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Citrulline and Skeletal Muscle
C H A P T E R
19 Citrulline and Skeletal Muscle Charlotte Breuillard, Arthur Goron and Christophe Moinard Laboratoire de bioenerge´tique fondamentale et applique´e, Universite´ Grenoble Alpes, Grenoble, France
Citrulline is a nonessential and nonproteinogenic amino acid that takes its name from watermelon (Citrullus vulgaris). For decades, citrulline was considered an intermediate of the urea cycle and as just one among many other amino acids. However, it began to arouse interest when Windmueller and Spaeth [1] showed the significance of intestinally produced citrulline. This particular feature was first used for diagnostic purposes, as citrulline proved to be the best marker of intestinal function [2]. Metabolic properties of this amino acid were subsequently demonstrated: first at cardiovascular level [as a precursor of nitric oxide (NO); see [3,4] for reviews] and then as a modulator of nitrogen homeostasis (see [5] for a recent review) when Osowska et al. [6] showed in a short bowel syndrome model that citrulline supplementation improves nitrogen balance. Moving forward, it was proposed that citrulline would be a regulator of muscle protein synthesis: in 2006, the same authors demonstrated, for the first time, the stimulatory effect of citrulline on muscle protein synthesis [7] in aged undernourished rats, using a model of protein energy restriction. In this model, the rats fed only 50% of spontaneous food intake for 12 weeks are then refed for 1 week with a diet corresponding to 90% of their spontaneous food intake and enriched with citrulline or a mix of nonessential amino acids (in order to make the diets isonitrogenous). Rats receiving the citrulline-enriched diet showed 80% increased muscle protein synthesis and a net muscle protein gain of 20%, without affecting myofibrillar proteolysis (estimated by urinary excretion of 3-methylhistidine). This action of citrulline on muscle protein synthesis is not limited to the aged rat: it has been demonstrated in another model of protein energy deficiency, a short fasting model in adult rats (fasted for 18 hours). Fasting results in a decrease of muscle protein synthesis (240%) that is fully restored by an oral bolus of citrulline [8]. In the same way, Ventura et al. [9] showed in adult female rats supplemented with citrulline and moderately feed-restricted (60% of their spontaneous food intake) for 2 weeks that citrulline increases the synthesis of myofibrillar proteins, unlike in feed-restricted rats not given citrulline supplementation.
Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00019-1
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© 2019 Elsevier Inc. All rights reserved.
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Finally, in a model of intrauterine growth restriction induced by maternal food restriction in rats, Bourdon et al. [10] demonstrated that supplementation with citrulline stimulates fetal growth (associated to an increase of protein synthesis). Furthermore, it appears that the positive effect of citrulline on muscular gain is maintained over time, since citrulline supplementation for 3 months in “healthy” aged (20month-old) rats enables an average 25% muscle gain specifically related to protein accretion and to an increase in muscle fiber size [11]. Interestingly, the protein accretion associated with citrulline intake is accompanied by an improvement of muscular function, thus establishing a continuum between metabolic action and clinical repercussions. In the protein energy restriction model in aged rats, citrulline increases maximum strength as well as animal motricity [12]. Muscular strength is preserved when moderately restricted adult female rats are supplemented with citrulline, but not without citrulline supplementation [9]. Nevertheless, all these data on the effects of citrulline on muscle protein synthesis and muscular function were observed in protein energy deficiency or malnutrition models, so confirmation was needed in healthy animals. Goron et al. [13] overcame this lack of data in a recent study where healthy adult rats received citrulline supplementation (1 g/kg/ day) or an isonitrogenous diet during 4 weeks. Just like in catabolic situations, citrulline increased muscle protein synthesis (133%), but without any effect on muscle mass and muscle protein content. Interestingly, despite this lack of effect on muscle protein content and mass, when citrulline supplementation is associated with exercise, the authors observed an improvement in performance (running time increased by 14%). Importantly, this ability of citrulline to modulate muscle protein synthesis has also been shown in humans. Oral supplementation with citrulline (10 g vs isonitrogenous placebo) improved muscle protein synthesis by 25% in healthy adults under a low-protein diet (8%) for 3 days [14], and this increased muscle protein synthesis appears to be insulinindependent, since