Amino acids, especially leucine, potently stimulate protein synthesis and reduce protein breakdown in healthy skeletal muscle and as a result have received considerable attention as potential treatments for muscle wasting. However, the normal anabolic response to amino acids is impaired during muscle-wasting conditions. Although the exact mechanisms of this anabolic resistance are unclear, inflammation and ROS are believed to play a central role. The nonessential amino acid glycine has anti-inflammatory and antioxidant properties and preserves muscle mass in calorie-restricted and tumor-bearing mice. We hypothesized that glycine would restore the normal muscle anabolic response to amino acids under inflammatory conditions. Relative rates of basal and leucine-stimulated protein synthesis were measured using SUnSET methodology 4 h after an injection of 1 mg/kg lipopolysaccharide (LPS). Whereas leucine failed to stimulate muscle protein synthesis in LPS-treated mice pretreated with l-alanine (isonitrogenous control), leucine robustly stimulated protein synthesis (+51%) in mice pretreated with 1 g/kg glycine. The improvement in leucine-stimulated protein synthesis was accompanied by a higher phosphorylation status of mTOR, S6, and 4E-BP1 compared with l-alanine-treated controls. Despite its known anti-inflammatory action in inflammatory cells, glycine did not alter the skeletal muscle inflammatory response to LPS in vivo or in vitro but markedly reduced DHE staining intensity, a marker of oxidative stress, in muscle cross-sections and attenuated LPS-induced wasting in C 2 C 12 myotubes. Our observations in male C57BL/6 mice suggest that glycine may represent a promising nutritional intervention for the attenuation of skeletal muscle wasting.

amino acids, especially leucine, potently stimulate protein synthesis and reduce protein breakdown in healthy skeletal muscle (34). As a result, leucine-rich amino acid supplementation has been proposed as a potential treatment for muscle-wasting conditions. However, to date, no long-term, isocaloric, placebo-controlled human study has shown beneficial effects of leucine supplementation on skeletal muscle mass or function (25). Despite many studies showing enhanced protein synthesis following intake of leucine-rich supplements in healthy skeletal muscle, leucine administration does not necessarily enhance protein synthesis under muscle-wasting conditions. This reduced (or complete lack of) protein synthetic response to amino acids has been termed “anabolic resistance” (46) and has been observed in many muscle-wasting conditions, including immobilization (19), aging (9), cancer cachexia (54, 55), and sepsis (17). The perturbation in normal amino acid-stimulated protein synthesis is considered a major contributor to the loss of muscle mass in a number of muscle-wasting conditions, and strategies to address anabolic resistance are important (9, 19, 40, 46).

Although the exact mechanisms responsible for anabolic resistance are unclear, the overproduction of proinflammatory cytokines and reactive oxygen species (ROS) associated with many muscle-wasting conditions is believed to play a central role (3, 9, 28). Inflammation and ROS inhibit the activity of the mechanistic target of rapamycin complex 1 (mTORC1) and decrease the phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1) (17). An ∼80% reduction in leucine-induced phosphorylation of mTOR and its substrates 4E-BP1 and S6K1 and the associated inhibition of leucine-stimulated protein synthesis have all been observed in skeletal muscle cells during inflammation (36). The anabolic resistance to leucine in inflammatory conditions is consistent with the disappointing results of clinical trials attempting to counteract muscle wasting in trauma patients using branched-chain amino acid-rich nutritional supplements (10). Given the failure of dietary amino acid administration as a strategy to overcome anabolic resistance and attenuate muscle wasting, we hypothesized that a more promising strategy to combat muscle wasting is to focus on restoring the normal anabolic response to amino acids.

The nonessential amino acid glycine is often considered biologically neutral and sometimes used as an isonitrogenous control in supplementation (feeding) studies. However, evidence that glycine has profound inhibitory effects on inflammatory cell activation has accumulated (56). Increased circulating concentrations of glycine can directly activate glycine-gated chloride (Cl−) channels (GlyR) expressed in inflammatory cells, such as macrophages (23), and reduce the production of proinflammatory cytokines (30). Consistent with this proposed mechanism, glycine administration blunts the increases in serum TNFα after hemorrhagic shock and LPS challenge (49). Recently, in a mouse model of cancer cachexia, we showed that glycine administration reduced the loss of muscle mass and strength by 50%, preserved food intake, and reduced the cancer-induced stimulation of muscle inflammation, macrophage infiltration, and production of reactive oxygen species (28). Glycine treatment in these mice also blunted the cancer-induced increase in skeletal muscle atrogin-1 mRNA expression and the reduction in eukaryotic initiation factor 3f (eIF3f) protein expression, a factor that mediates translation initiation, suggesting that glycine helps to maintain the muscle's protein synthetic machinery (28). As such, dietary glycine could represent an effective anti-inflammatory agent to restore the normal anabolic response to nutrition and counteract inflammation-induced muscle wasting.

