“Nutraceuticals” in relation to human skeletal muscle and exercise

Abstract

Skeletal muscles have a fundamental role in locomotion and whole body metabolism, with muscle mass and quality being linked to improved health and even lifespan. Optimizing nutrition in combination with exercise is considered an established, effective ergogenic practice for athletic performance. Importantly, exercise and nutritional approaches also remain arguably the most effective countermeasure for muscle dysfunction associated with aging and numerous clinical conditions, e.g., cancer cachexia, COPD, and organ failure, via engendering favorable adaptations such as increased muscle mass and oxidative capacity. Therefore, it is important to consider the effects of established and novel effectors of muscle mass, function, and metabolism in relation to nutrition and exercise. To address this gap, in this review, we detail existing evidence surrounding the efficacy of a nonexhaustive list of macronutrient, micronutrient, and “nutraceutical” compounds alone and in combination with exercise in relation to skeletal muscle mass, metabolism (protein and fuel), and exercise performance (i.e., strength and endurance capacity). It has long been established that macronutrients have specific roles and impact upon protein metabolism and exercise performance, (i.e., protein positively influences muscle mass and protein metabolism), whereas carbohydrate and fat intakes can influence fuel metabolism and exercise performance. Regarding novel nutraceuticals, we show that the following ones in particular may have effects in relation to 1) muscle mass/protein metabolism: leucine, hydroxyl β-methylbutyrate, creatine, vitamin-D, ursolic acid, and phosphatidic acid; and 2) exercise performance: (i.e., strength or endurance capacity): hydroxyl β-methylbutyrate, carnitine, creatine, nitrates, and β-alanine.

skeletal muscle represents the largest organ in the body, comprising ~40% of whole body mass (123). The functions of skeletal muscle extend beyond the widely recognized role of locomotion, serving as the body’s largest tissue for glucose storage and utilization (101, 121) and a primary site of lipid metabolism (104). Muscle also stores ~40% of total body amino acids (AA), which can act as a source of fuel and an AA substrate for other tissues in times of illness or fasting via release of glucogenic, ketogenic AA (264). Changes in muscle mass are regulated by dynamic turnover of the muscle protein pool (~1–1.5%/day), with skeletal muscle mass remaining constant when muscle protein synthesis (MPS) and muscle protein breakdown (MPB) are in balance (8). During situations of muscle growth [e.g., resistance exercise training (RET) combined with AA substrate], net MPS exceeds MPB (8). Conversely, net MPB is greater than MPS in conditions of muscle loss (e.g., bed rest, cachexia, and sarcopenia (75)); in humans, such wasting conditions are typically due predominantly to reduced MPS under fasted and/or fed conditions (191)). In addition to the regulation of muscle and function being clinically relevant, optimal strategies to promote growth, maintenance of muscle mass, and exercise performance (i.e., strength and endurance capacity) are of great interest to performance scientists. Therefore, a major area of interest surrounds the role of macronutrients, micronutrients, and nutraceuticals that influence muscle metabolism and function.

The consumption of nutritional supplements with “ergogenic” claims occurs in many populations, including athletes (186), the elderly (24), chronic disease sufferers (78), and sedentary (201) adults, often without sound empirical evidence. As such, there is a need to review the continually growing area of nutrients/nutraceuticals and associated mechanisms on aspects of skeletal muscle health to formulate evidence-based recommendations. Indeed, previous reviews have summarized the effects of multiple nutrient/nutraceutical compounds on aspects of skeletal muscle metabolism and exercise performance (53, 171). Often such reviews target a specific population (e.g., athletes), end point (i.e., aerobic performance), or dosing regime (e.g., timing and amount). As such, the present review adopts a more wide-ranging scope, including data irrespective of age, training status, or other independent variables, to highlight universal skeletal muscle effects of each nutritional compound.

Herein, we detail existing evidence for a nonexhaustive list of established and emerging nutrients in relation to some or all of the following end points: 1) muscle mass, 2) metabolism (protein and fuel), and 3) exercise performance (i.e., strength and endurance capacity). Since nutrition and exercise are the two key modifiable lifestyle factors for maintaining muscle health, this review will critique available literature examining the muscular responses to nutrient supplementation alone, nutrient supplementation plus acute exercise, and chronic nutrient supplementation combined with chronic exercise training (i.e., >1 bout of exercise). We shall include responses to both resistance exercise (RE)/RET and endurance exercise (EE)/endurance exercise training (EET) since exercise mode may differentially influence muscular responses to nutrition. Finally, because of the emerging nature of some nutrients, where mechanisms have not been well defined in humans, data from other models (e.g., cell/rodents) have been drawn upon where necessary. Therefore, this review should be of interest to scientists, clinicians, and athletes aiming to optimize muscle mass and function in clinical and athletic populations. Out of the scope of this review are a selection of established nutrients with purported effects on muscle (e.g., caffeine and green tea) due to the large volume of existing review literature available. Furthermore, some emerging nutrients (e.g., tomatidine and minerals) have been omitted from this review due to the paucity of existing literature. Therefore, we direct readers to the following publications for further reading regarding nutrients not discussed herein (53), in particular, caffeine (96), green tea (114), tomatidine (69), and minerals (209). Since we have not performed a systematic analysis, we apologize to those whose work we have not alluded to. Finally, in reading this review, we urge readers to refer to Supplemental Table S1 (all Supplemental Material for this article is available online at the AJP-Endocrinology and Metabolism website) as a resource, which illustrates source data relating to the impact(s) upon skeletal muscle mass, metabolism, and performance.

Definitions of Macro/Micronutrients and “Nutraceuticals”

From the outset, it is important that we define what is meant when we refer to macronutrients, micronutrients, and nutraceuticals, since the classification can be misinterpreted due to obscure classification boundaries. Proteins, fats, and carbohydrate (CHO) are required by the body in large amounts (i.e., g·kg⁻¹·day⁻¹), and therefore they are termed macronutrients (139). Micronutrients are defined as vitamins and trace elements (minerals) (212, 213) that are essential to our diet, albeit in small amounts (i.e., mg·kg⁻¹·day⁻¹), to maintain normal physiological and metabolic function. “Nutraceuticals” is an emerging term within the scientific literature that has not been well defined. A recent review defined a nutraceutical as a nutrient compound “with added extra health benefits” (i.e., in addition to the basic nutritional value contained in foods) (210). For the purpose of this review, we define a nutraceutical as “a compound that alone or in tandem with exercise impacts major physiological end point(s)” e.g., effectors of whole body metabolism, skeletal muscle mass, and/or whole body/muscle function.

