1. Introduction
Diminished bioavailability of nitric oxide (NO), the gaseous signaling molecule involved in the regulation of numerous vital biological functions, contributes to the development of multiple age- and lifestyle-related risk factors and diseases including hypertension, atherosclerosis, insulin resistance, type 2 diabetes (T2D), and cardiovascular disease [
1,
2,
3,
4]. In endothelial cells, NO is synthesized from
l-arginine (precursor) by endothelial-nitric oxide synthase (eNOS) generating NO and
l-citrulline (products) [
5,
6,
7]. In addition to reductions in NO synthesis, elevations in reactive oxygen species (ROS), especially superoxide (O
2−), can reduce the bioavailability of NO through the generation of peroxynitrite (ONOO
−), which further promotes endothelial dysfunction that is commonly associated with cardiometabolic diseases [
6]. Thus, augmenting
l-arginine levels in the circulation may represent a potential therapeutic mechanism to increase NO synthesis and bioavailability. However, oral
l-arginine supplementation is largely ineffective due to gastrointestinal and hepatic extraction of
l-arginine [
8] (
Figure 1), as well as a dose-dependent presentation of gastrointestinal distress [
9]. Alternatively, oral
l-citrulline supplementation consistently increases plasma and tissue levels of
l-arginine and NO bioavailability [
10,
11,
12].
l-Citrulline is a neutral, non-essential [
14] alpha-amino acid that is an important component of the urea cycle in the liver and kidneys [
15]. As a non-protein amino acid,
l-citrulline is rarely found in food, but is highly concentrated in watermelon [
16]. The concentration of
l-citrulline in watermelon grown in the United States can range from 1.6 to 3.5 g/kg of fresh watermelon [
16,
17,
18]. As such, consumption of approximately 1–1.5 kg/day (2.2–3.3 lbs/day) of fresh watermelon would be needed to achieve the minimum effective dose of
l-citrulline (3 g/day) and 3.3–5.0 kg/day (7.3–16.5 lbs/day) of fresh watermelon would be needed to achieve the maximum effective dose of
l-citrulline (10 g/day) [
16,
17,
18]. Given the growing evidence that endothelial dysfunction has its origins in deficient
l-arginine-NO metabolism and given the relative ineffectiveness of
l-arginine supplementation on NO metabolism, researchers have begun to explore the potential therapeutic benefits of
l-citrulline.
l-citrulline is typically supplemented using pharmaceutical/nutraceutical grade
l-citrulline,
l-citrulline conjugated with malate (1:1 ratio), or as watermelon extract.
l-citrulline, as with many other NO-boosting supplements, has received much interest for its potential cardiovascular and anti-hypertensive capabilities [
19]. Although recent reviews have eloquently reviewed the impact of citrulline supplementation in health and disease [
14,
20,
21], the present review focuses on
l-citrulline’s NO-dependent and NO-independent effects on cardiometabolic outcomes. Moreover, we specifically summarize current literature regarding the benefits of both pharmaceutical/nutraceutical grade
l-citrulline and watermelon supplements (juice, water, extract, etc.) on vascular and metabolic physiology and their potential therapeutic impact on cardiometabolic health. We also discuss recent studies that have begun to examine
l-citrulline’s direct and indirect effects on clinically relevant aspects of skeletal muscle and adipose tissue metabolism, which are key mediators for the development of cardiometabolic disorders.
3. Pharmacokinetics, Transport and Metabolism
Historically, researchers have considered
l-glutamine within enterocytes as the major precursor for the synthesis of
l-citrulline and subsequent release into systemic circulation [
27]. However, recent data suggest that
l-glutamine’s contribution to
l-citrulline biosynthesis may be overestimated [
28]. On the other hand, there is evidence that enterocytes take up orally ingested
l-citrulline and effectively transport the neutral amino acid through the gastrointestinal tract to the portal circulation, likely using the Na
+-dependent, neutral amino acid, including the ASC or B
0,+-amino acid transporters located in the enterocytes within the jejunum and ileum [
15,
29,
30]. Quantitatively,
l-citrulline is not extracted by the gastrointestinal tract or liver (net uptake~0) [
31], which likely facilitates greater down-stream production of NO through the recycling of
l-citrulline to the NO precursor
l-arginine (
Figure 1). In contrast, orally ingested
l-arginine is subject to moderate-to-high rates of first-pass extraction both in intestine and liver, likely due to their high expression of arginase [
32,
33,
34], increasing arginine catabolism and limiting systemic increases in circulating levels following its ingestion [
8].
