Leucine as a treatment for muscle wasting: a critical review.

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YCLNU2431_proof ■ 10 October 2014 ■ 1/9

Clinical Nutrition xxx (2014) 1e9

Contents lists available at ScienceDirect

Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu

Review

Leucine as a treatment for muscle wasting: A critical review Q4

Koopman* Daniel J. Ham, Marissa K. Caldow, Gordon S. Lynch, Rene Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Victoria 3010, Australia

a r t i c l e i n f o

s u m m a r y

Article history: Received 16 May 2014 Accepted 22 September 2014

Amino acids are potent modulators of protein turnover and skeletal muscle cells are highly sensitive to changes in amino acid availability. During amino acid abundance increased activity of mTORC1 drives protein synthesis and growth. In skeletal muscle, it has been clearly demonstrated that of all the amino acids, leucine is the most potent stimulator of mTORC1 and protein synthesis in vitro and in vivo. As such, leucine has received considerable attention as a potential pharmaconutrient for the treatment of numerous muscle wasting conditions. However, despite a multitude of studies showing enhanced acute protein synthesis with leucine or leucine-rich supplements in healthy individuals, additional leucine intake does not necessarily enhance protein synthesis during muscle wasting conditions. In addition, long-term, placebo controlled, iso-caloric studies in humans consistently show no beneficial effect of leucine supplementation on skeletal muscle mass or function. This review, critically evaluates the therapeutic potential of leucine to attenuate the skeletal muscle wasting associated with ageing, cancer and immobilization/bed rest. It also highlights the impact of inflammation on amino acid sensing, mTOR activation and stimulation of protein synthesis and challenges the underlying hypothesis that the acute activation of mTOR and stimulation of protein synthesis by leucine increases in muscle mass over time. We conclude that leucine, as a standalone nutritional intervention, is not effective in the prevention of muscle wasting. Future work should focus on identifying and utilizing other nutrients or treatments that sensitize skeletal muscle to leucine, thereby enhancing its therapeutic potential for muscle wasting conditions. © 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Keywords: Protein synthesis Protein breakdown mTOR Amino acids

1. Introduction Skeletal muscle mass is determined by the balance between protein synthesis and protein degradation. Skeletal muscle mass is increased (muscle grows or hypertrophies) when a chronic alteration in this balance favors protein synthesis. Conversely, skeletal muscle wasting (loss of muscle or atrophy), occurs when protein degradation exceeds protein synthesis [1]. Muscle wasting is a serious complication of a wide range of diseases and conditions such as ageing, disuse, muscular dystrophy, chronic heart failure, sepsis, and many cancers [2], which impairs the quality of life of those affected. The mechanistic target of rapamycin (mTOR) plays a central role in regulating the balance between protein synthesis and protein breakdown and is therefore important for controlling skeletal muscle hypertrophy and atrophy. mTOR is a master regulator of cell size; integrating signals from nutrients (e.g. amino acids), growth factors (e.g. insulin & IGF-I), energy status (ATP) and stress to drive * Corresponding author. Tel.: þ61 3 8344 0243; fax: þ61 3 8344 5818. E-mail address: [email protected] (R. Koopman).

cell growth or activate energy sparing processes (for a more detailed review see [3,4]). Nutrients are the dominant input, since amino acids are both necessary and sufficient for mTOR activation. mTOR exists as two protein complexes, mTOR complex 1 (mTORC1) containing raptor and mTORC2 containing rictor. mTORC1 is rapamycin-sensitive, activated by amino acids and considered the master regulator of protein synthesis. Upon activation, mTORC1 phosphorylates and activates two parallel signaling pathways involved in the control of translation. S6 kinase 1 (S6K1) phosphorylation leads to activation of the ribosomal protein S6, while phosphorylation of the eukaryotic initiation factor 4E (eIF4E)binding protein (4EBP1) releases its inhibition of the translation initiation factor eIF-4E, allowing initiation of translation and the synthesis of new proteins (Fig. 1). Growth factors stimulate mTORC1 by a well-defined mechanism involving the phosphorylation of Akt which leads to the phosphorylation and subsequent inhibition of both tuberous sclerosis complex protein 2 (TSC2) and the proline-rich Akt substrate 40 kDa (PRAS40), allowing the small GTPase Ras homolog enriched in brain (Rheb) to bind to mTORC1 and promote its kinase activity. However, growth factors cannot efficiently activate mTORC1 without

http://dx.doi.org/10.1016/j.clnu.2014.09.016 0261-5614/© 2014 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Please cite this article in press as: Ham DJ, et al., Leucine as a treatment for muscle wasting: A critical review, Clinical Nutrition (2014), http:// dx.doi.org/10.1016/j.clnu.2014.09.016

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D.J. Ham et al. / Clinical Nutrition xxx (2014) 1e9

Fig. 1. Leucine modulates protein synthesis and protein breakdown through two distinct mechanisms, converging on the PI3K-Akt-mTOR pathway. Leucine-stimulated insulin release activates Akt through the insulin receptor and IRS. Leucine also stimulates mTORC1 through an insulin independent mechanism that may involve the Ras-related GTPase (Rag), Vps34 or MAP4K3. mTORC1 activation stimulates protein synthesis through the phosphorylation of S6K1 and 4EBP1. Inflammation and oxidative stress, common during muscle wasting, attenuate leucine-stimulated mTORC1 activation, while chronic mTORC1 activation leads to impaired insulin signaling through a negative feedback loop involving S6K1 and IRS.

