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1,25(OH) 2 -vitamin D 3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes

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Abstract

In recent years, there has been a growing body of evidence pointing to an effect of vitamin D on muscle mass and function. Our aim was to investigate the combined effect of 1,25(OH)2 -vitamin D3 (1,25(OH)2 D3 ) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube. After differentiation, myotubes were cultured in 1,25(OH)2 D3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by l-[1-(13) C]valine and puromycin in presence or not of leucine and insulin, and protein FSR was determined by measuring tracer enrichments and puromycin incorporation in proteins, respectively. Protein expression and phosphorylation state of insulin receptor (IR), Akt, GSK3, mTOR, p70 S6 kinase, rpS6, and 4EBP1 were measured by Western blot. Transcript levels of IR and 1,25(OH)2 D3 receptor (VDR) were determined by qPCR. 1,25(OH)2 D3 (10 nM) with leucine and insulin increased protein FSR in C2C12 myotubes (14-16%). IR and VDR mRNA expression was increased with 1,25(OH)2 D3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)2 D3 . 1,25(OH)2 D3 sensitizes the Akt/mTOR-dependant pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in murine C2C12 skeletal myotubes.

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... C2C12 myoblasts (ATCC, Manassas, VA, USA) were grown to 80-90% confluence in DMEM supplemented with 10% fetal calf serum at 37 °C in a 5% CO₂-humidified atmosphere. Cells were then induced to differentiate into myotubes by switching to DMEM containing 2% heat-inactivated horse serum [23,24]. After 5 days of differentiation, C2C12 myotubes were serum-and amino acid-starved in HBSS for 2 h. ...
... C2C12 myoblasts (ATCC, Manassas, VA, USA) were grown to 80-90% confluence in DMEM supplemented with 10% fetal calf serum at 37 • C in a 5% CO 2 -humidified atmosphere. Cells were then induced to differentiate into myotubes by switching to DMEM containing 2% heat-inactivated horse serum [23,24]. After 5 days of differentiation, C2C12 myotubes were serum-and amino acid-starved in HBSS for 2 h. ...
... In preliminary experiments, and in accordance with Crossland et al. [26], we observed a linear rate of puromycin incorporation in neosynthesized peptides in C2C12 cells until 4 h of incubation. Therefore, to increase puromycin labeling and sensitivity between the different plasma treatments, C2C12 myotubes were treated with plasmas for 4 h in the presence of 1 µM puromycin, then homogenized in an ice-cold lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM NaPPi, 25 mM β-glycerophosphate, 100 mM NaF, 2 mM Na orthovanadate, 10% glycerol, 1% Triton X-100) containing a protease-inhibitor cocktail (Sigma-Aldrich, Saint-Quentin-Fallavier, France) as previously described [23,24,27]. Puromycin incorporation into nascent polypeptides was measured by separating 15 µg of denatured proteins by SDS-PAGE on a polyacrylamide gel until the dye front was 1.5 cm from the bottom of the gel [23,26]. ...
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As older adults tend to reduce their intake of animal-source proteins, plant-source proteins may offer valuable resources for better protein intake. The aim of this study was to assess whether the pea proteins can be used to achieve blood amino acid levels that stimulate muscle protein synthesis. We measured variations in plasma amino acid concentrations in young and older adults given pea (NUTRALYS® S85 Plus) or whey proteins either alone or in a standardized meal. The effect of amino acid concentrations on protein synthesis in C2C12 myotubes was determined. In terms of results, plasma amino acid concentrations reflected the difference between the amino acid contents of whey and pea proteins. Blood leucine showed a greater increase of 91 to 130% with whey protein compared to pea protein, while the opposite was observed for arginine (A greater increase of 147 to 210% with pea compared to whey). Culture media prepared with plasmas from the human study induced age-dependent but not protein-type-dependent changes in myotube protein synthesis. In conclusion, pea and whey proteins have the same qualities in terms of their properties to maintain muscle protein synthesis. Pea proteins can be recommended for older people who do not consume enough animal-source proteins.
... This regulatory mechanism can facilitate the remodeling of muscle tissue toward a fiber-like structure with metabolic properties favoring oxidation over glycolysis [81]. Noteworthy, mitochondria are fundamental in the progression of atrophy [82]. Mitochondria, major contributors to superoxide anion release, are regulated by PGC-1α, a transcriptional coactivator crucial for mitochondrial biogenesis and oxidative stress regulation [81]. ...
... Mitochondria, major contributors to superoxide anion release, are regulated by PGC-1α, a transcriptional coactivator crucial for mitochondrial biogenesis and oxidative stress regulation [81]. High PGC-1α levels inhibit the transcriptional activity of FOXO3a, which controls various atrophy-related genes, promoting muscle tissue remodeling towards a metabolically oxidative composition [82,83]. Conversely, FOXO factors inhibit cell cycle progression and activate apoptosis [84]. ...
... Furthermore, Akt modulates muscle synthesis via the mammalian target of rapamycin (mTOR). In a mouse skeletal myostatic tube experimental model, 1α,25(OH)2D3 induces the Akt/mTOR-dependent pathway, thereby facilitating protein synthesis activation [82]. Sirtuin-1 is a crucial regulator of biogenesis, inflammation, apoptosis, oxidative stress, cellular senescence, and mitochondrial activity. ...
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Pathological states marked by oxidative stress and systemic inflammation frequently compromise the functional capacity of muscular cells. This progressive decline in muscle mass and tone can significantly hamper the patient’s motor abilities, impeding even the most basic physical tasks. Muscle dysfunction can lead to metabolic disorders and severe muscle wasting, which, in turn, can potentially progress to sarcopenia. The functionality of skeletal muscle is profoundly influenced by factors such as environmental, nutritional, physical, and genetic components. A well-balanced diet, rich in proteins and vitamins, alongside an active lifestyle, plays a crucial role in fortifying tissues and mitigating general weakness and pathological conditions. Vitamin D, exerting antioxidant effects, is essential for skeletal muscle. Epidemiological evidence underscores a global prevalence of vitamin D deficiency, which induces oxidative harm, mitochondrial dysfunction, reduced adenosine triphosphate production, and impaired muscle function. This review explores the intricate molecular mechanisms through which vitamin D modulates oxidative stress and its consequent effects on muscle function. The aim is to evaluate if vitamin D supplementation in conditions involving oxidative stress and inflammation could prevent decline and promote or maintain muscle function effectively.
... Salles et al. investigated the combined effects of 1,25D with insulin and leucine on the protein synthesis rate in C2C12 cells [55]. Following 5 days of differentiation, myotubes were treated with 0, 1, or 10 nM 1,25D for 72 h, followed by stimulation with insulin (100 mM) plus leucine (5 mM) for 30 min. ...
... The studies performed confirmed the presence of vitamin D receptor (VDR) and vitamin D signaling components, CYP24A1 and CYP27B1, in skeletal muscle cells during the different stages of myogenesis [8,42,50]. They also found that 1,25D promoted protein anabolism and hypertrophy increased the crosssectional area of skeletal muscle cells and explored its molecular mechanisms in skeletal muscles [8,38,55]. 1,25D was also found to enhance the effects of leucine and insulin, which promote protein anabolism in muscle cells [55,86,87]. Overall, the studies suggest that it played a regulatory role in muscles due to its different effects in various stages of myogenesis. ...
... They also found that 1,25D promoted protein anabolism and hypertrophy increased the crosssectional area of skeletal muscle cells and explored its molecular mechanisms in skeletal muscles [8,38,55]. 1,25D was also found to enhance the effects of leucine and insulin, which promote protein anabolism in muscle cells [55,86,87]. Overall, the studies suggest that it played a regulatory role in muscles due to its different effects in various stages of myogenesis. ...
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Vitamin D deficiency, prevalent worldwide, is linked to muscle weakness, sarcopenia, and falls. Muscle regeneration is a vital process that allows for skeletal muscle tissue maintenance and repair after injury. PubMed and Web of Science were used to search for studies published prior to May 2023. We assessed eligible studies that discussed the relationship between vitamin D, muscle regeneration in this review. Overall, the literature reports strong associations between vitamin D and skeletal myocyte size, and muscle regeneration. In vitro studies in skeletal muscle cells derived from mice and humans showed vitamin D played a role in regulating myoblast growth, size, and gene expression. Animal studies, primarily in mice, demonstrate vitamin D’s positive effects on skeletal muscle function, such as improved grip strength and endurance. These studies encompass vitamin D diet research, genetically modified models, and disease-related mouse models. Relatively few studies looked at muscle function after injury, but these also support a role for vitamin D in muscle recovery. The human studies have also reported that vitamin D deficiency decreases muscle grip strength and gait speed, especially in the elderly population. Finally, human studies reported the benefits of vitamin D supplementation and achieving optimal serum vitamin D levels in muscle recovery after eccentric exercise and surgery. However, there were no benefits in rotator cuff injury studies, suggesting that repair mechanisms for muscle/ligament tears may be less reliant on vitamin D. In summary, vitamin D plays a crucial role in skeletal muscle function, structural integrity, and regeneration, potentially offering therapeutic benefits to patients with musculoskeletal diseases and in post-operative recovery.
