Outcomes (n = 6) are presented as means SEM. (p 0.05; One\way Anova + Bonferroni test). Table S1. Direct effect of citrulline (5 mM) on mitochondrial function Table S2. Direct effect of citrulline (5 mM) on mitochondrial respiration in permeabilized muscle fibers Table S3. Effects of citrulline or arginine (5 mM) on anaerobic glycolysis and \oxidation in amino acid/serum deficiency JCSM-10-919-s001.docx (981K) GUID:?5A272710-E968-49E6-92E2-31A027369AF8 Abstract Background Animal studies and clinical data support the interest of citrulline as a promising therapeutic for sarcopenia. Citrulline is known to stimulate muscle protein synthesis, but how it affects energy metabolism to support the highly energy\dependent protein synthesis machinery is poorly understood. Methods Here, we used myotubes derived from primary culture of mouse myoblasts to study the effect of citrulline on both energy metabolism and protein synthesis under different limiting conditions. Results When serum/amino acid deficiency or energy stress (mild uncoupling) were applied, citrulline stimulated muscle protein synthesis by +22% and +11%, respectively. Importantly, this increase was not associated with enhanced energy status (ATP/ADP ratio) or mitochondrial respiration. We further analysed the share of mitochondrial respiration and thus of generated ATP allocated to different metabolic pathways by using specific inhibitors. Our results indicate that addition of citrulline allocated an increased share of mitochondrially generated ATP to the protein synthesis machinery under conditions of both serum/amino acid deficiency (+28%) and energy stress (+21%). This reallocation was not because of reduced ATP supply to DNA synthesis or activities of sodium and calcium cycling ion pumps. Conclusions Under certain stress conditions, citrulline increases muscle protein synthesis by specifically reallocating mitochondrial fuel to the protein synthesis machinery. Because ATP/ADP ratios and thus Gibbs free energy of ATP hydrolysis remained globally constant, this reallocation may be linked to decreased activation energies of one or several ATP (and GTP)\consuming reactions involved in muscle protein synthesis. model, Le Plnier and muscles of two 4\week\old male mice as previously described.18 Experiments were performed on cells kept in culture for not more than 1 month (between 18C22 passages). Cells were plated at low density (100 cells/cm2) on 0.02% gelatin\coated dishes (cold water fish skin; Sigma\Aldrich, Saint\Quentin\Fallavier, France) and grown in complete medium composed of DMEM\Ham’s BQR695 F12 (Gibco, Invitrogen, Saint\Aubin, France), 2% Ultroser G (BiosepraPall Corporation, Saint\Germain\en\Laye, France), 20% foetal calf serum (Sigma\Aldrich), and antibiotic\antimycotic solution (10 000 U/mL penicillin G sodium, 10 000 g/mL streptomycin sulfate, and 25 g/mL amphotericin B; Gibco). To differentiate muscle cells into myotubes, myoblasts were plated on Matrigel\coated dishes (Dutscher, Brumath, France) in complete medium and, after 6 h adhesion, switched to DMEM\Ham’s F12 medium +2% horse serum (Sigma\Aldrich) at day 2. At day 5, myotubes were incubated either in amino acid\free and serum\free medium (Eurobio, Courtaboeuf, France) or in DMEM\Ham’s F12 medium +2% horse serum for 16 h. Myotubes were incubated under different conditions: (i) standard condition for 16 h BQR695 with no further addition (Ctrl+), or addition of leucine (LEU, 5 mM), CIT (5 mM), or ARG (5 mM). (ii) Serum\deprived and amino acid\deprived for 16 h with no further addition (AA/serum?), or addition of LEU (AA/serum? + LEU), CIT (AA/serum? + CIT), or ARG (AA/serum? + ARG). (iii) Standard condition for 16 h and addition of DNP, or further addition of LEU (DNP + LEU), CIT (DNP + CIT), or ARG (DNP + ARG). Before completion of the 16 h incubation period, the given amino acids (5 mM) were added for 90 min, and DNP (10 M) for 15 min (i.e. 60 min after addition of a given amino acid). LEU was used as a positive control (as LEU is known to be a strong activator of muscle protein synthesis) and ARG was used as a negative control (as ARG is not known to stimulate muscle protein synthesis and allows to evaluate the nitrogen load effect).7 Myotube protein synthesis and immunoblotting Myotube protein synthesis was quantified by the SUnSET method.19 Briefly, after the different treatments, puromycin (1 M) was added to the medium for 30 min. Cells were then collected, lysed in a RIPA buffer, and centrifuged at 3000 for 10 min at 4C, and the soluble protein fraction was collected. Immunoblotting of puromycin and analysis was then performed as previously described (quantification was made from the ratio puromycin\incorporated protein staining/Ponceau stain).20 Total BQR695 protein Ponceau staining of blots are illustrated in for 10 min at 4C, neutralized to pH 6C7 with 