We tested the potential of glycine to restore basal protein metabolism and the normal anabolic response to amino acids using a well-established model of acute inflammation in skeletal muscle. Inflammation was induced in C57BL/6 mice by administering 1 mg/kg lipopolysaccharide (LPS) via intraperitoneal injection, and the relative rates of basal and leucine-stimulated protein synthesis were measured 4 h later. Although it is well known that the clinical course and progression of disease in murine LPS models is much faster than in humans, the LPS model represents a well-recognized, simple, and reproducible endotoxemic model of acute inflammation (18, 45). Moreover, this protocol has been shown previously to reduce basal protein synthesis and completely abolish the protein-synthetic response to leucine (37). We hypothesized that pretreatment with 1 g/kg glycine would reduce inflammation and preserve the anabolic response to leucine (0.5 g/kg) in LPS-treated mice.

METHODS Animals. All experiments were approved by the Animal Ethics Committee of the University of Melbourne and conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes as stipulated by the National Health and Medical Research Council (Australia). Twenty-week-old male C57BL/6 mice with an average body mass of 37 ± 1 g (range: 30–45 g) were allocated into one of three experimental groups: a saline-treated control group (n = 13), a group injected with LPS [intraperitoneal (ip)] and pretreated with alanine (n = 15), and a group injected with LPS and pretreated with glycine (n = 15). Glyine or alanine (1 g/kg) was administered ip 30 min prior to LPS administration. Based on our observations of a consistent lack of effect of l-alanine supplementation on skeletal muscle across multiple in vitro (24, 26) and in vivo (6, 27, 28) studies, we used l-alanine as our amino acid control. Groups were matched for body mass, and food was removed 1 h before the administration of LPS (or saline in the control group). Each group was further separated into “basal” (n = 6–7) and “fed” (n = 7–8) groups. The three fed groups received 0.5 g/kg leucine dissolved in saline via ip injection 3 h after LPS injection and 1 h before the mice were euthanized, whereas basal groups received an injection of saline at the same time point. All mice were obtained from the Animal Resources Centre (Canning Vale, Western Australia, Australia) and housed in the Biological Research Facility at the University of Melbourne under a 12:12 h light-dark cycle. Animals were monitored throughout the experimental period (5 h) for adverse signs and symptoms. Protein synthesis. To determine the relative rate of protein synthesis, SUnSET methodology was utilized as described previously (22). Briefly, puromycin was administered to mice (ip) at a dose of 0.04 μmol/kg 30 min after an injection of 0.5 g/kg leucine or an equivalent volume of saline. Exactly 30 min after puromycin administration, mice were euthanized via cervical dislocation and the tibialis anterior (TA) and quadriceps (QUAD) muscles rapidly dissected, blotted on filter paper, and frozen (<1 min). The right TA muscle was mounted in embedding medium and frozen in thawing isopentane, whereas the QUAD muscle was frozen directly in liquid nitrogen and stored at −80°C for subsequent analyses. Real-time RT-PCR. Tnfα, Il6, Ccl2, Ccl5, Socs3, Trim63 (Murf1), Fbxo32 (atrogin-1), Bnip3, and Gapdh mRNA expression were determined by real-time quantitative PCR. Total RNA was extracted from 10 to 20 mg of muscle using a commercially available kit according to the manufacturer's instructions (RNeasy Fibrous Tissue Mini Kit; Qiagen). RNA quality and concentration were determined using the Nanodrop 2000 (Thermo-Fisher Scientific). First-strand cDNA was generated using 100 ng of total RNA using the SuperScript VILO cDNA Synthesis Kit according to the manufacturer's instructions (Life