... Bauer and colleagues reported 292 an effect of co-supplementation with whey protein with vitamin D on lean mass(11). We 293 propose that correcting vitamin D deficiency or insufficiency likely underpins this co-294 supplementation effect(53). Vitamin D can improve muscle mass through genomic and non-295 genomic pathways(54). ...
... Vitamin D can improve muscle mass through genomic and non-295 genomic pathways(54). The main mechanisms of the anabolic effects of vitamin D on muscle 296 anabolism are not fully understood; nevertheless, its positive influence on muscle protein 297 anabolism has been shown(53,55).298Analyzing all included RCT showed no significant change in muscle strength gains with whey 299 protein supplementation; however, significant increases were seen in subgroups who engaged in 300 RE and consumed whey protein doses higher than 20g. In another meta-analysis, the effects of 301 protein supplementation on muscle strength were mainly observed in RE-trained participants 302(56). ...
... Physical function could be affected by protein and/or 335 vitamin D ingestion. Optimizing vitamin D status is an important factor affecting muscle mass336 and function(71), and it can boost muscle strength and function by improving muscle anabolism 337 when combined with leucine(53).338 One of our main findings is that whey protein plus vitamin D affected muscle strength and 339 physical function in the subgroup without RE. ...
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Background: The effects of supplementation with whey protein alone or with vitamin D on sarcopenia-related outcomes in older adults are unclear. Objective: We aimed to assess the effect of whey protein supplementation alone or with vitamin D on LM, strength, and function in older adults with or without sarcopenia or frailty. Methods: We searched PubMed, Web of Sciences, and SCOPUS databases. Randomized controlled trials (RCT) that investigated the effect of whey protein supplementation with/without vitamin D on sarcopenia outcomes in healthy and sarcopenic/frail older adults were included. Standardized mean differences (SMD) were calculated for LM, muscle strength, and physical function data. Results: Analysis showed that whey protein supplementation had no effect on LM and muscle strength; nevertheless, a significant improvement was found in physical function (SMD=0.561, 95% confidence interval [CI] 0.256:0.865, n=33), especially gait speed (GS). On the other hand, whey protein supplementation significantly improved LM (SMD= 0.982, 95%CI 0.228:1.736, n=11), ALM, physical function (SMD= 1.211, 95% CI 0.588:1.834, n=16), and GS in sarcopenic/frail older adults. In contrast, co-supplementation with vitamin D enhanced LM gains (SMD=0.993, 95% CI 0.112:1.874, n=11), muscle strength (SMD=2.005, 95% CI 0.975:3.035, n=11), and physical function (SMD=3.038, 95% CI 2.196:3.879, n= 18) significantly. Muscle strength and physical function improvements after whey protein supplementation plus vitamin D were observed without RE and short study duration subgroups. Also, the combination of whey protein and vitamin D with RE did not enhance the effect of RE. Conclusion: Whey protein supplementation improved LM and function in sarcopenic/frail older adults but had no positive effect in healthy older persons. In contrast, our meta-analysis shows that co-supplementation with whey protein and vitamin D is effective, especially for healthy older adults, which is likely due to the correction of vitamin D insufficiency or deficiency. The study protocol was registered at https://inplasy.com as INPLASY202240167.
... C2C12 myoblasts (ATCC, Manassas, VA) were grown to 80-90% confluence in DMEM supplemented with 10% fetal calf serum at 37°C in a 5% CO 2humidified atmosphere. Cells were then induced to differentiate into myotubes by switching to DMEM containing 2% heat-inactivated horse serum 15,34 . Human primary skeletal muscle myoblasts (Lot#: SLSK002) were purchased from Zenbio inc. ...
... (New York City, NY) and cultured following the company's instructions. After 5 days of differentiation, C2C12 myotubes or human skeletal myotubes were cultured for 3 days with 0, 1, or 10 nM of 1,25(OH)2 vitamin D3 dissolved in ethanol 15,34 . ...
... Western blot analysis. C2C12 myotubes and mice gastrocnemius muscles were homogenized in an ice-cold lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM NaPPi, 25 mM β-glycerophosphate, 100 mM NaF, 2 mM Na orthovanadate, 10% glycerol, 1% Triton X-100) containing a protease-inhibitor cocktail (1%) (Sigma # P8340) as we previously described 15,34,35 . Denatured proteins were separated by SDS-PAGE on 4-15% precast polyacrylamide gel (BIO-RAD, Marnes-la-Coquette, France) and transferred to a polyvinylidene membrane (Millipore, Molsheim, France). ...
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Skeletal muscle mitochondrial function is the biggest component of whole-body energy output. Mitochondrial energy production during exercise is impaired in vitamin D-deficient subjects. In cultured myotubes, loss of vitamin D receptor (VDR) function decreases mitochondrial respiration rate and ATP production from oxidative phosphorylation. We aimed to examine the effects of vitamin D deficiency and supplementation on whole-body energy expenditure and muscle mitochondrial function in old rats, old mice, and human subjects. To gain further insight into the mechanisms involved, we used C2C12 and human muscle cells and transgenic mice with muscle-specific VDR tamoxifen-inducible deficiency. We observed that in vivo and in vitro vitamin D fluctuations changed mitochondrial biogenesis and oxidative activity in skeletal muscle. Vitamin D supplementation initiated in older people improved muscle mass and strength. We hypothesize that vitamin D supplementation is likely to help prevent not only sarcopenia but also sarcopenic obesity in vitamin D-deficient subjects. Vitamin D has an impact on mitochondrial biogenesis and oxidative activity in skeletal muscle both in vitro and in vivo, and vitamin D supplementation in older people may improve muscle mass and strength.
... In addition to protein consumption, another nutrient that has been studied related to muscle mass growth is vitamin D. Tanner and Harwell (2015) conducted a review about the relationship between vitamin D deficiency and muscle health, relating vitamin D deficiency with diffuse musculoskeletal pain, myopathy, sarcopenia and falls (Salles et al., 2013;Tanner and Harwell, 2015). ...
... Vitamin D deficiency is commonly found in older adults patients, especially in older adults people living in long-term care facilities (Visser et al., 2006). Salles et al. (2013) suggest a positive effect of vitamin D supplementation on muscle protein metabolism when combined with insulin and leucine, through sensitization of the akt/mTOR-dependent pathway, resulting in an additional activation of protein synthesis (Salles et al., 2013). ...
... Vitamin D deficiency is commonly found in older adults patients, especially in older adults people living in long-term care facilities (Visser et al., 2006). Salles et al. (2013) suggest a positive effect of vitamin D supplementation on muscle protein metabolism when combined with insulin and leucine, through sensitization of the akt/mTOR-dependent pathway, resulting in an additional activation of protein synthesis (Salles et al., 2013). ...
Article
Background: The increase in life expectancy and in the number of individuals over 60 years old brings new demands to health professionals and services based on the physiological changes that occur in this population. The aging process results in changes in body composition, increasing body fat and reducing muscle mass, in addition to a reduction in bone mass. Aim: The aim of this study was to examine the effect of whey protein and vitamin D supplementation on body composition and skeletal muscle in older adults living in long-term care facilities. Methods: This study is a double-blind randomized controlled trial. Thirty older adults (>60 years old) were randomized and allocated in three groups: group receiving resistance training and supplementation receiving resistance training, whey protein and vitamin D; group received resistance and placebo training receiving resistance training and placebo, and control group without any intervention. Body composition was measured by dual-energy X-ray absorptiometry at baseline, 12 weeks, and 24 weeks. Results: The mean age was 74.87 (± 8.14) years. A significant difference ( p = 0.042) was observed between the group receiving resistance training and supplementation and control groups in relation to lean mass increase (kg) at 24 weeks. After 24 weeks of intervention, there was a significant increase in Relative index of muscle mass for the two groups that underwent resistance training, group received resistance and placebo training ( p = 0.042) and group receiving resistance training and supplementation ( p = 0.045), in relation to the control. Conclusion: Combined supplementation of whey protein and vitamin D with resistance training can significantly improve lean mass, total mass, and relative index of muscle mass in institutionalized older adults.
... Protein synthesis is tightly controlled by the Akt / mTOR pathway, which is activated by anabolic factors such as insulin and amino acids (60). Vitamin D may interfere with the insulin signaling pathway and insulin sensitivity (61,62), and promote skeletal muscle protein anabolism (61). Vitamin D-and leucine-enriched nutrition may aid in the prevention and treatment of muscle mass and strength loss in vivo because of synergistic effects of leucine and vitamin D on protein synthesis in C2C12 myotubes (61). ...
... Protein synthesis is tightly controlled by the Akt / mTOR pathway, which is activated by anabolic factors such as insulin and amino acids (60). Vitamin D may interfere with the insulin signaling pathway and insulin sensitivity (61,62), and promote skeletal muscle protein anabolism (61). Vitamin D-and leucine-enriched nutrition may aid in the prevention and treatment of muscle mass and strength loss in vivo because of synergistic effects of leucine and vitamin D on protein synthesis in C2C12 myotubes (61). ...
... Vitamin D may interfere with the insulin signaling pathway and insulin sensitivity (61,62), and promote skeletal muscle protein anabolism (61). Vitamin D-and leucine-enriched nutrition may aid in the prevention and treatment of muscle mass and strength loss in vivo because of synergistic effects of leucine and vitamin D on protein synthesis in C2C12 myotubes (61). Therefore, synergy between vitamin D and milk protein may help to prevent or counteract the loss of muscle mass and strength during aging. ...
