Servane Le Plénier

Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?

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, nxa0=xa010) or food deprived for 18xa0h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, nxa0=xa010), l-leucine (1.35xa0g/kg, LEU, nxa0=xa010), l-citrulline (1.80xa0g/kg CIT, nxa0=xa010) or isonitrogenous non-essential amino acids (NEAA, nxa0=xa010). After gavage, the rats were injected with a flooding dose of [13C] valine to determine MPS. The rats were killed 50xa0min 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, Pxa0<xa00.05). 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, Pxa0<xa00.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEUxa0>xa0CIT). 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.

Overexpression of ornithine aminotransferase: consequences on amino acid homeostasis.

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 microg protein for wild-type (WT) mice; P < 0.05) but there were no differences in kinetic parameters (i.e. Km and maximum rate of reaction (Vmax)) 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.

Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats

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.

Effect of an immune-enhancing diet on lymphocyte in head-injured rats: What is the role of arginine?

ObjectiveThe benefit of immune-enhancing diets (IEDs) in the intensive care unit remains controversial. Considering their complexity, the role of each component, in particular arginine (Arg), in their properties is largely unknown. The aim of this study was to determine the role of arginine in the immunomodulatory effects of an IED (Crucial®) in head-injured rats.DesignThirty-four rats were randomized into five groups: AL (ad libitum), HI (head-injured), HI-STD (HI + standard enteral nutrition, EN), HI-STD-Arg (HI + standard EN + Arg in equimolar concentration to Arg in IED), and HI-IED (HI + IED). These isocaloric and isonitrogenous diets were administered over 4u202fdays. After death, the thymus was removed and weighed. The density of CD25, CD4 and CD8 on lymphocytes from blood and from Peyer patches was evaluated. Mesenteric lymph nodes, liver and spleen were cultured for analysis of enterobacterial translocation and dissemination.Measurements and resultsHI induced an atrophy of the thymus which was not corrected by the standard diet (HI 0.27u202f±u202f0.03, HI-STD 0.35u202f±u202f0.03 vs. AL 0.49u202f±u202f0.02u202fg; pu202f<u202f0.05). However, the standard diet supplemented with arginine limited the thymic atrophy and the IED restored thymus weight. CD25 density and interleukin-2 production were increased only in the HI-STD-Arg and HI-IED groups (pu202f<u202f0.05). Head injury induced enterobacterial translocation and dissemination which were blunted only in the HI-STD-Arg group (pu202f<u202f0.05).ConclusionsIn this rat HI model, arginine appears to be safe, contributes to axa0large extent to the immunomodulatory effects of the IED, and seems to limit enterobacterial translocation and dissemination more efficiently alone than in an IED.

Citrulline Supplementation Induces Changes in Body Composition and Limits Age-Related Metabolic Changes in Healthy Male Rats

BACKGROUNDnAging is associated with profound metabolic disturbances, and citrulline may be of use to limit them.nnnOBJECTIVEnThe aim of this work was to evaluate the long-term effect of citrulline supplementation on metabolism in healthy aged rats.nnnMETHODSnTwenty-month-old male rats were randomly assigned to be fed (ad libitum) for 12 wk with either a citrulline-enriched diet (1 gu2009⋅u2009kg(-1)u2009⋅u2009 d(-1)) or a standard diet [rendered isonitrogenous by addition of nonessential amino acids (NEAAs)]. Motor activity and muscle strength were measured, body composition was assessed, and muscle metabolism (protein structure, mitochondrial exploration, and transductional factors) and lipid metabolism (lipoprotein composition and sensitivity to oxidative stress) were explored.nnnRESULTSnCompared with the NEAA-treated group, citrulline supplementation was associated with lower mortality (0% vs. 20%; P = 0.05), 9% higher lean body mass (P < 0.05), and 13% lower fat mass (P < 0.05). Compared with the NEAA-treated group, citrulline-treated rats had greater muscle mass (+14-48% depending on type of muscle; P < 0.05 for tibialis, gastrocnemius, and plantaris). Susceptibility to oxidation of lipoproteins, as measured by the maximal concentration of 7-ketocholesterol after copper-induced VLDL and LDL oxidation, was lower in citrulline-treated rats than in NEAA-treated rats (187 ± 8 μmol/L vs. 243 ± 7 μmol/L; P = 0.0005).nnnCONCLUSIONSnCitrulline 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.

Combining citrulline with atorvastatin preserves glucose homeostasis in a murine model of diet-induced obesity

NO is a crucial regulator of energy and lipid metabolism, whose homeostasis is compromised during obesity. Combination of citrulline and atorvastatin potentiated NO production in vitro. Here we have assessed the effects of this combination in mice with diet‐induced obesity (DIO).