Established Macronutrients and Exercise

Providing a mixed macronutrient feed containing protein, CHO, and fat stimulates MPS (200). The absolute stimulation of MPS is highly dependent on the AA content, with the provision of AA alone being sufficient to maximally stimulate MPS (15); this effect is entirely attributable to the essential AA (EAA) (218). Of the EAA, the branched-chain AA (BCAA) provide the most potent anabolic stimulation (9), particularly leucine (9, 256). This stimulation of MPS by AA is highly dose dependent and saturable, with maximal stimulation provided by between 20 and 40 g of high-quality protein (166, 167, 230, 263) or 10–20 g of EAA (58). Furthermore, this MPS stimulation is finite, where following an initial lag period of ~30 min during intravenous infusion (or ~45–60 min following oral ingestion to allow for the digestion, absorption, and transport of AA into the systemic circulation) the rate of MPS is increased approximately two- to threefold, reaching a maximum by 1.5–3 h. Subsequently, rates of MPS return to baseline (~2–3 h postingestion) despite continued plasma and muscle AA availability and elevated anabolic signaling (7). Thereafter, muscle remains refractory to further stimulation for an as yet undefined period, a phenomenon coined “muscle full” (7). This ~2- to 3-h period of MPS stimulation can be extended, depending on the type and dose of AA and macronutrient coingestion in combination with resistance exercise (RE) (51). The timing of protein ingestion in close proximity with the performance of acute RE, which when performed alone stimulates MPS for ~48 h (190), is thought to be important. This is because there is an enhanced sensitivity of the muscle to the anabolic properties of AA for ≥24 h postexercise (36), synergistically impacting MPS. However, protein ingestion before (236), during (14), and 1 or 3 h (199) after RE have all elicited similar postexercise increases in MPS.

The mechanisms underlying the anabolic effects to nutrition involve both the stimulation of MPS (200) and suppression of MPB (255); however, it is generally accepted that increases in MPS are the primary driver (8). Following transportation into the muscle cell, leucine in particular stimulates mammalian target of rapamycin complex 1 (mTORC1) (9), which is considered a key regulator of cell growth. mTORC1 activation leads to the phosphorylation of the downstream translation eukaryotic initiation factor 4E-binding protein (4E-BP1) and 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) (see Fig. 1), stimulating the binding of eukaryotic initiation factors 4A (eIF4A) and 4E (eIF4E) to 4G (eIF4G) to form the 4F (eIF4F) complex (135). The eIF4F complex promotes the assembly of the 48S preinitiation complex via mediating the binding of mRNA to the 43S preinitiation complex, thereby promoting MPS (135). Currently the AA sensor coupling intracellular AA signaling to mTORC1 remains to be fully defined, although Rag GTPases (207), leucyl-tRNA synthetase (105), and sestrin2 (265) are all proposed candidates. This has led to intense interest in the development of novel leucine-enriched supplementation regimes to aid maintenance of muscle mass (44, 249). Unlike dietary protein, neither fats nor CHO lead to a direct stimulation of MPS (91, 95, 138); nonetheless, they can influence the bioavailability of AA when provided as part of a mixed meal, slowing plasma AA appearance and increasing AA retention (84) without blunting muscle anabolism (95). Finally, CHO (as well as AA; see Refs. 172 and 173) are insulin secretagogues, positively impacting net muscle anabolism via inhibition of MPB (255) (rather than stimulation of MPS; see Refs. 102 and 255).

Fig. 1.

Proposed metabolism and mechanisms of action for nutrients/nutraceuticals. Solid arrows, activation; Solid verticle line perpendicular to solid horizontal line, inhibition; dashed arrows, purported activation; dashed vertical line perpendicular to dashed horizontal line, purported suppression; question mark, unknown. n-3 PUFA, n-3 polyunsaturated fatty acids; RXR, retinoid X receptor; 4E-BP1, eukaryotic initiation factor 4E-binding protein-1; AA, amino acids; AMPK, 5′-AMP-activated protein kinase; AO, antioxidants; CARNS, carnosine synthase; CHO, carbohydrate; CK, creatine kinase; EDG-2, endothelial differentiation gene; eEF2, eukaryotic elongation factor 2; eIF4E, eukaryotic initiation factor 4E; HMB, β-hydroxy-β-methylbutyrate; MPS, muscle protein synthesis; mTORC1, mammalian target of rapamycin complex 1; $\text{N}{\text{O}}_3^{-}$, nitrate; $\text{N}{\text{O}}_2^{-}$, nitrite; NO, nitric oxide; OCTN2, organic cation transporter 2; PA, phosphatidic acid; PAT1, proton-coupled amino acid transporter 1; PEPT2, peptide transporter 2; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; RPS6, ribosomal protein S6; SLC6AS, solute carrier family 6, member 8; TauT, taurine transporter; UA, ursolic acid; VDR, vitamin D receptor; VDRE, vitamin D response elements; VitD, vitamin D; VitD₃, active vitamin D.

Exercise combined with feeding extends the stimulation of MPS (59), thereby delaying the “muscle full” set-point (8). It is the cumulative stimulation of muscle protein turnover with repeated bouts of exercise and feeding that drives exercise-induced skeletal muscle remodeling and hypertrophy (29). The impact of macronutrient supplements on exercise adaptation is multifarious. It is established that CHO intake helps to spare muscle and liver glycogen stores while also leading to a more rapid recovery of these stores postexercise (47, 162). The benefits of chronic protein supplementation alongside exercise are more inconsistent, with a number of studies showing positive (120, 134, 259) or negligible findings (149, 198, 242). However, a recent meta-analysis suggested that, overall, protein supplementation does lead to an augmentation of muscle mass and strength gains during chronic RET (49). To conclude, it is now well established that macronutrients play key roles in promoting muscle mass maintenance/ growth and functional adaptations. Future work should focus on identifying the underlying cellular mechanisms and associated refractory period of “muscle full.”

Emerging Nutraceuticals and Exercise

Leucine metabolites.

Leucine as a BCAA can be metabolized within muscle, engendering the possibility that its metabolites harbor anabolic effects. For instance, the keto-acid derivative of leucine metabolism, α-ketoisocaproate (α-KIC), was shown to stimulate MPS when provided by infusion; however, this effect could simply be due to KIC being reversibly transaminated to leucine (74). However, there is good evidence of anabolic activities of the more distal leucine metabolite β-hydroxy-β-methylbutyrate (HMB) produced via cytosolic α-KIC dioxygenase (174). Ingestion of ~3 g of HMB in humans elicited comparable increases in MPS to 3 g of leucine while also suppressing MPB independently of insulin (256). Similarly to leucine, the stimulation of MPS by HMB is attributable to enhanced mTORC1 signaling (256). To understand the insulin-independent suppression of MPB associated with HMB, numerous molecular targets associated with different proteolytic pathways [beclin 1, calpain 1, muscle RING finger 1 (MuRF1), muscle atrophy F-box, and cathepsin L] have been investigated, although no detectable changes in the protein abundance or posttranslational modifications were observed (256). Although it has been shown previously that there is a disparity between protein breakdown and the abundance in proteolytic proteins (102), it should be noted that only small amounts of HMB (~5%) are generated from normal leucine metabolism (239a), meaning that to obtain 3 g of HMB (a commonly supplemented amount) one would have to consume 60 g of leucine (260). Thus, when supplementing with physiological doses of leucine, it is unlikely that HMB is the main anabolic constituent, hence the practical use of HMB as a stand-alone nutritional supplement.