Article
An intervention study was conducted to investigate the effects of daily 1,000-IU vitamin D-fortified milk intake on skeletal muscle mass, power, physical function and nutrition status in 26 healthy people and 8 older adults living in a nursing home. The serum 25-hydroxyvitamin D [25(OH)D] level was 13.4 ± 0.8 ng / mL and it markedly increased to 29.6 ± 0.9 ng / mL after daily 1000-IU vitamin D-fortified milk intake for 6 months. Handgrip strength (kg) also significantly increased in the 21-50 years and total groups, and male subjects, and the timed up and go test significantly improved in the 21-50 years and total groups, and female subjects after 6-month vitamin D intake. However, there were no significant differences between baseline and post-treatment in the Barthel Index (BI), walking speed (m / sec) or skeletal muscle mass (kg, % of BW, kg / m2). Therefore, the present study suggested that vitamin D-fortified milk intake is effective at improving muscle strength and physical function in Japanese, although further studies are needed, particularly for older adults. J. Med. Invest. 68 : 249-255, August, 2021
... Evidence suggests that Vit-D directly stimulates muscle protein synthesis in Vit-D deficient rats (Birge and Haddad 1975;Wassner et al. 1983) and increases intramyonuclear VDR concentration and muscle fiber size in Vit-D-deficient humans (Ceglia et al. 2013). In murine myotubes (Salles et al. 2013) and human myoblasts (Romeu Montenegro et al. 2019), the stimulating effect of Vit-D on protein synthesis is associated with activation of Akt/mTORdependent pathway. ...
... Translation initiation, a crucial step in muscle protein synthesis, is controlled by the Akt/mTOR pathway (Bodine et al. 2001) which is stimulated by insulin (Biolo et al. 1995) and leucine (Haegens et al. 2012). In vitro, Vit-D potentiates the effect of leucine and insulin on the Akt/mTOR pathway, leading to a 14-35% greater increase in the rate of protein synthesis (Salles et al. 2013;Romeu Montenegro et al. 2019). After each RT session, all our participants ingested 20 g of whey protein. ...
... Due to high leucine content, this amount stimulates muscle protein synthesis for the initial 4 h of recovery in young men with unknown vitamin D status (Witard et al. 2014), and acute increases in muscle protein synthesis correlate with the extent of muscle hypertrophy occurring as a result of prolonged RT program (Damas et al. 2016). Considering the findings of Salles et al. (2013), Romeu Montenegro et al. (2019) and Damas et al. (2016), we expected to see greater increases in LBM and muscle strength in our VD group compared to PLC group, but this was not the case. The effect of Vit-D supplementation on Akt/mTOR pathway activation in Vit-D deficient men involved in RT remains to be measured in further studies. ...
Article
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Purpose Vitamin D (Vit-D) supplementation has been shown to increased muscle strength in young adults. It remains unclear if Vit-D supplementation enhances the efficacy of resistance training (RT). This study tested the hypothesis that Vit-D supplementation would enhance the RT-induced increases in muscle strength and lean body mass (LBM) in Vit-D deficient young men. Methods Thirty-nine men (baseline serum 25(OH)D < 50 nmol L‒1) were quasi-randomly assigned to one of the two groups that performed a 12-week supervised RT program concomitant with either Vit-D (8000 IU daily; VD) or placebo (PLC) supplementation. Results During 12-week RT, energy and nutrient (except Vit-D) intake and training loads did not differ in the two groups. Serum 25(OH)D levels increased from 36.3 ± 9.2 to 142.4 ± 21.9 nmol L‒1 (P < 0.05) in VD group and remained unchanged between 36.3 ± 8.9 and 29.4 ± 6.6 nmol L‒1 (P > 0.05) in PLC group. Muscle strength (1-repetition maximum) increased (P < 0.05) to an equal extent in the two groups in 5 exercises performed on RT equipment, whereas strength gains in chest press and seated row were greater (P < 0.05) in PLC compared to VD group. Total and regional LBM (measured by DXA scan) increased (P < 0.05) equally in the two groups. Android fat mass decreased (P < 0.05) in VD group only. Conclusion Vit-D supplementation does not enhance the efficacy of RT in terms of muscle strength and LBM gains in Vit-D deficient young healthy men.
... Significant muscle atrophy, decreased muscle strength, reduced muscle fiber size, lower bone density, and disturbance in the regulation of myogenic regulatory factors were observed in mice in which VDR has been knocked out and in mice that received a diet with a low vitamin D content, compared to the control group (51,52). It has been reported that vitamin D increases the phosphorylation of Akt and GSK3β and thus improves insulin signaling by regulating the insulin receptor, which itself can activate the protein synthesis process in skeletal muscle (53). Since vitamin D has an antioxidant effect, evidence shows that in conditions of vitamin D deficiency, the enzyme antioxidant defense is reduced and increases proteolysis in skeletal muscle tissue (54). ...
... Furthermore, vitamin D influences the production and secretion of insulin, affects the survival of β-cells of the pancreas, and increases the secretion of insulin from β-cells of the pancreas (56,57). Vitamin D not only affects the production and secretion of insulin from pancreatic beta cells but also influences the response of peripheral tissues to insulin or, in other words, the insulin signaling pathway in skeletal muscles (53). It has been reported that vitamin D raises insulin sensitivity by binding 1,25(OH) 2D3 to VDR, inducing insulin-receptor substrate expression in target tissues and activating PPARδ (58). ...
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Context: Resistance training (RT) and vitamin D are both utilized as interventions for muscle hypertrophy. Multiple studies have examined the impact of RT with and without vitamin D supplementation on muscle hypertrophy. There is currently no existing review study that has specifically investigated the combined effect of RT and vitamin D supplementation on skeletal muscle hypertrophy. This review article compiled studies examining the impact of both RT and vitamin D interventions on muscle hypertrophy. Evidence Acquisition: Three databases (PubMed, ScienceDirect, and Google Scholar) were searched until September 9, 2022. Only original articles written in English were included. The findings of the studies were analyzed and categorized into two sections: cross-sectional areas and muscle performance. Results: Very limited studies examined the effect of RT and vitamin D on hypertrophy and skeletal muscle function. The impact of RT and vitamin D on signaling pathways of protein synthesis and skeletal muscle hypertrophy was investigated separately, and the simultaneous effect of these two interventions was explored in a few studies. Conclusions: The proteins involved in the signaling pathway of muscle hypertrophy were examined in only three animal studies. Therefore, more field/clinical trials in the human model should investigate the effect of these two interventions on the degree of hypertrophy and muscle function. Besides, both in human studies using muscle biopsy and in animal studies at the cellular level, the expression of genes and proteins involved in signaling pathways affecting protein synthesis and muscle hypertrophy should be studied further to determine the simultaneous effect of these two interventions.
... Interestingly, high PGC-1α levels also avoid transcriptional activity of FOXO3a. This observation indicates that mitochondria might participate in the atrophy progression [109]. In fact, FOXO factors control several atrophy-related genes recognized as "atrophy patterns" present in various atrophy types and stimulate the expression of many and enhance protein degradation [110]. ...
... In addition, Akt regulates muscle synthesis via mTOR. In murine C2C12 skeletal myotubes, 1α,25(OH) 2 D 3 stimulates the Akt/mTOR-dependent pathway, inducing activation of their protein synthesis [109] (Figure 1). ...
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Skeletal muscle dysfunction is frequently associated with chronic obstructive pulmonary disease (COPD), which is characterized by a permanent airflow limitation, with a worsening respiratory disorder during disease evolution. In COPD, the pathophysiological changes related to the chronic inflammatory state affect oxidant–antioxidant balance, which is one of the main mechanisms accompanying extra-pulmonary comorbidity such as muscle wasting. Muscle impairment is characterized by alterations on muscle fiber architecture, contractile protein integrity, and mitochondrial dysfunction. Exogenous and endogenous sources of reactive oxygen species (ROS) are present in COPD pathology. One of the endogenous sources of ROS is represented by mitochondria. Evidence demonstrated that vitamin D plays a crucial role for the maintenance of skeletal muscle health. Vitamin D deficiency affects oxidative stress and mitochondrial function influencing disease course through an effect on muscle function in COPD patients. This review will focus on vitamin-D-linked mechanisms that could modulate and ameliorate the damage response to free radicals in muscle fibers, evaluating vitamin D supplementation with enough potent effect to contrast mitochondrial impairment, but which avoids potential severe side effects.
... 135 According to a study by Ottestad et al, 136 protein- Vitamin D and leucine have been shown to have synergistic effects in promoting protein anabolism. [137][138][139] Therefore, combining whey protein with vitamin D supplementation may further improve gait speed in the senior people with sarcopenia. 140 It is important to note that the synergistic effects of multiple nutrients, including vitamin D, with protein sources, may produce even more significant results. ...
