Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc þ /À mice, such as the well-characterized Apc Min/ þ model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc D14/ þ mice to that of the classical Apc Min/ þ . The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation D14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated b-catenin, overexpressed the b-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a b-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc D14/ þ model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.

Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), L: -leucine (1.35 g/kg, LEU, n = 10), L: -citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [(13)C] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P < 0.05). Administration of amino acids (NEAA, LEU or CIT) completely abolished this decrease (NEAA, CIT, LEU vs. Fed, NS). Food deprivation affected the phosphorylation status of the mTORC1 pathway and AMP kinase (Saline vs. Fed, P < 0.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEU > CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.

Citrulline treatment in male aged rats favorably modulates body composition and protects against lipid oxidation and, thus, emerges as an interesting candidate to help prevent the aging process.

(CIT) is an endogenous amino acid produced by the intestine. Recent literature has consistently shown CIT to be an activator of muscle protein synthesis (MPS). However, the underlying mechanism is still unknown. Our working hypothesis was that CIT might regulate muscle homeostasis directly through the mTORC1/PI3K/MAPK pathways. Because CIT undergoes both interorgan and intraorgan trafficking and metabolism, we combined three approaches: in vivo, ex vivo, and in vitro. Using a model of malnourished aged rats, CIT supplementation activated the phosphorylation of S6K1 and 4E-BP1 in muscle. Interestingly, the increase in S6K1 phosphorylation was positively correlated (P Ͻ 0.05) with plasma CIT concentration. In a model of isolated incubated skeletal muscle from malnourished rats, CIT enhanced MPS (from 30 to 80% CIT vs. Ctrl, P Ͻ 0.05), and the CIT effect was abolished in the presence of wortmannin, rapamycin, and PD-98059. In vitro, on myotubes in culture, CIT led to a 2.5-fold increase in S6K1 phosphorylation and a 1.5-fold increase in 4E-BP1 phosphorylation. Both rapamycin and PD-98059 inhibited the CIT effect on S6K1, whereas only LY-294002 inhibited the CIT effect on both S6K1 and 4E-BP1. These findings show that CIT is a signaling agent for muscle homeostasis, suggesting a new role of the intestine in muscle mass control. eukaryotic initiation factor 4E-binding protein 1; mitogen-activated protein kinase; phosphatidylinositol 3-kinase; muscle; myotube; amino acids; protein synthesis; mammalian target of rapamycin CITRULLINE (CIT) is a nonprotein amino acid. It takes its name from the watermelon (citrullus vulgaris). CIT is known mainly as an intermediary of ureagenesis in periportal hepatocytes (24) and at the whole body level is produced almost exclusively by enterocytes (4). Intestinal CIT production is controlled largely by dietary protein supply (37). CIT has also emerged as an important regulator of nitrogen homeostasis in both humans and animals, as reviewed recently (6). For example, in pioneering work, we used a model of aged malnourished rats to demonstrate that CIT-enriched diet stimulated muscle protein synthesis (MPS) (ϩ80%), and led to a net muscle protein gain (ϩ20%) (10, 33). Interestingly, this metabolic effect was accompanied by muscle histological change and an increase in maximum strength as well as increased traction in treated animals (11). Similar results were obtained in healthy aged rats, in which CIT increased muscle protein content (28). This was also found in other situations such as fasting or caloric restriction in adults rats (22,38). Finally, our experimental work showing the ability of CIT to modulate MPS was confirmed in humans; in a crossover trial, Jourdan et al. (20) showed that CIT administration to healthy volunteers fed a hypoprotein diet increased MPS compared with a nonessential amino acid mixture. Thus all these studies confirm the ability of CIT to modulate MPS in vivo, but no work has yet determined the underlying mechanisms of CIT action (an increase in nitrogen...

Low calorie diets are designed to reduce body weight and fat mass, but they also lead to a detrimental loss of lean body mass, which is an important problem for overweight people trying to lose weight. In this context, a specific dietary intervention that preserves muscle mass in people following a slimming regime would be of great benefit. Leucine (LEU) and Citrulline (CIT) are known to stimulate muscle protein synthesis (MPS) in post-prandial and post-absorptive state, respectively. This makes them interesting bioactive components to test in the context of dietary restriction. We tested the concept of combining LEU and CIT in adult female rats. We postulated that the sequential administration of LEU (mixed in chow) and CIT (given in drinking water before a rest period) could be beneficial for preservation of muscle function during food restriction. Sixty female rats (22 weeks old) were randomized into six groups: one group fed ad libitum with a standard diet (C) and five food-restricted groups (60 % of spontaneous intake for 2 weeks) receiving a standard diet (R group), a CIT-supplemented diet (0.2 or 1 g/kg/day, CIT0.2 group and CIT1 group, respectively), a LEU-supplemented diet (1.0 g/kg/day) or a CIT + LEU-supplemented diet (CIT + LEU 1.0 g/kg/day each). At the end of the experiment, body composition, muscle contractile properties and muscle protein synthesis (MPS) rate were studied in the tibialis anterior muscle. Dietary restriction tended to decrease MPS (R: 2.5 ± 0.2 vs. C: 3.4 ± 0.4 %/day, p = 0.06) and decrease muscle strength (R: 3,045 ± 663 vs. C: 5,650 ± 661 A.U., p = 0.03). Only CIT administration (1 g/kg) was able to restore MPS (CIT1: 3.4 ± 0.3 vs. R: 2.5 ± 0.2 %/day, p = 0.05) and increase muscle maximum tetanic force (CIT1: 441 ± 15 vs. R: 392 ± 22 g, p = 0.05) and muscle strength (CIT1: 4,259 ± 478 vs. R: 3,045 ± 663 A.U., p = 0.05). LEU had no effect and CIT + LEU supplementation had few effects, limited to adipose mass and fatigue force. The results of this study highlight the ability of CIT alone to preserve muscle function during dietary restriction. Surprisingly, LEU antagonized some effects of CIT. The mechanisms involved in this antagonistic effect warrant further study.

