be measured because the isotope label in the carboxyl position is released as CO2 when leucine is irreversibly committed to oxidation with the decarboxylation of alpha-ketoisocaproate (aKIC). As such the appearance of the 13C label in breath CO2 provides a direct measure of leucine oxidation. Finally, when a tracer of an essential amino acid (EAA) such as phenylalanine is used that is neither synthesized nor oxidized (unlike the BCAAs) in muscle, net balance, uptake and dilution of the tracer can be equated not only to rates of muscle synthesis but also to breakdown. Thus, human muscle protein synthesis and breakdown can be simultaneously measured in vivo. It is precisely this di erence between synthesis and breakdown (i.e. the net balance) that is the significant parameter relevant to the net gain (anabolism) or loss (catabolism) of muscle. When tracer methods are linked with sophisticated genetic or pharmacological approaches, and/or temporal observations of intramuscular signaling, we gain information on how signaling pathways and extracellular ligands are linked to alterations in human muscle proteostasis.

The mechanisms regulating changes in proteostasis in human aging, disease and trauma are complex and involve interplay between the systemic milieu (i.e. central hormones, Section 5.2.1), the immediate extracellular milieu (autocrine/ paracrine signals, Section 5.2.2) and those within the cell (i.e. metabolites, 2nd messengers etc.). The rest of the chapter is devoted to discussing what is understood about the regulation of muscle proteostasis by the previously discussed extracellular and intracellular signals in humans. The main focus is upon the primary environmental factors long known to e ect muscle size, nutrients and contraction, and on the regulation of muscle proteostasis in aging, disuse, disease and acute trauma.

5.3.1Nutrients as Regulators of Muscle Proteostasis in Man

Starvation and chronic malnutrition have long been known to cause weight loss with associated muscle wasting, thus nutrients are widely appreciated as key extracellular signals in the regulation of muscle proteostasis.26 In a healthy, weight-stable, weight-bearing human being the dynamic equilibrium between muscle protein synthesis and breakdown ensures that muscle mass remains constant. This occurs through two mechanisms. The first involves stimulation of muscle protein synthesis over basal rates for a period after feeding. Indeed, it was initially shown that muscle protein synthesis rates are increased about 2-fold above basal rates (which equate to about 0.05% h–1 in mixed human muscle) after feeding a mixed macronutrient meal (i.e. carbohydrate, fats, amino acids). Later the nutrients causing the stimulation were identified with AA (outlined in Section 5.2.2.8) now recognized as the nutritionally active constituents responsible for stimulating muscle synthesis. Because provision of EAA, those that cannot be synthesized in vivo, alone has equal e cacy in the stimulation of muscle synthesis to that of all AA or a mixed meal, we now recognize EAA as the active constituent. This key role of EAA in the regulation of proteostasis makes sense from an evolutionary perspective since the instruction to build muscle is received only when amino acids that cannot be

synthesized in vivo are ingested. Work from the authors’ labs recently charted the time course of the response to AA. After feeding enough AA to maximally stimulate muscle synthesis (equating to about 20 g of EAA), the response switches o after about 1.5 h, despite continued AA availability. We have termed this the ‘‘muscle full’’ phenomenon on the basis that the increases in fractional synthesis rates of muscle protein are outlasted by substrate availability. The mechanism for this is not known, but it is likely that the muscle gauges its own amino acid requirements based upon losses incurred during fasting periods. The second route by which feeding stimulates muscle protein accretion is via reducing muscle protein breakdown. This is because both mixed meal and AA feeding stimulates insulin release from pancreatic b cells. Consequently, the reduction in protein breakdown after feeding (–50%) is at least in part due to the anti-proteolytic e ects of insulin.

As a consequence, in vivo the two mechanisms combine such that food has anabolic e ects on both arms of proteostasis i.e. substantial increases in muscle protein synthesis are observed in response to delivery of AA and moderate reductions in muscle protein breakdown are observed in response to delivery of insulin. As a result, negative net balance observed in the post-absorptive state (i.e. muscle protein breakdown exceeds synthesis) is transiently reversed. This small gain in muscle protein o sets loss during fasted periods. As such, if all else is equal muscle mass remains constant.