Technologies). Quantitative PCR was performed in duplicate using the Bio-Rad CFX384 PCR system (Bio-Rad Laboratories, Gladesville, New South Wales, Australia) with reaction volumes of 10 μl containing Sso Advanced Universal SYBR Green Supermix (Bio-Rad Laboratories), forward and reverse primers, and cDNA template (2 ng/μl). For in vivo analysis, gene expression was quantified by normalizing raw C q values to the cDNA content of each sample and expressed as arbitrary units (AU) (26). Normalizing C q values to cDNA content, which is not influenced by experimental intervention, is increasingly used as an alternative to reference genes such as Gapdh, β-actin, and 18S, which have proved unsuitable for normalization in muscle samples due to their variability under different stress conditions (e.g., exercise and denervation) (39, 41, 43). For in vitro analysis, gene expression was normalized to Gapdh, which was not altered during the experiment. Primers were designed using the NCBI primer Basic Local Alignment Search Tool (BLAST), and specificity was confirmed using the BLAST. A melting point dissociation curve was generated by the PCR instrument for all PCR products to confirm the presence of a single amplified product. Primers are listed in Table 1. Table 1. Primer sequences used Gene GeneBank Accession No. Sense Primer (5′-3′) Antisense Primer (5′-3′) Trim63 NM_009066 AGGTGTCAGCGAAAAGCAGT CCTCCTTTGTCCTCTTGCTG Fbxo32 AF_441120 GTTTTCAGCAGGCCAAGAAG TTGCCAGAGAACACGCTATG Bnip3 NM_009760 GGGTTTTCCCCAAAGGAATA TGACCACCCAAGGTAATGGT Tnfa NM_013693 GGCCTTCCTACCTTCAGACC AGCAAAAGAGGAGGCAACAA Il6 NM_031168 CCGGAGAGGAGACTTCACAG TCCACGATTTCCCAGAGAAC Socs3 NM_007707 GCTGGCCAAAGAAATAACCA AGCTCACCAGCCTCATCTGT Gapdh NM_001289726 AACTTTGGCATTGTGGAAGG ACACATTGGGGGTAGGAACA Ccl2 NM_011333 AGATGCAGTTAACGCCCCAC GACCCATTCCTTCTTGGGGT Ccl5 NM_013653 CCTCACCATATGGCTCGGAC ACGACTGCAAGATTGGAGCA Skeletal muscle histology. Serial sections (5 μm) were cut transversely through the TA muscle using a refrigerated (−20°C) cryostat (CTI Cryostat; IEC, Needham Heights, MA). We employed SUnSET methodology combined with fiber-type staining to determine mean myofiber type-specific puromycin staining intensity, as described previously (21, 22, 32). Briefly, after fixation in −20°C acetone, to mask the endogenous IgG background signal, slides were blocked using a Mouse on Mouse blocking kit (Vector Laboratories, Burlingame, CA) for 1 h in PBS containing 0.5% BSA and 0.5% Triton X-100. Sections were then reacted with antibodies raised against laminin (1:50), puromycin (1:1,000; Clone 12D10; Merck Millipore, Kilsyth, Victoria, Australia), and MyHCIIa (N2.261, 1:25; developed by Prof. Helen M. Blau; obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa Department of Biology, Iowa City, IA). Following 3 × 5 min washes in PBS, slides were incubated in secondary antibodies for laminin (goat anti-rabbit IgG1 Alexa 488, 1:200), puromycin (goat anti-mouse IgG2a Alexa 555, 1:500), and N2.261 (goat anti-mouse IgG1 Alexa 350, 1:80). For measurements of superoxide radicals, slides were incubated for 30 min at 37°C in PBS containing 2 μM dihydroethidiumbromide (DHE; 0.1% DMSO). Digital images of stained sections were obtained using an upright microscope with a camera (Axio Imager D1; Carl Zeiss, Wrek Göttingen, Germany) controlled by AxioVision AC software (AxioVision AC Rel. 4.8.2; Carl Zeiss Imaging Solutions, Wrek Göttingen; Germany). Three images per sample taken in equivalent locations were quantified using AxioVision 4.8.2 software, as described previously (32). To allow accurate comparisons between groups for puromycin and DHE, one sample from each of the six groups (CON, LPS + ALA, and LPS + GLY; basal and leucine stimulated) plus a puromycin-free negative control were mounted on each slide. All slides were stained at the same time, and care was taken to ensure that each slide was