insulin levels were not different between groups. More recently, Bouillanne et al. [15] demonstrated for the first time that 21-day oral supplementation with citrulline (10 g/day) in undernourished patients increases lean body mass (15% 10%) and reduces fat mass (29% in women). Finally, the effect of citrulline on protein synthesis seems to be muscle-specific, as three clinical studies have studied the effects of citrulline supplementation on whole body protein synthesis under different conditions, and none of them was able to show a positive effect of citrulline [14,16,17]. While the capacity of citrulline to modulate muscle protein synthesis has been well demonstrated in several situations, the precise mechanisms of action involved remain unclear. However, a new study has partially remedied this deficit. Le Plenier et al. [18] showed, using a model of isolated incubated rat muscle (epitrochlearis) with or without citrulline in the culture medium, that muscle protein synthesis was higher with citrulline, demonstrating a direct action of citrulline on muscle protein synthesis. Moreover, this increase in muscle protein synthesis could be linked to stimulation of the mTORC1 (mammalian target of rapamycin complex 1) pathway—the main pathway for the regulation of protein synthesis. In this same study and in the same model of isolated incubated rat muscle, the addition of rapamycin, an mTORC1 inhibitor, blunted the positive effect of citrulline on muscle protein synthesis. Moreover, the stimulation of mTORC1 by citrulline has been confirmed in vivo in the model of protein energy undernutrition described earlier, since rats refed with citrulline showed higher activation of S6K1 (p70 ribosomal protein S6 III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE
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kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) in the muscle [18]. These data have been confirmed by Goron et al. [13] who showed that citrulline supplementation in adult healthy rats during 4 weeks increases phosphorylation of S6K1 (157%) and 4EBP1 (146%) in muscle. These results therefore appear to connect the ability of citrulline to increase muscle protein synthesis to a stimulatory action on the mTORC1 pathway, via activation of S6K1 and 4E-BP1 by phosphorylation. In addition, this stimulation of mTORC1 is partially Akt/PI3K (phosphatidylinositol 3-kinase)-independent, as citrulline maintains 4E-BP1 activation by phosphorylation even when the Akt/PI3K pathway, upstream of mTORC1, is inhibited [18]. Finally, although citrulline clearly stimulates overall muscle protein synthesis, it has a more complex action on protein expression. Indeed, citrulline is able to stimulate the expression of specific muscle proteins and inhibit the expression of others. This citrullinemediated modulation of specific proteins was recently listed in a general review by Bourgoin-Voillard et al. [19]. In particular, citrulline more specifically stimulates the expression of myofibrillar proteins and modulates enzymes that drive energy metabolism. Indeed, again in the same model of protein energy malnutrition as described earlier, a differential proteomics approach showed that at muscle level, citrulline leads to overexpression of the enzymes involved in glycogenolysis (i.e., glycogen phosphorylase) and glycolysis (i.e., phosphoglucomutase 1,6-phosphofructokinase, triosephosphate isomerase, β-enolase and pyruvate kinase M1/M2 isozymes) and lower expression of some enzymes of the Krebs cycle (i.e., isocitrate dehydrogenase and succinate dehydrogenase) and the mitochondrial respiratory chain (i.e., NADH dehydrogenase complex, NADH ubiquinone oxidoreductase, and ATP synthase) [20]. Another proteomics study in “healthy” aged rats showed that citrulline supplementation stimulates the expression of mitochondrial biogenesis factor (TFAM: Mitochondrial transcription factor A) as well as mitochondrial complex I activity [11]. A more recent study has confirmed the effect of citrulline supplementation on energy metabolism: Goron et al. [13] showed that citrulline supplementation in adult rats during 4 weeks stimulates several enzymes involved in the pathways generating acetyl-CoA. These properties of citrulline were further confirmed in vitro. In myotubes derived from primary myoblasts, deprived of amino acids and serum during 16 hours, citrulline (5 mM) is able to stimulate protein synthesis and reallocate ATP to the protein synthesis machinery (unpublished data). Thus, the stimulation of protein synthesis could be related to the energy reallocation toward protein synthesis, but the underlying mechanisms remain to be established. It is now well established that citrulline has positive effects on muscle protein synthesis, yet there is still surprisingly little data in the literature on the effects of citrulline on proteolysis. Ham et al. [21] have demonstrated in immobilized mice that citrulline supplementation downregulates Bnip3, a