The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.

Citrulline (Cit, C6H13N3O3), which is a ubiquitous amino acid in mammals, is strongly related to arginine. Citrulline metabolism in mammals is divided into two fields: free citrulline and citrullinated proteins. Free citrulline metabolism involves three key enzymes: NO synthase (NOS) and ornithine carbamoyltransferase (OCT) which produce citrulline, and argininosuccinate synthetase (ASS) that converts it into argininosuccinate. The tissue distribution of these enzymes distinguishes three "orthogonal" metabolic pathways for citrulline. Firstly, in the liver, citrulline is locally synthesized by OCT and metabolized by ASS for urea production. Secondly, in most of the tissues producing NO, citrulline is recycled into arginine via ASS to increase arginine availability for NO production. Thirdly, citrulline is synthesized in the gut from glutamine (with OCT), released into the blood and converted back into arginine in the kidneys (by ASS); in this pathway, circulating citrulline is in fact a masked form of arginine to avoid liver captation. Each of these pathways has related pathologies and, even more interestingly, citrulline could potentially be used to monitor or treat some of these pathologies. Citrulline has long been administered in the treatment of inherited urea cycle disorders, and recent studies suggest that citrulline may be used to control the production of NO. Recently, citrulline was demonstrated as a potentially useful marker of short bowel function in a wide range of pathologies. One of the most promising research directions deals with the administration of citrulline as a more efficient alternative to arginine, especially against underlying splanchnic sequestration of amino acids. Protein citrullination results from post-translational modification of arginine; that occurs mainly in keratinization-related proteins and myelins, and insufficiencies in this citrullination occur in some auto-immune diseases such as rheumatoid arthritis, psoriasis or multiple sclerosis.

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Previous experimental studies have highlighted that citrulline (CIT) could be a promising pharmaconutrient. However, its pharmacokinetic characteristics and tolerance to loading have not been studied to date. The objective was to characterise the plasma kinetics of CIT in a multiple-dosing study design and to assess the effect of CIT intake on the concentrations of other plasma amino acids (AA). The effects of CIT loading on anabolic hormones were also determined. Eight fasting healthy males underwent four separate oral loading tests (2, 5, 10 or 15 g CIT) in random order. Blood was drawn ten times over an 8 h period for measurement of plasma AA, insulin and growth hormone (Gh). Urine samples were collected before CIT administration and over the next 24 h. None of the subjects experienced side effects whatever the CIT dose. Concerning AA, only CIT, ornithine (ORN) and arginine (ARG) plasma concentrations were affected (maximum concentration 146 (SEM 8) to 303 (SEM 11) mmol/l (ARG) and 81 (SEM 4) to 179 (SEM 10) mmol/l (ORN); time to reach maximum concentration 1·17 (SEM 0·26) to 2·29 (SEM 0·20) h (ARG) and 1·38 (SEM 0·25) to 1·79 (SEM 0·11) h (ORN) according to CIT dose). Even at high doses, urinary excretion of CIT remained low (,5 %). Plasma insulin and Gh were not affected by CIT administration. Short-term CIT administration is safe and well-tolerated. CIT is a potent precursor of ARG. However, at the highest doses, CIT accumulated in plasma while plasma ARG levels increased less than expected. This may be due to saturation of the renal conversion of CIT into ARG.Pharmacokinetics: Arginine: Ornithine: Insulin: Growth hormone Citrulline (CIT) is an amino acid whose name is derived from Citrullus vulgaris (commonly known as watermelon) from which it was first isolated in the 1930 s (for a recent review, see Curis et al.( 1) ). Until recently, CIT had not attracted much interest in the scientific community because (i) it is a non-proteic amino acid and (ii) it was considered only as an intermediate of the urea cycle (2) . In the early 1980 s, Windmueller & Spaeth (3) demonstrated that the small intestine releases large amounts of CIT which is mainly taken up by the kidney (of note, CIT is not taken up by the liver) and, in turn, arginine (ARG) was released in amounts equivalent to about 75 % of the CIT taken up. Then, Castillo et al. (4,5) were the first to characterise the CIT and ARG in vivo kinetics at the whole-body level in healthy subjects. These findings allowed the suggestion of an ARG -CIT -ARG inter-organ cycle which can be seen (6) as a mechanism for protecting dietary ARG from excessive liver degradation (because CIT is not taken up by the liver (7) ) and thus maintaining protein homeostasis. Concurrently, it was also demonstrated that CIT was the endproduct of the NO synthase reaction (8) .The role of the intestine as a key regulator of CIT production was further emphasised in situations where intestinal function is altered (i.e. short-bowel syndrome, coeliac disease, radiation-induced intestinal ...

