A Leucine-enriched Diet Enhances Overload-induced Growth and Markers of Protein Synthesis in Aged Rat Skeletal Muscle

Introduction: The hypertrophic response to overload in fast-twitch skeletal muscle is impaired in aged humans and rats, and impaired protein synthesis pathway activation is hypothesized to be a contributing factor. Muscle growth occurs when protein synthesis exceeds protein degradation. Dietary supplementation of the essential amino acid leucine has been shown to enhance protein synthesis in both young and aged skeletal muscle. Leucine acts in part by activating mammalian target of rapamycin (mTOR; a key upstream regulator of protein synthesis pathways) as well as by attenuating the activation of 5\u27-AMP-activated protein kinase (AMPK; a negative regulator of mTOR and protein synthesis). During the aging process, AMPK Thr172 phosphorylation (and thus its activation) is increased, purportedly inhibiting gains in muscle mass and strength. Although dietary leucine supplementation has been shown to enhance strength gains in response to resistance training in young humans, the potential for leucine supplementation to enhance overload-induced muscle hypertrophy in aged humans or animal models has not been examined. Thus, the aim of this study was to determine whether dietary leucine supplementation can enhance markers of protein synthesis and rescue hypertrophy in overloaded fast-twitch skeletal muscles of aged rats to levels comparable to their younger counterparts. It was hypothesized that dietary leucine supplementation during 7 days of fast-twitch plantaris muscle overload would enhance plantaris muscle hypertrophy in aged rats to levels observed in young adult rats not receiving leucine. It was also hypothesized that dietary leucine supplementation during the overload period would suppress AMPK phosphorylation and enhance markers of protein synthesis [70 kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), and eukaryotic elongation factor 2 (eEF2)] in the overloaded fast-twitch plantaris muscles of the aged rats to levels observed in young adult rats not receiving leucine. Methods: Young adult (8 mo.) and old (33 mo.) male Fisher 344 x Brown Norway F1 Hybrid (FBN) rats underwent a 1-week unilateral overload of the fast-twitch plantaris muscles via tenotomy of the synergistic gastrocnemius muscle. Within each age group, animals were matched for body weight and separated into either a dietary leucine supplementation group (normal rat chow supplemented by an additional 5% leucine content in place of 5% of the carbohydrate content; n = 7/age group) or placebo group (normal rat chow; n = 6/age group). The leucine groups started the leucine-enriched diet 2 days prior to, and throughout, the overload intervention. All animals had ad libitum access to water and chow during the entire experiment; no differences in daily calorie consumption were observed between the placebo vs. leucine groups within each age group. At the end of the overload period, sham-operated and overloaded plantaris muscles were harvested and analyzed via western blotting for the phosphorylations of AMPK, p70S6k, rpS6, and eEF2. A 2x2x2 ANOVA with repeated measures was used for analyses of the effects of age, dietary intervention, and overload (the repeated measure) on muscle hypertrophy. A 2x2 ANOVA was used to measure the percent changes in hypertrophy and western blot analyses. Post-hoc comparisons were accomplished via a Fisher\u27s Least Significant Difference test, with statistical significance being set at p ≤ 0.05. Results: Dietary leucine enrichment significantly (p ≤ 0.05) enhanced overload-induced fast-twitch plantaris muscle hypertrophy in old, but not in young adult, animals. A similar effect was also observed in the slow-twitch soleus muscles, but western blotting analyses are only presented for the fast-twitch plantaris muscles. Sham and overloaded plantaris muscle AMPK phosphorylation (Thr172) was significantly higher in aged animals receiving normal chow compared to young adult animals; however, leucine supplementation in old animals reduced this AMPK phosphorylation to levels similar to young adult animals. Phospho-p70S6k (Thr389) and phospho-rpS6 (Ser235/Ser236) were significantly lower in old vs. young overloaded muscles under placebo conditions, but leucine partially restored both p70S6k and rpS6 phosphorylations in old overloaded muscles to that of young adult overloaded muscles. Overload significantly increased total eEF2 content and decreased inhibitory eEF2 phosphorylation (Thr56; normalized to total eEF2) in young adult muscles regardless of leucine supplementation. Total eEF2 content was unaffected by overload in old placebo muscles, but leucine supplementation in old animals non-significantly (p = 0.09) restored the overload-induced increase in total eEF2 content. Muscle eEF2 phosphorylation was unaffected by overload or leucine supplementation in old animals. Discussion: These novel findings indicate that a leucine-enriched diet may potentially enhance overload-induced growth of aged fast-twitch muscle, in part by enhancing pathways known to stimulate protein synthesis. This is in accord with previous findings of leucine’s stimulating effect on protein synthesis in both young adult and aged skeletal muscle under resting conditions. The fact that leucine supplementation enhanced overload-induced hypertrophy only in the old (and not the young) animals may reflect the high growth stimulus of the chronic overload model. That is, the balance of protein synthesis/degradation rates under such a large chronic growth stimulus may not be the limiting factor in young animals, in which muscle growth is not impaired (i.e., synthesis/degradation rates may reach futile levels, and another factor such as sarcomere assembly may be limiting). However, the impaired balance of protein synthesis/degradation rates may be the limiting factor to growth in aged muscle, and leucine may correct this imbalance to restore muscle growth to levels observed in young animals