proautophagic gene strongly stimulated by immobilization. In addition, LC3BII:LC3BI protein ratio, a marker of autophagosome number, is increased during immobilization but restored when the mouse is supplemented with citrulline. This result could tie into work by Faure et al. [20] showing that a 5-day citrulline complement decreases proteasome activator complex subunit 1. However, despite interesting but fragmentary data, the regulation of muscle proteolysis by citrulline remains to be explored. Finally, in a very preliminary set of data [22], we focused our attention on a still underexplored aspect of muscle protein metabolism, i.e., muscle secreted proteins. Indeed, a new aspect of muscle protein metabolism has emerged in the past few years. Several III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE
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authors have pointed out the ability of muscle to secrete specific proteins in response to various stimuli, in particular exercise (see Pedersen et al. [23,24] for reviews). We observed that citrulline is able to modify the patterns of proteins secreted by muscle cells in cultures by decreasing the production of five proteins and increasing the secretion of four proteins compared to amino acid-free media [22]. Four of these proteins (calumenin, cystatin C, fetuin-A, and transcobalamin) are involved in citrulline-modulated functions, i.e., cardiovascular functions (see [3,4] for reviews), brain functions [25,26], and protein synthesis. This proteomics study therefore suggests, for the first time, that the effect of citrulline on general metabolism could hinge on its effect on muscle, opening up an important new field for research. As citrulline has key properties in muscle functions, there is major interest in the effects of citrulline on exercise in humans. Surprisingly though, only a few studies have evaluated the potential benefit of citrulline on performance, and the results seem to be different according to duration of supplementation, type of exercise, and form of citrulline given. Indeed, an acute citrulline supplementation seems to have zero if not deleterious effect on anaerobic or aerobic exercise performance [27,28]. However, the results change with chronic citrulline administration: a 7-day citrulline ingestion leads to increased time to exhaustion and total amount of work completed in high-intensity cycling exercise, and decreased time to complete a 4-km cycling time trial associated with better subjective feelings of muscle fatigue [29,30]. It was proposed that is due to effects of citrulline on vascular function (see [3,4] for reviews). These data are in line with the fact that citrulline preserves splanchnic perfusion in the gut and attenuates intestinal disorders during exercise in athletes [31]. However, the athletes usually consumed citrulline as malate salt. Several studies have been performed with this chemical form to evaluate the properties of citrulline. The effects of citrulline malate were beneficial on performance, recovery, and immune system, which is altered after exercise [32 37]. The effects on performance could be due to the ability of citrulline to increase the use of amino acids during exercise, especially branched-chain amino acids [38]. However, results found with citrulline malate supplementation must be interpreted with caution, as it is impossible to know whether such effects are due to the citrulline itself or to the malate, which is an intermediate of the Krebs cycle [39].
CONCLUSION The beneficial value of citrulline to stimulate protein synthesis to maintain muscle mass, and thus improve muscle functionality, has been incontrovertibly demonstrated, particularly in situations of protein deficiency. An original hypothesis, proposed by Moinard et al. [40], is that citrulline, which is synthesized and released by the intestine mainly in postabsorptive state or in the situation of low protein intake, could maintain a minimal level of protein synthesis to preserve muscle proteins during the postabsorptive state, unlike leucine, a well-known amino acid activator of protein synthesis during the postprandial state. Citrulline and leucine could be the two essential amino acids that control nitrogen balance and muscle protein composition, depending on nutritional state. This elegant hypothesis needs to be confirmed by further studies.
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To conclude, despite the recent surge in data on the effects of citrulline on muscle, further explorations are still needed in order to gain a complete overview of the role of citrulline in muscle metabolism and the mechanisms involved. Moreover, promising data on the effect of citrulline on proteins secreted by muscle cells open up important avenues for research on the indirect action of citrulline, and of nutrients in general, on general metabolism, through its effects on muscle. Conflict of Interest C. Moinard and C. Breuillard are shareholders in Citrage.
