory protein 1 (Crisp1) as a novel adipokine that is only secreted from male iWAT in response to exercise. Crisp1 expression is upregulated by testosterone and functions to increase glucose and fatty acid uptake. Our finding that adaptations to iWAT with exercise training are dramatically greater in male mice has potential clinical implications for understanding the different metabolic response to exercise training in males and females and demonstrates the importance of investigating both sexes in studies of adipose tissue biology.

Nicotinamide riboside does not alter mitochondrial respiration, content or morphology in skeletal muscle from obese and insulin-resistant men.

KEY POINTS: This is the first long-term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. ABSTRACT: Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle-aged, obese, insulin-resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo-controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high-resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady-state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin-resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people.

Six Weeks of Low-Load Blood Flow Restricted and High-Load Resistance Exercise Training Produce Similar Increases in Cumulative Myofibrillar Protein Synthesis and Ribosomal Biogenesis in Healthy Males.

Purpose: High-load resistance exercise contributes to maintenance of muscle mass, muscle protein quality, and contractile function by stimulation of muscle protein synthesis (MPS), hypertrophy, and strength gains. However, high loading may not be feasible in several clinical populations. Low-load blood flow restricted resistance exercise (BFRRE) may provide an alternative approach. However, the long-term protein synthetic response to BFRRE is unknown and the myocellular adaptations to prolonged BFRRE are not well described. Methods: To investigate this, 34 healthy young subjects were randomized to 6 weeks of low-load BFRRE, HLRE, or non-exercise control (CON). Deuterium oxide (D2O) was orally administered throughout the intervention period. Muscle biopsies from m. vastus lateralis were collected before and after the 6-week intervention period to assess long-term myofibrillar MPS and RNA synthesis as well as muscle fiber-type-specific cross-sectional area (CSA), satellite cell content, and myonuclei content. Muscle biopsies were also collected in the immediate hours following single-bout exercise to assess signaling for muscle protein degradation. Isometric and dynamic quadriceps muscle strength was evaluated before and after the intervention. Results: Myofibrillar MPS was higher in BFRRE (1.34%/day, p < 0.01) and HLRE (1.12%/day, p < 0.05) compared to CON (0.96%/day) with no significant differences between exercise groups. Muscle RNA synthesis was higher in BFRRE (0.65%/day, p < 0.001) and HLRE (0.55%/day, p < 0.01) compared to CON (0.38%/day) and both training groups increased RNA content, indicating ribosomal biogenesis in response to exercise. BFRRE and HLRE both activated muscle degradation signaling. Muscle strength increased 6-10% in BFRRE (p < 0.05) and 13-23% in HLRE (p < 0.01). Dynamic muscle strength increased to a greater extent in HLRE (p < 0.05). No changes in type I and type II muscle fiber-type-specific CSA, satellite cell content, or myonuclei content were observed. Conclusions: These results demonstrate that BFRRE increases long-term muscle protein turnover, ribosomal biogenesis, and muscle strength to a similar degree as HLRE. These findings emphasize the potential application of low-load BFRRE to stimulate muscle protein turnover and increase muscle function in clinical populations where high loading is untenable.

Effects of 3-hydroxybutyrate and free fatty acids on muscle protein kinetics and signaling during LPS-induced inflammation in humans: anticatabolic impact of ketone bodies.

Background: Acute inflammation, and subsequent release of bacterial products (e.g. LPS), inflammatory cytokines, and stress hormones, is catabolic, and the loss of lean body mass predicts morbidity and mortality. Lipid intermediates may reduce protein loss, but the roles of free fatty acids (FFAs) and ketone bodies during acute inflammation are unclear. Objective: We aimed to test whether infusions of 3-hydroxybutyrate (3OHB), FFAs, and saline reduce protein catabolism during exposure to LPS and Acipimox (to restrict and control endogenous lipolysis). Design: A total of 10 healthy male subjects were randomly tested 3 times, with: 1) LPS, Acipimox (Olbetam) and saline, 2) LPS, Acipimox, and nonesterified fatty acids (Intralipid), and 3) LPS, Acipimox, and 3OHB, during a 5-h basal period and a 2-h hyperinsulinemic, euglycemic clamp. Labeled phenylalanine, tyrosine, and urea tracers were used to estimate protein kinetics, and muscle biopsies were taken for Western blot analysis of protein metabolic signaling. Results: 3OHB infusion increased 3OHB concentrations (P < 0.0005) to 3.5 mM and decreased whole-body phenylalanine-to-tyrosine degradation. Basal and insulin-stimulated net forearm phenylalanine release decreased by >70% (P < 0.005), with both appearance and phenylalanine disappearance being profoundly decreased. Phosphorylation of eukaryotic initiation factor 2α at Ser51 was increased in skeletal muscle, and S6 kinase phosphorylation at Ser235/236 tended (P = 0.074) to be decreased with 3OHB infusion (suggesting inhibition of protein synthesis), whereas no detectable effects were seen on markers of protein breakdown. Lipid infusion did not affect phenylalanine kinetics, and insulin sensitivity was unaffected by interventions. Conclusion: During acute inflammation, 3OHB has potent anticatabolic actions in muscle and at the whole-body level; in muscle, reduction of protein breakdown overrides inhibition of synthesis. This trial was registered at clinicaltrials.gov as NCT01752348.