2 N KOH + 0.3 M MOPS, and.To differentiate muscle cells into myotubes, myoblasts were plated on Matrigel\coated dishes (Dutscher, Brumath, France) in complete medium and, after 6 h adhesion, switched to DMEM\Ham’s F12 medium +2% horse serum (Sigma\Aldrich) at day 2. on mitochondrial respiration in permeabilized muscle fibers Table S3. Effects of citrulline or arginine (5 mM) on anaerobic glycolysis and \oxidation in amino acid/serum deficiency JCSM-10-919-s001.docx (981K) GUID:?5A272710-E968-49E6-92E2-31A027369AF8 Abstract Background Animal studies and clinical data support the interest of citrulline as a promising therapeutic for sarcopenia. Citrulline is known to stimulate muscle protein synthesis, but how it affects energy metabolism to support the highly energy\dependent protein synthesis machinery is poorly understood. Methods Here, we used myotubes derived from primary culture of mouse myoblasts to study the effect of citrulline on both energy metabolism and protein synthesis under different limiting conditions. Results When serum/amino acid deficiency or energy stress (mild uncoupling) were applied, citrulline stimulated muscle protein synthesis by +22% and +11%, respectively. Importantly, this increase was not associated with enhanced energy status (ATP/ADP ratio) or mitochondrial respiration. We further analysed the share of mitochondrial respiration and thus of generated ATP allocated to different metabolic pathways by using specific inhibitors. Our results indicate that addition of citrulline allocated an increased share of mitochondrially generated ATP to the protein synthesis machinery under conditions of both serum/amino acid deficiency (+28%) and energy stress (+21%). This reallocation was not because of reduced ATP supply to DNA synthesis or activities of sodium and calcium cycling ion pumps. Conclusions Under certain stress conditions, citrulline increases muscle protein synthesis by specifically reallocating mitochondrial fuel to the protein synthesis machinery. Because ATP/ADP ratios and thus Gibbs free energy of ATP hydrolysis remained globally constant, this reallocation may be linked to decreased activation energies of one or several ATP (and GTP)\consuming reactions involved in muscle protein synthesis. model, Le Plnier and muscles of two 4\week\old male mice as previously described.18 Experiments were performed on cells kept in culture for not more than 1 month (between 18C22 passages). Cells were plated at low density (100 cells/cm2) on 0.02% gelatin\coated dishes (cold water fish skin; Sigma\Aldrich, BQR695 Saint\Quentin\Fallavier, France) and grown in complete medium composed of DMEM\Ham’s F12 (Gibco, Invitrogen, Saint\Aubin, France), 2% Ultroser G (BiosepraPall Corporation, Saint\Germain\en\Laye, France), 20% foetal calf serum (Sigma\Aldrich), and antibiotic\antimycotic solution (10 000 U/mL penicillin G sodium, 10 000 g/mL streptomycin sulfate, and 25 g/mL amphotericin B; Gibco). To differentiate muscle cells into myotubes, myoblasts were plated on Matrigel\coated dishes (Dutscher, Brumath, France) in complete medium and, after 6 h adhesion, switched to DMEM\Ham’s F12 medium +2% horse serum (Sigma\Aldrich) at day 2. At day 5, myotubes were incubated either BQR695 in amino acid\free and serum\free medium (Eurobio, Courtaboeuf, France) or in DMEM\Ham’s F12 medium +2% horse serum for 16 h. Myotubes were incubated under different conditions: (i) standard condition for 16 h with no further addition (Ctrl+), or addition of leucine (LEU, 5 mM), CIT (5 mM), or ARG (5 mM). (ii) Serum\deprived and amino acid\deprived for 16 h with no further addition (AA/serum?), or addition of LEU (AA/serum? + LEU), CIT (AA/serum? + CIT), or ARG (AA/serum? + ARG). (iii) Standard condition for 16 h and addition of DNP, or further addition of LEU (DNP + LEU), CIT (DNP + CIT), or ARG (DNP + ARG). Before completion of the 16 h incubation period, the given amino acids (5 mM) were added for 90 min, and DNP (10 M) for 15 min (i.e. 60 min after addition of a given amino acid). LEU was used as a positive control (as LEU is known to be a strong activator of muscle protein synthesis) and ARG was used as a negative control (as ARG is not known to stimulate muscle protein synthesis and allows Rabbit polyclonal to INPP5A to evaluate the nitrogen load effect).7 Myotube protein synthesis and immunoblotting Myotube protein synthesis was quantified by the SUnSET method.19 Briefly, after the different treatments, puromycin (1 M) was added to the medium for 30 min. Cells were then collected, lysed in a RIPA buffer, and centrifuged at 3000 for 10 min at 4C, and the soluble protein fraction was collected. Immunoblotting of puromycin and analysis was then performed as previously described (quantification was made from the ratio puromycin\incorporated protein.
Outcomes (n = 6) are presented as means SEM