Indeed, longer-term studies have found that HMB preserved muscle mass during periods of disuse (65), whereas year-long supplementation of HMB (plus arginine and lysine) in the elderly led to improved preservation of lean body mass, possibly due to an augmentation in muscle protein turnover (10), although since HMB was administered as part of a nutritional cocktail it is impossible to delineate whether HMB was solely responsible for the effects on lean body mass. However, recent meta-analysis of 287 elderly participants (147 HMB-supplemented and 140 controls) found that HMB supplementation led to greater gains in muscle mass compared with controls, indicating that HMB is an effective ergogenic aid, at least in the elderly population, for preventing the loss of lean body mass (268). These anabolic properties of HMB have also been suggested to facilitate favorable RET adaptations. For example, supplementation of HMB (3 g/day) with RET for between 4 and 7 wk led to heightened increases in muscle strength (181), lean body mass (261), and fat free mass (174) compared with RET alone. However, not all studies have reported positive effects; for instance, RET for 1 mo combined with between 3 and 6 g/day of HMB did not change parameters of body composition in RE-trained males (140). In this latter case, HMB was provided in its calcium form (CaHMB) (140), which compared with the free acid form (FA-HMB) may have lower bioavailability and, therefore, might not enhance anabolism to the same extent (although this premise remains to be tested) (82).

Another ergogenic effect of HMB is the purported ability to attenuate exercise-induced muscle damage. For example, oral HMB supplementation (3 g/day for 6 wk) in EE athletes attenuated the increase in creatine phosphokinase and lactate dehydrogenase (plasma markers of exercise-induced muscle damage) after a 20-km time trial run compared with placebo (136). This protective effect of HMB may be due to HMB being a precursor of de novo cholesterol synthesis (175), which is critical for cell membrane (sarcolemmal) maintenance. Thus, HMB may maintain muscle membrane integrity during bouts of damaging exercise.

Furthermore, HMB has been shown to be efficacious for improving EE performance. For example, Vukovich and Dreifort (246) reported that HMB in combination with EE prolonged the time to reach the onset of blood lactate accumulation and V̇o_2peak, albeit via an unknown mechanism. Others have investigated markers of endurance performance following high-intensity interval training (HIIT) with or without HMB supplementation. To exemplify, following 5 wk of HIIT-based running in combination with 3 g/day ca-HMB, V̇o_2max improved more compared with placebo (144). These authors speculated that the performance benefits were attributable to the preservation of the cell membrane; however, membrane stability was not measured in the study, and thus no mechanistic conclusions can be drawn. Furthermore, HMB in untrained participants potentiated the effects of HIIT on physical working capacity at the onset of neuromuscular fatigue compared with HIIT training alone (163).

In summary, the literature supports a role for HMB supplementation in promoting 1) muscle mass, demonstrated by the preservation or increase in muscle mass when combined with RET; 2) muscle metabolism, since HMB stimulates MPS and inhibits MPB; and 3) aerobic and strength performance. However, data reporting negligible effects of HMB do exist (140, 214); prior exercise training history and/or being accustomed to an exercise stimulus may determine the effectiveness of the intervention. This is supported by evidence that HMB supplementation combined with RET in trained individuals had no effect on muscle strength or lean body mass vs. placebo (214). Further research is warranted that rigorously investigates 1) the mechanisms regulating the insulin-independent suppression of MPB associated with HMB supplementation, 2) the effects of novel and accustomed exercise in combination with HMB on endurance performance, and 3) the effects of EET and HMB on muscle mass.

Creatine.

Creatine (Cr) is an endogenously formed metabolite synthesized from arginine, glycine, and methionine (20). Found almost exclusively in skeletal muscle, Cr levels can be increased via endogenous synthesis in the liver and pancreas or exogenously from foodstuff, particularly meat and fish (43, 99). Following oral consumption of Cr, Cr is absorbed into the systemic circulation and is taken up by skeletal muscle via the sarcolemal Na⁺/Cl⁻-dependent transporter soluble carrier family 6 member 8 (126). Intramuscular Cr can then be phosphorylated to phosphocreatine in a reversible reaction facilitated by the enzyme creatine kinase. During high energy demands, the phosphate of phosphocreatine plus free ADP is used for ATP synthesis (126). Another fate of intramuscular Cr is the conversion to the end product creatinine, which due to its muscle exclusivity correlates with muscle mass (110). Creatinine diffuses out of the muscle cell and is removed from the body via urine (126). Oral Cr administration (20–30 g/day for ≥2 days) increases total muscle Cr stores by >20%, of which 20–30% is stored in the form of phosphocreatine (PCr) (107). The greatest Cr loading effects are seen in those with the lowest basal Cr pool levels, i.e., vegetarians (99); thus basal muscle Cr levels are an important determinant of Cr uptake (43, 107). The ergogenic effects of Cr are facilitated by elevated resting PCr, which sustains PCr-mediated ATP resynthesis during intense anaerobic exercise (42) primarily in fatigue-susceptible type II fibers (43), thus improving acute high-intensity performance. Increased basal muscle PCr levels also expedite the replenishment of PCr stores during recovery from intense exercise, leading to improved performance over repeated bouts of sprint exercise (43, 99). For example, 20 g/day of Cr for 5 days led to sustained isokinetic torque compared with placebo during repeated bouts of maximal voluntary contractions (100). Similar results have been obtained when different exercise modes such as cycling are employed (18, 70). In contrast, some studies have shown no effect of Cr supplementation on exercise performance (55, 170, 219, 234). For example, despite increased total muscle Cr following 5 days of 30-g Cr (and 30-g dextrose) supplementation, there were no improvements in sprint exercise performance (219). A lack of ergogenic effect may be attributable to the small total muscle Cr levels of ~12 mmol/kg dry wt (219), where previous reports show that a total Cr of >20 mmol/kg dry mass results in ergogenic benefit (42). Factors affecting the extent to which muscle Cr stores increase are not well known, although preexisting muscle Cr, exercise (107), and CHO ingestion (98) may be potential factors. Also with regard to performance, Cr supplementation improves the rate of functional recovery following exercise (54), which might be mediated by Cr promoting gene expression, thereby aiding MPS during the recovery periods (54, 258), ultimately increasing the deposition of newer functional proteins for improved functional recovery. Indeed, Cr supplementation will also increase muscle PCr, which might increase local rephosphorylation from ADP to ATP (54), thus providing more energy for contraction. As such, performance during successive bouts is maximized (i.e., can work at higher training loads), which in turn may contribute to the gains in strength observed when combined with RET (31, 63, 66).