the presence of amino acids [3]. Amino acids activate mTORC1 through a growth factor-independent mechanism that may involve MAP4K, the type III PI3-Kinase Vps34 or the conformation of RAG GTPases in mTORC1 [4]. As such, modulating protein metabolism through amino acid supplementation has received considerable attention as a potential treatment for muscle wasting conditions. 2. Leucine as a modulator of muscle protein metabolism 2.1. Protein synthesis In a landmark study using rat diaphragm muscle, Buse and Reid [5] discovered that the essential branched chain amino acid leucine played a unique role in the modulation of skeletal muscle metabolism. Unlike other amino acids tested, increasing extracellular leucine concentration from 0.1 to 0.5 mM rapidly stimulated protein synthesis and reduced protein breakdown. Importantly, these concentrations are broadly consistent with plasma leucine concentrations in the basal state (~0.1 mM) and after the administration of a leucine rich supplement (~0.4e0.5 mM) [6,7]. In humans, plasma, but not intramuscular amino acid concentrations are correlated strongly with changes in muscle protein synthesis [8]. Furthermore, a similar increase in muscle protein synthesis was observed when food-deprived rats were infused with either mixed amino acids or the equivalent amount of BCAA (9 mg leucine,

7.5 mg isoleucine and 7.3 mg valine) over a 1 h period [9]. Anthony et al. [10] found that of the three BCAAs, leucine was unique in its ability to stimulate protein synthesis when a dose of 1.35 g kg1 body weight was administered to food-deprived rats. Numerous subsequent studies in rodents have demonstrated that the administration of leucine, either alone [11], as part of a mixed meal [12], or contained within a leucine-rich protein (i.e. whey) [13] effectively stimulates muscle protein synthesis and this effect persists over prolonged treatment periods [13,14]. Although studies in humans are less prolific, a number of groups have shown that ingestion of a leucine-rich meal effectively stimulates postprandial muscle protein synthesis in humans [7,15,16]. Furthermore, in healthy young men, Wilkinson et al. [17] observed a 110% increase in skeletal muscle protein synthesis after a small (3.42 g) oral dose of free leucine. Interestingly, the authors reported a similar response in muscle protein metabolism to 2.42 g of the leucine metabolite ß-hydroxy-b-methylbutyrate (HMB). While the contribution of HMB to the modulatory effects of leucine on muscle protein metabolism are beyond the scope of this review, the research certainly warrants further attention. Therefore, mechanisms involved in the regulatory effect of leucine on protein metabolism may include: 1) increasing substrate availability; 2) increasing the secretion of anabolic hormones such as insulin; 3) directly modulating anabolic signaling pathways in skeletal muscle and; 4) potential secondary effects of metabolites such as HMB.


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When consumed or infused in very large doses, amino acids particularly leucine, stimulate insulin secretion from the pancreas by allosterically activating glutamate dehydrogenase in the b-cell and via oxidative decarboxylation [6,15,18]. Small increases in insulin have also been reported following the ingestion of a single bolus of just 3.42 g free leucine [17]. However, insulin is unlikely to be the main stimulator of protein synthesis in skeletal muscle as leucine also stimulates protein synthesis in vitro when insulin levels are constant [5,19]. In addition, the observations that essential amino acids (EAA) show a dose-dependent stimulation of muscle protein synthesis in the absence of increased plasma insulin [20], and that carbohydrate ingestion does not significantly increase protein synthesis [21], suggest that insulin is permissive rather than modulatory [22]. Indeed, Greenhaff et al. [22] showed that increasing insulin in the range of ~30e150 mU/ml does not further stimulate muscle protein synthesis in healthy young individuals. 2.2. Protein breakdown The impact of leucine on skeletal muscle anabolism is not solely reliant on the stimulation of protein synthesis, but rather a combination of increased protein synthesis and/or decreased protein breakdown. In healthy men a leucine dose of ~0.14 g kg1 body weight infused over a 7-h period reduces muscle protein breakdown by 35e40% without increasing protein synthesis [23]. In contrast to protein synthesis, muscle protein degradation seems to be very responsive to relatively small changes in insulin concentrations. Insulin levels of 15 mU/ml can almost maximally reduce muscle protein breakdown [24] and there seems to be no further inhibition above 30 mU/ml [22]. These data suggest that only a slight increase in insulin concentration is required to maximally reduce protein breakdown which is achieved with the intake of a small meal in healthy young men [25]. As such, the insulin response required to facilitate the regulation of protein metabolism by leucine is likely to be achieved with the nutrient content of a normal meal. It has been speculated that the relationship between plasma insulin levels and protein turnover is altered during conditions of mild insulin resistance [18]. Indeed, a reduced capacity of insulin to stimulate postprandial muscle perfusion in elderly humans has been associated with impaired amino acid delivery to the muscle and attenuated anabolic signaling [26]. Thus, the notion that leucine can improve protein metabolism during certain conditions by increasing circulating insulin and thereby overcoming insulin resistance has received considerable attention. The mechanisms involved in leucine and BCAA-induced insulin release appear to remain unimpeded in glucose intolerant patients and as such a number of studies have observed positive effects of leucine and BCAA supplementation on glycemic control and glucose metabolism in patients with type 2 diabetes [27]. However, studies have linked the development of insulin resistance to increased BCAA catabolism in obese humans and chronic BCAA supplementation in rats fed a high fat diet [28]. This group showed that a BCAA-induced chronic activation of mTORC1 led to impaired insulin signaling through a negative feedback loop involving the inhibition of IRS1 by S6K1 [28]. As such, despite potential short term benefits, long term supplementation with high doses of leucine may negatively impact insulin signaling in some populations. 2.3. Dose and administration Many regimes of leucine administration involving different doses and co-nutrients have been used to investigate the effect of leucine on acute changes in protein metabolism. In a