Protein energy malnutrition is common in the elderly, especially in hospitalized patients. The development of strategies designed to correct such malnutrition is essential. Our working hypothesis was that poor response to nutrition with advancing age might be related to splanchnic sequestration of amino acids, which implies that fewer amino acids reach the systemic circulation. Administration of citrulline, which is not taken up by the liver, can offer a means of increasing whole body nitrogen availability and, hence, improve nutritional status. Thirty old (19 mo) rats were submitted to dietary restriction (50% of food intake) for 12 wk. They were randomized into three groups: 10 rats (R group) were killed and 20 others refed (90% of food intake) for 1 wk with a standard diet (NEAA group) or a citrulline-supplemented diet (Cit group). Before being killed, the rats were injected with [(13)C]valine, and the absolute protein synthesis rate (ASR) was measured in the tibialis using the flooding-dose method. When the rats were killed, the tibialis was removed for protein content analysis. Blood was sampled for amino acid and insulin analysis. The standard diet did not have any effect on protein synthesis or on the protein content in the muscle. Citrulline supplementation led to higher protein synthesis and protein content in muscle (117 +/- 9, 120 +/- 14, and 163 +/- 4 mg/organ for protein content in R, NEAA, and Cit groups, P < 0.05). The ASR were 0.30 +/- 0.04, 0.31 +/- 0.04, and 0.56 +/- 0.10 mg/h in the three groups, respectively (R and NEAA vs. Cit, P < 0.05). Insulinemia was significantly higher in the Cit group. For the first time, a realistic therapeutic approach is proposed to improve muscle protein content in muscle in frail state related to malnutrition in aging.

Citrulline possesses a highly specific metabolism that bypasses splanchnic extraction because it is not used by the intestine or taken up by the liver. The administration of citrulline may be used to deliver available nitrogen for protein homeostasis in peripheral tissues and as an arginine precursor synthesized de novo in the kidneys and endothelial and immune cells. Fresh research has shown that citrulline is efficiently transported across the intestinal luminal membrane by a set of transporters belonging to the B⁰,⁺, L, and b⁰,⁺ systems. Several pharmacokinetic studies have confirmed that citrulline is efficiently absorbed when administered orally. Oral citrulline could be used to deliver arginine to the systemic circulation or as a protein anabolic agent in specific clinical situations, because recent data have suggested that citrulline, although not a component of proteins, stimulates protein synthesis in skeletal muscle through the mammalian target of rapamycin signaling pathway. Hence, citrulline could play a pivotal role in maintaining protein homeostasis and is a promising pharmaconutrient in nutritional support strategies for malnourished patients, especially in aging and sarcopenia.

Citrulline (Cit) is a non-essential amino acid whose metabolic properties were largely ignored until the last decade when it began to emerge as a highly promising nutrient with many regulatory properties, with a key role in nitrogen homeostasis. Because Cit is not taken up by the liver, its synthesis from arginine, glutamine, ornithine and proline in the intestine prevents the hepatic uptake of the two first amino acids which activate the urea cycle and so prevents amino acid catabolism. This sparing effect may have positive spin-off for muscle via increased protein synthesis, protein content and functionality. However, the mechanisms of action of Cit are not fully known, even if preliminary data suggest an implication of mTOR pathway. Further exploration is needed to gain a complete overview of the role of Cit in the control of nitrogen homeostasis.

Citrulline (CIT) is an amino acid that is not involved in protein synthesis but that is tightly linked to arginine (ARG) metabolism. CIT displays a very specific metabolism: In the 1980s, Windmuller demonstrated that the small intestine releases CIT, which is mainly taken up by the kidney and metabolized into ARG. Because CIT is not taken up by the liver, this ARG-CIT-ARG cycle can be seen as a means of protecting dietary ARG from liver degradation and of sustaining protein homeostasis. These observations have led to the concept that plasma CIT concentration would be a good marker of intestinal failure in short bowel syndrome. Hence, in massive intestinal resection, citrullinemia is greatly reduced, and this is proportional to the severity of the intestinal disease. This concept was then extended to other situations in which the intestinal function is compromised. The data strongly suggest that CIT may be a conditionally essential amino acid in situations where the intestinal function is compromised. Recent data support this idea. Thus, CIT supplementation is able to restore nitrogen balance, generate large amounts of ARG in rats with short bowel syndrome, and increase muscle protein content (+20%) as well as muscle protein synthesis (+90%) in elderly malnourished rats. Finally, recent data indicate that CIT per se could be able to stimulate muscle protein synthesis. Hence, CIT could play a pivotal role in maintaining protein homeostasis, and the determination of the underlying mechanisms involved in its action should be important for the development of new nutritional strategies in malnourished patients with compromised intestinal functions.