Файл: Extracellular and Intracellular Signaling (книга).pdf

‘‘building blocks’’ of muscle proteins, but also directly stimulate intracellular signaling pathways that regulate mRNA translation. Evidence for this is derived from studies in which the provision of single essential amino acids (EAA), such as leucine, is su cient for the stimulation of muscle synthesis, even in the absence of other AA as substrate for peptide synthesis. Leucine, of all the EAA, seems to have the most potent capacity to stimulate muscle synthesis and inhibit lysosome-based muscle protein degradation. Precisely how the EAA stimulate proteostasis-regulating signals remains poorly understood. Unlike peptide hormones and local factors, there does not appear to be a receptor in the plasma membrane as genetic loss of the transporter system (for leucine: system L, a Na1-independent, glutamine-dependent exchanger) abolishes intracellular signaling responses to EAA. Thus EAA presumably act like glucocorticoids, TH and testosterone by binding to proteins once inside the cell. However, there are no known transcription factors or EAA response elements within the DNA and it is not known whether leucine itself or metabolites generated during its catabolism are responsible for the e ects. For example, it remains to be investigated whether deamination of leucine to aKIC via branch chain amino acid transferase or oxidative decarboxylation to isovaleryl CoA via branch chain keto-acid dehydrogenase is required for leucine-induced phosphoproteome activity. Currently, it is believed that AA promote synthesis while also inhibiting degradation, presumably via coordinated action of Akt/ FKHR and mTORc1-mediated inhibition of autophagy (see Section 5.3.1.1 for more detail); though the mechanism is unknown and multiple mechanisms may exist as leucine and aKIC appear to inhibit lysosome-based degradation via distinct pathways.

5.3 Regulation of Muscle Proteostasis in Humans

Since the early 1980s, many of the advances made surrounding the regulation of muscle proteostasis in humans are a consequence of the development of methods for detecting stable isotopically labeled amino acid ‘‘tracers’’ by gas chromatography combustion isotope ratio mass spectrometry (GC-C- IRMS).25 Constant improvements mean that these tracer approaches have extensive application even for studies of muscle proteostasis over periods as short as 30–60 min. Labeling amino acids such as the branch chain amino acid leucine with 13C, for example [1,2-13C2]leucine, allows measurement of fractional synthetic rates (muscle protein synthesis rates) of incorporation of these amino acids into functional muscle proteins from biopsy tissue. Increased sensitivity and precision of gas chromatography mass spectrometry (GC-MS) now allow measurement of the incorporation of deuterium labeled amino acids into protein i.e. d5 phenylalanine. Fractionation of proteins from these tissues further allows measurement of fractional synthetic rates of incorporation into di erent cellular compartments such as mitochondria, sarcoplasm and myofibers. Using these same tracers, the catabolism of the branch chain amino acids (BCAA), the only amino acids that may be oxidized in skeletal muscle, can also

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).

5.3.2Muscular Activity (i.e. Exercise) as a Regulator of Muscle Proteostasis

Repeated muscle use, in the form of manual labor, and lack of muscle use, in response to cessation of work or convalescence, have long been known to a ect muscle size; increasing and decreasing it respectively.29 We now appreciate that some form of muscular activity, even weight bearing per se, is essential for the maintenance of muscle mass. Indeed, it has become apparent that aside from nutrients the most potent regulators of proteostasis in postnatal skeletal muscle are Newton’s gravity and active muscle contraction (NB: in modern Western society this is largely only exercise). As examples, significant loss of soleus muscle cross-sectional area (CSA) of about 15–26%, depending on fiber type, is evident following removal of Newton’s force in the form of 17 days in Low Earth Orbit onboard a NASA Space Shuttle. Conversely, a 5% increase in muscle CSA is evident in leg muscles after fewer than ten individual bouts of heavy resistance exercise. These observations detail the importance of ambulatory activity in the maintenance of muscle mass and also demonstrate chronic high-force contractions stimulate muscle growth. Although muscle mass is ultimately regulated by the balance of protein synthesis and degradation, the prevailing view is that changes in protein synthesis are most critical to development of atrophy and hypertrophy since,whenexposedtospaceflightorexercise, the magnitudeofchangesinsynthesis are much larger than those of degradation. Consequently, the consensus is that changes in protein synthesis are facilitative, while changes in breakdown are adaptive.