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doseeresponse study in rats, a leucine dose of 0.14 g kg1 is sufficient to maximally stimulate protein synthesis [11]. Others support that a similar leucine dose (~0.12 g kg1 lean body mass) is sufficient to induce mTORC1 and S6K1 activation and optimally increase muscle protein synthesis in humans [29]. In healthy young men, smaller oral doses of leucine (3.4e5 g) have also been shown to effectively stimulate myofibrillar protein synthesis when consumed as part of a low-protein mixed macronutrient supplement [16] or as a single bolus of free leucine [17]. Different leucine administration routes and regimes may also impact whether protein balance is modulated by a stimulation of protein synthesis or a reduction in protein breakdown. For example, in healthy men a leucine dose of 0.14 g kg1 body weight infused over a 7-h period reduces muscle protein breakdown by 35e40% without increasing protein synthesis [23]. On the other hand, administration of a lower amount of leucine (0.05 g kg1 body weight) delivered as a flooding dose potently stimulates muscle protein synthesis in humans [30]. The leucine dose required to stimulate protein synthesis can also differ depending on the physiological properties of the muscle. For example, the protein synthetic response to low doses of leucine is reduced in muscles from aged compared with young rats whereas higher doses stimulate protein synthesis to a similar degree in muscles from young and aged rats [31]. Due to the wide variation in administration regimes, routes, doses, nutrient composition and subject pools used to study the potential therapeutic benefits of leucine on skeletal muscle mass, it is prudent to demonstrate the acute effect of a chosen leucine or leucine-rich treatment on muscle protein synthesis in the subject pool to be tested. Like other essential nutrients, leucine is required to sustain life and a diet completely devoid of leucine leads to rapid weight loss and ultimately death in rodents [32]. Similarly, leucine deprivation increases protein breakdown in cultured skeletal muscle cells by inducing autophagy and lysosomal-dependent proteolysis [33]. As such, the importance of adequate leucine intake should not be underestimated and is considered a basic requirement of life. However, taken together, these data clearly demonstrate that supplemental leucine, beyond this basic requirement for life, acutely modulates protein metabolism by increasing substrate availability, increasing the secretion of anabolic hormones such as insulin, and directly modulating anabolic signaling pathways in skeletal muscles of healthy humans. This well demonstrated capacity of leucine to directly activate mTORC1, stimulate protein synthesis, reduce protein breakdown and thus acutely improve protein balance, has led many to the logical conclusion that over time, the summation of these acute increases in protein balance would lead to muscle mass accretion, augment training adaptations and counteract skeletal muscle wasting. However, despite the strong data to support an acute modulation of protein metabolism by leucine, there is little evidence to support a beneficial effect of leucine as a treatment for muscle wasting conditions. It is important therefore to critically evaluate the therapeutic potential of leucine to preserve muscle mass and physical function during immobilization/bed rest, ageing and cancer cachexia. 3. Leucine: a treatment for skeletal muscle wasting? 3.1. Immobilization and bed rest During immobilization and bed rest muscle wasting results from both increased protein breakdown, which is upregulated within the first 24 h, and decreased protein synthesis [34]. While reduced basal protein synthesis may or may not contribute to the loss of muscle mass during the early stages of inactivity, after the first ~10 d of immobilization nearly all of the muscle loss is thought to


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Protein, free valine 4 thigh muscle size and strength. and isoleucine 4 single fiber size and power. Control: ~1 g kg1 day1 Treatment: ~1.6 g kg1 day1 60 d bed Yes YES (CHO reduced rest (added to a meal) in treatment group) 24 healthy women 0.45 g kg1.day1 protein þ free >3.6 g (8 per group) leucine (3.6 g), valine (1.8 g) and isoleucine (1.8 g). Trappe et al., (38, 39)

90 g sucrose, EAA and 2.1 g glycine 9.3 g 13 healthy males Paddon-Jones et al., (43) & Fitts et al., (21)

3 16.5 g EAA and 30 g sucrose dissolved in a diet soft drink

28 d bed Yes rest (diet soft drink)

Control: ~0.7 g kg No Control: ~2500 kcal; Treatment: treatment: ~3000 kcal ~1.3 g kg1.day1

1

Leucine Duration Placebo Author

Subjects

Treatment

Isocaloric

Daily protein intake

.day

1

Other nutrients

[lean leg mass, [ leg strength, [ power in type II single fibers of the vastus lateralis.