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This article reviews the mechanisms and prevention strategies associated with vitamin D and sarcopenia in older adults. As a geriatric syndrome, sarcopenia is defined by a notable decline in skeletal muscle mass and strength, which increases the risk of adverse health outcomes such as falls and fractures. Vitamin D, an essential fat-soluble vitamin, is pivotal in skeletal muscle health. It affects muscle function through various mechanisms, including regulating calcium and phosphorus metabolism, promoting muscle protein synthesis, and modulation of muscle cell proliferation and differentiation. A deficiency in vitamin D has been identified as a significant risk factor for the development of sarcopenia in older adults. Many studies have demonstrated that low serum vitamin D levels are significantly associated with an increased risk of sarcopenia. While there is inconsistency in the findings, most studies support the importance of vitamin D in maintaining skeletal muscle health. Vitamin D influences the onset and progression of sarcopenia through various pathways, including the promotion of muscle protein synthesis, the regulation of mitochondrial function, and the modulation of immune and inflammatory responses. Regarding the prevention and treatment of sarcopenia, a combination of nutritional, exercise, and pharmacological interventions is recommended. Further research should be conducted to elucidate the molecular mechanism of vitamin D in sarcopenia, to study genes related to sarcopenia, to perform large-scale clinical trials, to investigate special populations, and to examine the combined application of vitamin D with other nutrients or drugs. A comprehensive investigation of the interconnection between vitamin D and sarcopenia will furnish a novel scientific foundation and productive strategies for preventing and treating sarcopenia. This, in turn, will enhance the senior people’s quality of life and health.
... Although the role of vitamin D/VDR signalling in skeletal muscle cell proliferation and differentiation remains controversial [6e9], it positively regulates mitochondrial respiration and ATP generation in skeletal muscle cells [10e12]. Vitamin D promotes protein synthesis by activating the Akt/mTOR pathway [13]. Vitamin D deficiency, diagnosed based on reduced serum 25-hydroxyvitamin D (25[OH]D) levels, has been associated with reduced muscle mass and strength [14,15]. ...
... The nature of whole foods compared with isolated protein sources generates additional variables beyond plasma amino acid kinetics that could have potentially modulated the anabolic response [57,58]. For example, the presence of dietary fiber in MYCO and CHLO [59,60], antioxidants and carotenoids in SPIR and CHLO [51,61] and/or large quantities of omega-3 PUFAs and vitamin D in CHLO [51] have all been linked with upregulating anabolic signaling pathways [62][63][64][65], thus plausibly explaining our inability to detect differences in postprandial MyoPS rates between these diverse protein-rich foods. Alternatively, despite peak leucine concentrations being~19% and 56% lower following the ingestion of CHLO (initially providing 2.1 g leucine) compared with MYCO (1.7 g leucine) and SPIR (2.3 g leucine), it may still have been sufficient to exceed the threshold required to robustly increase MyoPS, or may have saturated the MyoPS response entirely [66]. ...
... To complete the evaluation of muscle protein synthesis, we evaluated the activation state of its main regulatory pathway, the p70 S6 kinase pathway. Plantaris muscles were homogenized in an ice-cold lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM NaPPi, 25 mM β-glycerophosphate, 100 mM NaF, 2 mM Na orthovanadate, 10% glycerol, 1% Triton X-100) containing a protease inhibitor cocktail (1%) as previously described [12,25,26]. Denatured proteins were separated by SDS-PAGE on a polyacrylamide gel and transferred to a polyvinylidene membrane (Millipore, Molsheim, France). ...
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Aging is associated with a decline in muscle mass and function, leading to increased risk for mobility limitations and frailty. Dietary interventions incorporating specific nutrients, such as pea proteins or inulin, have shown promise in attenuating age-related muscle loss. This study aimed to investigate the effect of pea proteins given with inulin on skeletal muscle in old rats. Old male rats (20 months old) were randomly assigned to one of two diet groups for 16 weeks: a ‘PEA’ group receiving a pea-protein-based diet, or a ‘PEA + INU’ group receiving the same pea protein-based diet supplemented with inulin. Both groups showed significant postprandial stimulation of muscle p70 S6 kinase phosphorylation rate after consumption of pea proteins. However, the PEA + INU rats showed significant preservation of muscle mass with time together with decreased MuRF1 transcript levels. In addition, inulin specifically increased PGC1-α expression and key mitochondrial enzyme activities in the plantaris muscle of the old rats. These findings suggest that dietary supplementation with pea proteins in combination with inulin has the potential to attenuate age-related muscle loss. Further research is warranted to explore the underlying mechanisms and determine the optimal dosage and duration of intervention for potential translation to human studies.
... Precision nutrition and sarcopenia 423 supplementation with leucine-enriched protein in older adults, vitamin D 3 was co-ingested within the supplement (87)(88)(89) . In vitro, vitamin D 3 treatment was shown to sensitise muscle cells to the stimulatory effects of leucine and insulin on MPS (92) suggesting that synergistic effects may occur with combined supplementation. Thus, further work is needed to ascertain if, and under which conditions, leucine and/or leucine-enriched protein may be beneficial in sarcopenia prevention and treatment. ...
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Diminished skeletal muscle strength and size, termed sarcopenia, contributes substantially to physical disability, falls, dependence and reduced quality of life among older people. Physical activity and nutrition are the cornerstones of sarcopenia prevention and treatment. The optimal daily protein intake required to preserve muscle mass and function among older adults is a topic of intense scientific debate. Older adults require protein intakes about 67 % higher than their younger counterparts to maximally stimulate postprandial muscle protein synthesis rates. In addition, evidence suggests a possible benefit of increasing protein intake above the population reference intake (0⋅83 g/kg/d) on lean mass and, when combined with exercise training, muscle strength. In addition to protein quantity, protein quality, the pattern of protein intake over the day and specific amino acids (i.e. leucine) represent key considerations. Long-chain n -3 PUFA (LC n- 3 PUFA) supplementation has been shown to enhance muscle protein synthesis rates, increase muscle mass and function and augment adaptations to resistance training in older adults. Yet, these effects are not consistent across all studies. Emerging evidence indicates that an older person's dietary, phenotypic and behavioural characteristics may modulate the efficacy of protein and LC n -3 PUFA interventions for promoting improvements in muscle mass and function, highlighting the potential inadequacy of a ‘one-size-fits-all’ approach. The application of personalised or precision nutrition to sarcopenia represents an exciting and highly novel field of research with the potential to help resolve inconsistencies in the literature and improve the efficacy of dietary interventions for sarcopenia.
... [4][5][6] The link between VitD and muscle strength can be explained by a potential anabolic effect of VitD on skeletal muscle, as myocytes express the vitamin D receptor (VDR) that enables VitD to stimulate skeletal muscle protein synthesis and enables myoblasts to differentiate into myocytes. [7][8][9] Furthermore, higher serum VitD levels are associated with reduced injury rates and better athletic performance. 3,6,10 Overtraining syndrome is a condition resulting from excessive training load associated with inadequate recovery and poor sleep quality, leading to performance decrements and fatigue. ...
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Overtraining syndrome is a condition resulting from excessive training load associated with inadequate recovery and poor sleep quality, leading to performance decrements and fatigue. Here we hypothesized that vitamin D (VitD) deficiency is a lead factor in the development of the overtraining syndrome. To test this hypothesis, two groups of 60‐week‐old C57BL/6 mice followed a 16‐week excessive eccentric‐based overtraining by excessive downhill running with or without dietary VitD depletion (EX and EX‐D⁻ groups). Two control groups were trained by uphill running at the same load with or without VitD depletion (CX and CX‐D⁻ groups). Handgrip strength decreased throughout the protocol for all groups but the decrease was sharper in EX‐D⁻ group (VitD × training, p = 0.0427). At the end of the protocol, the mass of Triceps brachii muscle, which is heavily stressed by eccentric contractions, was reduced in eccentric‐trained groups (training effect, p = 0.0107). This atrophy was associated with a lower concentration of the anabolic myokine IL‐15 (training effect, p = 0.0314) and a tendency to a higher expression of the atrogene cathepsin‐L (training effect, p = 0.0628). VitD depletion led to a 50% decrease of the fractional protein synthesis rate in this muscle (VitD effect, p = 0.0004) as well as decreased FGF21 (VitD effect, p = 0.0351) and increased osteocrin (VitD effect, p = 0.038) concentrations that would lead to metabolic defects. Moreover, the proportion of anti‐inflammatory Th2 lymphocytes was significantly decreased by the combination of eccentric training with VitD depletion (vitD × training, p = 0.0249) suggesting a systemic inflammation. Finally, exploratory behavior time of mice was decreased by VitD depletion (VitD effect, p = 0.0146) suggesting a cognitive dysfunction. Our results suggest that VitD deficiency exacerbates the effects of overtraining.
... For example, Trendelenburg et al. [92] show that myostatin reduces Akt/mTOR signaling complex 1 (TORC1)/p70 S6 kinase (p70S6K) signaling, inhibiting myoblast differentiation and reducing myotube size. Intriguingly, 1α,25(OH) 2 D 3 sensitizes the Akt/mTOR signaling pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in C2C12 myotubes [95]. Recently, however, Olsson et al. [9] report that myostatin expression remains unchanged in response to 1α,25(OH) 2 D 3 in human muscle precursor cells. ...
... Therefore, it seems likely that the previously reported effects may have resulted from additional components in the supplementation procedure [13]. For example, vitamin D is involved in vari- ous anabolic pathways in skeletal muscle [30], and it also increases the stimulatory effect of leucine on protein synthesis through mTOR-mediated signaling in skeletal myotubes [31]. In addition, creatine supplementation at a dose of 3 g per day for 4 weeks effectively increases the total creatine content in muscles [32]. ...