incubated for the same time. Images were taken for all samples in a single sitting, and all microscope settings and exposure times were kept identical throughout image collection. The staining intensity was corrected using the background fluorescence signal from a negative control sample. Immunoblotting. Quadriceps muscle samples (∼20 mg) were homogenized using ice-cold lysis buffer (20 mM Tris·HCl, 5 mM ethylenediamine-tetraacetic acid, 10 mM Na-pyrophosphate, 100 mM NaF, 2 mM Na 3 VO 4 , and 1 mM PMSF) containing protease and phosphatase inhibitors (Sigma-Aldrich, Castle Hill, New South Wales, Australia). Protein (30 μg) was separated by 4–15% SDS-PAGE using Criterion TGX Stain-Free Precast Gels (Bio-Rad Laboratories). Western blots for phosphorylated mTOR (Ser2448, no. 5536), S6 (Ser235/236, no. 4858), 4E-BP1 (Thr37/46; no. 2855), ERK1/2 (Thr202/Tyr204, no. 9101), Akt (Ser473, no. 9271), STAT3 (Tyr705, no. 9131), eEF2 (T56, 1:1,000 dilution; Eurogentec, Seraing, Belgium), p38 MAPK (Thr180/Tyr182), total mTOR (no. 2972), S6 (no. 2217), 4E-BP1 (no. 9644), ERK1/2 (no. 9102), Akt (no. 9272), STAT3 (no. 9132), eEF2 (no. 2332), light chain 3B (LC3B; no. 2775) (1:1,000 dilution unless stated otherwise; all Cell Signaling Technology, Beverly, MA), eIF3F (no. 600-401-934; Rockland Immunochemicals, Limerick, PA), suppressor of cytokine signaling 3 (SOCS3; clone 1B2; Millipore, Bayswater, Victoria, Australia), and IL-6 (GTX27737; GeneTex, Irvine, CA) were performed as described previously (6). For 4-hydroxynonenal (4HNE) (ab46545; Abcam, Melbourne, Victoria, Australia), gels were transferred to a nitrocellulose membrane, blocked in 5% milk-TBST, and incubated overnight at 4°C in 5% milk-TBST containing 4HNE (1:1,000). For SUnSET protein synthesis measurements, Western blots were performed as described previously (22). Briefly, after blocking, membranes were incubated overnight at 4°C with anti-puromycin (Clone 12D10; Millipore) and diluted 1:5,000 in 1% BSA-TBST. The following day, membranes were washed for 3 × 5 min in TBST and then incubated for 1 h at RT in horseradish peroxidise-conjugated secondary antibodies (goat anti-mouse IgG Fc 2a) diluted 1:50,000 in 5% milk-TBST. For each membrane, the standard curve for serial dilutions of a single puromycin-positive sample with a single puromycin-negative muscle sample (100, 75, 50, 25, and 0%) was used to normalize changes in staining density. The volume density of puromycin staining was quantified for the entire lane and divided by total protein volume density using Criterion TGX Stain-Free Precast Gels (Bio-Rad Laboratories) for the same lane. The 0% puromycin standard was used to subtract background staining before relative rates of protein synthesis were calculated using the quantified volume density for the five dilution standards. The same five standards were run on each membrane to allow normalization between membranes. An r2 of ≥0.98 was observed for each standard curve (Fig. 1, A and B). Data were subsequently normalized to 1 for ease of visualization. All other Western blot data were normalized to total protein. Fig. 1.Glycine counteracts LPS-induced anabolic resistance to leucine. Standard curve for serial dilutions of representative Western blot (A) and puromycin-positive with puromycin-negative protein sample (B). Quantification of puromycin-labeled proteins (protein synthesis) corrected using representative Western blots (C) and puromycin standard curve (D). Significant differences (P < 0.05) and trends (P ≤ 0.10) are shown where appropriate. Main effects for treatment or leucine are reported in the top left corner of each graph where appropriate. **Trend or significant difference at P < 0.05, P < 0.01, or P < 0.001 between basal and leucine-stimulated conditions for the respective group. CON, saline (control); ALA, alanine; GLY, glycine. Download figureDownload PowerPoint