Result

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Table 1 Leucine and leucine rich supplementation during prolonged bed rest.

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result from a blunted anabolic response to food intake [35e37]. Since leucine and leucine-rich amino acids are potent stimulators of muscle protein synthesis, numerous investigators have examined whether administration of leucine-rich amino acid mixtures can overcome this anabolic resistance and reduce inactivityinduced muscle wasting. A very high dose of free leucine (2.7 g kg1 day1) delivered orally to rats once per day, attenuated the loss of soleus muscle mass, fiber cross-sectional area and strength after 7 days of unilateral hindlimb immobilization [38]. Despite the high leucine dose, rates of protein synthesis were not restored to normal. Rather, the attenuation of muscle wasting was attributed to an inhibition of the early upregulation of genes involved in muscle breakdown, specifically the two key ubiquitin E3 ligase genes atrogin-1 and MuRF1 [38]. However, it is unclear whether the beneficial effects of leucine treatment observed in the rat soleus muscle, a small postural muscle that contributes very little to the overall muscle mass, over a short immobilization period would translate into meaningful changes in the mass of larger muscles with prolonged immobilization. Furthermore, it is important to recognize the inherent differences between rodents and humans in muscle protein metabolism and the relative contributions that changes in rates of protein synthesis and protein breakdown may make to the development of disuse-induced atrophy [39]. Aside from the wellrecognized elevations in protein turnover in rodents, a disproportionately large amount of a rodent's life is spent growing and as a result the majority of intervention studies are performed during the growth phase. In the context of leucine supplementation this is important since muscle protein synthesis in both rodents and humans is insulin sensitive during growth, but lost during adulthood in humans [22,40]. An early study in humans reported promising improvements in muscle mass and strength after 28 days of bed rest in healthy male subjects using the combination of 16.5 g of EAA and 30 g sucrose taken three times a day between meals (total leucine dose of 9.3 g per day) [41,42]. However, while the treatment group received a combined daily energy intake of ~3000 kcal and maintained body weight across the 28 d treatment period, subjects in the control group had a daily energy intake of only ~2500 kcal and lost 2.4 kg (2.8%) body weight [41,42]. A summary of long-term immobilization and bed rest studies in humans is provided in Table 1. The implications of this limitation were highlighted by Biolo et al. [35], who demonstrated that healthy young volunteers consuming 80% of their total energy requirements during 14 d of bed rest lost significantly more lean mass than those consuming 100% of their energy requirements. It is important to note, that inactivity is often associated with reduced energy intake in bedridden individuals and in the elderly [35] and it therefore plays an important role in the extent of muscle wasting. In a follow-up study, Biolo et al. [43] showed that consuming 120% of energy requirements also increased the loss of muscle mass. As such, studies where daily energy intake differs between the treatment and control groups make it difficult to attribute differences in study outcomes to the specific treatment. In a study of female subjects undergoing 60 days of bed rest, Trappe et al. [44], investigated the potential protective effects of exercise and a leucine-enriched high-protein diet on the size and functional capacity of single fiber segments taken from needle biopsies of the vastus lateralis muscle. The control and exercise groups maintained a diet containing ~1.0 g kg1 day1 protein, while the nutritional intervention group received ~1.6 g kg1 day1 including 3.6 g day1 of free leucine, 1.8 g day1 of free valine and 1.8 g day1 of free isoleucine spread over the three meals of the day. The exercise intervention effectively prevented the ~15e20% reduction in fiber diameter and 20e40% reduction in force and


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power in both MHC type I and type IIa muscle fibers. In contrast, the leucine-enriched high protein diet did not provide similar protection against the reductions in muscle fiber size and function. In a separate report on the same study, similar results were presented for whole muscle size and function, with the leucine-rich high protein diet again providing no protection from the deleterious effects of bed rest [45]. Given the synergistic effects of exercise training and increased protein intake, it is disappointing that an ‘exercise þ leucine’ group was not included in this study. However, in a 28 d bed rest study, Brooks et al. [46], observed no additional effect of a 15 g EAA þ 30 g sucrose supplement taken once per day when combined with exercise compared to exercise alone [46]. In summary, rodent studies suggest that leucine may provide some protection against muscle wasting during the early stages of immobilization and bed rest by reducing the expression of genes regulating muscle breakdown. Despite the differences in insulin sensitivity between growing rodents and the adult human, it is unclear how applicable these findings are to humans. Indeed, evidence from long-term, iso-caloric trials in humans does not support the notion that increasing EAA intake can counteract muscle wasting. The mechanisms responsible for the failure of leucine and leucine-rich treatments to overcome anabolic resistance and reduce muscle wasting are currently unclear, but may involve local inflammation, which can directly interfere with anabolic signaling [47]. 3.2. Ageing Normal healthy ageing is associated with a progressive loss of muscle mass. An impaired protein synthetic response to anabolic stimuli such as nutrition and exercise is believed to contribute, at least in part, to this progressive loss of muscle mass. Cuthbertson et al. [20], observed blunted phosphorylation of mTOR and muscle protein synthesis in response to a bolus ingestion of EAAs in elderly (70 y) compared to young (28 y) men, with no differences in rates of basal protein synthesis. This ‘anabolic resistance’ only became evident with EAA doses of 10e20 g, while sensitivity to smaller doses of EAA (2.5e5 g) was preserved. However, since these initial observations, some evidence of anabolic resistance has been observed following small doses (7 g) of EAA [48] but in elderly subjects administered liberal doses of high quality protein (20e35 g, equivalent to ~10e15 g EAA), anabolic resistance is not typically observed [49]. In any case, muscles from the elderly can still increase protein synthesis in response to nutrition, and increasing the potency of this stimulus through leucine rich diets has received considerable attention. Rieu et al. [13], observed an increase in postprandial muscle protein synthesis in elderly rats fed leucine rich meals. Importantly, the increased anabolic response to this leucine rich meal was maintained after a 30 d treatment period. However, despite the

