Influence of intramuscular steroid receptor content and fiber capillarization on skeletal muscle hypertrophy.

Multiple intramuscular variables have been proposed to explain the high variability in resistance training induced muscle hypertrophy across humans. This study investigated if muscular androgen receptor (AR), estrogen receptor α (ERα) and ß (ERß) content and fiber capillarization are associated with fiber and whole-muscle hypertrophy after chronic resistance training. Male (n = 11) and female (n = 10) resistance training novices (22.1 ± 2.2 years) trained their knee extensors 3×/week for 10 weeks. Vastus lateralis biopsies were taken at baseline and post the training period to determine changes in fiber type specific cross-sectional area (CSA) and fiber capillarization by immunohistochemistry and, intramuscular AR, ERα and ERß content by Western blotting. Vastus lateralis volume was quantified by MRI-based 3D segmentation. Vastus lateralis muscle volume significantly increased over the training period (+7.22%; range: -1.82 to +18.8%, p < 0.0001) but no changes occurred in all fiber (+1.64%; range: -21 to +34%, p = 0.869), type I fiber (+1.33%; range: -24 to +41%, p = 0.952) and type II fiber CSA (+2.19%; range: -23 to +29%, p = 0.838). However, wide inter-individual ranges were found. Resistance training increased the protein expression of ERα but not ERß and AR, and the increase in ERα content was positively related to changes in fiber CSA. Only for the type II fibers, the baseline capillary-to-fiber-perimeter index was positively related to type II fiber hypertrophy but not to whole muscle responsiveness. In conclusion, an upregulation of ERα content and an adequate initial fiber capillarization may be contributing factors implicated in muscle fiber hypertrophy responsiveness after chronic resistance training.

Pro-Brain-Derived Neurotrophic Factor (BDNF), but Not Mature BDNF, Is Expressed in Human Skeletal Muscle: Implications for Exercise-Induced Neuroplasticity.

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.

Evidence for Simultaneous Muscle Atrophy and Hypertrophy in Response to Resistance Training in Humans.

PURPOSE: Human skeletal muscle has the profound ability to hypertrophy in response to resistance training (RT). Yet, this has a high energy and protein cost and is presumably mainly restricted to recruited muscles. It remains largely unknown what happens with non-recruited muscles during RT. This study investigated the volume changes of 17 recruited and 13 non-recruited muscles during a 10-week single-joint RT program targeting upper arm and upper leg musculature. METHODS: Muscle volume changes were measured by manual or automatic 3D segmentation in 21 RT novices. Subjects ate ad libitum during the study and energy and protein intake were assessed by self-reported diaries. RESULTS: Post-training, all recruited muscles increased in volume (range: +2.2% to +17.7%, p < 0.05) while the non-recruited adductor magnus (mean: -1.5 ± 3.1%, p = 0.038) and soleus (-2.4 ± 2.3%, p = 0.0004) decreased in volume. Net muscle growth (r = 0.453, p = 0.045) and changes in adductor magnus volume (r = 0.450, p = 0.047) were positively associated with protein intake. Changes in total non-recruited muscle volume (r = 0.469, p = 0.037), adductor magnus (r = 0.640, p = 0.002), adductor longus (r = 0.465, p = 0.039) and soleus muscle volume (r = 0.481, p = 0.032) were positively related to energy intake (p < 0.05). When subjects were divided into a HIGH or LOW energy intake group, overall non-recruited muscle volume (-1.7 ± 2.0%), adductor longus (-5.6 ± 3.7%), adductor magnus (-2.8 ± 2.4%) and soleus volume (-3.7 ± 1.8%) decreased significantly (p < 0.05) in the LOW but not the HIGH group. CONCLUSIONS: To our knowledge, this is the first study documenting that some non-recruited muscles significantly atrophy during a period of resistance training. Our data therefore suggest muscle mass reallocation, i.e., that hypertrophy in recruited muscles takes place at the expense of atrophy in non-recruited muscles, especially when energy and protein availability are limited.