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[18] Le Ple´nier S, Goron A, Sotiropoulos A, Archambault E, Guihenneuc C, Walrand S, et al. Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies. Am J Physiol Endocrinol Metab 2017;312:E27 36. [19] Bourgoin-Voillard S, Goron A, Seve M, Moinard C. Regulation of the proteome by amino acids. Proteomics 2016;16:831 46. [20] Faure C, Morio B, Chafey P, Le Ple´nier S, Noirez P, Randrianarison-Huetz V, et al. Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats. Proteomics 2013;13:2191 201. [21] Ham DJ, Kennedy TL, Caldow MK, Chee A, Lynch GS, Koopman R. Citrulline does not prevent skeletal muscle wasting or weakness in limb-casted mice. J Nutr 2015;145:900 6. [22] Breuillard, C., Goron, A., Cunin, V., Bourgoin-Voillard, S., Se`ve, M., Moinard, C. (2017). Modulation of muscle protein synthesis by amino acids: consequences on the secretome—a preliminary in vitro study. In: XXXIXth ESPEN congress, The Hague, The Netherlands. September 2017. Clin Nutr. 36 (S1), S157. [23] Pedersen BK. Muscle as a secretory organ. Compr Physiol. 2013;3:1337 62. [24] Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a secretory organ. Nat Rev Endocrinol. 2012;8:457 65. [25] Moinard C, Tliba L, Diaz J, Le Ple´nier S, Nay L, Neveux N, et al. Citrulline stimulates locomotor activity in aged rats: implication of the dopaminergic pathway. Nutrition 2017;38:9 12. [26] Marquet-de Rouge´ P, Clamagirand C, Facchinetti P, Rose C, Sargueil F, Guihenneuc-Jouyaux C, et al. Citrulline diet supplementation improves specific age-related raft changes in wild-type rodent hippocampus. Age 2013;35:1589 606. [27] Hickner RC, Tanner CJ, Evans CA, Clark PD, Haddock A, Fortune C, et al. L-citrulline reduces time to exhaustion and insulin response to a graded exercise test. Med Sci Sports Exerc 2006;38:660 6. [28] Cutrufello PT, Gadomski SJ, Zavorsky GS. The effect of l-citrulline and watermelon juice supplementation on anaerobic and aerobic exercise performance. J Sports Sci 2015;33:1459 66. [29] Bailey SJ, Blackwell JR, Lord T, Vanhatalo A, Winyard PG, Jones AM. L-Citrulline supplementation improves O2 uptake kinetics and high-intensity exercise performance in humans. J Appl Physiol 2015;119:385 95. [30] Suzuki T, Morita M, Kobayashi Y, Kamimura A. Oral L-citrulline supplementation enhances cycling time trial performance in healthy trained men: double-blind randomized placebo-controlled 2-way crossover study. J Int Soc Sports Nutr 2016;13:6. [31] van Wijck K, Wijnands KAP, Meesters DM, Boonen B, van Loon LJC, Buurman WA, et al. L-Citrulline improves splanchnic perfusion and reduces gut injury during exercise. Med Sci Sports Exerc 2014;46:2039 46. [32] Bendahan D, Mattei JP, Ghattas B, Confort-Gouny S, Le Guern ME, Cozzone PJ. Citrulline/malate promotes aerobic energy production in human exercising muscle. Br J Sports Med 2002;36:282 9. [33] Pe´rez-Guisado J, Jakeman PM. Citrulline malate enhances athletic anaerobic performance and relieves muscle soreness. J Strength Cond Res Natl Strength Cond Assoc. 2010;24:1215 22. [34] Glenn JM, Gray M, Wethington LN, Stone MS, Stewart RW, Moyen NE. Acute citrulline malate supplementation improves upper- and lower-body submaximal weightlifting exercise performance in resistance-trained females. Eur J Nutr 2017;56:775 84. [35] Wax B, Kavazis AN, Weldon K, Sperlak J. Effects of supplemental citrulline malate ingestion during repeated bouts of lower-body exercise in advanced weightlifters. J Strength Cond Res Natl Strength Cond Assoc. 2015;29:786 92. [36] Glenn JM, Gray M, Jensen A, Stone MS, Vincenzo JL. Acute citrulline-malate supplementation improves maximal strength and anaerobic power in female, masters athletes tennis players. Eur J Sport Sci. 2016;16:1095 103. [37] Sureda A, Cordova A, Ferrer MD, Tauler P, Perez G, Tur JA, et al. Effects of L-citrulline oral supplementation on polymorphonuclear neutrophils oxidative burst and nitric oxide production after exercise. Free Radic Res 2009;43:828 35. [38] Sureda A, Co´rdova A, Ferrer MD, Pe´rez G, Tur JA, Pons A. L-citrulline-malate influence over branched chain amino acid utilization during exercise. Eur J Appl Physiol 2010;110:341 51. [39] Wagenmakers AJ. Muscle amino acid metabolism at rest and during exercise: role in human physiology and metabolism. Exerc Sport Sci Rev 1998;26:287 314. [40] Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis. J Nutr 2007;137:1621S 5S.