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increase in protein synthesis, skeletal muscle mass was not improved in rats receiving a leucine rich diet. Additionally, a relatively high dose of leucine (4% of total diet) administered to ageing rats over a 40 week treatment period from the age of 6 months provided no protection against the loss of lean mass [50]. Early studies of prolonged leucine-rich supplementation in elderly humans showed promising results [51,52]. Borsheim et al. [51], observed a significant increase in lean body mass (1.3%) and strength (22%) in elderly subjects (>65 y) supplemented with 11 g of essential amino acids and 1.1 g of arginine twice per day (total of 5.6 g leucine). However, since this study did not include a control group, i.e. all subjects received the amino acid supplement, it is difficult to ascribe any of the reported effects specifically to the treatment. A summary of long-term ageing studies in humans is provided in Table 2. In a similar study, healthy elderly women (68 y) were supplemented with 7.5 g of EAAs twice per day (4.0 g leucine) for 12 weeks [52]. Dillon et al. [52] reported a very small, but significant increase in lean mass (3.9% or 1.7 kg of lean tissue assessed using DEXA) over this period but no change in muscle function. Since protein intake was not reported in this study, the contribution of a potential deficiency in protein intake in these subjects cannot be excluded. In contrast, a well-controlled prolonged leucine supplementation study in healthy elderly humans did not show any improvement in muscle mass or strength compared to iso-energetic controls when 2.5 g of free leucine was included into breakfast, lunch and dinner (7.5 g day1) for 3 months [53]. Based on the authors' estimation of habitual leucine intake, this dose represents an increase from ~2.1 g per meal to ~4.6 g per meal or ~0.038 g kg1 to ~0.084 g kg1 lean mass per meal. As the authors did not measure the capacity of this dose to augment rates of protein synthesis in their subjects, it could be speculated that an inadequate dose of leucine was provided. However, recent data suggest that increasing the leucine content of a low-protein mixed macronutrient supplement from 0.75 g to 5 g improves the post exercise protein synthetic response in healthy young humans [16]. Similarly, a single bolus of 3.42 g free leucine was sufficient to double the myofibrillar protein synthetic rate in healthy young males [17]. Furthermore, increasing the leucine content of a 6.7 g EAA supplement from 1.7 g to 2.7 g was sufficient to stimulate protein synthesis in elderly subjects [54]. Although not conclusive, these data suggest that the leucine supplementation regime employed by Verhoeven et al. [53] would have likely improved the acute protein synthetic response in their subjects. Following criticisms about the use of healthy subjects and the timing of the intervention, the same group evaluated the effect of leucine treatment, using the same protocol administered to patients with type 2 diabetes over a period of 6 months. Similarly, they found no improvement in muscle mass or strength with leucine treatment [55]. Together, these results do not support a role

Table 2 Leucine and leucine rich supplementation studies in elderly humans. Author

Subjects

Treatment

Leucine Duration

Placebo

Isocaloric Daily protein intake

Other Result nutrients

Bohrsheim 12 (7 female, 5 male) et al., (43) elderly, glucose intolerant subjects (67 ± 6 yr)

11 g EAA þ Arg 2 per day between meals

7.9 g

4 months No control group

N/A

~1 g kg1 day1 2.2 g day [LBM (1.3%), [ leg strength arginine (þ22%), [physical function

Dillon 14 elderly women et al., (18) (68 ± 2 yr)

2 7.5 g EAA

2.78 g

3 months YES (lactose)

YES

Not measured

Verhoeven 30 healthy elderly men et al., (45) (71 ± 4 yr)

3 2.5 g leucine 7.5 g

3 months YES YES (wheat flour)

~1 g kg1 day1 No

4LBM, 4 Lean leg mass

Leenders 60 elderly males (71 ± 1 yr) 3 2.5 g leucine 7.5 g et al., (46) with type 2 diabetes

6 months YES YES (wheat flour)