Carnosine synthase deficiency aggravates neuroinflammation in multiple sclerosis.

Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, CARNS1 is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur, and neither endogenous nor exogenous HCDs protect against cuprizone-induced demyelination. In conclusion, the loss of CARNS1 from demyelinated MS lesions can aggravate disease progression through weakening the endogenous protection against neuroinflammation.

Extensive profiling of histidine-containing dipeptides reveals species- and tissue-specific distribution and metabolism in mice, rats, and humans.

AIM: Histidine-containing dipeptides (HCDs) are pleiotropic homeostatic molecules with potent antioxidative and carbonyl quenching properties linked to various inflammatory, metabolic, and neurological diseases, as well as exercise performance. However, the distribution and metabolism of HCDs across tissues and species are still unclear. METHODS: Using a sensitive UHPLC-MS/MS approach and an optimized quantification method, we performed a systematic and extensive profiling of HCDs in the mouse, rat, and human body (in n = 26, n = 25, and n = 19 tissues, respectively). RESULTS: Our data show that tissue HCD levels are uniquely produced by carnosine synthase (CARNS1), an enzyme that was preferentially expressed by fast-twitch skeletal muscle fibres and brain oligodendrocytes. Cardiac HCD levels are remarkably low compared to other excitable tissues. Carnosine is unstable in human plasma, but is preferentially transported within red blood cells in humans but not rodents. The low abundant carnosine analogue N-acetylcarnosine is the most stable plasma HCD, and is enriched in human skeletal muscles. Here, N-acetylcarnosine is continuously secreted into the circulation, which is further induced by acute exercise in a myokine-like fashion. CONCLUSION: Collectively, we provide a novel basis to unravel tissue-specific, paracrine, and endocrine roles of HCDs in human health and disease.

Acute balenine supplementation in humans as a natural carnosinase-resistant alternative to carnosine.

Balenine possesses some of carnosine's and anserine's functions, yet it appears more resistant to the hydrolysing CN1 enzyme. The aim of this study was to elucidate the stability of balenine in the systemic circulation and its bioavailability in humans following acute supplementation. Two experiments were conducted in which (in vitro) carnosine, anserine and balenine were added to plasma to compare degradation profiles and (in vivo) three increasing doses (1-4-10 mg/kg) of balenine were acutely administered to 6 human volunteers. Half-life of balenine (34.9 ± 14.6 min) was respectively 29.1 and 16.3 times longer than that of carnosine (1.20 ± 0.36 min, p = 0.0044) and anserine (2.14 ± 0.58 min, p = 0.0044). In vivo, 10 mg/kg of balenine elicited a peak plasma concentration (Cmax) of 28 µM, which was 4 and 18 times higher than with 4 (p = 0.0034) and 1 mg/kg (p = 0.0017), respectively. CN1 activity showed strong negative correlations with half-life (ρ = - 0.829; p = 0.0583), Cmax (r = - 0.938; p = 0.0372) and incremental area under the curve (r = - 0.825; p = 0.0433). Overall, balenine seems more resistant to CN1 hydrolysis resulting in better in vivo bioavailability, yet its degradation remains dependent on enzyme activity. Although a similar functionality as carnosine and anserine remains to be demonstrated, opportunities arise for balenine as nutraceutical or ergogenic aid.

Can muscle typology explain the inter-individual variability in resistance training adaptations?