III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE
19 Citrulline and Skeletal Muscle Charlotte Breuillard, Arthur Goron and Christophe Moinard Laboratoire de bioenerge´tique fondamentale et applique´e, Universite´ Grenoble Alpes, Grenoble, France
Citrulline is a nonessential and nonproteinogenic amino acid that takes its name from watermelon (Citrullus vulgaris). For decades, citrulline was considered an intermediate of the urea cycle and as just one among many other amino acids. However, it began to arouse interest when Windmueller and Spaeth [1] showed the significance of intestinally produced citrulline. This particular feature was first used for diagnostic purposes, as citrulline proved to be the best marker of intestinal function [2]. Metabolic properties of this amino acid were subsequently demonstrated: first at cardiovascular level [as a precursor of nitric oxide (NO); see [3,4] for reviews] and then as a modulator of nitrogen homeostasis (see [5] for a recent review) when Osowska et al. [6] showed in a short bowel syndrome model that citrulline supplementation improves nitrogen balance. Moving forward, it was proposed that citrulline would be a regulator of muscle protein synthesis: in 2006, the same authors demonstrated, for the first time, the stimulatory effect of citrulline on muscle protein synthesis [7] in aged undernourished rats, using a model of protein energy restriction. In this model, the rats fed only 50% of spontaneous food intake for 12 weeks are then refed for 1 week with a diet corresponding to 90% of their spontaneous food intake and enriched with citrulline or a mix of nonessential amino acids (in order to make the diets isonitrogenous). Rats receiving the citrulline-enriched diet showed 80% increased muscle protein synthesis and a net muscle protein gain of 20%, without affecting myofibrillar proteolysis (estimated by urinary excretion of 3-methylhistidine). This action of citrulline on muscle protein synthesis is not limited to the aged rat: it has been demonstrated in another model of protein energy deficiency, a short fasting model in adult rats (fasted for 18 hours). Fasting results in a decrease of muscle protein synthesis (240%) that is fully restored by an oral bolus of citrulline [8]. In the same way, Ventura et al. [9] showed in adult female rats supplemented with citrulline and moderately feed-restricted (60% of their spontaneous food intake) for 2 weeks that citrulline increases the synthesis of myofibrillar proteins, unlike in feed-restricted rats not given citrulline supplementation.
Nutrition and Skeletal Muscle DOI: https://doi.org/10.1016/B978-0-12-810422-4.00019-1
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© 2019 Elsevier Inc. All rights reserved.
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Finally, in a model of intrauterine growth restriction induced by maternal food restriction in rats, Bourdon et al. [10] demonstrated that supplementation with citrulline stimulates fetal growth (associated to an increase of protein synthesis). Furthermore, it appears that the positive effect of citrulline on muscular gain is maintained over time, since citrulline supplementation for 3 months in “healthy” aged (20month-old) rats enables an average 25% muscle gain specifically related to protein accretion and to an increase in muscle fiber size [11]. Interestingly, the protein accretion associated with citrulline intake is accompanied by an improvement of muscular function, thus establishing a continuum between metabolic action and clinical repercussions. In the protein energy restriction model in aged rats, citrulline increases maximum strength as well as animal motricity [12]. Muscular strength is preserved when moderately restricted adult female rats are supplemented with citrulline, but not without citrulline supplementation [9]. Nevertheless, all these data on the effects of citrulline on muscle protein synthesis and muscular function were observed in protein energy deficiency or malnutrition models, so confirmation was needed in healthy animals. Goron et al. [13] overcame this lack of data in a recent study where healthy adult rats received citrulline supplementation (1 g/kg/ day) or an isonitrogenous diet during 4 weeks. Just like in catabolic situations, citrulline increased muscle protein synthesis (133%), but without any effect on muscle mass and muscle protein content. Interestingly, despite this lack of effect on muscle protein content and mass, when citrulline supplementation is associated with exercise, the authors observed an improvement in performance (running time increased by 14%). Importantly, this ability of citrulline to modulate muscle protein synthesis has also been shown in humans. Oral supplementation with citrulline (10 g vs isonitrogenous placebo) improved muscle protein synthesis by 25% in healthy adults under a low-protein diet (8%) for 3 days [14], and this increased muscle protein synthesis appears to be insulinindependent, since insulin levels were not