To date only a few studies have systematically investigated the pharmacokinetics of
l-citrulline supplementation [
11,
12]. Following oral
l-citrulline ingestion, circulating
l-arginine concentrations peak after ~1–2 h [
11,
12]. As has been shown for both
l-arginine and
l-ornithine, circulating concentrations of
l-citrulline return to baseline within 8 h [
11]). The higher activity and bioavailability of
l-citrulline, compared to
l-arginine, is due to several factors. For example, 0.75 grams of
l-citrulline ingested twice daily (1.5 g total) increased the
l-arginine area under the curve to a similar degree as ingesting 1.6 g of
l-arginine twice daily (3.2 g total) (271 vs. 289 μmol·h·L
−1) [
12]. Orally ingested
l-citrulline is absorbed by the enterocytes of the small intestine. However, the lack of gastrointestinal distress from
l-citrulline compared to
l-arginine supplementation may suggest that
l-citrulline uptake utilizes a differing transport system.
l-arginine is mainly transported across the intestinal membrane through Na+-independent cationic amino acid transporters (CAT-1, 2 and 3) [
35].
l-citrulline transport has been demonstrated in enterocytes, macrophages, glial cells, and aortic smooth muscle, with the highest K
m (4.1 ± 0.9 mM) reported in the enterocytes [
19,
30,
36]. While a specific
l-citrulline transporter has not been identified, the B
0-transporters have been suggested to play a role in the Na
+ dependent transport of
l-citrulline across the enterocytes [
30].
Clinical dose ranging and tolerability studies have also been conducted for
l-citrulline supplementation. One such human study demonstrated a tolerance of up to 15 g
l-citrulline per day in healthy volunteers [
11]. By comparison, high-doses of
l-arginine (~13 g) can induce significant gastrointestinal complications [
9,
21]. However, at 15 g doses of
l-citrulline, a lower fractional absorption rate and plasma retention of
l-citrulline was observed, potentially due to saturation of its transporters (e.g., ASC or B
0,+-amino acid transporters) or reduced renal conversion of
l-citrulline to
l-arginine. As such, the authors suggested a dose of 10 g
l-citrulline for clinical use [
11]. However, for increasing circulating
l-arginine concentrations, doses of
l-citrulline as low as 3 g have been shown to be effective [
12]. Thus, the minimum effective dose is ~3 g/day, whereas the maximal effective dose may be as high as 10 g/day.
The metabolism of orally ingested
l-citrulline is mainly confined to the biosynthesis of
l-arginine. Researchers previously thought that most dietary
l-citrulline was synthesized via the consumption of
l-glutamine through a transamination reaction in the enterocyte of the gastrointestinal tract [
37]. However, a recent study using a labeled 2,3,3,4,4 [
2H
5] glutamine tracer provided evidence that the contribution of
l-glutamine to
l-citrulline may be rather modest [
28]. Since
l-citrulline metabolism in the liver is somewhat compartmentalized to the urea cycle, exogenous
l-citrulline typically bypasses hepatic metabolism. Circulating
l-citrulline, released from the gut, is absorbed by the proximal tubular cells of the kidney [
38].
l-Citrulline is rapidly acted upon by cytosolic arginosuccinate synthase and converted into arginosuccinate which is then converted into
l-arginine by arginosuccinate lyase (see
Figure 1). This partial urea cycle meets the demand of the body’s
l-arginine requirement. De novo synthesis of
l-arginine from
l-citrulline is essential for downregulating urea formation in the liver during periods of low protein intake to increase nitrogen retention [
39].
Another potential source of
l-citrulline is its synthesis via the NO cycle (
Figure 2).
l-arginine is required for NO formation via eNOS, iNOS, and nNOS. Hydrolysis of the intermediate nitrosoarginine from
l-arginine yields NO and
l-citrulline [
15]. This is particularly useful in endothelial cells via eNOS [
40,
41] and activated macrophages via iNOS [
42] to sustain
l-citrulline as a precursor to
l-arginine to produce NO [
36] (
Figure 1). However, the recycling of
l-citrulline to
l-arginine does not appear as viable in cell types that have suboptimal uptake of
l-citrulline from plasma, such as aortic smooth muscle cells. Indeed, aortic smooth muscle cells take up
l-citrulline at a relatively slow rate compared to
l-arginine, due in part to its transport through a low affinity (K
m 1.6 mM) transporter [
36]. Consistent with this notion, Hattori and colleagues reported that physiologically high concentrations of
l-citrulline are necessary to maximally stimulate iNOS activity in cultured smooth muscle cells [
43]. Under inflammatory conditions (Lipopolysaccharides and Interferon-γ stimulation),
l-arginine itself is capable of inducing NOS via its increased transport.