Considerable inter-individual heterogeneity exists in the muscular adaptations to resistance training. It has been proposed that fast-twitch fibres are more sensitive to hypertrophic stimuli and thus that variation in muscle fibre type composition is a contributing factor to the magnitude of training response. This study investigated if the inter-individual variability in resistance training adaptations is determined by muscle typology and if the most appropriate weekly training frequency depends on muscle typology. In strength-training novices, 11 slow (ST) and 10 fast typology (FT) individuals were selected by measuring muscle carnosine with proton magnetic resonance spectroscopy. Participants trained both upper arm and leg muscles to failure at 60% of one-repetition maximum (1RM) for 10 weeks, whereby one arm and leg trained 3×/week and the contralateral arm and leg 2×/week. Muscle volume (MRI-based 3D segmentation), maximal dynamic strength (1RM) and fibre type-specific cross-sectional area (vastus lateralis biopsies) were evaluated. The training response for total muscle volume (+3 to +14%), fibre size (-19 to +22%) and strength (+17 to +47%) showed considerable inter-individual variability, but these could not be attributed to differences in muscle typology. However, ST individuals performed a significantly higher training volume to gain these similar adaptations than FT individuals. The limb that trained 3×/week had generally more pronounced hypertrophy than the limb that trained 2×/week, and there was no interaction with muscle typology. In conclusion, muscle typology cannot explain the high variability in resistance training adaptations when training is performed to failure at 60% of 1RM. KEY POINTS: This study investigated the influence of muscle typology (muscle fibre type composition) on the variability in resistance training adaptations and on its role in the individualization of resistance training frequency. We demonstrate that an individual's muscle typology cannot explain the inter-individual variability in resistance training-induced increases in muscle volume, maximal dynamic strength and fibre cross-sectional area when repetitions are performed to failure. Importantly, slow typology individuals performed a significantly higher training volume to obtain similar adaptations compared to fast typology individuals. Muscle typology does not determine the most appropriate resistance training frequency. However, regardless of muscle typology, an additional weekly training (3×/week vs. 2×/week) increases muscle hypertrophy but not maximal dynamic strength. These findings expand on our understanding of the underlying mechanisms for the large inter-individual variability in resistance training adaptations.

The ergogenic effect of acute carnosine and anserine supplementation: dosing, timing, and underlying mechanism.

Background: Recent studies suggest that acute-combined carnosine and anserine supplementation has the potential to improve the performance of certain cycling protocols. Yet, data on optimal dose, timing of ingestion, effective exercise range, and mode of action are lacking. Three studies were conducted to establish dosing and timing guidelines concerning carnosine and anserine intake and to unravel the mechanism underlying the ergogenic effects. Methods: First, a dose response study A was conducted in which 11 men randomly received placebo, 10, 20, or 30 mg.kg-1 of both carnosine and anserine. They performed 3x maximal voluntary isometric contractions (MVC), followed by a 5 x 6 s repeated cycling sprint ability test (RSA), once before the supplement and 30 and 60 minutes after. In a second study, 15 men performed 3x MVCs with femoral nerve electrical stimulation, followed by an RSA test, once before 30 mg.kg-1 carnosine and anserine and 60 minutes after. Finally, in study C, eight men performed a high intensity cycling training after randomly ingesting 30 mg.kg-1 of carnosine and anserine, a placebo or antihistamines (reduce post-exercise blood flow) to investigate effects on muscle perfusion. Results: Study A showed a 3% peak power (p = 0.0005; 95% CI = 0.07 to 0.27; ES = 0.91) and 4.5% peak torque (p = 0.0006; 95% CI = 0.12 to 0.50; ES = 0.87) improvement on RSA and MVC, with 30 mg.kg-1 carnosine + anserine ingestion 60 minutes before the performance yielding the best results. Study B found no performance improvement on group level; however, a negative correlation (r = -0.54; p = 0.0053; 95% CI = -0.77 to -0.19) was found between carnosinase enzyme activity (responsible for carnosine and anserine breakdown) and performance improvement. No effect of the supplement on neuromuscular function nor on muscle perfusion was found. Conclusions: These studies reveal that acute ingestion of 30 mg.kg-1 of both carnosine and anserine, 60 minutes before a high intensity exercise, can potentially improve performance, such as short cycling sprints or maximal muscle contractions. Subjects with lower carnosinase activity, and thus a slower breakdown of circulating dipeptides, appear to benefit more from this ergogenic effect. Finally, neither the involvement of a direct effect on neuromuscular function, nor an indirect effect on recovery through increased muscle perfusion could be confirmed as potential mechanism of action. The ergogenic mechanism therefore remains elusive.

