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2 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Evansville, IN 47712 and 3 Mead Johnson Nutritionals, Evansville, IN 47721
* To whom correspondence should be addressed. E-mail: tganthon{at}iupui.edu.
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)4 (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 food-deprived 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/GβL, 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.
Activation of AMPK can be mimicked using 5-aminoimidazole-4-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).
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Although the present data bring us one step closer to understanding the leucine/BCAA signal transduction pathway, much work remains. First, the significant drop in plasma insulin caused by AICAR may have contributed to the complete blocking of leucine-stimulated MPS and mTOR signaling. A future study designed to clamp plasma insulin during AICAR treatment would help clarify this possibility. Second, AICAR increased plasma BCAA concentrations in both the basal and leucine-stimulated rats, consistent with the report that AICAR activates myofibrillar protein degradation in C2C12 myotubes (10). Thus, coordination of MPS and degradation by AMPK may be relevant to understanding leucine resistance in muscle. Third, amino acid deprivation modulates the binding of Ras homolog enhanced in brain to mTOR in cells in culture (5), but how oral leucine administration alters this interaction in skeletal muscle is unknown. Fourth, the roles of the class III phosphatidylinositol 3-kinase, hVps34 (5), and the recently identified Rag GTPases (11) are additional targets of interest for evaluation in animal tissues. Finally, as pointed out by Pruznak et al. (2), not all mechanisms identified in cells in culture are operational in whole tissues. Along these lines, it is important to consider that muscle fiber types differ with regard to their capacity for AMPK signaling. Indeed, it is reported that in Sprague-Dawley rats, the concentration of the AMPK catalytic subunits (
1 and 2) differs significantly among the different muscle fiber types, even within the gastrocnemius, a predominantly fast-twitch muscle used in the current study (12). Assessing muscle leucine resistance according to fiber type may reveal novel insights about the role of AMPK in regulating MPS by leucine.
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FOOTNOTES |
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4 Abbreviations used: AICAR, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside; AMPK, adenosine monophosphate-activated protein kinase; EIF, eukaryotic initiation factor; MPS, muscle protein synthesis; mTOR, mammalian target of rapamycin, mTORC1, mammalian target of rapamycin complex 1; Raptor, regulatory associated protein of mTOR; PRAS40, proline-rich Akt substrate of 40 kDa.
Manuscript received 14 August 2008. Initial review completed 28 August 2008. Revision accepted 22 September 2008.
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LITERATURE CITED |
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