Anabolic effects of leucine-rich whey protein, carbohydrate, and soy protein with and without ß-hydroxy-ß-methylbutyrate (HMB) during fasting-induced catabolism: A human randomized crossover trial.

BACKGROUND: Protein-rich beverages are widely used clinically to preserve muscle protein and improve physical performance. Beverages with high contents of leucine or its keto-metabolite ß-hydroxy-ß-methylbutyrate (HMB) are especially anabolic in muscle, but it is uncertain whether this also applies to catabolic conditions such as fasting and whether common or separate intracellular signaling cascades are involved. OBJECTIVE: To compare a specific leucine-rich whey protein beverage (LWH) with isocaloric carbohydrate- (CHO), soy protein (SOY), and soy protein +3 g HMB (HMB) during fasting-induced catabolic conditions. DESIGN: Eight healthy lean male subjects underwent four interventions (LWH, CHO, SOY, and HMB) using a randomized crossover design. Each trial included a 36 h fast and consisted of a 3 h basal fasting period and a 4 h 'sipping' period. RESULTS: Forearm net balances of phenylalanine (NBphe, measure of net protein loss) improved for all groups (p < 0.05), but more prominently so for LWH and HMB compared with SOY (p < 0.05). Muscle protein phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets eukaryotic translation factor 4E-binding protein 1 (4EBP1) and ribosomal S6 kinase 1 (S6) were distinctly increased during LWH consumption (p < 0.05). The ratio between autophagy protein microtubule-associated protein 1 light chain-3ß II and I (LC3II/LC3I, a measure of autophagy activity) was decreased during LWH and SOY intake compared with the fasting period (p < 0.05). CONCLUSION: LWH and HMB have superior anabolic effects on muscle protein kinetics after 36 h of fasting, and LWH distinctly activates the mTOR pathway. These novel findings suggest that leucine-rich whey protein and/or HMB are specifically beneficial during fasting-induced catabolic conditions.

Stress hormone release is a key component of the metabolic response to lipopolysaccharide: studies in hypopituitary and healthy subjects.

OBJECTIVE: Acute and chronic inflammatory and metabolic responses are generated by lipopolysaccharide (LPS) during acute illness and in the pathogenesis of the metabolic syndrome, type 2 diabetes and cardiovascular disease, but whether these responses depend on intact pituitary release of hormones are not clearly identified. We compared the metabolic effects of LPS in hypopituitary patients (HPs) (in the absence of growth hormone (GH) and ACTH responses) and healthy control subjects (CTR) (with normal pituitary hormone responses). DESIGN: Single-blind randomized. METHODS: We compared the effects of LPS on glucose, protein and lipid metabolism in eight HP and eight matched CTR twice during 4-h basal and 2-h hyperinsulinemic-euglycemic clamp conditions with muscle and fat biopsies in each period during infusion with saline or LPS. RESULTS: LPS increased cortisol and GH levels in CTR but not in HP. Also, it increased whole-body palmitate fluxes (3-fold) and decreased palmitate-specific activity (SA) 40-50% in CTR, but not in HP. G(0)/G(1) Switch Gene 2 (G0S2 - an inhibitor of lipolysis) adipose tissue (AT) mRNA was decreased in CTR. Although LPS increased phenylalanine fluxes significantly more in CTR, there was no difference in glucose metabolism between groups and intramyocellular insulin signaling was unaltered in both groups. CONCLUSIONS: LPS increased indices of lipolysis and amino acid/protein fluxes significantly more in CTR compared with HP and decreased adipocyte G0S2 mRNA only in CTR. Thus, in humans intact pituitary function and appropriate cortisol and GH release are crucial components of the metabolic response to LPS.