Resistance exercise, in which each e ort is performed against a specific opposing force, is singly the most potent hypertrophic stimulus in adult human muscle. This is evidenced by the gross musculature achieved in bodybuilders engaged in routinely lifting heavy weights. The mechanism by which resistance training promotes muscle hypertrophy is chiefly through inducing transient increases in muscle protein synthesis after each exercise bout. The amplitude and duration of increases in muscle synthesis after exercise ranges B50–300% and lasts B4–48 h. Though largely unexplored, such a wide range in responsiveness is likely the function of exercise protocol (e.g. intensity/duration), nutritional status (e.g. post-absorptive/fed), subject characteristics (e.g. age, sex), training status and measurement duration. The crucial role for the stimulation of muscle synthesis by exercise in regulating adaptation is demonstrated by data that show that the amplitude of post-exercise increases in muscle synthesis are qualitatively predictive of long-term adaptation (i.e. muscle hypertrophy). After resistance exercise, increases in muscle protein breakdown are also observed and these can exceed the magnitude of increases in synthesis. Consequently, resistance exercise in post-absorptive conditions creates a net catabolic state despite increases in synthesis. Importantly this catabolic state is prevented when AA are ingested in close proximity to exercise. This incapacity to stimulate muscle growth without exogenous AA makes sense because one cannot achieve muscle growth without su cient building materials. This also highlights the important interaction between exogenous AA availability and muscular activity in the regulation of muscle proteostasis.

Interestingly, it seems that muscle protein synthesis is increased only in the ‘‘remodeling period’’ after resistance exercise (as described above) but is actually suppressed during muscular activity. It is likely this suppression is a direct result of cellular energy stress (i.e. ATP turnover) because the degree of suppression relates directly to the contraction duty-cycle. Consequently, the adaptive cellular response is to switch o ATP consuming processes such as mRNA translation (which requires 4 ATP per peptide bond) and synchronously switch on catabolic pathways (ATP creating pathways). This highlights the close links between cellular energy status and muscle proteostasis.

5.3.2.1Extracellular–Intracellular Signaling and Muscular Activity

The molecular mechanism(s) by which contractile activity modulates proteostasis is poorly defined.30 Most of the various intramuscular signaling molecules have been reported to be activated in response to exercise in some form or other. However, most recent work supports the notion that ostensibly anabolic hormones (i.e. testosterone, growth hormone and insulin-like growth factor 1) do not play a significant role, at least in alterations in short-term anabolic signaling and muscle proteostasis after muscular activity. There are several examples of this. Firstly, when the acute responses of muscle protein synthesis to resistance exercise are compared in arm muscles under conditions that either do generate increases in systemic hormones (i.e. previous intense exercise of large muscle groups) or do not (i.e. no prior exercise) the synthetic responses are identical. This is in spite of gross di erences in systemic concentrations of anabolic hormones such as testosterone and growth hormone. Furthermore, muscle hypertrophy after chronic exercise training under these conditions is identical and thus adaptation is independent of the systemic environment. Secondly, when both legs of the same person are trained, one using a resistance training protocol and one using an endurance protocol, both adapt distinctly. That is, the resistance-exercised leg hypertrophies while the endurance trained leg does not, but instead becomes fatigue resistant. Thus the molecular mechanism involved appears to be largely within a given muscle rather than from the systemic milieu (i.e. hormones). It also appears that known local factors (autocrine/paracrine) may not play a significant role in the acute responses in muscle proteostasis to contractile activity. For example, while changes in mTORc1 signaling (associated with a variety of peptide growth factors, such as IGFs; see Section 5.2.2.1) are observed in response to contraction, these changes are extremely fast, detectable within seconds, and are PI3K-independent (i.e. not going through receptor -4PI3K -4Akt -4mTORc1 pathways). This observation almost certainly excludes short-term receptormediated IGF-1/MGF inputs. Perhaps even more strikingly, a functional IGF-1 receptor is not necessary for load-induced skeletal muscle hypertrophy. Further evidence excludes other local factors as having a role because the medium in which stretched muscle is bathed (i.e. conditioned medium) does not induce the same anabolic signaling changes as stretch, for example