In addition to energetic impacts, evidence supports a role for chronic Cr supplementation, typically provided as a loading dose (i.e., ~5 days of 20–30 g) followed by maintenance doses (~5 g) (32), for increasing muscle mass (25, 31, 245). For example, 12 wk of RET plus Cr (25 g/day for the 1st wk, followed by a maintenance dose of 5 g/day for the rest of the training duration) resulted in significantly greater fat-free mass, strength, and fiber cross-sectional area gains compared with placebo (245). Similarly, 14 wk of whole body RET (3 times/week) combined with Cr (5 g/day plus 2 g of dextrose) led to significantly greater gains in fat-free mass (31). Furthermore, a recent meta-analysis concluded that Cr supplementation combined with RET elicited further increases in fat-free mass compared with RET alone (albeit in older adults) (66). This meta-analysis reported a weighted mean difference of 1.33 kg for RET combined with Cr (66) compared with 0.69 kg for RET with protein (49), demonstrating the potent ergogenic effect of Cr on fat-free mass. The mechanisms regulating the effects of Cr on muscle mass remain to be fully elucidated, although it is known that acute provision of Cr does not directly stimulate MPS either with (152) or without RE (153). However, Cr did augment the satellite cell (SC) response following RE (178), which may contribute to hypertrophic gains since increased SC content is observed following chronic RET (241). Although the contribution of SC to hypertrophy is still debated (158), theoretically, the nucleus content in hypertrophying muscle fibers becomes diluted such that additional nuclei are required for continued growth. As such, SCs fuse and donate nuclei to the preexisting muscle fibers, thereby increasing the transcriptional capacity of the muscle cell and thus the potential for growth (30). Additionally, augmented PCr availability and ATP resynthesis during intense exercise likely permits greater work output. Greater work may be a factor that stimulates greater muscle gene expression, thereby promoting muscle mass accretion observed with Cr supplementation (32, 204, 257). It is possible that changes in fat-free mass may be in part attributable to the osmotic potential of elevated intracellular Cr, leading to myocellular water retention (204, 273). This potential increase in cell volume from Cr-induced fluid retention may then act as an anabolic signal, activating intracellular signaling cascades that maintain cellular function (204). For example, the attachment complex protein focal adhesion kinase, which is critical for osmosensing and hypertrophic signaling (56), is upregulated following Cr supplementation (204).

To summarize, Cr supplementation is capable of increasing total muscle Cr stores, which improves performance via maintaining PCr-mediated ATP resynthesis, although not all studies have shown improved exercise performance. Beyond performance, chronic Cr supplementation combined with RET is capable of stimulating muscle mass accretion. Although acute effects of Cr supplementation on MPS are not shown, potentiating RET capacity and enhanced recovery likely mediates increased muscle mass. Further studies are needed to firmly establish factors that determine the variability of Cr storage in muscle, since this could have implications for optimizing the dosing regime of Cr.

Carnitine.

Carnitine is synthesized endogenously from AA precursors and can also be obtained exogenously from the diet, particularly red meat, with the majority of whole body carnitine (95%) being stored in skeletal muscles (26). Carnitine has well-documented roles in regulating the translocation of long-chain fatty acids into the mitochondrial matrix for subsequent β-oxidation (223). This process is regulated via the mitochondrial enzyme carnitine palmitoyltransferase (CPT) 1 catalyzing the esterification of carnitine with long-chain acyl-CoA (223). The long-chain acylcarnitine is transported across the mitochondrial membrane into the mitochondrial matrix concurrently with the exchange of free carnitine from the mitochondrial matrix (94). Inside the mitochondrial matrix, acylcarnitine is transesterified to long-chain acyl-CoA and free carnitine via CPT2 (223). Subsequently, the long-chain acyl-CoA is able to undergo β-oxidation. Readers are directed toward the review by Stephens et al. (223) for a more comprehensive overview regarding the role of carnitine in fatty acid translocation.

Therefore, increasing muscle carnitine content could hypothetically enhance fat oxidation while sparing glycogen, therein posing an attractive ergogenic strategy for delaying fatigue during prolonged aerobic exercise and aiding body weight control by promoting fat oxidation. However, a number of studies have failed to increase muscle carnitine via intravenous infusion despite increasing plasma carnitine availability (225). Similarly, oral consumption of carnitine acutely (220) and chronically (247) failed to increase muscle carnitine levels. It is likely that the poor bioavailability of oral carnitine and rapid urinary clearance (106) explain, at least partly, why carnitine supplementation alone does not increase muscle carnitine stores (225). Consequently, several strategies have been tested to stimulate muscle carnitine accretion; concurrent hyperinsulinemia and hypercarnitinemia increased human muscle carnitine content by ~15% (225), and carnitine plus CHO supplementation promoted muscle carnitine accretion (211). Mechanisms by which insulin can facilitate increased muscle carnitine are purported to be due to insulin increasing Na⁺-dependent active transport of carnitine into the muscle via organic cation transporter (OCTN2) (225). Similarly, Na⁺-dependent uptake of AA (274) and Cr (97) by skeletal muscle is increased by insulin, thereby supporting the proposed mechanisms of carnitine uptake (225). However, CHO in addition to protein blunts the stimulation of muscle carnitine uptake (211). This was previously suggested to be related to AA inhibiting carnitine intestinal absorption (233); however, since the combination of CHO and protein led to greater plasma and urinary carnitine vs. CHO alone, this suggests otherwise (211). The precise mechanisms underlying the blunting effect of protein on carnitine uptake into skeletal muscle remain to be fully identified.

By increasing muscle carnitine content, human fuel metabolism can be manipulated. For example, acute increases in resting skeletal muscle carnitine content led to an inhibited glycolytic flux (denoted by reduced lactate) and CHO oxidation (demonstrated via reduced pyruvate dehydrogenase complex activity) concurrent with increased muscle glycogen and long-chain acyl-CoA accumulation (224). Therefore, these studies support the notion that carnitine can enhance fat oxidation while sparing glycogen. A subsequent study by the same group found a 30% increase in muscle carnitine content following dietary carnitine (1.36 g) and CHO (80 g) twice a day for 6 mo and an ~55% reduction in glycogen use during low-intensity exercise (30 min cycling at 50% V̇o_2max) compared with controls (250). Additionally, following 3 mo of supplementation, carnitine and CHO feeding prevented the 2-kg increase in body mass, which was seen in the control group (250). The authors speculate that the lack of increase in body mass in the carnitine group may be due to carnitine-induced increases in long-chain fatty acid oxidation (250).

Subsequent studies have supported the role of carnitine combined with CHO for the prevention of fat gain, which was associated with increased fat oxidation during low-intensity exercise (227). Conversely, increased CHO but not fat oxidation during steady-state exercise has been reported following 2 wk of carnitine supplementation (3 g/day carnitine and tartrate combined with CHO meals) (1), and 1 mo of carnitine intake (3 g/day carnitine and tartrate) had no effect on substrate oxidation during steady-state exercise (27). These findings conflict with those reported at rest and differ from hypotheses suggesting that limited carnitine availability may limit fat oxidation during exercise (224). Interestingly, in the study by Broad et al. (27), there was no mention of daily carnitine supplementation being coingested with supplemental CHO, which is critical for increasing muscle carnitine stores (226). Therefore, the protocol might have been suboptimal for increasing muscle carnitine stores, which was not measured within the study, and thus may explain the negligible effect of carnitine on substrate utilization.

