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L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism.

Ham DJ, Gleeson BG, Chee A, Baum DM, Caldow MK, Lynch GS, Koopman R - PLoS ONE (2015)

Bottom Line: The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models.The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase.In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform.

View Article: PubMed Central - PubMed

Affiliation: Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H2O2) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.

No MeSH data available.


Related in: MedlinePlus

L-citrulline protects C2C12 muscle myotubes from wasting.Myotube diameter after 5 h incubation in HEPES buffered saline (HBS) with increasing concentrations of L-citrulline or isomolar concentrations of L-alanine (B) and representative images (20× objective) at the optimal dose of 2.5 mM (A). Myotube diameter for cells incubated in differentiation media (DM) or: HBS for 5 h (C) or; serum free (SF) media for 48 h (D). In each model, cells were treated with vehicle (PBS), 2.5 mM L-arginine or 2.5 mM L-citrulline (n = 5–8 per group). Values are means ± SE. For (B) comparisons were made using a two-way ANOVA (treatment × dose) with Fisher’s LSD post-hoc test. Significant differences are displayed where appropriate. For (C-D), Comparisons were made using a one-way ANOVA with Tukey’s post hoc test. Different letters denote significant differences (P<0.05) between groups, where a>b>c.
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pone.0141572.g001: L-citrulline protects C2C12 muscle myotubes from wasting.Myotube diameter after 5 h incubation in HEPES buffered saline (HBS) with increasing concentrations of L-citrulline or isomolar concentrations of L-alanine (B) and representative images (20× objective) at the optimal dose of 2.5 mM (A). Myotube diameter for cells incubated in differentiation media (DM) or: HBS for 5 h (C) or; serum free (SF) media for 48 h (D). In each model, cells were treated with vehicle (PBS), 2.5 mM L-arginine or 2.5 mM L-citrulline (n = 5–8 per group). Values are means ± SE. For (B) comparisons were made using a two-way ANOVA (treatment × dose) with Fisher’s LSD post-hoc test. Significant differences are displayed where appropriate. For (C-D), Comparisons were made using a one-way ANOVA with Tukey’s post hoc test. Different letters denote significant differences (P<0.05) between groups, where a>b>c.

Mentions: Murine C2C12 myoblasts (Cryosite distribution, NSW, Australia) were plated in 6 or 12 well plates and cultured in DMEM (Life Technologies, Australia) containing 10% (v/v) fetal calf serum (Life Technologies) and antimycotic antibiotic solution (100 unit/ml penicillin/streptomycin, Life Technologies) at 37°C in an atmosphere of 5% CO2. Upon confluency, the media was changed to DMEM containing 2% (v/v) horse serum (Life Technologies) for 5 days to promote formation of mature multinucleated myotubes [14]. To induce wasting, cells were: 1) washed once in HEPES buffered saline (HBS) and then incubated in HBS for 5 h, as previously described [14]; 2) washed once in serum free DMEM (Life Technologies, Australia) and then incubated in serum free DMEM for 48 h (SF); 3) incubated in DMEM containing 1 μg.ml-1 LPS for 24 h; or 4) incubated in DMEM containing 25 μM H2O2 for 24 h. L-arginine, leucine and lysine free DMEM was purchased from Life Technologies (Australia) and appropriate concentrations of lysine, and L-arginine or leucine were added back into the media to obtain L-arginine-free DMEM and leucine-free DMEM. All amino acids were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Based on an initial dose-ranging experiment (Fig 1), 2.5 mM of L-citrulline or control amino acids (L-alanine and L-arginine) was chosen for all future experiments.


L-Citrulline Protects Skeletal Muscle Cells from Cachectic Stimuli through an iNOS-Dependent Mechanism.

Ham DJ, Gleeson BG, Chee A, Baum DM, Caldow MK, Lynch GS, Koopman R - PLoS ONE (2015)