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Introduction: To increase the total carnitine (TC) content in muscles, LC should be co-ingested with carbohydrates to induce an insulin response. Leucine has an insulin secretagogue effect. Therefore, the primary aim of this study was to examine the effects of 24 weeks of LC and leucine supplementation on the skeletal muscle TC content, muscle mass, and strength in active college-aged subjects. The secondary aim was to determine the activation of the Akt/mTOR signaling pathway in skeletal muscles after supplementation. Methods: Over the 24 weeks, the participants were supplemented with either 1 g of L-carnitine-L-tartrate and 3 g of leucine per day (LC + L group; n = 7) or 4 g of leucine per day (L group; n = 7) as a placebo. Before and 24 weeks after the initiation of the study protocol, the free carnitine (FC) and TC content in plasma and muscle samples, as well as body composition and muscle strength were measured. In addition, the phosphorylation of the Akt/mTOR pathway proteins in muscles was evaluated. Results: Plasma FC and TC content increased in LC + L group after 24 weeks of supplementation (p = 0.003 and 0.010, respectively). However, the skeletal muscle FC and TC content were not affected by the supplementation protocol. No changes were noted in the body mass and composition; serum insulin-like growth factor-1 concentration; and phosphorylation of the signaling pathway proteins Akt, mTOR and p70S6K. Conclusion: LC supplementation may have the potential to exert beneficial effects in muscle atrophy. Therefore, additional research is necessary to investigate the effect of various LC supplementation protocols.
... L-Leucine concentration of 1 mM was based on previous data showing increased protein synthesis in C2C12 myotubes. Vitamin D3 concentration of 10 nM was selected because of Salles et al. [42] showed significant increase in protein synthesis with 1 and 10 nM vitamin D3 together with insulin and leucine as anabolic stimuli. For muscle cell assays, EPA and DHA were dissolved in 100% ethanol and further diluted in PBS + 2.5% essentially fatty acid free BSA (Sigma-Aldrich), vitamin D3 was dissolved in 100% ethanol, L-leucine in differentiation medium. ...
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Purpose: Muscle-wasting and treatment-related toxicities negatively impact prognosis of colorectal cancer (CRC) patients. Specific nutritional composition might support skeletal muscle and enhance treatment support. In this in vitro study we assess the effect of nutrients EPA, DHA, L-leucine and vitamin D3, as single nutrients or in combination on chemotherapy-treated C2C12-myotubes, and specific CRC-tumor cells. Materials and methods: Using C2C12-myotubes, the effects of chemotherapy (oxaliplatin, 5-fluorouracil, oxaliplatin+5-fluorouracil and irinotecan) on protein synthesis, cell-viability, caspase-3/7-activity and LDH-activity were assessed. Addition of EPA, DHA, L-leucine and vitamin D3 and their combination (SNCi) were studied in presence of above chemotherapies. Tumor cell-viability was assessed in oxaliplatin-treated C26 and MC38 CRC cells, and in murine and patient-derived CRC-organoids. Results: While chemotherapy treatment of C2C12-myotubes decreased protein synthesis, cell-viability and increased caspase-3/7 and LDH-activity, SNCi showed improved protein synthesis and cell viability and lowered LDH activity. The nutrient combination SNCi showed a better overall performance compared to the single nutrients. Treatment response of tumor models was not significantly affected by addition of nutrients. Conclusions: This in vitro study shows protective effect with specific nutrition composition of C2C12-myotubes against chemotherapy toxicity, which is superior to the single nutrients, while treatment response of tumor cells remained.
... Although results were non-significant, they indicated an increased functionality of the muscle to produce force [25]. Studies investigating the additive effect of VD on protein utilization found enhanced protein synthesis in murine C2C12 skeletal myotubes [26], but the effect of VD on MPS in humans is less clear [27]. ...
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There is increasing evidence that dietary protein intake with leucine and vitamin D is an important factor in muscle protein synthesis. This study investigated the combined effects of consuming whey protein and vitamin D3 in the evening before bedtime or in the morning after sleeping on muscle mass and strength. Healthy, untrained males (N = 42; Age = 18–24 year) were randomly assigned into three groups: before bedtime, after sleeping, and control. Subjects underwent a 6-week resistance training program in combination with supplements that provided 25 g whey protein and 4000 IU vitamin D3 for the before bedtime and after sleeping groups and a 5 g maltodextrin placebo for the control group. A significant increase in serum vitamin D was observed in both before bedtime and after sleeping groups. All groups experienced a significant gain in leg press. However, the control group did not experience significant improvements in muscle mass and associated blood hormones that were experienced by the before bedtime and after sleeping groups. No significant differences in assessed values were observed between the before bedtime and after sleeping groups. These findings suggest that the combination of whey protein and vitamin D supplements provided either before or after sleep resulted in beneficial increases in muscle mass in young males undergoing resistance training that exceeded the changes observed without these supplements.
... However, to our knowledge, no studies have explored the influence of VD 3 on the absorption capacity of AAs and small peptides in animal intestines. In rat skeletal muscle cells, GSK3 enhanced SLC38A2 mRNA expression (Stretton et al., 2019), and VD 3 increased GSK3 contents in a C2C12 cell line (Salles et al., 2013). Moreover, low levels of insulin decreased SLC7A1 expression in umbilical cord veins endothelial cells (Gonz alez et al., 2011). ...
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Vitamin D3 (VD3), an essential nutrient for animals, has been demonstrated to stimulate the uptake of certain amino acids. However, the role of VD3 in the intestine, the primary site for digestion and absorption of nutrients, remains poorly characterized. Here, the grass carp (Ctenopharyngodon idella) was studied to assess the influence of different doses of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) on growth performance, intestinal morphology, digestive absorption, amino acid transport, and potential signaling molecule levels in a feeding experiment. As a result, dietary VD3 improved growth performance, intestinal structure, and digestive and brush border enzyme activities. Additionally, most intestinal free amino acids and their transporters were upregulated after VD3 intake, except for Ala, Lys, Asp, Leu, solute carrier (SLC) 7A7, SLC1A5, and SLC1A3 mRNA in different segments, Leu and SLC6A14 mRNA in the proximal intestine, and SLC7A5 mRNA in the mid and distal intestine. In the crucial target of rapamycin (TOR) signal pathway of amino acid transport, the gene and protein expression of TOR, S6 kinase 1, and activating transcription factor 4 were elevated, whereas 4E-binding protein 1 was decreased, further suggesting an advanced amino acid absorption capacity in the fish due to VD3 supplementation. Based on percentage weight gain, feed efficiency, and trypsin activity, the VD3 requirements of on-growing grass carp were estimated to be 968.33, 1,005.00, and 1,166.67 IU/kg, respectively. Our findings provide novel recommendations for VD3 supplementation to promote digestion and absorption capacities of fish, contributing to the overall productivity of aquaculture.
... Accordingly, Salles et al. have demonstrated that calcitriol can directly activate Akt and improve insulin sensitivity in C2C12 cells. 33 Furthermore, the inhibition of GSK-3β activity through Akt is sufficient to stimulate myogenic differentiation. GSK-3β KO animals show an early expression of MyoD and myogenin in disuse-atrophied skeletal muscle compared with wild-type animals. ...
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Background: Fetal stage is a critical developmental window for the skeletal muscle, but little information is available about the impact of maternal vitamin D (Vit. D) deficiency (VDD) on offspring lean mass development in the adult life of male and female animals. Methods: Female rats (Wistar Hannover) were fed either a control (1000 IU Vit. D3/kg) or a VDD diet (0 IU Vit. D3/kg) for 6 weeks and during gestation and lactation. At weaning, male and female offspring were randomly separated and received a standard diet up to 180 days old. Results: Vitamin D deficiency induced muscle atrophy in the male (M-VDD) offspring at the end of weaning, an effect that was reverted along the time. Following 180 days, fast-twitch skeletal muscles [extensor digitorum longus (EDL)] from the M-VDD showed a decrease (20%; P < 0.05) in the number of total fibres but an increase in the cross-sectional area of IIB (17%; P < 0.05), IIA (19%; P < 0.05) and IIAX (21%; P < 0.05) fibres. The fibre hypertrophy was associated with the higher protein levels of MyoD (73%; P < 0.05) and myogenin (55% %; P < 0.05) and in the number of satellite cells (128.8 ± 14 vs. 91 ± 7.6 nuclei Pax7 + in the M-CTRL; P < 0.05). M-VDD increased time to fatigue during ex vivo contractions of EDL muscles and showed an increase in the phosphorylation levels of IGF-1/insulin receptor and their downstream targets related to anabolic processes and myogenic activation, including Ser 473 Akt and Ser 21/9 GSK-3β. In such muscles, maternal VDD induced a compensatory increase in the content of calcitriol (two-fold; P < 0.05) and CYP27B1 (58%; P < 0.05), a metabolizing enzyme that converts calcidiol to calcitriol. Interestingly, most morphological and biochemical changes found in EDL were not observed in slow-twitch skeletal muscles (soleus) from the M-VDD group as well as in both EDL and soleus muscles from the female offspring. Conclusions: These data show that maternal VDD selectively affects the development of type-II muscle fibres in male offspring rats but not in female offspring rats and suggest that the enhancement of their size and fatigue resistance in fast-twitch skeletal muscle (EDL) is probably due to a compensatory increase in the muscle content of Vit. D in the adult age.