Intact pituitary function is decisive for the catabolic response to TNF-α: studies of protein, glucose and fatty acid metabolism in hypopituitary and healthy subjects.

CONTEXT: TNF-α generates inflammatory responses and insulin resistance, lipolysis, and protein breakdown. It is unclear whether these changes depend on intact hypothalamo-pituitary stress hormone responses to trigger the release of cortisol and growth hormone. OBJECTIVE: To define differential effects of TNF-α on glucose, protein, and lipid metabolism in hypopituitary patients (without intact hypothalamo-pituitary axis) and healthy controls. DESIGN: Randomized, placebo controlled, single-blinded. Setting, Participants, and Intervention: We studied eight hypopituitary (HP) patients and eight matched control subjects [control volunteers (CTR)] twice during 4-h basal and 2-h hyperinsulinemic clamp conditions with isotope dilution during infusion of saline or TNF-α(12 ng/kg/h) for 6 h. MAIN OUTCOME MEASURES: Phenylalanine, urea, palmitate, and glucose fluxes and fat biopsies in basal and clamp periods. RESULTS: TNF-α infusion significantly increased cortisol and GH levels in CTR but not in HP. TNF-α increased phenylalanine fluxes in both groups, with the increase being significantly greater in CTR, and raised urea flux by 40 % in CTR without any alteration in HP. Endogenous glucose production (EGP) was elevated in CTR compared to HP after TNF-α administration, whereas insulin sensitivity remained similarly unaffected in both groups. TNF-α increased whole body palmitate fluxes and decreased palmitate specific activity in CTR, but not in HP without statistical difference between groups. We did not detect significant effects TNF-α on lipase expression or regulation in fat. CONCLUSIONS: TNF-α increased both urea and amino acid fluxes and EGP significantly more in CTR compared to HP, suggesting that increases in endogenous cortisol and GH release are significant components of the metabolic response to TNF-α.

Direct effects of TNF-α on local fuel metabolism and cytokine levels in the placebo-controlled, bilaterally infused human leg: increased insulin sensitivity, increased net protein breakdown, and increased IL-6 release.

Tumor necrosis factor-α (TNF-α) has widespread metabolic actions. Systemic TNF-α administration, however, generates a complex hormonal and metabolic response. Our study was designed to test whether regional, placebo-controlled TNF-α infusion directly affects insulin resistance and protein breakdown. We studied eight healthy volunteers once with bilateral femoral vein and artery catheters during a 3-h basal period and a 3-h hyperinsulinemic-euglycemic clamp. One artery was perfused with saline and one with TNF-α. During the clamp, TNF-α perfusion increased glucose arteriovenous differences (0.91 ± 0.17 vs. 0.74 ± 0.15 mmol/L, P = 0.012) and leg glucose uptake rates. Net phenylalanine release was increased by TNF-α perfusion with concomitant increases in appearance and disappearance rates. Free fatty acid kinetics was not affected by TNF-α, whereas interleukin-6 (IL-6) release increased. Insulin and protein signaling in muscle biopsies was not affected by TNF-α. TNF-α directly increased net muscle protein loss, which may contribute to cachexia and general protein loss during severe illness. The finding of increased insulin sensitivity, which could relate to IL-6, is of major clinical interest and may concurrently act to provide adequate tissue fuel supply and contribute to the occurrence of systemic hypoglycemia. This distinct metabolic feature places TNF-α among the rare insulin mimetics of human origin.

Direct effects of locally administered lipopolysaccharide on glucose, lipid, and protein metabolism in the placebo-controlled, bilaterally infused human leg.