different between groups. More recently, Bouillanne et al. [15] demonstrated for the first time that 21-day oral supplementation with citrulline (10 g/day) in undernourished patients increases lean body mass (15% 10%) and reduces fat mass (29% in women). Finally, the effect of citrulline on protein synthesis seems to be muscle-specific, as three clinical studies have studied the effects of citrulline supplementation on whole body protein synthesis under different conditions, and none of them was able to show a positive effect of citrulline [14,16,17]. While the capacity of citrulline to modulate muscle protein synthesis has been well demonstrated in several situations, the precise mechanisms of action involved remain unclear. However, a new study has partially remedied this deficit. Le Plenier et al. [18] showed, using a model of isolated incubated rat muscle (epitrochlearis) with or without citrulline in the culture medium, that muscle protein synthesis was higher with citrulline, demonstrating a direct action of citrulline on muscle protein synthesis. Moreover, this increase in muscle protein synthesis could be linked to stimulation of the mTORC1 (mammalian target of rapamycin complex 1) pathway—the main pathway for the regulation of protein synthesis. In this same study and in the same model of isolated incubated rat muscle, the addition of rapamycin, an mTORC1 inhibitor, blunted the positive effect of citrulline on muscle protein synthesis. Moreover, the stimulation of mTORC1 by citrulline has been confirmed in vivo in the model of protein energy undernutrition described earlier, since rats refed with citrulline showed higher activation of S6K1 (p70 ribosomal protein S6 III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE
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kinase 1) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) in the muscle [18]. These data have been confirmed by Goron et al. [13] who showed that citrulline supplementation in adult healthy rats during 4 weeks increases phosphorylation of S6K1 (157%) and 4EBP1 (146%) in muscle. These results therefore appear to connect the ability of citrulline to increase muscle protein synthesis to a stimulatory action on the mTORC1 pathway, via activation of S6K1 and 4E-BP1 by phosphorylation. In addition, this stimulation of mTORC1 is partially Akt/PI3K (phosphatidylinositol 3-kinase)-independent, as citrulline maintains 4E-BP1 activation by phosphorylation even when the Akt/PI3K pathway, upstream of mTORC1, is inhibited [18]. Finally, although citrulline clearly stimulates overall muscle protein synthesis, it has a more complex action on protein expression. Indeed, citrulline is able to stimulate the expression of specific muscle proteins and inhibit the expression of others. This citrullinemediated modulation of specific proteins was recently listed in a general review by Bourgoin-Voillard et al. [19]. In particular, citrulline more specifically stimulates the expression of myofibrillar proteins and modulates enzymes that drive energy metabolism. Indeed, again in the same model of protein energy malnutrition as described earlier, a differential proteomics approach showed that at muscle level, citrulline leads to overexpression of the enzymes involved in glycogenolysis (i.e., glycogen phosphorylase) and glycolysis (i.e., phosphoglucomutase 1,6-phosphofructokinase, triosephosphate isomerase, β-enolase and pyruvate kinase M1/M2 isozymes) and lower expression of some enzymes of the Krebs cycle (i.e., isocitrate dehydrogenase and succinate dehydrogenase) and the mitochondrial respiratory chain (i.e., NADH dehydrogenase complex, NADH ubiquinone oxidoreductase, and ATP synthase) [20]. Another proteomics study in “healthy” aged rats showed that citrulline supplementation stimulates the expression of mitochondrial biogenesis factor (TFAM: Mitochondrial transcription factor A) as well as mitochondrial complex I activity [11]. A more recent study has confirmed the effect of citrulline supplementation on energy metabolism: Goron et al. [13] showed that citrulline supplementation in adult rats during 4 weeks stimulates several enzymes involved in the pathways generating acetyl-CoA. These properties of citrulline were further confirmed in vitro. In myotubes derived from primary myoblasts, deprived of amino acids and serum during 16 hours, citrulline (5 mM) is able to stimulate protein synthesis and reallocate ATP to the protein synthesis machinery (unpublished data). Thus, the stimulation of protein synthesis could be related to the energy reallocation toward protein synthesis, but the underlying mechanisms remain to be established. It is now well established that citrulline has positive effects on muscle protein synthesis, yet there is still surprisingly little data in the literature on the effects of citrulline on proteolysis. Ham et al. [21] have demonstrated in immobilized mice that citrulline supplementation downregulates Bnip3, a proautophagic gene strongly stimulated by