~1 g kg1 day1 No

4LBM, 4 Lean leg mass, 4 leg strength

No

[LBM (3.9%), 4 leg strength


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for leucine in the treatment of age-related loss of muscle mass and function. 3.3. Cancer cachexia Cancer cachexia is the wasting of muscle and fat associated with tumor bearing. It is a multifactorial muscle wasting syndrome with a complex interplay between the tumor and host inflammatory response [56]. Protein synthesis is reduced in the muscles of both cachectic human patients [57] and cachectic mice [58]. Activation of proteins within the PI3K-AKT-mTOR pathway are reduced at various levels during cachexia depending on the type of tumor, the specific factors released by the tumor, the presence of inflammation and the extent of cachexia. Reduced activation of 4EBP1 and S6K1 contribute to reduced protein synthesis during the early stages of cachexia, while reductions in Akt and mTOR contribute to a further reduction in protein synthesis during the later stages of cachexia [58]. The mechanisms responsible for the loss of muscle mass during cancer cachexia are reviewed in detail elsewhere [59], but anorexia, muscle inflammation and factors secreted by the tumor, such as proteolysis inducing factor (PIF) and angiotensin II (Ang II), may all contribute to the alterations in muscle protein metabolism during cancer cachexia. While there have not been long term intervention studies with leucine in human cancer patients, leucine supplementation in tumor bearing mice have provided a small, but significant protection of skeletal muscle mass [60,61]. In a C26 mouse model of cancer cachexia, Peters et al. [60], observed a ~9% increase in tibialis anterior and gastrocnemius muscle mass with a relatively high leucine dose of 1.3 g kg1 day1. To put this in perspective, the preservation of muscle mass is equivalent to a reduction in the tumor-induced loss of muscle mass of ~25%. Likewise, in a MAC16 mouse model of cancer cachexia, Eley et al. [61], observed increased soleus muscle mass over a 4e5 day treatment period after the onset of cachexia. Again, it is somewhat surprising that the mass of muscles other than the small, postural, soleus muscle were not reported in this study. Furthermore, the impact of a reduced tumor burden cannot be ruled out since tumor volume was significantly lower in leucine treated animals [61]. Surprisingly, protein synthesis was increased and protein breakdown reduced with leucine treatment, while valine treatment also increased protein synthesis, but did not reduce protein breakdown or soleus muscle mass. The increase in protein synthesis with valine in this study is surprising and generally not observed in skeletal muscle [10,19]. Interestingly, in a separate study, 0.25 g kg1 of the leucine metabolite HMB was 60% more effective than 1 g kg1 leucine in preventing the loss of body mass over a 4 day period in MAC16 tumor bearing mice [62]. Taken together, these findings are consistent with those showing that high doses of leucine attenuate the loss of muscle mass during the early stages of immobilization-induced wasting through inhibition of protein breakdown rather than stimulation of protein synthesis. Again, it is important to remember interspecies differences in protein metabolism and the increased sensitivity of rodent skeletal muscle to insulin. As such, if the observed improvements in muscle mass are the result of insulin action then the benefits of leucine supplementation in tumor bearing rodents is unlikely to translate to adult humans. An additional consideration in cancer cachexia is what effect the treatment will have on the tumor. It is of course possible that an anabolic treatment may promote tumor growth. Currently there is a lack of evidence to support or refute a specific effect of leucine supplementation in the growth of tumors. In C26 tumor-bearing mice, leucine supplementation did not affect tumor mass [60], while in an MAC16 mouse model, tumor volume was decreased by both leucine and valine supplementation [61]. However, since

mTORC1 plays a central role in the regulation of tumor growth and leucine is a potent regulator of this pathway, strategies aimed at depriving melanoma cells of leucine can restrict growth of the tumor in vivo and cause cells to die in vitro [63]. Other amino acids have also been shown to alter the growth of tumors in mouse models. We have recently shown that the non-essential amino acid glycine reduced tumor mass by ~30%, while supplementation with the arginine precursor citrulline increased tumor mass by ~40% in C26 tumor bearing mice [56]. As such, careful monitoring of tumor size would be prudent during amino acid treatment in humans. The limited data from studies in animal models of cancer cachexia suggests that high doses of a leucine rich supplement can stimulate protein synthesis, reduce protein breakdown, and may provide a small degree of protection from muscle wasting. These small improvements in muscle mass are more likely to result from a decrease in protein breakdown rather than an increase in protein synthesis. However, given the limited impact of even high doses of leucine on muscle mass and the role of mTORC1 signaling and leucine availability in tumor growth, it may be more appropriate to focus on other more effective nutritional treatments to reduce muscle wasting in cancer. In summary, the evidence supporting a central role for leucine in the acute stimulation of protein synthesis in rodent and human studies is overwhelming. However, aside from studies where the effects of caloric intake and insufficient dietary amino acid intake cannot be ruled out, leucine supplementation as a treatment for muscle wasting has yielded disappointing results. In fact, there are currently no well-designed, prolonged, iso-caloric trials in humans consuming sufficient dietary protein, that show a beneficial effect of leucine supplementation on muscle mass during inactivity and age-related muscle wasting. Theoretically, the cumulative effect of repeated acute increases in protein synthesis should, with sufficient time, result in meaningful changes in skeletal muscle mass. The fact that this does not occur is perplexing. Given the complexities and assumptions associated with quantitative measures of muscle protein metabolism utilizing isotopically labeled amino acids, it is possible that methodological issues contribute to the discrepancy between acute and chronic responses. If we assume that these measures are broadly accurate, the lack of translation questions the value of acute measures of protein synthesis for evaluating the potential effectiveness of a treatment. Indeed, Mitchell et al. [64], observed no correlation between acute post-exercise measures of myofibrillar protein synthesis and the magnitude of resistance training-induced hypertrophy. Similarly, hypertrophy was not correlated with acute increases in the phosphorylation status of Akt, mTOR or S6 [64]. Currently, the evidence supporting or refuting a specific role for the modulation of protein metabolism by leucine for cancer cachexia, beyond addressing the reduction in BCAA availability, is insufficient. However, other concerns regarding the effects of leucine and mTORC1 on tumor growth, and the mild increases in mass observed in animal models may prevent further development in this area. 4. Lost in translation or a faulty premise? 4.1. Anabolic resistance While it has been demonstrated clearly in healthy humans that increasing plasma and muscle leucine concentration potently stimulates protein synthesis, this is not necessarily the case during catabolic conditions. The reduced protein synthetic response to food intake has been termed ‘anabolic resistance’ [25]. Anabolic resistance to amino acids has been observed in many muscle wasting conditions, including immobilization [37], ageing [20],


