Joshua C. Anthony

AMPing Down Leucine Action in Skeletal Muscle

In recent years, several investigative teams have tested leucine as a therapeutic agent to support muscle growth. Although some studies have shown that BCAA or leucine support muscle protein synthesis (MPS) (1), others have found that leucine is ineffective at stimulating muscle protein anabolism under certain conditions, e.g. sepsis, alcohol intoxication, and glucocorticoid excess in aged rats. During these catabolic states, MPS is either precluded or blunted in response to leucine, leading the authors of the current highlighted paper, Pruznak et al. (2), which was published in the October, 2008 issue of The Journal of Nutrition, to describe the muscle as being in a state of leucine resistance. Understanding the molecular basis for why muscle is unresponsive to supplemental leucine can be used to identify novel therapeutic targets and/or optimize current nutritional support strategies. The BCAA are known for their unique ability to stimulate MPS independently of other amino acids. When administered orally, leucine alone stimulates MPS in exercised or fooddeprived rats (3). The mechanism for the anabolic effect involves stimulation of mRNA translation initiation by increasing eukaryotic initiation factor (EIF) 4F complex assembly and signaling via mammalian target of rapamycin (mTOR) (3). The mTOR kinase facilitates cell growth and proliferation by nutrients and insulin through its association with the regulatory proteins Raptor (regulatory associated protein of mTOR), mLST8/GbL, and PRAS40 (proline-rich Akt substrate of 40 kDa) in a complex referred to as mTOR complex 1 (mTORC1) (4,5). Over the past decade, many laboratories have sought to define how leucine stimulates mTORC1 activity. Although it is clear that leucine does not activate the insulin/insulin-like growth factor/phosphatidylinositol 3-kinase/protein kinase B signal transduction pathway, identification of the relevant molecules upstream of mTORC1 remains elusive. To understand why leucine is ineffective at inducing MPS under certain stress conditions in vivo, Pruznak et al. (2) examined the relationship between leucine and AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that serves as a major energy sensor in the cell. Activated when ATP becomes limiting, AMPK stimulates catabolic pathways and inhibits anabolic pathways in an effort to supply ATP for cell survival. As such, AMPK is a negative upstream regulator of mTORC1, reducing MPS under conditions of energetic stress such as hypoxia, intense exercise, or glucose starvation (6). AMPK inhibits mTORC1 signaling in 2 ways: via phosphorylation of Raptor, which leads to decreased mTOR kinase activity, and via phosphorylation of tuberin (also known as tuberous sclerosis complex 2) at Ser1345 (7), converting the mTOR activator, Ras homolog enhanced in brain, to its inactive form. Activationof AMPK canbemimickedusing5-aminoimidazole4-carboxamide-1–4-ribofuranoside (AICAR), a cell-permeable compound whose phosphorylated metabolite activates AMPK without perturbing the cellular concentrations of ATP, ADP, or AMP in skeletal muscle (8). In the October issue, Pruznak et al. (2) utilizes AICAR treatment in rats to very effectively block the stimulation of skeletal MPS, EIF4F complex assembly, and mTORC1 signaling by oral administration of leucine (Fig. 1). Importantly, the overall block in leucine action is more complete than that achieved by other inhibitors tested previously in rats (e.g. rapamycin), demonstrating that cellular energy state plays a dominant role in the regulation of MPS by amino acids in vivo. These findings are supported by recently published work showing that leucine stimulation of mTOR and MPS is inhibited by AICAR treatment of C2C12 cells (9). Pruznak et al. (2) further advance our understanding of how leucine transmits mTORC1 signaling in vivo by identifying PRAS40 as a mediator of leucine’s effects. PRAS40 is a Raptorbinding protein that is phosphorylated directly by mTORC1 at several sites, including Ser183, and by protein kinase B at Thr246. Whereas studies using cells in culture show that phosphorylation of PRAS40 at Ser183 is modulated by amino acid-activated mTORC1 (4), the study of Pruznak et al. (2) finds that leucine enhances PRAS40 phosphorylation at the insulin-activated Thr246 site. Interpretation of this result should be viewed cautiously, because it is possible that the transient rise in insulin following leucine administration may have contributed to 1 Author disclosures: T. Anthony and J. Anthony, no conflicts of interest.