Effects of Dietary Leucine Supplementation on Muscle Mass and Markers of Protein Degradation in Overloaded Skeletal Muscles of Young Adult and Aged Rats

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'-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 Ser[superscript]318/321. However AMPK inhibits Akt's 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 Thr[superscript]172 phosphorylation (and thus its activation) is increased purportedly inhibiting gains in muscle mass and strength. Although dietary leucine supplementation has been shown to enhance muscle hypertrophy 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. 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. Dietary leucine enrichment significantly (p [equal to or less than] 0.05) enhanced overload-induced plantaris muscle hypertrophy in old but not in young adult animals. 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. These 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.