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cancer cachexia [57,65] and sepsis [47]. Although the exact mechanisms responsible for anabolic resistance are unclear the overproduction of pro-inflammatory cytokines and ROS production associated with many muscle wasting conditions is believed to play a central role [20,56,66]. Inflammation and ROS inhibit mTORC1 activity and decrease the phosphorylation of 4EBP1 and S6K1 [47]. An ~80% reduction in leucine-induced phosphorylation of mTOR and its substrates 4EBP1 and S6K1 and an inhibition of leucinestimulated protein synthesis have all been observed in skeletal muscle cells during inflammation [67]. Interestingly, there seems to be a specific inhibition of amino acid sensing by mTORC1, since leucine fails to stimulate mTORC1 in a caecal ligation and puncture model of sepsis in rats, while the potent stimulatory effect of the growth factor insulin-like growth factor I (IGF-I) remains largely unaffected [52]. Additionally, increases in the concentration of free leucine both in muscle and plasma have been reported during catabolic conditions such as fasting, uncontrolled diabetes, limb immobilization and cancer cachexia [5]. In contrast to normal healthy muscle, this rise in leucine concentration during catabolic states fails to stimulate protein synthesis. After 14 days of unilateral knee immobilization in young healthy subjects, Glover et al. [37], reported a ~40% increase in basal muscle intracellular leucine content in the immobilized leg, and a similar increase in amino acid concentration following amino acid ingestion, but a 27% reduction in post-absorptive muscle protein synthesis. These data strongly support a reduced anabolic sensitivity of skeletal muscle to amino acids during muscle wasting conditions. Ultimately, the inability of leucine to stimulate muscle protein synthesis during inflammation could also be the result of any number of alterations to mTOR, its binding partners or the proteins responsible for its cellular localization that prevents the interaction of mTOR and Rheb [47]. Anabolic resistance to amino acids is considered a major contributor to muscle wasting, and strategies to address anabolic resistance are a focus for many researchers [20,25,34,37]. 4.2. Counteracting anabolic resistance There are two major strategies to try and overcome anabolic resistance. The first involves providing a greater anabolic stimulus, which forms the major rationale for increasing dietary leucine intake during muscle wasting conditions. The second involves attenuation of the factors causing the reduction in normal anabolic signaling; for example using anti-inflammatory approaches and antioxidants [34]. While both make rational arguments, current evidence suggests the latter approach is more effective than the former. While protein synthesis can be acutely increased in immobilized [37], cachectic [65] and aged [20] muscles by a large leucine-rich dose of amino acids, the response is often attenuated compared with that in healthy control subjects. Irrespective of the capacity of amino acids to stimulate protein synthesis during muscle wasting conditions, based on current evidence, acute stimulation of protein synthesis by leucine does not lead to meaningful changes in skeletal muscle mass over time. This fact, especially in the light of robust increases in protein synthesis and activation of mTORC1 with leucine treatment, raises a more pertinent question: can chronic activation of mTORC1 increase muscle mass? Genetic overexpression of Akt in mouse skeletal muscle has been shown repeatedly to induce marked skeletal muscle hypertrophy (for review see [68]). Similarly, activation of Akt through IGF-I and other growth factors potently stimulates skeletal muscle hypertrophy, even during inflammatory states [67]. Studies using rapamycin treatment have demonstrated that Akt-induced skeletal muscle hypertrophy is dependent on activation of mTORC1.