Statistical analyses. All values were expressed as means ± SE. Data were normalized to the appropriate control group for ease of visualization, unless otherwise stated. Data were tested for normality and homogeneity of variance using a Shapiro-Wilk and Levene's test, respectively. Two-way ANOVAs (treatment × basal/fed) were used to compare between groups where appropriate, whereas one-way ANOVAs were used for all other comparisons. Tukey's post hoc test was used to determine significant main effects, whereas Fisher's least significant difference test was used to compare between individual groups for interactions. For transparency, both significant differences (P < 0.05) and trends (P < 0.1) are reported where appropriate.

RESULTS Glycine counteracts LPS-induced anabolic resistance to leucine. Under basal conditions, protein synthesis (as measured by Western blot) tended to be lower after LPS administration than in CON (ALA: −21 ± 6%; GLY: −24 ± 5%, P < 0.10; Fig. 1, C and D). Immunohistochemical analysis of mean fiber type-specific puromycin staining intensity revealed that the tendency for lower basal protein synthesis with LPS treatment was localized to type IIb/x fibers (ALA: P = 0.09; GLY: P < 0.05), whereas puromycin staining intensity in IIa fibers was unaffected by LPS (Fig. 2, A and B). Type IIa fibers made up ∼15% of fibers analyzed, and this proportion was not different between groups (Fig. 2C). Fig. 2.LPS attenuates leucine-stimulated puromycin staining intensity in both type IIa and IIb skeletal muscle fibers. Representative individual images of laminin, N2.261 (MyHCIIa), and puromycin (protein synthesis) and the 3 images merged (A), quantification of fiber type-specific puromycin staining intensity (B), and %fiber type (C) in glycine (GLY) and alanine (ALA; control amino acid) treated mice 4 h after an injection of either saline (CON) or LPS (1 mg/kg) and 1 h after an injection of either saline (basal) or leucine. Significant differences (P < 0.05) and trends (P ≤ 0.10) are shown where appropriate. Main effects for treatment or leucine are reported in the top left corner of each graph where appropriate. # and *Trend or significant difference at P < 0.05, P < 0.01, or P < 0.001 between basal and leucine-stimulated conditions for the respective group. Download figureDownload PowerPoint

The tendency for a lower basal relative rate of protein synthesis was associated with lower activation of some (but not all) signaling proteins involved in the regulation of protein synthesis (Fig. 3). In the Akt-mTORC1 pathway, we observed a lower basal ratio of phosphorylated to total Akt (p-Akt/Akt, ALA: −52 ± 7%; GLY: 50 ± 7%, P < 0.001) and p-4E-BP1 to 4E-BP1 (ALA: −37 ± 8%; GLY: −51 ± 7.3%, P < 0.01) but not p-mTOR/mTOR or p-S6/S6 after LPS administration. Elongation factor eEF2 phosphorylation was unchanged by LPS (Fig. 3). Fig. 3.Glycine improves leucine-stimulated mammalian target of rapamycin (mTOR) complex 1 activation. Quantification of the ratio of phosphorylated to total protein for mTOR (A), S6 (B), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1; C), eukaryotic elongation factor 2 (eEF2; D), Akt (E), and ERK (F) and representative Western blots (G) in glycine (GLY)- and alanine (ALA; control amino acid)-treated mice 4 h after an injection of either saline (CON) or LPS (1 mg/kg) and 1 h after an injection of either saline (basal) or leucine. Significant differences (P < 0.05) and trends (P ≤ 0.10) are shown where appropriate. Main effects for treatment or leucine are reported in the top left corner of each graph where appropriate. Download figureDownload PowerPoint