5.3.1.1AA as Extracellular–Intracellular Signals

The intramuscular regulatory signals governing how AA and insulin stimulate muscle protein synthesis and reduce degradation have been the subject of much scrutiny.27,28 With regard to protein synthesis, most work has centered on activities of phosphoproteins that, independent of cell type, (in)directly regulate the initiation and elongation phases of mRNA translation; so-called anabolic signals. Although the initiating events remain unknown, currently the most proximal step for sensing of AA involves stimulation of Ras-related guanosine triphosphate hydrolases (RAGs) and/or the class III PI3Ks, vacuolar protein sorting 34 (Vps34). These elements ultimately converge to increase signaling through the mammalian target of rapamycin complex 1 (mTORc1). The initiating events for insulin are better understood with insulin binding to its receptor initiating signaling via an insulin receptor/IRS pathway to Akt. Akt phosphorylates glycogen synthase kinase 3 beta (GSK3b), the key enzyme responsible for glycogen synthesis, and then GSK3b phosphorylates and inhibits eukaryotic initiation factor 2B (eIF2Be) at serine 535. The net e ect is a stimulation of eIF2B. The guanine nucleotide exchange factor activity of eIF2B serves a key role in translation by catalyzing the recycling of eIF2 methyl tRNA between consecutive rounds of peptide-chain initiation. Finally, Akt both directly (via phosphorylation) and indirectly (via tuberous sclerosis complex 1/2 and proline rich Akt substrate (PRAS40)) stimulates mTORc1 activity. Consequently, both AA and insulin signaling converge on mTORc1;

a heterotrimeric complex consisting of regulatory associated protein of TOR (raptor), PRAS40, mLST8 and mTOR and a central regulator of growth in all eukaryotic cells.

Activation of mTORc1 triggers a series of ‘‘anabolic’’ signaling events. The best characterized substrates of mTORc1 are 4E-BP1, whose phosphorylation promotes eukaryotic initiation factor 4F (eIF4F) complex assembly (an assumed requirement for cap-dependent translation) and p70S6K1, which phosphorylates, among other substrates, eukaryotic initiation factor 4B (eIF4B), which facilitates unwinding of tertiary mRNA structure and eukaryotic elongation factor 2 kinase (eEF2K), which promotes peptide elongation by recruiting charged tRNA to the ribosome. Though it is less well established how insulin suppresses protein degradation, Akt phosphorylates FKHR transcription factors and in doing so regulates their nuclearcytoplasmic translocation thereby preventing transcription of pro-proteolytic genes. It is therefore assumed to be this mechanism by which insulin suppresses muscle proteolysis, though it may also be through stimulating the inhibitory e ects of mTOR on autophagy.

Confirming the key role of mTORc1 in human muscle is the observation that Rapamycin, an immunosuppressant and potent mTORc1 inhibitor, robustly attenuates the stimulation of muscle protein synthesis and associated anabolic signaling in response to leucine. Thus it appears that AA and insulin action via mTORc1 principally serves to increase the e ciency of translation (i.e. number of mRNA translated per ribosome). Confirming distinct actions of AA and insulin upon muscle proteostasis is the observation that inhibiting postprandial increases in insulin does not suppress increases in muscle protein synthesis. Thus it appears that the physiological role of insulin is primarily in the suppression of protein breakdown in adult humans.

While the pathways described above are su cient to account for the in vivo e ects of AA and insulin upon muscle proteostasis, it is possible that there is a transcriptional component to the anabolic response to feeding. For example, performing euglycemic hyperinsulinemic clamps in humans acutely modulates B800 transcripts in adult human muscle. Moreover, activation of mTORc1 by AA also modulates expression of rRNA and other transcripts. In support of this, there have been a number of reports of pro-anabolic transcriptional responses to feeding. Decreases in myostatin mRNA has been reported in humans after feeding, which could relieve inhibition of mTORc1 signaling. Furthermore, increases in AA transporter expression could facilitate influx of AA to intracellular pools. Nonetheless, early work using actinomycin D (a transcriptional inhibitor) failed to repress the acute synthesis response to leucine. Thus, both insulin and AA probably work to facilitate both shortterm and long-term changes in muscle proteostasis. In the acute phase described above changes are largely via mTORc1 e ects on translational e ciency and possibly protein degradation. In the longer term, changes are due to increased transcription of AA transporters, intramuscular 2nd messenger signals and perhaps the transcriptional/translational machinery (i.e. preserving anabolic capacity).