L-citrulline protects C2C12 muscle myotubes from wasting.Myotube diameter after 5 h incubation in HEPES buffered saline (HBS) with increasing concentrations of L-citrulline or isomolar concentrations of L-alanine (B) and representative images (20× objective) at the optimal dose of 2.5 mM (A). Myotube diameter for cells incubated in differentiation media (DM) or: HBS for 5 h (C) or; serum free (SF) media for 48 h (D). In each model, cells were treated with vehicle (PBS), 2.5 mM L-arginine or 2.5 mM L-citrulline (n = 5–8 per group). Values are means ± SE. For (B) comparisons were made using a two-way ANOVA (treatment × dose) with Fisher’s LSD post-hoc test. Significant differences are displayed where appropriate. For (C-D), Comparisons were made using a one-way ANOVA with Tukey’s post hoc test. Different letters denote significant differences (P<0.05) between groups, where a>b>c.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4625972&req=5

pone.0141572.g001: L-citrulline protects C2C12 muscle myotubes from wasting.Myotube diameter after 5 h incubation in HEPES buffered saline (HBS) with increasing concentrations of L-citrulline or isomolar concentrations of L-alanine (B) and representative images (20× objective) at the optimal dose of 2.5 mM (A). Myotube diameter for cells incubated in differentiation media (DM) or: HBS for 5 h (C) or; serum free (SF) media for 48 h (D). In each model, cells were treated with vehicle (PBS), 2.5 mM L-arginine or 2.5 mM L-citrulline (n = 5–8 per group). Values are means ± SE. For (B) comparisons were made using a two-way ANOVA (treatment × dose) with Fisher’s LSD post-hoc test. Significant differences are displayed where appropriate. For (C-D), Comparisons were made using a one-way ANOVA with Tukey’s post hoc test. Different letters denote significant differences (P<0.05) between groups, where a>b>c.
Mentions: Murine C2C12 myoblasts (Cryosite distribution, NSW, Australia) were plated in 6 or 12 well plates and cultured in DMEM (Life Technologies, Australia) containing 10% (v/v) fetal calf serum (Life Technologies) and antimycotic antibiotic solution (100 unit/ml penicillin/streptomycin, Life Technologies) at 37°C in an atmosphere of 5% CO2. Upon confluency, the media was changed to DMEM containing 2% (v/v) horse serum (Life Technologies) for 5 days to promote formation of mature multinucleated myotubes [14]. To induce wasting, cells were: 1) washed once in HEPES buffered saline (HBS) and then incubated in HBS for 5 h, as previously described [14]; 2) washed once in serum free DMEM (Life Technologies, Australia) and then incubated in serum free DMEM for 48 h (SF); 3) incubated in DMEM containing 1 μg.ml-1 LPS for 24 h; or 4) incubated in DMEM containing 25 μM H2O2 for 24 h. L-arginine, leucine and lysine free DMEM was purchased from Life Technologies (Australia) and appropriate concentrations of lysine, and L-arginine or leucine were added back into the media to obtain L-arginine-free DMEM and leucine-free DMEM. All amino acids were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Based on an initial dose-ranging experiment (Fig 1), 2.5 mM of L-citrulline or control amino acids (L-alanine and L-arginine) was chosen for all future experiments.

Bottom Line: The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models.The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase.In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform.

View Article: PubMed Central - PubMed

Affiliation: Basic and Clinical Myology Laboratory, Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
Dietary L-citrulline is thought to modulate muscle protein turnover by increasing L-arginine availability. To date, the direct effects of increased L-citrulline concentrations in muscle have been completely neglected. Therefore, we determined the role of L-citrulline in regulating cell size during catabolic conditions by depriving mature C2C12 myotubes of growth factors (serum free; SF) or growth factors and nutrients (HEPES buffered saline; HBS). Cells were treated with L-citrulline or equimolar concentrations of L-arginine (positive control) or L-alanine (negative control) and changes in cell size and protein turnover were assessed. In myotubes incubated in HBS or SF media, L-citrulline improved rates of protein synthesis (HBS: +63%, SF: +37%) and myotube diameter (HBS: +18%, SF: +29%). L-citrulline treatment substantially increased iNOS mRNA expression (SF: 350%, HBS: 750%). The general NOS inhibitor L-NAME and the iNOS specific inhibitor aminoguanidine prevented these effects in both models. Depriving myotubes in SF media of L-arginine or L-leucine, exacerbated wasting which was not attenuated by L-citrulline. The increased iNOS mRNA expression was temporally associated with increases in mRNA of the endogenous antioxidants SOD1, SOD3 and catalase. Furthermore, L-citrulline prevented inflammation (LPS) and oxidative stress (H2O2) induced muscle cell wasting. In conclusion, we demonstrate a novel direct protective effect of L-citrulline on skeletal muscle cell size independent of L-arginine that is mediated through induction of the inducible NOS (iNOS) isoform. This discovery of a nutritional modulator of iNOS mRNA expression in skeletal muscle cells could have substantial implications for the treatment of muscle wasting conditions.

No MeSH data available.


Related in: MedlinePlus