In response to the administration of 0.5 g/kg leucine, protein synthesis (35 ± 12%, P < 0.01; Fig. 1), p-mTOR/mTOR (30 ± 6%), and p-S6/S6 (186 ± 43%) were robustly higher in the CON group, and there was a main effect for slightly higher p-Akt/Akt in all groups. In the LPS-treated ALA group, p-mTOR/mTOR, p-S6/S6, and p-4E-BP1/4E-BP1 were all higher under leucine-stimulated than basal conditions and were not different from values observed in leucine-stimulated CON mice (Fig. 3). However, leucine failed to stimulate protein synthesis in the ALA group, which was −32 ± 4% (P < 0.001; Fig. 1D) lower than in CON mice. In contrast, leucine-stimulated protein synthesis was 51 ± 9% higher than basal protein synthesis in LPS-treated GLY mice. However, leucine-stimulated protein synthesis in LPS-treated GLY mice was not significantly different from basal CON levels and tended to be lower than leucine-stimulated protein synthesis in CON mice (−15 ± 5%, P = 0.06). Compared with the ALA group, GLY treatment also improved the leucine-stimulated phosphorylation of mTOR, S6, and 4E-BP1 (Fig. 3). Interestingly, the improvement in leucine-stimulated mTORC1 signaling and protein synthesis in the GLY group was associated with a markedly higher phosphorylation status of ERK compared with all other groups (Fig. 1E). Using an immunohistochemical approach, mean puromycin staining intensity in both IIa (+43 ± 14%, P < 0.02) and IIb/x (+40 ± 16%, P = 0.09) fibers was higher following leucine administration in the CON group (Fig. 2). In contrast, leucine failed to improve mean puromycin staining intensity irrespective of fiber type in the ALA group. Compared with basal conditions in the GLY group, mean puromycin staining intensity under leucine-stimulated conditions was higher in type IIb (111 ± 59%, P < 0.05) but not type IIa fibers and still tended to be lower than under leucine-stimulated conditions in the CON group. Glycine reduces protein breakdown signaling following LPS under leucine-stimulated conditions. Four hours after an injection of 1 mg/kg LPS, the mRNA expression of genes involved in proteasomal muscle breakdown (i.e., atrogin-1 and MuRF1) was not markedly altered, although atrogin-1 (P < 0.01) mRNA was lower following leucine administration (Fig. 4, A and B). Despite the lack of effect of glycine on atrogin-1, higher levels of eIF3f, a key target of atrogin-1, were observed in the GLY group compared with the ALA group, with no effect of LPS or leucine treatment (Fig. 4C). GLY also protected the leucine-stimulated suppression of autophagic muscle protein breakdown signaling following LPS treatment. Bnip3 mRNA tended (P = 0.06) to be lower following leucine administration, and the GLY group was lower than CON (P < 0.05) and tended to be lower than ALA (P = 0.10). Furthermore, the ratio of lipidated to nonlipidated LC3B (LC3BII/LC3BI) was markedly higher in both ALA and GLY groups following LPS administration under basal conditions (Fig. 4E), indicating higher autophagosome formation. Compared with basal conditions, LC3BII/LC3BI tended to be lower under leucine-stimulated conditions in the CON group (P = 0.06) and was markedly lower in the GLY group (P < 0.001) but unaltered in the ALA group. Together, these data indicate a reduction in protein breakdown signaling under leucine-stimulated conditions in the GLY group. Fig. 4.Glycine restores the leucine-induced inhibition of autophagic signaling. mRNA expression of Fbxo32 (atrogin-1; A), Trim63 (MurF1; B), and Bnip3 (D) and quantification of protein expression for eukaryotic initiation factor 3f (eIF3f; C) and the ratio of LC3BII to LC3BI (E) and representative Western blots (F) in glycine (GLY)- and alanine (ALA; control amino acid)-treated mice 4 h after an injection of either saline (CON) or LPS (1 mg/kg) and 1 h after an injection of either saline (basal) or leucine. Significant differences (P < 0.05) and trends (P ≤ 0.10) are shown where appropriate. Main effects for treatment or leucine are reported in the top left corner of each graph where appropriate. # and ***Trend or a significant difference at P < 0.05, P < 0.01, or P < 0.001 between basal and leucine-stimulated conditions for the respective group. Download figureDownload PowerPoint

Glycine reduces oxidative stress but not inflammatory signaling in skeletal muscle. Compared with CON, DHE staining intensity was significantly higher following LPS injection in the ALA group, with no effect of leucine administration (Fig. 5, A and B). In contrast, DHE staining intensity was lower in the GLY group than both the CON and ALA groups, with no effect of leucine. An accumulation of 4HNE-labeled proteins was not observed 4 h after LPS administration and was not different between any groups (Fig. 5C). Interestingly, the phosphorylation status of p38 MAPK, a potential link between oxidative stress and impaired protein metabolism, was markedly higher (P < 0.01) in the ALA group after leucine administration than in any other group. Fig. 5.LPS promotes and glycine reduces oxidative stress in skeletal muscle. Representative images of dihydroethidiumbromide staining, a marker of reactive oxygen species (A), and quantification of staining intensity (B), quantification of 4HNE-labeled proteins (C), the ratio of phosphorylated to total 38 MAPK (D), and representative Western blots (E) for glycine (GLY)- and alanine (ALA; control amino acid)-treated mice 4 h after an injection of either saline (CON) or LPS (1 mg/kg) and 1 h after an injection of either saline (basal) or leucine. Significant differences (P < 0.05) and trends (P ≤ 0.10) are shown where appropriate. Main effects for treatment or leucine are reported in the top left corner of each graph where appropriate. Download figureDownload PowerPoint