immobilization. In addition, LC3BII:LC3BI protein ratio, a marker of autophagosome number, is increased during immobilization but restored when the mouse is supplemented with citrulline. This result could tie into work by Faure et al. [20] showing that a 5-day citrulline complement decreases proteasome activator complex subunit 1. However, despite interesting but fragmentary data, the regulation of muscle proteolysis by citrulline remains to be explored. Finally, in a very preliminary set of data [22], we focused our attention on a still underexplored aspect of muscle protein metabolism, i.e., muscle secreted proteins. Indeed, a new aspect of muscle protein metabolism has emerged in the past few years. Several III. NUTRITION AS A THERAPEUTICAL TOOL FOR SKELETAL MUSCLE
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authors have pointed out the ability of muscle to secrete specific proteins in response to various stimuli, in particular exercise (see Pedersen et al. [23,24] for reviews). We observed that citrulline is able to modify the patterns of proteins secreted by muscle cells in cultures by decreasing the production of five proteins and increasing the secretion of four proteins compared to amino acid-free media [22]. Four of these proteins (calumenin, cystatin C, fetuin-A, and transcobalamin) are involved in citrulline-modulated functions, i.e., cardiovascular functions (see [3,4] for reviews), brain functions [25,26], and protein synthesis. This proteomics study therefore suggests, for the first time, that the effect of citrulline on general metabolism could hinge on its effect on muscle, opening up an important new field for research. As citrulline has key properties in muscle functions, there is major interest in the effects of citrulline on exercise in humans. Surprisingly though, only a few studies have evaluated the potential benefit of citrulline on performance, and the results seem to be different according to duration of supplementation, type of exercise, and form of citrulline given. Indeed, an acute citrulline supplementation seems to have zero if not deleterious effect on anaerobic or aerobic exercise performance [27,28]. However, the results change with chronic citrulline administration: a 7-day citrulline ingestion leads to increased time to exhaustion and total amount of work completed in high-intensity cycling exercise, and decreased time to complete a 4-km cycling time trial associated with better subjective feelings of muscle fatigue [29,30]. It was proposed that is due to effects of citrulline on vascular function (see [3,4] for reviews). These data are in line with the fact that citrulline preserves splanchnic perfusion in the gut and attenuates intestinal disorders during exercise in athletes [31]. However, the athletes usually consumed citrulline as malate salt. Several studies have been performed with this chemical form to evaluate the properties of citrulline. The effects of citrulline malate were beneficial on performance, recovery, and immune system, which is altered after exercise [32 37]. The effects on performance could be due to the ability of citrulline to increase the use of amino acids during exercise, especially branched-chain amino acids [38]. However, results found with citrulline malate supplementation must be interpreted with caution, as it is impossible to know whether such effects are due to the citrulline itself or to the malate, which is an intermediate of the Krebs cycle [39].
CONCLUSION The beneficial value of citrulline to stimulate protein synthesis to maintain muscle mass, and thus improve muscle functionality, has been incontrovertibly demonstrated, particularly in situations of protein deficiency. An original hypothesis, proposed by Moinard et al. [40], is that citrulline, which is synthesized and released by the intestine mainly in postabsorptive state or in the situation of low protein intake, could maintain a minimal level of protein synthesis to preserve muscle proteins during the postabsorptive state, unlike leucine, a well-known amino acid activator of protein synthesis during the postprandial state. Citrulline and leucine could be the two essential amino acids that control nitrogen balance and muscle protein composition, depending on nutritional state. This elegant hypothesis needs to be confirmed by further studies.
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To conclude, despite the recent surge in data on the effects of citrulline on muscle, further explorations are still needed in order to gain a complete overview of the role of citrulline in muscle metabolism and the mechanisms involved. Moreover, promising data on the effect of citrulline on proteins secreted by muscle cells open up important avenues for research on the indirect action of citrulline, and of nutrients in general, on general metabolism, through its effects on muscle. Conflict of Interest C. Moinard and C. Breuillard are shareholders in Citrage.
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