... Although the role of vitamin D/VDR signalling in skeletal muscle cell proliferation and differentiation remains controversial [6e9], it positively regulates mitochondrial respiration and ATP generation in skeletal muscle cells [10e12]. Vitamin D promotes protein synthesis by activating the Akt/mTOR pathway [13]. Vitamin D deficiency, diagnosed based on reduced serum 25-hydroxyvitamin D (25[OH]D) levels, has been associated with reduced muscle mass and strength [14,15]. ...
Article
Funding Acknowledgements Type of funding sources: None. Introduction Sarcopenia is associated with poor functional status and clinical outcomes in heart failure (HF) patients. Although recent observational studies showed the relationship between lower serum vitamin D levels and the development of poor physical function in community-dwelling older adults, involvement of vitamin D status in the development of sarcopenia in HF patients remain unclear. This study aimed to investigate the impact of serum vitamin D concentrations on sarcopenia in patients with HF. Methods We retrospectively enrolled 269 consecutive patients [median age of 73 years (interquartile range 63-82 years); 35% female] admitted to our institute for diagnosis and management of HF, and received the dual-energy X-ray absorptiometry (DEXA) method during the period from 1 September 2018 to 30 September 2021. The 25-hydroxyvitamin D [25(OH)D] was detected by a chemiluminescence immunoassay (CLIA) technology. The diagnosis of sarcopenia was made according to the criteria of Asia Working Group for Sarcopenia incorporating reduced skeletal muscle mass (appendicular skeletal muscle index [ASMI], <7.00 kg/m2 in males and <5.40 kg/m2 in females), and lower muscle strength (handgrip strength, <28 kg in males and <18 kg in females) and/or poor physical performance (gait speed, <1.0 m/s; chair stand test time, ≥12 s; short physical performance battery, ≤9 points). Results Of 269 patients, 116 (43%) patients had sarcopenia. An adjusted logistic regression model with a restricted cubic spline function showed that the odds ratio (OR) for sarcopenia increased as the serum 25(OH)D levels decreased. When the value that corresponded to an upper limit of 95% confidence interval (CI) for an OR of 1.0 was defined as the cut-off value of 25(OH)D levels for predicting sarcopenia, it was 18 ng/mL (Figure 1A). A multivariate logistic regression model was fit to calculate the propensity score (PS) for the 25(OH)D levels being <18 ng/mL based on covariates such as age, sex, and N-terminal pro B-type natriuretic peptide. (C-statistics 0.761). The inverse probability of treatment weighting (IPTW) was computed using PS to minimize differences in potential confounding factors between patients with a low serum 25(OH)D levels (<18 ng/mL) and those with a high serum 25(OH)D levels (≥18 ng/mL, Figure 1B). Results of the multivariate logistic regression analysis in the IPTW-weighted patients showed that a low serum 25(OH)D was independently associated with presence of sarcopenia (adjusted OR 2.03, 95% CI 1.31-3.16, p<0.01). In addition, patients with a low serum 25(OH)D had a significantly lower muscle strength and poor physical performance, but not ASMI, than those with a high serum 25(OH)D (Figure 2). Conclusion Decreased serum 25(OH)D levels are associated with decline in muscle strength and physical performance in HF patients. Serum 25(OH)D levels of <18 ng/mL may be a novel risk factor of sarcopenia in HF patients.
... Indeed, multiple trials have addressed the impact of its supplementation on different muscle outcomes (mass, strength and power), but only a small benefit on muscle strength has been demonstrated, with a higher and clinically meaningful efficacy in specific subgroups of patients, such as those aged ≥ 65 years and those presenting serum insufficiency (<30 nmol/L) [35]. On the other hand, while there is more convincing evidence that vitamin D has anabolic properties in myotubes and rodents, including a synergic stimulation with leucine [34,52], findings on an independent effect on protein synthesis in humans are inconsistent. In an 8-week double-blind placebo-controlled RCT (interventions: (1) vitamin D, 2000 IU/day; (2) conjugated linoleic acid, 4000 mg/day; (3) both nutrients; (4) placebo [corn oil]) conducted in 32 sedentary older adults (age range, 60-85 years) with suboptimal serum vitamin D (<35 ng/mL), Van Vliet and colleagues reported no effect of supplementation on MPS or handgrip strength [44]. ...
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Sarcopenia has been recognized as a muscle disease, with adverse consequences on health. Updated recommendations, aimed at increasing awareness of sarcopenia and its accompanying risks, have been produced to urge the early detection and treatment of this disease. Recommended treatment is based on an individually tailored resistance exercise training program, the optimization of protein intake using high-quality protein sources (i.e., whey protein) in order to provide a high amount of essential amino acids—particularly leucine—and addressing vitamin D deficiency/insufficiency. The purpose of this review is to collate and describe all of the relevant efficacy studies carried out with a muscle-targeted oral nutritional supplementation (MT-ONS)—namely a whey-protein-based, leucine- and vitamin D-enriched formula aimed at optimizing their intake and satisfying their requirements—in different patient populations and clinical settings in order to determine if there is enough evidence to recommend prescription for the treatment of sarcopenia or its prevention in high-risk patient populations. Trials using a MT-ONS with or without a concomitant physical exercise program were systematically searched (up to June 2021), and those addressing relevant endpoints (muscle mass, physical performance and function) were critically reviewed. In total, 10 articles providing efficacy data from eight trials were identified and narratively reviewed. As far as older patients with sarcopenia are concerned, MT-ONS has been pertinently tested in six clinical trials (duration 4–52 weeks), mostly using a high-quality randomized controlled trial design and demonstrating efficacy in increasing the muscle mass and strength, as well as the physical performance versus iso-caloric placebo or standard practice. Consistent results have been observed in various clinical settings (community, rehabilitation centers, care homes), with or without adjunctive physical exercise programs. A positive effect on markers of inflammation has also been shown. A muscle-protein-sparing effect, with benefits on physical performance and function, has also been demonstrated in patients at risk of losing skeletal muscle mass (three trials), such as older patients undergoing weight loss or intensive rehabilitation programs associated with neurological disability (Parkinson’s disease). MT-ONS has demonstrated not only a significant efficacy in clinical variables, but also a positive impact on healthcare resource consumption in the rehabilitation setting (length of stay and duration of rehabilitation). In summary, MT-ONS, alone or in association with an appropriate exercise program, is an effective therapy for older patients with sarcopenia and should be offered as a first-line treatment, not only to improve clinical outcomes but also to reduce healthcare resource consumption, particularly in patients admitted to a rehabilitation center.
... For example, in several trials that reported improvements in muscle mass and/or physical performance follow-ing supplementation with LEU-PRO in older adults, vitamin D 3 was co-ingested within the supplement. [25][26][27]34 Vitamin D 3 treatment was previously shown to sensitize muscle cells to the stimulatory effects of leucine and insulin on muscle protein synthesis in culture 35 and vitamin D supplementation may improve strength in older adults. 36 Our metabolomic data revealed that, interestingly, at the group level, spectral regions related to LDL and VLDL were higher PRE-supplementation in the LEU-PRO+n-3 group compared with POST-supplementation, an observation that is consistent with human kinetics studies showing reduced VLDL production following LC n-3 PUFA supplementation. ...
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Background: Precision nutrition is highly topical. However, no studies have explored the interindividual variability in response to nutrition interventions for sarcopenia. The purpose of this study was to determine the magnitude of interindividual variability in response to two nutrition supplementation interventions for sarcopenia and metabolic health, after accounting for sources of variability not attributable to supplementation. Methods: A 24 week, randomized, double-blind, placebo-controlled trial tested the impact of leucine-enriched protein (LEU-PRO), LEU-PRO plus long-chain n-3 PUFA (LEU-PRO+n-3) or control (CON) supplementation in older adults (n = 83, 71 ± 6 years) at risk of sarcopenia. To estimate the true interindividual variability in response to supplementation (free of the variability due to measurement error and within-subject variation), the standard deviation of individual responses (SDR ) was computed and compared with the minimally clinically important difference (MCID) for appendicular lean mass (ALM), leg strength, timed up-and-go (TUG), and serum triacylglycerol (TG) concentration. Clinically meaningful interindividual variability in response to supplementation was deemed to be present when the SDR positively exceeded the MCID. The probability that individual responses were clinically meaningful, and the phenotypic, dietary, and behavioural determinants of response to supplementation were examined. Results: The SDR was below the MCID for ALM (LEU-PRO: -0.12 kg [90% CI: -0.38, 0.35], LEU-PRO+n-3: -0.32 kg [-0.45, 0.03], MCID: 0.21 kg), TUG (LEU-PRO: 0.58 s [0.18, 0.80], LEU-PRO+n-3: 0.73 s [0.41, 0.95], MCID: 0.9 s) and TG (LEU-PRO: -0.38 mmol/L [-0.80, 0.25], LEU-PRO+n-3: -0.44 mmol/L [-0.63, 0.06], MCID: 0.1 mmol/L), indicating no meaningful interindividual variability in response to either supplement. The SDR exceeded the MCID (19 Nm) for strength in response to LEU-PRO (25 Nm [-29, 45]) and LEU-PRO+n-3 (23 Nm [-29, 43]) supplementation but the effect was uncertain, evidenced by wide confidence intervals. In the next stage of analysis, similar proportions of participant responses were identified as very likely, likely, possibly, unlikely, and very unlikely to represent clinically meaningful improvements across the LEU-PRO, LEU-PRO+n-3, and CON groups (P > 0.05). Baseline LC n-3 PUFA status, habitual protein intake, and numerous other phenotypic and behavioural factors were not determinants of response to LEU-PRO or LEU-PRO+n-3 supplementation. Conclusions: Applying a novel, robust methodological approach to precision nutrition, we show that there was minimal interindividual variability in changes in ALM, muscle function, and TG in response to LEU-PRO and LEU-PRO+n-3 supplementation in older adults at risk of sarcopenia.