Thus, insulin-stimulated carnitine uptake is capable of increasing muscle carnitine stores which promotes fat oxidation, spares muscle glycogen, and thereby improves endurance performance. Further work is required to fully elucidate the mechanisms regulating the blunting of carnitine uptake when combined with CHO and protein.

n-3 polyunsaturated fatty acids.

n-3 Polyunsaturated fatty acids (n-3 PUFA) contain a double bond at the third carbon atom from the end of the carbon chain. Abundantly found in walnuts and oily fish, there are three types of n-3 PUFA: 1) α-linoleic acid (ALA), 2) eicosapentaenoic acid (EPA), and 3) docosahexaenoic acid (DHA). n-3 PUFA serve well-established roles as critical components of cell membranes and as substrates for lipid signaling (37). Early evidence demonstrated a role for n-3 PUFA in muscle anabolism when n-3 PUFA-enriched feed provided to growing steers increased the phosphorylation of anabolic signaling and the nonoxidative whole body disposal of AA, which was representative of increased whole body protein synthesis (85). Additionally, fish oil containing 18% EPA attenuated the loss of skeletal muscle following 30% burn in guinea pigs, which may be mediated by EPA reducing inflammatory related prostanoids (4). Hence, there is interest in the application of n-3 PUFA as a nutritional supplement in humans. It has been suggested that fish oil supplementation in humans may increase muscle n-3 PUFA content (160), have anti-inflammatory properties (128) via reduced leukotriene B4 formation (an inducer of inflammation) (79), and attenuate the loss of muscle mass in disease states, possibly via reductions in proinflammatory cytokines (203). Furthermore, n-3 PUFA might potentiate anabolic responses to nutrition in skeletal muscle. In support of this, 8 wk of n-3 PUFA supplementation (1.86 g of EPA + 1.5 g DHA/day) was shown to augment hyperaminoacidemia/hyperinsulinemia-induced increases in mixed MPS compared with corn oil controls in young, middle-aged, and older adults (215, 216). Indeed, enhanced phosphorylation of mTORC1 and the downstream target p70S6K1 were observed in young, middle-aged, and older adults (215, 216). However, MPS increases were observed in the context of hyperaminoacidemia and hyperinsulinemia, which may not be physiologically obtainable. Moreover, supplementation of n-3 PUFA for 3 (151) and 6 mo (217) led to increases in muscle mass and function in older adults. A recent study in C₂C₁₂ skeletal muscle cells found a 25% increase in MPS following EPA that was not observed following DHA (131), suggesting that EPA may be the more anabolic constituent of n-3 PUFA. Interestingly, both EPA and DHA stimulated p70S6K1, and thus EPA might stimulate MPS via a p70S6K1-independent mechanism (131).

Despite being less well defined, these positive effects of n-3 PUFA on muscle appear to be recapitulated when combined with exercise (202). Supplementation during 3 mo of RET promoted increases in muscle strength in older women (202), suggesting that n-3 PUFA could have a positive role on muscle protein metabolism by enhancing the anabolic response to RE (90). Despite recent contrasting findings that chronic fish oil supplementation failed to increase muscle anabolism in younger people under rested and exercise-trained conditions (161), the lack of pre- and postintervention measurements confound interpretation of these results. Additionally, positive findings regarding the efficacy of n-3 PUFA supplementation have been observed largely in older adults. Because aging associates with blunted anabolic responses to AA and exercise, the muscular benefits of n-3 PUFA may be more pronounced in those in whom anabolic responses are already suboptimal.

Although the combination of EE and n-3 PUFA has not been investigated in the context of muscle mass and protein metabolism, there is sound evidence to suggest that n-3 PUFA supplementation may alter fuel metabolism by improving metabolic flexibility, i.e., the ability to switch between using fat or CHO as a fuel source. For example, 6 g/day of fish oil for 3 wk led to a 35% increase in fat oxidation following a glucose or fructose bolus (61). In the context of exercise, 3 wk of fish oil supplementation (6 g/day) led to a nonsignificant trend for greater fat oxidation during an acute bout of cycling (90 min at 60% O₂ output), a possible compensatory response for the lower CHO oxidation (62). Further studies have found significantly greater fat oxidation during EE in humans following 3 wk of fish oil supplementation (119). Although each of these studies lacked comprehensive investigation into the mechanisms regulating changes in metabolic flexibility, n-3 PUFA have been shown to mediate the upregulation of genes regulating mitochondrial biogenesis, such as peroxisome proliferator-activated receptor-α and -γ and the transcription factor nuclear respiratory factor 1 in mice (146), offering a potential explanation for these findings. Additionally, rats fed a low-fat diet supplemented with DHA had higher oxygen consumption and apparent K_m for ADP in permeabilized muscle fibers compared with placebo, which was indicative of improved mitochondrial function (103). Thus, effects on mitochondrial biogenesis and function may underpin the synergistic effects of n-3 PUFA and EE-associated metabolic adaptation.

Collectively, n-3 PUFA supplementation beneficially affects muscle protein metabolism, which may contribute to chronic gains in muscle mass, and also shows promise for impacting metabolic flexibility. Further human research that investigates the effects of EPA and DHA individually on aspects of skeletal muscle health is warranted to establish which is the main anabolic constituent.

Nitrates.

Nutrients that contain dietary inorganic nitrates (e.g., beetroot and lettuce) or related precursors (e.g., arginine) can increase nitric oxide (NO) availability, which is capable of modulating muscle-related processes, including contraction, glucose homeostasis, blood flow (127), and satellite cell activation (5, 35). Following oral ingestion of dietary nitrate-rich foods, nitrate ($\text{N}{\text{O}}_3^{-}$) is reduced to nitrite ($\text{N}{\text{O}}_2^{-}$) via nitrate reductases within the mouth (68). Subsequently, $\text{N}{\text{O}}_2^{-}$ is converted into NO and additional reactive nitrogen species in the acidic environment of the stomach (2). Oral $\text{N}{\text{O}}_3^{-}$^- increases plasma $\text{N}{\text{O}}_3^{-}$ and $\text{N}{\text{O}}_2^{-}$ levels, indicating that nitrates are bioavailable. With regard to muscle protein turnover, these compounds are thought to promote anabolism via improving blood flow (through increased NO production), thus enhancing nutrient delivery to the muscle, providing more substrates for MPS. However, it has been shown on several occasions that enhanced muscle blood flow does not augment anabolic responses in young or older males (164, 187–189). Nonetheless, dietary arginine (the principle substrate for endothelial nitric oxide synthase for endogenous production of NO) supplementation did increase the weight of the soleus and EDL muscle in obese rats (125). However, in humans, Luiking et al. (154) and Tang et al. (232) found that oral arginine (10 g), of which ~70% is bioavailable following ingestion, had no effect on muscle blood flow or MPS when provided alone or in combination with AA or acute RE. In contrast, vasodilatory effects of arginine have been shown when administered by intravenous (iv) infusion at higher doses (30 g) (23). By comparison, the peak in plasma arginine was considerably lower following 10 g of oral arginine (~225 µmol/l) (232) vs. 30 g iv infused arginine (~6,223 µmol/) (23), and thus the dose of arginine used by Luiking et al. (154) and Tang et al. (232) may not have been sufficient to increase plasma arginine to an amount that elicits effects on vasodilation. In fact, these authors projected that, on the premise of 70% bioavailability, a total of ~43 g of oral arginine would have been required to reach similar plasma levels reported following iv infusion (232). An alternative may be to utilize the arginine precursor citrulline (156), which bypasses splanchnic extraction (267). Supplementation of citrulline in rodents was shown to stimulate MPS (179) via the mTORC1 pathway (193). However, similar effects have not been observed in humans, since there was no additional impact of citrulline (10 g), when coingested with whey, on MPS or blood flow with or without acute RE vs. whey combined with nonessential AA (52). Finally, flavanols such as in cocoa (39, 109) also promote vasodilation via NO pathways (80, 132). It was reported recently that despite an acute dose of cocoa flavanols (350 mg) increasing macro- and microvascular blood flow, this was not associated with enhanced muscle anabolic responses to nutrition (188), suggesting that in healthy individuals nutrient delivery is not rate limiting for muscle anabolism (189).