7

Similarly, whole body mTOR knockout is embryonically lethal, whereas muscle specific knockout of mTOR or raptor, which forms part of mTORC1, results in severe myopathy [68]. Surprisingly, few studies have attempted to genetically overexpress or activate the mTORC1 pathway more directly, with mixed results. Using electroporation, Goodman et al. [69], overexpressed Rheb in mouse muscle, which increased activation of Rheb and mTOR and muscle hypertrophy in the transfected fibers. In contrast, muscle specific knockout of TSC1 leads to sustained activation of mTOR without muscle hypertrophy, and these mice develop a late-onset myopathy related to impaired autophagy [70]. Perhaps a more relevant genetic model is the mitochondrial branched chain amino acid transferase (BCATm) null mouse. BCATm is responsible for the catabolism of BCAAs, and whole body deletion of BCATm chronically increases plasma BCAAs between 5and 10-fold compared to wild-type mice [71]. The increase in BCAA availability is associated with an increased phosphorylation of the mTORC1 substrates 4EBP1 and S6K1, and an increase in muscle protein synthesis. However, despite the increased availability of amino acids, mTORC1 activity and protein synthesis, neither muscle mass nor lean body mass is altered in BCATm-null mice compared with wild type mice. Rather, the chronic increase in BCAA availability leads to futile cycles of protein turnover [71]. These studies highlight the importance of mTORC1 and the synthesis of new proteins in the maintenance of muscle mass and promotion of skeletal muscle hypertrophy. Additionally, increasing the availability of amino acids appears to be an effective approach for stimulating mTORC1 and increasing protein synthesis. However, observations in BCATm-null mice suggest that even sustained amino acid stimulated protein synthesis is not sufficient to promote skeletal muscle accretion in isolation. It is well recognized that many factors play a role in the modulation of skeletal muscle mass and a concomitant change in the regulation of growth factors and hormones, satellite cells and neuromuscular factors may be required for the translation of acute increases in protein synthesis into chronic increases in muscle mass [18]. Furthermore, sustained activation of mTORC1 is unlikely to increase skeletal muscle mass and may impair other essential cellular functions, such as autophagy. While attempts to overcome anabolic resistance and increase muscle mass through increasing amino acid availability has been unsuccessful, the importance of a normal anabolic response to nutrition, especially amino acids, should not be underestimated. Indeed, recent work focusing on restoring normal protein metabolism in disease states, by reducing the production of proinflammatory cytokines and ROS have proved more successful. We have shown recently that the non-essential amino acid glycine reduces inflammation and ROS and attenuates the loss of skeletal muscle mass by ~50% in a C26 mouse model of cancer cachexia [56]. Likewise, the anti-inflammatory drug ibuprofen has been shown to improve the anabolic response to food intake and reduce muscle wasting in aged rats [72]. As such, strategies that focus on restoring the normal anabolic response to nutrition rather than increasing the anabolic stimulus may be more promising therapeutic targets for muscle wasting during ageing, immobilization and cancer cachexia. 5. Summary and conclusion In summary, the evidence supporting a central role for leucine in the acute stimulation of protein synthesis in rodent and human studies is overwhelming. Likewise the importance of maintaining a sufficient intake of leucine is indisputable. However, there are currently no well-designed, long-term, iso-caloric experiments in humans consuming sufficient dietary protein that show a beneficial


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8


effect of leucine supplementation on muscle mass during inactivity, cancer cachexia or sarcopenia. The lack of translation of the robust acute muscle protein synthetic response to leucine into meaningful changes in skeletal muscle mass in long-term animal and human studies leads us to conclude that acute stimulation of mTORC1 and muscle protein synthesis by leucine does not lead to changes in muscle mass. Support for this conclusion comes from BCATm-null mice which have markedly increased plasma and muscle BCAA concentrations and increased protein synthesis without an increase in muscle mass. While the regulation of muscle mass is dependent on a sufficient supply of nutrients, the extra provision of nutrients (even leucine) in isolation do not appear to be capable of further modulating muscle mass.

[17]

[18] [19]

[20]

[21]

[22]

Sources of support [23]

Supported by grants from the European Society for Clinical Nutrition (ESPEN). RK was supported by a C.R. Roper Research Fellowship from the Faculty of Medicine, Dentistry and Health Sciences of The University of Melbourne.

[24]

[25]

Disclosure summary [26]

The authors have nothing to disclose. Conflict of interest

[27]

None declared. [28]

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Impact of supplementation with amino acids or their metabolites on muscle wasting in patients with critical illness or other muscle wasting illness: a systematic review.

Qualitative Ultrasound in Acute Critical Illness Muscle Wasting.

Biofeedback as a treatment for childhood hyperactivity: a critical review of the literature.

Emerging desalination technologies for water treatment: a critical review.

Muscle as a metamaterial operating near a critical point.

Dropping out of treatment: a critical review.

Cancer-induced muscle wasting: latest findings in prevention and treatment.

Muscle strength and golf performance: a critical review.

Treatment of skeletal muscle injury: a review.

A metabolic link to skeletal muscle wasting and regeneration.

Muscle wasting: is mitochondrial dysfunction a key target?

The emerging role of skeletal muscle oxidative metabolism as a biological target and cellular regulator of cancer-induced muscle wasting.

Melatonin as a treatment for gastrointestinal cancer: a review.

Myostatin inhibitors as therapies for muscle wasting associated with cancer and other disorders.

Selective androgen receptor modulators for the prevention and treatment of muscle wasting associated with cancer.

Ultrasound for the assessment of peripheral skeletal muscle architecture in critical illness: a systematic review.

Valine, isoleucine, and leucine. A new treatment for phenylketonuria.

Thrombolysis: the need for a critical review.

Green Adsorbents for Wastewaters: A Critical Review.

Treatment of epileptic seizures in brain tumors: a critical review.

Behavioral and cognitive treatment methods: a critical comparative review.

Decellularized and Engineered Tendons as Biological Substitutes: A Critical Review.

Effects of psychological treatment on cancer patients: a critical review.

Interleukin-27 as a Novel Therapy for Inflammatory Bowel Disease: A Critical Review of the Literature.