A Leucine-enriched Diet Enhances Overload-induced Growth and Suppresses Markers of Protein Degradation in Aged Rat Skeletal Muscle

Introduction: The hypertrophic response to overload in fast-twitch skeletal muscle is impaired in aged humans and rats, and upregulation of protein degradation pathways are hypothesized to be a contributing factor. Muscle growth occurs when protein synthesis is greater than protein degradation. Dietary supplementation of the essential amino acid leucine has been shown to reduce protein degradation in both young and aged skeletal muscle. Specifically, leucine acts in part by attenuating 5\u27-AMP-activated protein kinase (AMPK) activation as well as the translocation of the forkhead box transcription factor 3A (FoxO3, known to promote transcription of mRNAs encoding degradation pathway proteins) to the nucleus. Akt (a promoter of muscle growth) prevents translocation of FoxO3 into the nucleus by phosphorylating FoxO3 phosphorylation at Ser318/321. However, AMPK, inhibits Akt\u27s phosphorylation of FoxO3, allowing it to enter the nucleus and increase transcription of protein degradation pathway genes encoding ubiquitin ligase proteins such as muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx, or Atrogin-1). During the aging process, AMPK Thr172 phosphorylation (and thus its activation) is increased, purportedly inhibiting gains in muscle mass and strength. Although dietary leucine supplementation has been shown to enhance strength gians in response to resistance training in young humans, the potential for leucine supplementation to enhance overload-induced muscle hypertrophy in aged humans or animal models has not been examined. Thus, the aim of this study was to determine whether dietary leucine supplementation can attenuate markers of protein degradation and rescue hypertrophy during overload in the fast-twitch skeletal muscles of aged rats to levels comparable to their younger counterparts. It was hypothesized that dietary leucine supplementation during 7 days of fast-twitch plantaris muscle overload would enhance plantaris muscle hypertrophy in aged rats to levels observed in young adult rats not receiving leucine. It was also hypothesized that dietary leucine supplementation during the overload period would alter markers of protein degradation (enhance FoxO3 phosphorylation and reduce the levels of AMPK phosphorylation, Atrogin-1 protein content, and MuRF1 protein content) in the overloaded fast-twitch plantaris muscles of the aged rats to levels observed in young adult rats not receiving leucine. Methods: Young adult (8 mo.) and old (33 mo.) male Fisher 344 x Brown Norway F1 Hybrid (FBN) rats underwent a 1-week unilateral overload of the fast-twitch plantaris muscles via tenotomy of the synergistic gastrocnemius muscle. Within each age group, animals were matched for body weight and separated into either a dietary leucine supplementation group (normal rat chow supplemented by an additional 5% leucine content in place of 5% of the carbohydrate content; n = 7/age group) or placebo group (normal rat chow; n = 6/age group). The leucine groups started the leucine-enriched diet 2 days prior to, and throughout, the overload intervention. All animals had ad libitum access to water and chow during the entire experiment; no differences in daily calorie consumption were observed between the placebo vs. leucine groups within each age group. At the end of the overload period, sham-operated and overloaded plantaris muscles were harvested and analyzed via western blotting for the phosphorylations of AMPK and FoxO3 as well as total levels of Atrogin-1 and MuRF1. A 2x2x2 ANOVA with repeated measures was used for analyses of the effects of age, dietary intervention, and overload (the repeated measure) on muscle hypertrophy. A 2x2 ANOVA was used to measure the percent changes in hypertrophy and western blot analyses. Post-hoc comparisons were accomplished via a Fisher\u27s Least Significant Difference test, with statistical significance being set at p ≤ 0.05. Results: Dietary leucine enrichment significantly (p ≤ 0.05) enhanced overload-induced fast-twitch plantaris muscle hypertrophy in old, but not in young adult, animals. A similar effect was also observed in the slow-twitch soleus muscles, but western blotting analyses are only presented for the fast-twitch plantaris muscles. Sham and overloaded plantaris muscle AMPK phosphorylation was significantly higher in aged animals receiving normal chow compared to young adult animals; however, leucine supplementation in old animals reduced this AMPK phosphorylation to levels similar to young adult animals. Compared to placebo, leucine also non-significantly (p = 0.07) enhanced FoxO3 phosphorylation in the overloaded muscles of both young adult and old animals (thus theoretically reducing FoxO3 translocation to the nucleus). Accordingly, leucine also non-significantly (p = 0.07) reversed the overload-induced increase (from a 22.8% increase to a 17.0% decrease) in Atrogin-1 content in aged muscles and non-significantly (p = 0.14) enhanced the overload-induced decrease in MuRF1 content in the muscles of both age groups. Discussion: These novel findings indicate that a leucine-enriched diet may potentially enhance overload-induced growth of aged fast-twitch muscle, in part by suppressing pathways known to stimulate protein degradation. This is in accord with previous findings of leucine’s suppressive effect on protein degradation in both young adult and aged skeletal muscle under resting conditions. The fact that leucine supplementation enhanced overload-induced hypertrophy only in the old (and not the young) animals may reflect the high growth stimulus of the chronic overload model. That is, the balance of protein synthesis/degradation rates under such a large chronic growth stimulus may not be the limiting factor in young animals, in which muscle growth is not impaired (i.e., synthesis/degradation rates may reach futile levels, and another factor such as sarcomere assembly may be limiting). However, the impaired balance of protein synthesis/degradation rates may be the limiting factor to growth in aged muscle, and leucine may correct this imbalance to restore muscle growth to levels observed in young animals