In contrast to muscle mass and strength-related studies, a plethora of research has investigated the effects of nitrates and EE on whole body metabolism and endurance performance. An early study by Larsen et al. (148) reported that sodium nitrate supplementation reduced the O₂ cost of submaximal cycling exercise, whereas similar results have been reported following nitrate-rich beetroot juice supplementation (11), which is indicative of improved aerobic metabolism or mechanical efficiency (147). In addition to metabolic improvements, nitrate supplementation provided in the form of 500 ml of beetroot juice improved 4- and 16.1-km cycling time trial performance in trained cyclists (145). These improvements are likely attributable to an enhanced rate of PCr recovery (239) increasing the rate of ATP synthesis, although this mechanism remains speculative at present. Emerging evidence from cell culture studies suggests that nitrate supplementation enhances mitochondrial biogenesis and oxidative metabolism via increased 5′-adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-γ coactivator-1α gene expression (240), although in vivo data is lacking. Although others have also reported nitrate-mediated improvements in EE performance (169, 269), several authors have shown no improvements (6, 48, 254). For example, consuming 140 ml of beetroot juice 2.5 h before a 1-h cycling time trial did not improve time trial performance in trained cyclists compared with placebo (48). These discrepant findings may be explained by methodological differences such as the dose of nitrates (since the increase in plasma $\text{N}{\text{O}}_3^{-}$ and $\text{N}{\text{O}}_2^{-}$ is somewhat dose dependent; see Ref. 270), control of nitrate intake, the source of nitrates provided, and the training status of the participants. For example, since numerous studies demonstrate nitrate supplementation to have no beneficial effect on performance in well-trained participants (6, 48, 254), it is likely that fitness status influences the ergogenic potential of nitrate supplementation (127). Indeed, higher plasma levels of $\text{N}{\text{O}}_2^{-}$ were present in trained vs. untrained participants pre- and post-acute exercise (195). This may be explained by higher nitric oxide synthase activity (159) and/or higher plasma nitrate values (195) in trained participants.

Thus it is established that nitrates reduce the O₂ cost of aerobic exercise. Further in vivo work is required to understand whether oral doses of arginine larger than those already tested can enhance vasodilation and effect protein metabolism across different ages. Furthermore, precise mechanisms regulating the nitrate-induced beneficial effect on O₂ cost remain to be delineated in vivo.

β-Alanine and carnosine.

β-alanine (BA) is a β-AA produced endogenously in the liver and found primarily in meat (238). BA is the rate-limiting precursor for the synthesis of carnosine, which is a dipeptide of BA and histidine that improves the muscle buffering capacity (222). BA supplementation has generated interest as an ergogenic aid since early studies found BA supplementation to be capable of increasing muscle carnosine stores by ~40–65%, demonstrating good bioavailability, a consistent and reproducible finding (16, 108, 222), although the extent to which carnosine content increases may be dependent on the dosing protocol (108). Other factors have been shown to cause muscle carnosine variability, including sex, age, dietary BA intake, vegetarianism (76), and fiber type distribution, since carnosine content is double in type II compared with type I fibers (106a). The regulation of muscle carnosine stores from dietary/supplemental sources is still under investigation (222). Oral BA may be transported across the gut via the H⁺-coupled PAT1 AA transporter (235), which increases plasma availability of BA for muscle carnosine synthesis. Transport of BA into skeletal muscle has been shown to be regulated via both peptide transporter 2 (67) and the taurine transporter (237), although this remains to be confirmed in humans. Once within the muscle cell, BA and sarcoplasmic histidine synthesize carnosine via carnosine synthase (222).

Increased muscle carnosine stores may increase RE work capacity via regulation of the muscle-buffering capacity during RE, and therefore, interest in the potential of BA supplementation for promoting RET adaptations has been gained (133). However, 10 wk of RET combined with 6.4 g/day BA did not enhance body mass or strength changes in 26 males despite increased muscle carnosine (133).

During high-intensity exercise, the buildup of H⁺ ions reduces the intramuscular pH, leading to fatigue that is likely due to acidosis-induced reductions in ATP generation (205). Increased muscle carnosine via BA supplementation is capable of reducing intramuscular acidity during high-intensity exercise, therefore enhancing exercise performance (57, 112, 229). For example, 4 and 10 wk of BA supplementation increased cycling capacity (total work done) in untrained males when cycling at 110% of maximum power (112), which is hypothesized to be due to improved intracellular buffering. In sprint-trained athletes, 4–5 wk of BA supplementation (4.8 g/day) led to increased knee torque but did not enhance sprint performance (64). Importantly, this study found increased muscle carnosine stores (+47%), demonstrating that it is possible to increase muscle carnosine even in trained athletes (64). Women supplemented with BA for 28 days delayed the onset of neuromuscular fatigue (denoted by improved ventilatory threshold, physical working capacity, and time to exhaustion), which was likely the result of improved intracellular buffering capacity (228).

BA supplementation is associated with paresthesia (i.e., flushing) following acute doses of ≥800 mg (60, 108). This side effect is deemed to be dose dependent and likely related to BA plasma kinetics (108). Compared with pure BA, slow-releasing BA capsules eliminate all paresthesia side effects, which is most likely explained by the attenuated BA plasma concentration and delayed time to peak (60), and thus offer a suitable alternative supplement option.

Therefore, BA supplementation may be implemented to increase muscle carnosine stores, which in turn enhances acute EE performance, which is mediated likely via an enhanced intracellular buffering capacity. However, the effects of BA combined with RET need to be studied further in vivo.

Micronutrients: Vitamins and Exercise

Vitamins are essential for many metabolic processes; however consuming vastly more or less than recommended can likely result in toxicity or deficiency, respectively (212), which can be detrimental for muscle health. For example, vitamin D (VitD) deficiency has been linked to muscle wasting (86), and as such, vitamins have been implicated in regulating muscle mass, metabolism, and performance, as discussed below.

Vitamin D.

VitD is a steroid hormone, the deficiency of which in humans throughout the world is reaching epidemic levels mostly because of reduced sun exposure (116). VitD deficiency is prevalent in many debilitating conditions, including osteoporosis and rickets (116, 117), and is associated with reduced muscle mass and strength (244). For example, rodent models have demonstrated that VitD deficiency induced muscle loss, a consequence of increased MPB and reduced MPS compared with controls (17). The VitD receptor (VDR) is present in many tissues, including muscle (89), which has led to increasing interest in the effects of VitD on muscle metabolism. Although conflicting reports exist regarding the presence of the VDR (192, 251), these discrepancies are most likely due to the use of nonvalidated antibodies, lack of controls, or differences in antibody specificity (89).