... In animal models, vitamin D supplementation has been demonstrated to activate the mammalian target of rapamycin/ S6 kinase (mTOR/S6K) pathway, which leads to increased muscle protein synthesis (MPS) (34) essential for increases in muscle protein accrual and size (35). Cell culture models have also reported that vitamin D enhances the stimulating effect of leucine and insulin on muscle protein synthesis rates (36) and promotes myogenic differentiation and reduces the expression of myostatin, a known negative regulator of muscle size (37). Vitamin D has also been reported to stimulate the expression of genes involved in the control of cellular growth (33,38). ...
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Purpose Low serum vitamin D status has been associated with reduced muscle mass in observational studies although the relationship is controversial and a causal association cannot be determined from such observations. Two-sample Mendelian randomization (MR) was applied to assess the association between serum vitamin D (25(OH)D) and total, trunk, arm and leg fat-free mass (FFM). Methods MR was implemented using summary-level data from the largest genome-wide association studies (GWAS) on vitamin D (n=73,699) and total, trunk, arm and leg FFM. Inverse variance weighted method (IVW) was used to estimate the causal estimates. Weighted median (WM)-based method, and MR-Egger, leave-one-out were applied as sensitivity analysis. Results Genetically higher serum 25(OH)D levels had a positive effect on total (IVW = Beta: 0.042, p = 0.038), trunk (IVW = Beta: 0.045, p = 0.023) and arm (right arm IVW = Beta: 0.044, p = 0.002; left arm IVW = Beta: 0.05, p = 0.005) FFM. However, the association with leg FFM was not significant (right leg IVW = Beta: 0.03, p = 0.238; left leg IVW = Beta: 0.039, p = 0.100). The likelihood of heterogeneity and pleiotropy was determined to be low (statistically non-significant), and the observed associations were not driven by single SNPs. Furthermore, MR pleiotropy residual sum and outlier test did not highlight any outliers. Conclusions Our results illustrate the potentially causal, positive effect of serum 25(OH)D concentration on total, trunk and upper body appendicular fat-free mass.
... In vitro studies using isolated myotubes and cultured muscle cell lines are one way of testing whether vitamin D and its metabolites have direct effects in muscle, bypassing the modulation of circulating calcium and phosphate mineral levels and the subsequent effects these changes may elicit in muscle. Various studies have investigated the direct effects of vitamin D using cell culture models, the majority of which have analyzed myotube formation and size, (9,26,(28)(29)(30)(31) the expression of proteins involved in muscle formation and function, insulin sensitivity, (32)(33)(34)(35) glucose (35) and lipid uptake and metabolism, (36,37) as well as mitochondrial activity. (36) Most of the cell studies have employed similar methods that include incubating either isolated mouse or human skeletal myoblasts or commonly used immortalized mouse or rat myoblast cell lines with active 1,25(OH) 2 D 3 . ...
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Muscle weakness has been recognised as a hallmark feature of vitamin D deficiency for many years. Until recently, the direct bio‐molecular effects of vitamin D on skeletal muscle have been unclear. Whilst in the past, some reservations have been raised regarding the expression of the vitamin D receptor in muscle tissue, this special issue review article outlines the clear evidence from preclinical studies for not only the expression of the receptor in muscle but also the roles of vitamin D activity in muscle development, mass and strength. Additionally, muscle may also serve as a dynamic storage site for vitamin D, and play a central role in the maintenance of circulating 25‐hydroxy vitamin D levels during periods of low sun exposure. This article is protected by copyright. All rights reserved.
... However, a recent systematic review found that supplementation with a combination of leucine and vitamin D was associated with a greater lean body mass [176]. At the biological level, 1,25(OH)2 vitamin D3 potentiates the stimulating effect of leucine and insulin, leading to further activation of protein synthesis in murine C2C12 skeletal myotubes [177]. Similarly, the ability of leucine to stimulate muscle protein synthesis was significantly amplified after the co-administration of antioxidants (a mixture containing rutin, vitamin E, vitamin A, zinc, and selenium). ...
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Vitamin deficiencies have a serious impact on healthy aging in older people. Many age-related disorders have a direct or indirect impact on nutrition, both in terms of nutrient assimilation and food access, which may result in vitamin deficiencies and may lead to or worsen disabilities. Frailty is characterized by reduced functional abilities, with a key role of malnutrition in its pathogenesis. Aging is associated with various changes in body composition that lead to sarcopenia. Frailty, aging, and sarcopenia all favor malnutrition, and poor nutritional status is a major cause of geriatric morbidity and mortality. In the present narrative review, we focused on vitamins with a significant risk of deficiency in high-income countries: D, C, and B (B6/B9/B12). We also focused on vitamin E as the main lipophilic antioxidant, synergistic to vitamin C. We first discuss the role and needs of these vitamins, the prevalence of deficiencies, and their causes and consequences. We then look at how these vitamins are involved in the biological pathways associated with sarcopenia and frailty. Lastly, we discuss the critical early diagnosis and management of these deficiencies and summarize potential ways of screening malnutrition. A focused nutritional approach might improve the diagnosis of nutritional deficiencies and the initiation of appropriate clinical interventions for reducing the risk of frailty. Further comprehensive research programs on nutritional interventions are needed, with a view to lowering deficiencies in older people and thus decreasing the risk of frailty and sarcopenia.
... Indeed, the leucine content and the timing of the supplementation were similar across all 3 studies, whereas, phenotypically, our participants (who had low muscle mass and/or handgrip strength) fell in-between the healthy and the sarcopenic participants in the 2 aforementioned trials. It is possible that the vitamin D component of the supplements in the 2 previous studies may have enhanced the beneficial effects of the LEU-PRO (63,64). ...
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Background: Leucine-enriched protein (LEU-PRO) and long-chain (LC) n-3 (ω-3) PUFAs have each been proposed to improve muscle mass and function in older adults, whereas their combination may be more effective than either alone. Objective: The impact of LEU-PRO supplementation alone and combined with LC n-3 PUFAs on appendicular lean mass, strength, physical performance and myofibrillar protein synthesis (MyoPS) was investigated in older adults at risk of sarcopenia. Methods: This 24-wk, 3-arm parallel, randomized, double-blind, placebo-controlled trial was conducted in 107 men and women aged ≥65 y with low muscle mass and/or strength. Twice daily, participants consumed a supplement containing either LEU-PRO (3 g leucine, 10 g protein; n = 38), LEU-PRO plus LC n-3 PUFAs (0.8 g EPA, 1.1 g DHA; LEU-PRO+n-3; n = 38), or an isoenergetic control (CON; n = 31). Appendicular lean mass, handgrip strength, leg strength, physical performance, and circulating metabolic and renal function markers were measured pre-, mid-, and postintervention. Integrated rates of MyoPS were assessed in a subcohort (n = 28). Results: Neither LEU-PRO nor LEU-PRO+n-3 supplementation affected appendicular lean mass, handgrip strength, knee extension strength, physical performance or MyoPS. However, isometric knee flexion peak torque (treatment effect: -7.1 Nm; 95% CI: -12.5, -1.8 Nm; P < 0.01) was lower postsupplementation in LEU-PRO+n-3 compared with CON. Serum triacylglycerol and total adiponectin concentrations were lower, and HOMA-IR was higher, in LEU-PRO+n-3 compared with CON postsupplementation (all P < 0.05). Estimated glomerular filtration rate was higher and cystatin c was lower in LEU-PRO and LEU-PRO+n-3 postsupplementation compared with CON (all P < 0.05). Conclusions: Contrary to our hypothesis, we did not observe a beneficial effect of LEU-PRO supplementation alone or combined with LC n-3 PUFA supplementation on appendicular lean mass, strength, physical performance or MyoPS in older adults at risk of sarcopenia. This trial was registered at clinicaltrials.gov as NCT03429491.
... However, the above two studies did not study the effect of vitamin D on bone health, lean mass and fat mass. Despite four RCTs [31,32,35,36] in this current review showing that BMI and BMIZ were lower in participants who received prenatal or postnatal vitamin D supplementation, it is important to note that children were studied around the usual age of BMI and adiposity rebound, which occur at approximately 4 and 6 years of age, respectively [35,47]. The longterm effects of vitamin D supplementation during early life on BMI and BMIZ are unclear. ...
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... 33,34 Similarly, calcitriol was found to significantly attenuate p-mTOR levels in the present study while significantly upregulating levels of p-AMPKa, which also concurs with a previous report. 35 Interestingly, in the UUO group, p-AMPKa levels showed a mild peak on day 3, but t-AMPKa did not show the same trend (data not shown). AMPKa is activated to curtail energy consumption while cellular energy is depleted. ...