OR-047 Mechanical stretch activates glycometabolism-related enzyme through estrogen in C2C12 myoblasts

Objective Exercise is involved with some Metabolic diseases, moderate exercise may improve glycometabolism and type 2 diabetes mellitus in menopausal female. Previous study showed that exercise increased the level of muscular estrogen in Ovariectomized rats, improved muscle mass and glycometabolism, it provided a reference to relief type 2 diabetes mellitus symptom. Until now, the effect of estrogen induced by exercise on muscular glycometabolism is not clear, the present study was designed to explore the effect of estrogen induced by mechanical stretch on glycometabolism in mouse C2C12 myoblasts. Methods The mouse C2C12 myoblasts in vitro were plated at BioFlex Culture Plate, and assigned randomly to the control group(C), stretch group(S), SA group. SA group was cultured in growth medium with 400μg/ml anastrozole(aromatase inhibitor), other groups were cultured in GM with DMSO for 36h, and then S, SA groups were stretched by Flexcell FX-5000TM system (magnitude 15%, frequency 1Hz, duration 6hours), Cellular proteins were extracted after 24h of stretch, ELISA assay was used to detect estradiol levels, we detected the expression of HK, PI3K, the ratio of p-AKT and AKT, GLUT4 protein level by Western blotting. Results Compared with the control group, a higher estradiol level was detected in stretch group(P<0.05), and the protein expression of HK, PI3K, the ratio of p-AKT and AKT, GLUT4 is higher(P<0.05) after stretching. The estradiol level and protein expression is lower in SAF group as compare to the stretch group(P<0.05). while there was no significant difference in estradiol level and protein expression between SF group and SAF group(P>0.05). Conclusions Estrogen induced by mechanical stretch can improve glycometabolism-related enzyme and protein expression of mouse C2C12 myoblasts

From mitochondria to sarcopenia: role of 17β-estradiol and testosterone

Sarcopenia, characterized by a loss of muscle mass and strength with aging, is prevalent in older adults. Although the exact mechanisms underlying sarcopenia are not fully understood, evidence suggests that the loss of mitochondrial integrity in skeletal myocytes has emerged as a pivotal contributor to the complex etiology of sarcopenia. Mitochondria are the primary source of ATP production and are also involved in generating reactive oxygen species (ROS), regulating ion signals, and initiating apoptosis signals in muscle cells. The accumulation of damaged mitochondria due to age-related impairments in any of the mitochondrial quality control (MQC) processes, such as proteostasis, biogenesis, dynamics, and mitophagy, can contribute to the decline in muscle mass and strength associated with aging. Interestingly, a decrease in sex hormones (e.g., 17β-estradiol and testosterone), which occurs with aging, has also been linked to sarcopenia. Indeed, 17β-estradiol and testosterone targeted mitochondria and exhibited activities in regulating mitochondrial functions. Here, we overview the current literature on the key mechanisms by which mitochondrial dysfunction contribute to the development and progression of sarcopenia and the potential modulatory effects of 17β-estradiol and testosterone on mitochondrial function in this context. The advance in its understanding will facilitate the development of potential therapeutic agents to mitigate and manage sarcopenia

PO-047 Expression of Aromatase and Synthesis of Sex Steroid Hormones in Skeletal Muscle Following Exercise Training in Ovariectomized Rats