Following sun exposure or consumption of VitD-rich dietary sources/supplements, circulating VitD bound to VitD-binding protein increases and transports to the liver, where hydroxylation (via 25-hydroxylase) generates 25-hydroxyvitamin D. A second hydroxylation in the kidney (via 1α-hydroxylase) produces the biologically active form of VitD [1,25(OH)₂D] (87). Mechanisms underpinning the effects of VitD on muscle metabolism are not fully understood but are believed to be in part related to the regulation of gene expression via the VDR or secondary messenger protein signaling (194). The binding of 1,25(OH)₂D to the VDR causes conformational changes, allowing VDR to heterodimerize with the retinoid X receptor. This complex then binds to VitD response elements on the DNA, promoting gene transcription (45, 87). 1,25(OH)₂D may also have nongenomic effects on intramuscular signaling by binding to a cell surface receptor (40), which in turn activates intracellular signaling pathways such as the Akt and mitogen-activated protein kinases (MAPK) pathway (33). For example, VitD treatment increased myotube size, downregulated myostatin (88), upregulated Akt (33), and sensitized the Akt/mTORC1 pathway and MPS responses to leucine and insulin (206) in muscle cell cultures. Thus, there is growing in vitro evidence for an anabolic role of VitD in skeletal muscle. In humans, supplementation of VitD has been proposed to increase muscle strength (13), function (83, 252), fiber area (46, 208, 221), and lean body mass (72) and reduce falls (83, 130), although a recent meta-analysis found no overall effects of VitD supplementation on muscle mass (13). Of importance, benefits of VitD supplements are observed particularly in the elderly or in those who are VitD deficient (13), which may be a potential explanation for some of the discrepant findings within the literature.

Since VitD supplementation has been suggested to promote muscle mass and function, concurrent VitD supplementation with RET may be expected to potentiate exercise-induced adaptations. Indeed, 4 mo of VitD₃ supplementation (1,920 IU/day + 800 mg/day calcium) in combination with lower body RET for 3 mo led to a greater reduction in myostatin mRNA expression, a negative regulator of muscle mass, and a greater change in the percentage of type IIa muscle fibers in young males (3). However, these changes did not translate into greater muscle strength or hypertrophy above RET alone (3). Elderly adults undertaking RET combined with VitD improved muscle quality (strength/cross-sectional area) more so than young males, thus demonstrating that elderly individuals may benefit more from VitD supplementation (3). VitD-insufficient (according to VitD previously reported ranges; see Ref. 118) overweight and obese adults did not augment gains in lean body mass compared with placebo following 3 mo of RET and 4,000 IU/day VitD₃ (41). This may be due to the fact that VitD is deposited in body fat, reducing bioavailability (266) and requiring greater levels of VitD supplementation to promote muscle anabolism in this population. Similarly, others reported no change in body composition after 9 mo supplementation of 400 IU/day and RET twice/wk in overweight males and females (34). Since no change in body composition was seen in the training only group either, these findings may result from low training adherence (~53%) (34).

Therefore, although there is some evidence to suggest an emerging role for the supplementation of VitD for the promotion of muscle mass and protein metabolism, more high-quality in vivo work is required. For example, investigations into the direct effect of VitD on MPS in humans are needed, as are more acute and chronic EE studies to understand the potential synergistic effects of VitD supplementation and exercise on muscle health. These studies need to be well controlled, accounting for basal VitD status, and should determine true VitD bioavailability.

Vitamins C and E (i.e., “antioxidants”).

High levels of free radicals (an atom with a single, unpaired electron) and reactive oxygen species (ROS) can disrupt protein homeostasis (196). This is likely due to ROS promoting catabolism via increases in the ubiquitin-conjugating activity (150) and diminishing anabolism via attenuation of MPS and signaling proteins (182), with evidence for these mechanisms arising from cell culture studies. Therefore, it is thought that consuming dietary antioxidants [i.e., vitamins C (VitC) and E (VitE)] that are capable of donating an electron to neutralize free radicals (168) may reduce ROS, thus minimizing disruption of protein homeostasis. For instance, a positive relationship was observed between VitC intake and appendicular lean body mass (209), which may be related to the fact that muscle is a major storage site for VitC (253).

However, physiological levels of ROS such as that produced during exercise (248) promote gene expression (e.g., manganese superoxide dismutase) (185) and cell signaling (e.g., c-Jun NH₂-terminal kinases and MAPKs) (92, 185) in healthy skeletal muscle. Thus, it may be hypothesized that provision of antioxidants combined with RET could hamper exercise-induced adaptations. Human studies assessing the interactions of RET and antioxidant supplementation have produced varied results, with support for positive (22, 143), negative (19, 184), and negligible (21, 184) effects of antioxidants. For example, greater gains in fat-free mass were observed following 6 mo of RET combined with VitC (1,000 mg/day) and VitE (600 mg/day) compared with RET alone, which was postulated to be a result of antioxidants increasing protein synthesis, although this was not measured (22, 143). However, 3-mo supplementation of daily VitC (1,000 mg) and VitE (235 mg) alongside whole body RET led to blunted gains in total lean body mass and muscle thickness (19). Ten weeks of whole body RET combined with 1,000 mg of VitC and 235 mg of VitE daily found negligible effects on acute MPS and muscle mass; however, the phosphorylation of anabolic signaling proteins was blunted compared with placebo (184). Supporting the lack of ability to potentiate exercise-induced adaptations, RET and antioxidants increased fat-free mass but no more than RET alone (21). This may be a result of the low participant numbers or due to the fact that the participants were not vitamin deficient, and therefore, it may be that additional vitamin intake provides little or no added benefits. The absorption of antioxidants, particularly VitC, may also be limited (21), further reducing the antioxidant-induced anabolic potential. Another factor that may explain the efficacy of antioxidant supplements is the age of the participants, since the elderly have an altered redox status (184), which could impact the efficacy of the antioxidants.

Detrimental and negligible interactions have also been reported following EE and antioxidant supplementation (183, 272). For example, daily VitC (1,000 mg) and VitE (235 mg) during an 11-wk EE training program consisting of steady state and HIIT in humans led to blunted increases in mitochondrial protein content, which was indicative of blunted mitochondrial biogenesis, although no differences were observed in V̇o_2max compared with placebo (183). Similarly, VitC hampered running time to exhaustion in rats, perhaps as a result of impaired mitochondrial biogenesis (93). Others have reported no alterations in EE-induced adaptations (measured as maximal O₂ consumption, power output, and workload at lactate threshold) following antioxidant supplementation (272). Differences in the antioxidant dosing regimens might explain some divergent findings between studies (183). Thus, although VitC and VitE are vital for maintaining health, the benefits of supplementation are debatable and likely to depend on the age group and deficiency status. The poor bioavailability described in several studies may further impact any benefits of supplementation (21).

Currently, it is difficult to conclude whether antioxidant supplementation is beneficial or detrimental for muscle mass, protein metabolism, and performance/adaptation. Close et al. (53) highlighted that confusion and misguided conclusions are often drawn due to inappropriate methodological techniques. For example, the lipid peroxidation markers thiobarbituric acid reactive substances can be the result of non-redox-related sources and are thus no longer recommended for use as oxidative stress markers (81), yet they are often published in the context of antioxidant supplementation (111, 155, 157). It is believed that diets rich in fruits and vegetables as opposed to large supplemental doses of antioxidants are preferable since no investigations to date support attenuations in adaptations to training in response to fruits and vegetables, which have naturally occurring antioxidants (53).