Oral anserine supplementation does not attenuate type-2 diabetes or diabetic nephropathy in BTBR ob/ob mice.

Carnosine, a naturally occurring dipeptide present in an omnivorous diet, has been shown to ameliorate the development of metabolic syndrome, type-2 diabetes (T2D) and early- and advanced-stage diabetic nephropathy in different rodent models. Anserine, its methylated analogue, is more bio-available in humans upon supplementation without affecting its functionality. In this work, we investigated the effect of oral supplementation with anserine or carnosine on circulating and tissue anserine and carnosine levels and on the development of T2D and diabetic nephropathy in BTBR ob/ob mice. BTBR ob/ob mice were either supplemented with carnosine or anserine in drinking water (4 mM) for 18 weeks and compared with non-supplemented BTBR ob/ob and wild-type (WT) mice. Circulating and kidney, but not muscle, carnosine, and anserine levels were enhanced by supplementation with the respective dipeptides in ob/ob mice compared to non-treated ob/ob mice. The evolution of fasting blood glucose, insulin, fructosamine, triglycerides, and cholesterol was not affected by the supplementation regimens. The albumin/creatine ratio, glomerular hypertrophy, and mesangial matrix expansion were aggravated in ob/ob vs. WT mice, but not alleviated by supplementation. To conclude, long-term supplementation with anserine elevates circulating and kidney anserine levels in diabetic mice. However, anserine supplementation was not able to attenuate the development of T2D or diabetic nephropathy in BTBR ob/ob mice. Further research will have to elucidate whether anserine can attenuate milder forms of T2D or metabolic syndrome.

Histamine H1 and H2 receptors are essential transducers of the integrative exercise training response in humans.

Exercise training is a powerful strategy to prevent and combat cardiovascular and metabolic diseases, although the integrative nature of the training-induced adaptations is not completely understood. We show that chronic blockade of histamine H1/H2 receptors led to marked impairments of microvascular and mitochondrial adaptations to interval training in humans. Consequently, functional adaptations in exercise capacity, whole-body glycemic control, and vascular function were blunted. Furthermore, the sustained elevation of muscle perfusion after acute interval exercise was severely reduced when H1/H2 receptors were pharmaceutically blocked. Our work suggests that histamine H1/H2 receptors are important transducers of the integrative exercise training response in humans, potentially related to regulation of optimal post-exercise muscle perfusion. These findings add to our understanding of how skeletal muscle and the cardiovascular system adapt to exercise training, knowledge that will help us further unravel and develop the exercise-is-medicine concept.

Carnosinase-1 overexpression, but not aerobic exercise training, affects the development of diabetic nephropathy in BTBR ob/ob mice.

Manipulation of circulating histidine-containing dipeptides (HCD) has been shown to affect the development of diabetes and early-stage diabetic nephropathy (DN). The aim of the present study was to investigate whether such interventions, which potentially alter levels of circulating HCD, also affect the development of advanced-stage DN. Two interventions, aerobic exercise training and overexpression of the human carnosinase-1 (hCN1) enzyme, were tested. BTBR ob/ob mice were either subjected to aerobic exercise training (20 wk) or genetically manipulated to overexpress hCN1, and different diabetes- and DN-related markers were compared with control ob/ob and healthy (wild-type) mice. An acute exercise study was performed to elucidate the effect of obesity, acute running, and hCN1 overexpression on plasma HCD levels. Chronic aerobic exercise training did not affect the development of diabetes or DN, but hCN1 overexpression accelerated hyperlipidemia and aggravated the development of albuminuria, mesangial matrix expansion, and glomerular hypertrophy of ob/ob mice. In line, plasma, kidney, and muscle HCD were markedly lower in ob/ob versus wild-type mice, and plasma and kidney HCD in particular were lower in ob/ob hCN1 versus ob/ob mice but were unaffected by aerobic exercise. In conclusion, advanced glomerular damage is accelerated in mice overexpressing the hCN1 enzyme but not protected by chronic exercise training. Interestingly, we showed, for the first time, that the development of DN is closely linked to renal HCD availability. Further research will have to elucidate whether the stimulation of renal HCD levels can be a therapeutic strategy to reduce the risk for developing DN.