Objective Age-related muscle wasting (sarcopenia) is accompanied by a decrease in estrogen levels which can compromise the health of aging women. Recent studies have shown that the key enzyme of estrogen synthesis (aromatase) is detected in the skeletal muscle. The purpose of this study was to investigate the effects of exercise on the expression of aromatase and the synthesis of sex steroid hormones in skeletal muscle following exercise training. Methods Fourteen female ovariectomized rats were divided into two groups, treadmill running (n=7) and sedentary (n=7) group. Exercise training on a treadmill (25 m/min, 60 min/day, 6 days/week) for 5 weeks. Immunofluorescence assay was used to detect estradiol and aromatase levels in soleus muscle and plantar muscle. Detected the expression of AKT, Aromatase, FoxO1, MyoD protein level by Western blotting. Results We found that in ovariectomized rats, exercise training significantly increased the soleus and plantar muscles mass. The level of aromatase expression and 17-b-estradiol (E2) were increased significantly in skeletal muscle following exercise training(P < 0.05). In addition, the down-stream Akt-FoxO1-MyoD signaling pathway was significantly regulated in both soleus and plantaris muscles following exercise(P< 0.05). Conclusions These results demonstrate that exercise training increased the expression of aromatase and local estrogen production in skeletal muscle, which potentially influences skeletal muscle in ovariectomized rats through activation of Akt-FoxO1-MyoD signaling pathway

The Current Status, Prospects, and Challenges of Shape Memory Polymers Application in Bone Tissue Engineering

Bone defects can occur after severe trauma, infection, or bone tumor resection surgery, which requires grafting to repair the defect when it reaches a critical size, as the bone’s self-healing ability is insufficient to complete the bone repair. Natural bone grafts or artificial bone grafts, such as bioceramics, are currently used in bone tissue engineering, but the low availability of bone and high cost limit these treatments. Therefore, shape memory polymers (SMPs), which combine biocompatibility, biodegradability, mechanical properties, shape tunability, ease of access, and minimally invasive implantation, have received attention in bone tissue engineering in recent years. Here, we reviewed the various excellent properties of SMPs and their contribution to bone formation in experiments at the cellular and animal levels, respectively, especially for the repair of defects in craniomaxillofacial (CMF) and limb bones, to provide new ideas for the application of these new SMPs in bone tissue engineering

a-Klotho Expression in Mouse Tissues Following Acute Exhaustive Exercise

a-Klotho, a multifunctional protein, has been demonstrated to protect tissues from\r\ninjury via anti-oxidation and anti-inflammatory effects. The expression of a-klotho is\r\nregulated by several physiological and pathological factors, including acute inflammatory\r\nstress, oxidative stress, hypertension, and chronic renal failure. Exhaustive exercise has\r\nbeen reported to result in tissue damage, which is induced by inflammation, oxidative\r\nstress, and energy metabolism disturbance. However, little is known about the effects\r\nof exhaustive exercise on the expression of a-klotho in various tissues. To determine the\r\neffects, the treadmill exhaustion test in mice was performed and the mice were sacrificed\r\nat different time points following exhaustive exercise. Our results confirmed that the\r\nfull-length (130 kDa) and shorter-form (65 kDa) a-klotho were primarily expressed in\r\nthe kidneys. Moreover, we found that, except for the kidneys and brain, other tissues\r\nprimarily expressed the shorter-form a-klotho, including liver, which was in contrast to\r\nprevious reports. Furthermore, the shorter-form a-klotho was decreased immediately\r\nfollowing the acute exhaustive exercise and was then restored to the pre-exercise level\r\nor even higher levels in the next few days. Our results indicate that a-klotho may play a\r\nkey role in the body exhaustion and recovery following exhaustive exercise

a-Klotho Expression in Mouse Tissues Following Acute Exhaustive Exercise

a-Klotho, a multifunctional protein, has been demonstrated to protect tissues frominjury via anti-oxidation and anti-inflammatory effects. The expression of a-klotho isregulated by several physiological and pathological factors, including acute inflammatorystress, oxidative stress, hypertension, and chronic renal failure. Exhaustive exercise hasbeen reported to result in tissue damage, which is induced by inflammation, oxidativestress, and energy metabolism disturbance. However, little is known about the effectsof exhaustive exercise on the expression of a-klotho in various tissues. To determine theeffects, the treadmill exhaustion test in mice was performed and the mice were sacrificedat different time points following exhaustive exercise. Our results confirmed that thefull-length (130 kDa) and shorter-form (65 kDa) a-klotho were primarily expressed inthe kidneys. Moreover, we found that, except for the kidneys and brain, other tissuesprimarily expressed the shorter-form a-klotho, including liver, which was in contrast toprevious reports. Furthermore, the shorter-form a-klotho was decreased immediatelyfollowing the acute exhaustive exercise and was then restored to the pre-exercise levelor even higher levels in the next few days. Our results indicate that a-klotho may play akey role in the body exhaustion and recovery following exhaustive exercise