CONTEXT: Accumulating evidence suggests that chronic exposure to lipopolysaccharide (LPS, endotoxin) may create a constant low-grade inflammation, leading to insulin resistance and diabetes. All previous human studies assessing the metabolic actions of LPS have used systemic administration, making discrimination between direct and indirect effects impossible. OBJECTIVE: We sought to define the direct, placebo-controlled effects of LPS on insulin resistance and protein and lipid metabolism in the infused human leg without systemic interference from cytokines and stress hormones. DESIGN: This was a randomized, placebo-controlled, single-blinded study. PARTICIPANTS AND INTERVENTION: We studied 8 healthy volunteers with bilateral femoral vein and artery catheters during a 3-hour basal and 3-hour hyperinsulinemic-euglycemic clamp period with bilateral muscle biopsies in each period during infusion with saline and LPS. RESULTS: Overall, LPS perfusion significantly decreased leg glucose uptake, and during the clamp LPS decreased glucose arteriovenous differences (0.65 ± 0.07 mmol/L vs 0.73 ± 0.08 mmol/L). Net palmitate release was increased by LPS, and secondary post hoc testing indicated increased palmitate isotopic dilution, although primary ANOVA tests did not reveal significant dilution. Leg blood flows, phenylalanine, lactate kinetics, cytokines, and intramyocellular insulin signaling were not affected by LPS. LPS thus directly inhibits insulin-stimulated glucose uptake and increases palmitate release in the perfused human leg without detectable effects on amino acid metabolism. CONCLUSIONS: These data strongly suggest that the primary metabolic effect of LPS is increased lipolysis and muscle insulin resistance, which, together with secondary insulin resistance, caused by systemic cytokine and stress hormone release may lead to overt glucose intolerance and diabetes.

Heat sensitive persons with multiple sclerosis are more tolerant to resistance exercise than to endurance exercise.

BACKGROUND: Heat sensitivity (HS) is reported by 58% of all persons with multiple sclerosis (MS), causing symptom exacerbation possibly limiting exercise participation. OBJECTIVE: The purpose of this study was to test the hypotheses that (a) a relationship between exercise-induced changes in core-temperature (C(temp)) and changes in symptom intensity exists, and (b) that resistance exercise (RE), as a consequence of a minor increase in core temperature, will induce a lesser worsening of symptoms than endurance exercise (EE) in HS persons with MS. METHODS: On two separate days, 16 HS persons with MS randomly completed a session of RE and EE, or EE and RE, respectively. Testing was conducted pre, post and one hour after exercise and consisted of Visual Analogue Scale (VAS) scoring (fatigue, spasticity, pain, strength, walking and balance), the 5-time sit-to-stand (5STS), the Multiple Sclerosis Functional Composite (MSFC) and Body Sway. Composite scores describing average subjective symptom intensity (SI) and total number of symptoms (SN) were calculated from VAS scores. RESULTS: C(temp) (0.9±0.4°C vs 0.3±0.1°C, p<0.001), SI (1.7±1.9 cm vs 0.6±1.5 cm, p<0.05) and SN (1.6±1.9 vs 0.6±2.1, p<0.05) increased significantly more during EE than RE. Changes in C(temp) correlated to changes in SI (r=0.50, p<0.01). No differences were observed in 5STS, MSFC and Body Sway scores after EE when compared to RE. CONCLUSION: An exercise-induced increase in C(temp) is associated with increased number and severity of perceived symptoms in HS persons with MS. Based on these findings it is expected that HS persons with MS do tolerate RE better than EE.

Muscle morphological and strength adaptations to endurance vs. resistance training.

Fascicle angle (FA) is suggested to increase as a result of fiber hypertrophy and furthermore to serve as the explanatory link in the discrepancy in the relative adaptations in the anatomical cross-sectional area (CSA) and fiber CSA after resistance training (RT). In contrast to RT, the effects of endurance training on FA are unclear. The purpose of this study was therefore to investigate and compare the longitudinal effects of either progressive endurance training (END, n = 7) or RT (n = 7) in young untrained men on FA, anatomical CSA, and fiber CSA. Muscle morphological measures included the assessment of vastus lateralis FA obtained by ultrasonography and anatomical CSA by magnetic resonance imaging of the thigh and fiber CSA deduced from histochemical analyses of biopsy samples from m. vastus lateralis. Functional performance measures included VO2max and maximal voluntary contraction (MVC). The RT produced increases in FA by 23 ± 8% (p < 0.01), anatomical CSA of the knee extensor muscles by 9 ± 3% (p = 0.001), and fiber CSA by 19 ± 7% (p < 0.05). RT increased knee extensor MVC by 20 ± 5% (p < 0.001). END increased VO2max by 10 ± 2% but did not evoke changes in FA, anatomical CSA, or in fiber CSA. In conclusion, the morphological changes induced by 10 weeks of RT support that FA does indeed serve as the explanatory link in the observed discrepancy between the changes in anatomical and fiber CSA. Contrarily, 10 weeks of endurance training did not induce changes in FA, but the lack of morphological changes from END indirectly support the fact that fiber hypertrophy and FA are interrelated.