Fragmented Dosing of ß-alanine Induces A Body Weight-Independent Pharmacokinetic Response.

Personalised dosing of performance-enhancing food supplements is a hot topic. ß-alanine is currently dosed using a fixed dose; however, evidence suggests that this might favour light compared to heavy subjects. A weight-relative dose seems to reverse this problem. In the present study, a novel dosing strategy was tested. A fragmented dose, composed of a fixed fragment of 800 mg and a weight-relative fragment of 10 mg/kg body weight, was compared to a fixed dose of 1600 mg and a weight-relative dose of 20 mg/kg body weight in a cohort of 20 subjects with a body weight ranging 48-139 kg (79.9 ± 24.4 kg). The results show that, following a fragmented dose, the influence of body weight on the pharmacokinetic response (iAUC) over a 210 min period was absent (r = -0.168; p = 0.478), in contrast to the fixed or weight-relative dose. The pharmacokinetic response also seemed more homogenous (CV% = 26%) following a fragmented dose compared to the fixed (33%) and the weight-relative dose (31%). The primary advantage of the easy-to-calculate fragmented dosing strategy is that it does not systematically favour or impair a certain weight group. Thorough dosage studies are lacking in the current field of sports and food supplements, therefore similar considerations can be made towards other (ergogenic) food supplements.

The Reconstitution of W' Depends on Both Work and Recovery Characteristics.

PURPOSE: This study aimed to investigate the effects of different work and recovery characteristics on the W' reconstitution and to test the predictive capabilities of the W'BAL model. METHODS: Eleven male participants (22 ± 3 yr, 55 ± 4 mL·kg⋅min) completed three to five constant work rate tests to determine CP and W'. Subsequently, subjects performed 12 experimental trials, each comprising two exhaustive constant work rate bouts (i.e., WB1 and WB2), interspersed by an active recovery interval. In each trial, work bout characteristics (P4 or P8, i.e., the work rate predicted to result in exhaustion in 4 and 8 min, respectively), recovery work rate (33% CP or 66% CP), and recovery duration (2, 4, or 6 min) were varied. Actual (W'ACT) and model-predicted (W'PRED) reconstitution values of W' were calculated. RESULTS: After 2, 4, and 6 min recovery, W'ACT averaged 46% ± 2.7%, 51.2% ± 3.3%, and 59.4% ± 4.1%, respectively (P = 0.003). W'ACT was 9.4% higher after recovery at 33% CP than at 66% CP (56.9% ± 3.9% vs 47.5% ± 3.2%) (P = 0.019). P4 exercise yielded a 11.3% higher W'ACT than P8 exercise (57.8% ± 3.9% vs 46.5% ± 2.7%) (P = 0.001). W'ACT was higher than W'PRED in the conditions P4-2 min (+29.7%), P4-4 min (+18.4%), and P8-2 min (+18%) (P < 0.01). A strong correlation (R = 0.68) between the rate of W' depletion and W' recovery was found (P = 0.001). CONCLUSION: This study demonstrated that both the work and recovery characteristics of a prior exhaustive exercise bout can affect the W' reconstitution. Results revealed a slower W' reconstitution when the rate of W' depletion was slower as well. Furthermore, it was shown that the current W'BAL model underestimates actual W' reconstitution, especially after shorter recovery.