Citrulline directly modulates muscle protein synthesis via the PI3K/MAPK/4E-BP1 pathway in a malnourished state: evidence from in vivo, ex vivo, and in vitro studies

Citrulline (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 < 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 < 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.

Citrulline stimulates locomotor activity in aged rats: Implication of the dopaminergic pathway

OBJECTIVESnA citrulline (CIT)-enriched diet improves locomotor activity in aged rats, but the underlying mechanism is unknown. The aim of this study was to determine the effect of CIT administration on locomotor activity and dopamine activity in healthy aged rats.nnnMETHODSnSixty adult (3-mo-old) and aged (20-mo-old) rats were divided into four groups (nxa0=xa015 each) stratified by age (adult versus old) and diet (control versus CIT; i.e., Ad-Control, Ad-CIT, Old-Control, Old-CIT) and fed for 4xa0d on either a CIT-enriched diet (5xa0g/kg daily; Ad-CIT and Old-CIT) or an isonitrogeneous control diet (Ad-Control and Old-Control). Locomotor activity was evaluated in a Y-maze. On day 5, animals were sacrificed and brain (striatum) was removed to determine total and phosphorylated forms of tyrosine hydroxylase (TH) by immunohistochemistry.nnnRESULTSnCIT restored locomotor activity in aged rats (arm visits: Old-CIT 28xa0±xa01 versus Old-Control 23xa0±xa01; Pxa0<xa00.05), associated with an increase in total TH (Old-CIT 668xa0±xa027 versus Old-Control 529xa0±xa022; Pxa0<xa00.05) and phosphorylated forms of TH (Old-CIT 1012xa0±xa039 versus Old-Control 589xa0±xa069; Pxa0<xa00.05).nnnCONCLUSIONnIn aged rats, CIT is able to stimulate locomotor activity via the dopaminergic pathway.

Evaluation of a new concept of immune-enhancing diet in a model of head-injured rat with infectious complications: A proof of concept study

Immune-enhancing diet (IED) utilization in critically ill septic patients is still debated. A new concept of IED has been proposed combining extra glutamine sequentially with either antioxidants or other amino acids, in order to match patient requirements according to their response to injury. We evaluated whether this new IED elicits a more favorable response to stress when compared with two existing IEDs both enriched in arginine but with different levels of anti-oxidants, in a validated rat model combining head injury (HI) and infectious complications. Forty-eight HI rats were randomized into four groups (nxa0=xa011-13 per group) to receive, for 4 days, standard enteral nutrition (S), one of the two existing IEDs (IED1, IED2), or the new IED (IED3; providing glutamine and antioxidants for two days and glutamine and specific amino acids for two days). Two days after HI, the rats received an enteral bolus of luminescent Escherichia coli Xen14 to induce infection, and bacterial dissemination was evaluated. Body weight (BW) was recorded daily. Four days after HI, animals were euthanized; blood was sampled; organs were weighed; cumulated nitrogen balance (CNB) and nitrogen efficiency were determined. IED3 was more efficient than IED1 and IED2 in improving BW recovery from D3 (D3 vs. D1, pxa0<xa00.05) after HI. It significantly improved CNB and net protein utilization (IED3 vs. S, IED1, IED2, pxa0<xa00.05). An IED with sequential administration of anti-oxidants and glutamine may be better suited to meeting nutritional requirements in severe catabolic states.

Nitric oxide production by peritoneal macrophages from aged rats: A short term and direct modulation by citrulline

Citrulline has anti-inflammatory properties and exerts beneficial effects on various impaired functions in aging. However, there are few data on citrulline action on immune function in aged populations. The objective of the study was to evaluate citrulline ability, after inxa0vivo and inxa0vitro administration, to modulate macrophage functions in aged rats and the possible pathways involved. Twenty-one-month-old Sprague-Dawley rats (nxa0=xa027) received a citrulline supplementation at 5xa0g/kg/d for 5xa0days, or an isonitrogenous diet, and peritoneal macrophages were cultured with or without LPS. In the inxa0vitro study, macrophages from 22-month-old rats (nxa0=xa016) were cultured with or without LPS, citrulline and inhibitors of different inflammatory pathways (nxa0=xa08/conditions). Nitric oxide (NO) and tumor necrosis factor α (TNFα) production were measured in both inxa0vivo and inxa0vitro studies. Citrulline decreased NO production variability by peritoneal macrophages after inxa0vivo administration (pxa0=xa00.0034) and downregulated NO production by 22% after inxa0vitro administration (95% CI: [6%; 35%]; pxa0=xa00.0394), without any direct effect on TNFα production. None of the transductional pathways explored seem to be involved. Citrulline slightly modulates NO production inxa0vivo and inxa0vitro, suggesting a possible action through modulation of arginine metabolism in macrophages rather than a direct transductional effect. The pleiotropic effects of citrulline in aging could be due, at least in part, to the anti-inflammatory effect of citrulline.