8. Adipose Tissue and Lipolysis Effects of l-citrulline
The
l-Arginine-NO pathway is highly involved in the breakdown and oxidation of fatty acids in adipose tissue, as well as differentiation of adipocytes [
160]. Obesity is associated with increased expression of iNOS, which may increase NO synthesis and contribute to metabolic dysregulation in adipocytes [
161]. iNOS appears to be a critical factor in regulating cytokine (tumor necrosis factor-alpha/TNF) induced lipolysis that occurs during chronic inflammation [
162]. Interestingly, selective iNOS inhibition increases TNF induced lipolysis [
162]. A more global investigation of NO-mediated lipolysis demonstrates that specific NO donors (nitrosothiols) can increase basal lipolysis [
163] but inhibit isoproterenol (beta-adrenergic) induced lipolysis [
164,
165]. These studies suggest there is a regulatory loop related to the availability of NO and its role in basal or beta-adrenergic stimulated lipolysis.
l-arginine supplementation has been shown to increase NO synthesis as well as isoproterenol stimulated lipolysis and fatty acid oxidation in the adipose tissue of ZDF rats [
166]. To date, only a few experiments have examined the impact of
l-citrulline on lipolysis [
167,
168,
169]. One study has demonstrated that a 24 h treatment of
l-citrulline (2.5 mmol/L) was effective at increasing basal lipolysis, reducing glyceroneogenesis, and increasing palmitate oxidation (
14C-palmitate) in white adipose tissue explants [
168]. The authors of this study reported increased expression of macrophage markers (f4/80 and CD68) and inflammatory cytokines (IL-6 and TNF) in the high fat diet (HFD) condition. It is well established that the chronic inflammation present during diet-induced obesity is characterized by the infiltration and alteration of macrophage populations in adipose tissue. Therefore, the lipolytic effects of
l-citrulline reported ex vivo could be mediated by the NO produced by adipose tissue macrophages and not the adipocytes.
Treatment of adipose explants with
l-citrulline has also been shown to increase the expression of UCP1, PPAR and PGC1-α in young (2–4 months), but not old (25 months) rats [
167]. The observation that
l-citrulline increases lipolysis and fatty acid oxidation while reducing re-esterification is somewhat unexpected. During lipolysis, there is a change in cellular energy demand that results in altered AMPK phosphorylation. In fact, AMPK levels are induced during lipolysis; however, this response is largely due to acylation of fatty acids (i.e. re-esterification) [
170]. Therefore, it would be counterintuitive that
l-citrulline would reduce re-esterification and increase energy consumption (fatty acid oxidation). A more plausible explanation for increased energy consumption observed with
l-citrulline treatment is the increased mitochondrial uncoupling that has been reported [
167]. A cell autonomous role for
l-citrulline in the adipocyte has yet to be explored.
9. Summary and Future Directions
Increasing numbers of studies now suggest that pharmaceutical/nutraceutical grade l-citrulline and watermelon extract supplementation can increase the bioavailability of l-arginine and subsequently lead to elevations in NO synthesis. Moreover, accumulating data suggest that short-term l-citrulline supplementation can reduce peripheral and central (aortic) blood pressures in pre- and hypertensive adults. There is also evidence that l-citrulline supplementation, when compared to l-arginine, attenuates blood pressure reactivity to acute sympathetic stimulation (e.g., cold pressor and isometric/intermittent handgrip exercise) and pre-clinical (animal) evidence that it may protect against acute endothelial dysfunction. The improvement in these indicators of vascular health and resilience, and the lack of adverse side effects make l-citrulline an attractive non-pharmaceutical agent for populations at heightened cardiometabolic risk. From a metabolic standpoint, l-citrulline has been shown to increase mitochondrial biogenesis and MPS in skeletal muscle, while pre-clinical data suggests that adipose tissue lipolysis is improved with l-citrulline. Thus, l-citrulline supplementation represents an attractive non-pharmacological approach for increasing NO bioavailability, which may have the potential to counteract many of the age- and/or lifestyle-related diseases currently plaguing our society.
A major limitation of
l-citrulline research to date is the short-term nature of most of the intervention periods of study. Long-term studies (>6 months) investigating the effects of
l-arginine supplementation have demonstrated a concerning lack of safety and efficacy with respect to improvements in blood pressure regulation [
171,
172]. Basic mechanistic studies in human umbilical endothelial cells have shown that repeated exposure to
l-arginine promotes oxidative stress by increased superoxide formation and reduced eNOS protein expression [
173]. It is thus plausible that prolonged exposure to
l-arginine promotes cellular tolerance and maladaptation. The safety and efficacy of long-term
l-citrulline supplementation therefore requires further investigation. Finally, except for pharmacokinetic studies, most
l-citrulline supplementation studies fail to report the plasma/serum concentrations that were achieved.
In conclusion,
l-citrulline is a unique amino acid that exerts its effects on cardiometabolic health via direct and indirect pathways. The variety of applications for which
l-citrulline has been utilized underscores the importance of
l-citrulline in vascular health, protein metabolism, and lipid metabolism. However, the direct role of
l-citrulline, outside of its function as a precursor of
l-arginine, is still not well characterized. The interaction of
l-citrulline with other pharmaceutical drugs for the treatment of hypertension, atherosclerosis, insulin resistance, T2D, and cardiovascular disease should also be investigated, as some of these drugs have been shown to affect citrulline metabolism [
13,
174,
175].