Emerging Nutraceuticals

Ursolic acid.

Despite the paucity of research at present, other novel nutraceuticals have gained recent attention for their potential to promote muscle mass, protein metabolism, and/or exercise adaptations. For example, the naturally occurring phytochemical ursolic acid (UA) found in apple peel has drawn attention ever since UA-supplemented mice gained 7% muscle weight (142), suggesting that UA may be capable of promoting muscle hypertrophy (71, 124, 141, 142). UA-induced hypertrophic effects are proposed to be due to the attenuation of the atrophy-related genes MuRF1 and atrogin-1 and the upregulation in IGF gene expression (142). Contrary to this, UA incubations in cell cultures were reported to inhibit leucine-stimulated mTORC1 signaling by inhibiting mTORC1 localization to the lysosome (180), a key step in AA-induced anabolic signaling (207). Research is warranted to detail the effects of UA on muscle metabolism in humans.

With regard to exercise interactions, UA injection following RE in rats stimulated p70S6K1 at 1 h and was maintained 6 h later, which began the descent to baseline in the exercise-only group, reflecting prolonged mTORC1 activity and thus anabolic potential when RE was combined with UA (177). Despite an unclear mechanism, these authors speculated that IGF-I may contribute to the UA-induced p70S6K1 activation, and previous work supports this hypothesis (142). Contrary, data in humans (not in the context of UA) show no change in IGF-I but increased anabolic signaling after acute RE (28). In RE-trained males, RET six times/wk (at 60–80% of 1-RM) for 2 mo combined with 450 mg/day UA improved leg strength but had no effect on lean body mass, although RET alone also had no effects on lean body mass (12). This may be due the fact that the participants had >3 yr RET experience, and hypertrophic responses predominate in the early stages of RET (29). To the authors’ knowledge, no evidence exists regarding UA supplementation combined with EE. An important issue to consider is the low and variable bioavailability of UA following oral ingestion, which is likely due to its lack of solubility in aqueous solutions (113). This could markedly impact its potential as a nutraceutical. However, recent efforts have been made to improve the bioavailability of UA and other triterpenoids by, for instance, using nanoliposomes to aid solubility (271). The varied and low bioavailability of UA in humans is demonstrated by the lack of UA content in some participants following a 1-g oral dose, and in those that did display UA content, it was observed only ≤12 h postconsumption (113). Additional findings show that oral UA ingestion (3 g) led to increased plasma UA 2 and 6 h postexercise (50). As such, the true bioavailability of UA in response to time and dose should be investigated further.

Phosphatidic acid.

Phosphatidic acid (PA) is a diacyl-glycerophospholipid found endogenously in mammalian cell membranes that can be obtained exogenously from raw cabbage (231). Both endogenous and exogenous PA are believed to positively influence muscle protein metabolism, whereby endogenous PA can be increased by RE and binds directly to mTORC1, influencing MPS. Exogenous PA indirectly stimulates mTORC1 activation (77, 165) via extracellular-signal regulated protein kinase-dependent (262) and phosphatidylinositol-3-kinase-independent (176) mechanisms and may also attenuate MPB via attenuation of atrophy-related genes (210). Exogenous PA in cultured muscle cells also prevented atrophy in the presence of the atrophy-inducing substances tumor necrosis factor-α (TNFα) and dexamethasone (122). Recently, acute PA supplementation in rodents tended to increase MPS in the fasted state; however, PA blunted the whey protein-induced rise in MPS (165). Possibly the addition of PA to whey alters the pathways of mTORC1 activation, thus shifting peak MPS (165); research is needed to understand the signaling responses of PA alone vs. PA plus whey. In a human case study, orally ingested PA metabolized into lysophosphatidic acid (LPA) and glycerophosphate increased plasma PA and LPA 30 min postingestion (of 1.5 g of PA), which plateaued at 1–3 h and remained elevated above baseline at 7 h (197). Thus, it seems PA is bioavailable in humans, although beyond 7 h postingestion the bioavailability is unknown, and further studies with a larger cohort are needed to determine the true bioavailability of PA. PA supplementation (750 mg daily) combined with 2 mo of supervised whole body RET in RE-trained males found increased lean body mass and cross-sectional area compared with the placebo group (129). Conversely, others have shown nonsignificant increases (+2.6%) in lean body mass despite utilizing a similar RET and supplementation program (115). The differential findings between these studies may be due to the fact that training was unsupervised in the later study. To our knowledge, no data assessing the interactions of PA plus EE currently exist.

Combined Nutraceuticals

Although not the focus of this review, it is worth speculating that combining nutraceuticals may provide multiple benefits to skeletal muscle health or potentiate skeletal muscle health benefits in response to exercise. Consequently, some studies have investigated the potential of combined nutritional “cocktails.” For example, a supplement containing PA, HMB, and VitD in combination with 2 mo of RET led to greater gains in lean body mass and strength compared with the placebo group, providing support that the combined supplement possessed anabolic properties (73). The combination of VitD, leucine, and whey twice daily in tandem with RET three times/wk for 13 wk prevented the loss of appendicular muscle mass during intentional weight loss in obese males and females (243). The caveat with implementing combined nutritional supplementation is that it is difficult to attribute changes in the end point to the responsible individual or combination of nutrients unless rigorous study designs are implemented with adequate control groups.

Conclusion and Future Directions

Although it is extremely unlikely that a single nutraceutical will prove to be a “magic bullet,” it is clear that certain nutraceuticals, under certain conditions, do indeed possess ergogenic potential. Of the nutrients discussed herein, strong evidence exists for leucine, HMB, and Cr for muscle mass, leucine and HMB for protein metabolism, carnitine for fuel metabolism and leucine, and HMB, carnitine, Cr, nitrates, and β-alanine for athletic (strength or endurance) performance. Further empirical in vivo evidence is required to firmly establish the currently emerging roles of VitD, UA, and PA for promoting muscle mass and n-3 PUFA, UA, and PA for muscle protein metabolism. This review highlights 1) the need for better-controlled, longer-duration human studies that investigate the role of individual nutrients on muscle mass, protein/fuel metabolism, and indices of exercise performance/adaptation, 2) the lack of in vivo “mechanistic” studies, and 3) the need to determine the bioavailability of emerging nutrients. As mentioned in the introduction to this review, please refer to Supplemental Table S1 for a summary outlining the outcomes of individual studies relating to nutraceutical supplementation(s).

GRANTS

C. S. Deane is a Ph.D student funded by Bournemouth University. D. J. Wilkinson is a postdoctoral research fellow funded through the MRC-ARUK Centre for Musculoskeletal Aging Research. The Medical Research Council-Arthritis Research UK (MRC-ARUK) Centre for Musculoskeletal Aging Research was funded by grants from the MRC (grant no. MR/K00414X/1) and ARUK (grant no.19891) awarded to the Universities of Nottingham and Birmingham.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.SD prepared figures; C.S.D., D.J.W., and B.E.P. drafted manuscript; C.S.D., D.J.W., B.E.P., K.S., T.E., and P.J.A. edited and revised manuscript; C.S.D., D.J.W., B.E.P., K.S., T.E., and P.J.A. approved final version of manuscript.