Citrulline (Cit) actions on muscle metabolism remain unclear. Those latter were investigated using a proteomic approach on Tibialis muscles from male Sprague-Dawley rats. At 23 months of age, rats were either fed ad libitum (AL group) or subjected to dietary restriction for 12 weeks. At the end of the restriction period, one group of rats was euthanized (R group) and two groups were refed for one week with a standard diet supplemented with nonessential amino acids group or Cit (CIT group). Results of the proteomic approach were validated using targeted Western blot analysis and assessment of gene expression of the related genes. Maximal activities of the key enzymes involved in mitochondrial functioning were also determined. Cit supplementation results in a significant increase in the protein expression of the main myofibrillar constituents and of a few enzymes involved in glycogenolysis and glycolysis (CIT vs. AL and R, p < 0.05). Conversely, the expression of oxidative enzymes from Krebs cycle and mitochondrial respiratory chain was significantly decreased (CIT vs. AL, p < 0.05). However, maximal activities of key enzymes of mitochondrial metabolism were not significantly affected, except for complex 1 which presented an increased activity (CIT vs. AL and R, p < 0.05). In conclusion, Cit supplementation increases expression of the main myofibrillar proteins and seems to induce a switch in muscle energy metabolism, from aerobia toward anaerobia.

Ornithine aminotransferase (OAT) is a reversible enzyme expressed mainly in the liver, kidney and intestine. OAT controls the interconversion of ornithine into glutamate semi-aldehyde, and is therefore involved in the metabolism of arginine and glutamine which play a major role in N homeostasis. We hypothesised that OAT could be a limiting step in glutamine -arginine interconversion. To study the contribution of the OAT enzyme in amino acid metabolism, transgenic mice that specifically overexpress human OAT in the liver, kidneys and intestine were generated. The transgene expression was analysed by in situ hybridisation and real-time PCR. Tissue (liver, jejunum and kidney) OAT activity, and plasma and tissue (liver and jejunum) amino acid concentrations were measured. Transgenic male mice exhibited higher OAT activity in the liver (25 (SEM 4) v. 11 (SEM 1) nmol/min per mg protein for wild-type (WT) mice; P, 0·05) but there were no differences in kinetic parameters (i.e. K m and maximum rate of reaction (V max )) between WT and transgenic animals. OAT overexpression decreased plasma and liver ornithine concentrations but did not affect glutamine or arginine homeostasis. There was an inverse relationship between ornithine levels and OAT activity. We conclude that OAT overexpression has only limited metabolic effects, probably due to the reversible nature of the enzyme. Moreover, these metabolic modifications had no effect on phenotype. Ornithine: Arginine: GlutamineOrnithine aminotransferase (OAT; L-ornithine 2-oxo acid aminotransferase; EC 2.6.1.13) is a pyridoxal-5 0 -phosphate-dependent mitochondrial matrix aminotransferase that catalyses the interconversion of ornithine into glutamate semi-aldehyde, which is in spontaneous equilibrium with its cyclic tautomer, pyrroline-5-carboxylate (P5C). In contrast to other aminotransferases, this reaction enables the reversible transfer of the ornithine v-amino group. It depends on two tyrosine residues leading to the interaction with ornithine and the protection of the v-amino group against hydrolysis (1) .OAT is localised at a crossing between two important metabolisms, with arginine and polyamine metabolism on one side and glutamate and proline metabolism on the other (Fig. 1).Although OAT is expressed constitutively in many tissues, it is mainly found in the liver, kidney (at higher levels in females) and the small intestine (2,3) where its response to hormones and variations in dietary protein intake is subject to complex regulation mechanisms (2,4,5) . The reaction is directed towards glutamate semi-aldehyde synthesis in the liver and kidneys and towards ornithine synthesis in the small intestine. Hepatic OAT expression is restricted to perivenous hepatocytes (4) and co-localised with glutamine synthetase expression (6) , which suggests that OAT plays a major role in N homeostasis. In the kidney, Levillain et al. (7) showed that OAT is distributed along the whole nephron, but its activity is higher in proximal tubules than in distal tubules. In the intestine, OAT is...

In this rat HI model, arginine appears to be safe, contributes to a large extent to the immunomodulatory effects of the IED, and seems to limit enterobacterial translocation and dissemination more efficiently alone than in an IED.