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Chapter
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By completing this chapter, the reader will be able to: Recognize and define the subject group of interest in the context of this book. Identify the individual roles caregivers fulfill. Relate the concept of caregiver burden to caregiving activities including post-intensive care syndrome (PICS-F). Describe individual consequences and effects of caregiving activities on the caregiver. Describe coping and support strategies that may be adopted to overcome negative aspects of caregiving. KeywordsCaregiverCarerCaretakerRelativesFamily members
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Purpose of review: To provide an updated overview of recent efficacy data on the use of muscle-targeted nutritional therapy, which should consider the optimization of protein and essential amino acids intakes, possibly in combination with supplementation with vitamin D (correction of deficiency/insufficiency status) and ω-3 fatty acids. Recent findings: Intervention studies conducted in the last years in different healthcare settings and heterogeneous patient populations support the use of muscle-targeted oral nutritional supplementation to improve muscle mass, function and physical performance in patients with sarcopenia. Higher efficacy is likely to be achieved in combination with individually tailored resistance exercise training programs and when nutritional therapy and the provision of specific nutrients result in an adequate protein-calorie balance. However, not only a reactive but also a pro-active application of this therapy could be proposed as evidence exists on the maintenance of or improvement in the same outcome variables in patients at risk of losing skeletal muscle mass. Summary: Based on available efficacy data, both a reactive and pro-active use of muscle-targeted nutritional therapy are promising and should be proposed. However, future research should be directed toward the management of patient populations characterized by substantial muscle wasting, as these have been frequently excluded from previous trials, perhaps to avoid confounding.
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PURPOSE: Vitamin D plays important roles in calcium homeostasis and bone metabolism. Since vitamin D receptors (VDRs) are located in a variety of organs, including skeletal muscle, vitamin D has potentially widespread effects. The purpose of this review was to summarize the current understanding of the effects of vitamin D on muscle function and exercise performance in athletes.METHODS: In this narrative review, we summarized previous studies by searching the literature in the PubMed, Google Scholar, and Science Direct databases.RESULTS: Vitamin D has been shown to regulate multiple actions in skeletal muscle tissue, such as myocyte proliferation and growth via genomic and non-genomic molecular pathways. Higher levels of vitamin D are associated with improved skeletal muscle function and exercise performance. Moreover, in some studies, vitamin D supplementation has beneficial effects on muscle strength in athletes, especially those who are vitamin D-deficient.CONCLUSIONS: Vitamin D appears to have beneficial effects on muscle and exercise performance in athletes. However, more studies are needed to clarify the action and dosage of vitamin D in athletes.
Chapter
Muscle erosion is one of the most important factors of disability in older people, but it may also occur early in life in case of chronic disease. The reduction in muscle mass and strength provokes an impaired mobility and increased risk for falls and fall‐related fractures. Sarcopenia is due predominantly to the atrophy of skeletal muscle fibers, mainly type II fibers. Many authors have argued that muscle loss in older persons partly depends upon inadequate nutritional intake and/or an impaired response of skeletal muscle to nutrients, example amino acids. Muscle protein turnover and amino acid transport in healthy young and older people were also determined during an oral administration of an amino acid mixture. The consumption of different protein sources and its effect on protein metabolism has been assessed in older women. The anabolic effect of resistance exercise should be used to amplify the anabolic action of dietary proteins on muscle protein synthesis.
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Purpose of review: Vitamin D is best known for its role in regulating calcium homeostasis and in strengthening bone. However, it has become increasingly clear that it also has important beneficial effects beyond the skeleton, including muscle. This review summarizes current knowledge about the role of vitamin D in skeletal muscle tissue and physical performance. Recent findings: Molecular mechanisms of vitamin D action in muscle tissue include genomic and nongenomic effects via a receptor present in muscle cells. Knockout mouse models of the vitamin D receptor provide insight into understanding the direct effects of vitamin D on muscle tissue. Vitamin D status is positively associated with physical performance and inversely associated with risk of falling. Vitamin D supplementation has been shown to improve tests of muscle performance, reduce falls, and possibly impact on muscle fiber composition and morphology in vitamin D deficient older adults. Summary: Further studies are needed to fully characterize the underlying mechanisms of vitamin D action in human muscle tissue, to understand how these actions translate into changes in muscle cell morphology and improvements in physical performance, and to define the 25-hydroxyvitamin D level at which to achieve these beneficial effects in muscle.
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this study set out to examine the effects of the treatment with 1,25-dihydroxyvitamin D3 (1,25D3) [150 IU/Kg (3.75 microg/Kg) one a day, for 15 days] to non-diabetic rats and in rats rendered diabetic by a single injection of streptozotocin [65 mg/kg]. treatment with 1,25D3 to non-diabetic rats did not affect the biochemical parameters measured in the plasma and urine of these animals. Likewise, insulin receptor expression in the kidney, liver, or adipose tissue and insulin-stimulated glucose transport in adipocytes from these animals were not affected either. Treatment with 1,25D3 to streptozotocin-induced diabetic rats did not correct the hyperglycemia, hypoinsulinemia, glycosuria or ketonemia induced by the diabetes, although it partially reversed the over-expression of the insulin receptor gene in the liver and adipose tissue, without altering the normal expression of this gene in the kidney. These effects were accompanied by a normalization of the number of insulin receptors without altering receptor affinity but improving the insulin response to glucose transport in adipocytes from these diabetic animals. Moreover, a computer search in the rat insulin receptor promoter revealed the existence of two candidate vitamin D response element (VDRE) sequences located at -256/-219 bp and -653/-620 bp, the first overlapped by three and the second by four AP-2-like sites. these genomic actions of 1,25D3 could represent beneficial effects associated with the amelioration of diabetes via mechanisms that possibly involve direct transcriptional activation of the rat insulin receptor gene. The candidate VDREs identified may respond to 1,25D3 via activation of the vitamin D receptor, although this remains to be investigated.
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In 242 community-dwelling seniors, supplementation with either 1000 mg of calcium or 1000 mg of calcium plus vitamin D resulted in a decrease in the number of subjects with first falls of 27% at month 12 and 39% at month 20. Additionally, parameters of muscle function improved significantly. The efficacy of vitamin D and calcium supplementation on risk of falling in the elderly is discussed controversially. Randomized controlled trials using falls as primary outcome are needed. We investigated long-term effects of calcium and vitamin D on falls and parameters of muscle function in community-dwelling elderly women and men. Our study population consisted of 242 individuals recruited by advertisements and mailing lists (mean [ +/- SD] age, 77 +/- 4 years). All serum 25-hydroxyvitamin D (25[OH]D) levels were below 78 nmol/l. Individuals received in a double blinded fashion either 1000 mg of calcium or 1000 mg of calcium plus 800 IU of vitamin D per day over a treatment period of 12 months, which was followed by a treatment-free but still blinded observation period of 8 months. Falls were documented using diaries. The study took place in Bad Pyrmont, Germany (latitude 52 degrees ) and Graz, Austria (latitude 46 degrees ). Compared to calcium mono, supplementation with calcium plus vitamin D resulted in a significant decrease in the number of subjects with first falls of 27% at month 12 (RR = 0.73; CI = 0.54-0.96) and 39% at month 20 (RR = 0.61; CI = 0.34-0.76). Concerning secondary endpoints, we observed significant improvements in quadriceps strength of 8%, a decrease in body sway of 28%, and a decrease in time needed to perform the TUG test of 11%. Combined calcium and vitamin D supplementation proved superior to calcium alone in reducing the number of falls and improving muscle function in community-dwelling older individuals.
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In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). In addition, IV-SUnSET detected food deprivation-induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV-SUnSET (IV-IHC-SUnSET). By combining IV-IHC-SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.
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1α,25(OH)(2)D(3), the active form of vitamin D(3), has been reported to regulate the cell biology of skeletal muscle. However, there has been some controversy about the expression of the vitamin D receptor (VDR) and thus the potential role of vitamin D(3) in skeletal muscle. In this study, we isolated and sequenced the full-length Vdr and Cyp27b1 transcripts in C2C12 myoblasts and myotubes. Western blots and immunocytochemistry confirmed protein expression in both myoblasts and myotubes clearly demonstrating that C2C12 cells express VDR and CYP27B1. To determine the vitamin D(3) action, we found that C2C12 myoblasts treated with either 1α,25(OH)(2)D(3) or 25(OH)D(3) inhibited cell proliferation and this was associated with increased Vdr expression. The observation that treatment of C2C12 myoblasts with the inactive form of vitamin D(3), [25(OH)D(3)], inhibited proliferation suggested that CYP27B1 was functionally active. We used small interfering RNA to knock down Cyp27b1 in myoblasts, and cells were treated with 25(OH)D(3). The growth-suppressive effects of 25(OH)D(3) were abolished, suggesting that CYP27B1 in myoblasts is necessary for the ability of 25(OH)D(3) to affect cell proliferation. Finally, we analyzed expression of VDR and CYP27B1 in regenerating skeletal muscle in vivo. We found that expression of VDR and CYP27B1 increased significantly at day 7 of regeneration, and these results confirm the expression of Vdr and Cyp27b1 in vivo and suggest a potential role for vitamin D(3) in skeletal muscle regeneration following injury.