Administration of LPS robustly increased the mRNA expression of Il-6 (∼80-fold), Tnfα (∼15-fold), Socs3 (∼35-fold), Ccl2 (∼65-fold), and Ccl5 (∼7-fold) and the phosphorylation status of STAT3 (Tyr705) in quadriceps muscle (Fig. 6, A–F). However, there was a tendency (P = 0.09) for a lower IL-6 protein concentration in the GLY group compared with the ALA group. Despite blunted DHE staining intensity in glycine-treated mice, inflammatory signaling was not different between the ALA and GLY groups under basal or leucine-stimulated conditions. Similarly, glycine treatment did not alter the inflammatory response to LPS in cultured C 2 C 12 skeletal muscle myotubes (Fig. 7, A–E). Unexpectedly, leucine administration in mice appeared to augment the inflammatory response to LPS, with a significant main effect for leucine observed for Tnfα and Socs3 and a trend for higher Il-6 (P = 0.11) and Ccl2 (P = 0.06) mRNA. Despite an unaltered inflammatory response, glycine attenuated the LPS-induced reduction in C 2 C 12 myotube diameter when cells were incubated in 1 μg/ml LPS for 24 h (Fig. 7, F and G). Fig. 6.Glycine does not reduce the inflammatory response to LPS in skeletal muscle. mRNA expression of Il-6 (A), Tnfα (B), suppressor of cytokine signaling 3 (Socs3; C), Ccl2 (D), and Ccl5 (E) in glycine (GLY)- and alanine (ALA)-treated mice 4 h after an injection of either saline (CON) or LPS (1 mg/kg) and 1 h after an injection of either saline (basal) or leucine and quantification of protein expression for IL-6 (G), SOCS3 (H), and the ratio of phosphorylated to total STAT3 (F) and representative Western blots (I). mRNA expression was normalized to cDNA content. Main effects for treatment or leucine are reported in the top left corner of each graph where appropriate. Download figureDownload PowerPoint

Fig. 7.Glycine preserves myotube diameter but does not reduce the inflammatory response to LPS in C 2 C 12 skeletal muscle cells. mRNA expression of Il-6 (A), Tnfα (B), Socs3 (C), Ccl2 (D), and Ccl5 (E) in mature 5-day-old myotubes treated with 2.5 mM glycine (GLY) or alanine (ALA) and then incubated in 1 μg/ml LPS for 0 (control) or 1, 2, or 4 h and representative images (F) and quantification (G) of myotube diameter after 24-h incubation in differentiation media treated with PBS (CON), LPS, and alanine (LPS + ALA) or LPS and glycine (LPS + GLY). mRNA expression was normalized to Gapdh expression. Different letters denote significant difference at the P < 0.05 level, where a > b > c and bc is not different from b or c. Download figureDownload PowerPoint



DISCUSSION The nonessential amino acid glycine improved mTORC1 signaling and effectively restored the protein synthetic response to leucine in skeletal muscles of LPS-treated mice. The improvement in protein metabolism was associated with a reduction in skeletal muscle ROS but did not alter skeletal muscle inflammatory signaling in vivo or in vitro. Glycine represents a promising nutritional intervention for the attenuation of skeletal muscle wasting. Glycine improves leucine-stimulated mTORC1 signaling and protein synthesis in LPS-treated mice. Consistent with previous observations in humans (52) and rats (36–38), we observed a tendency (P < 0.1) for lower basal protein synthesis 4 h after LPS administration in both ALA and GLY groups compared with saline-treated CON mice (Fig. 1). Using immunohistochemical techniques, a trend for reduced basal puromycin staining intensity was observed in type IIb/x but not IIa fibers (Fig. 2), as seen previously in rodents administered LPS (51). In line with this observation, the phosphorylation status of a number of positive regulators of protein synthesis (Akt and 4E-BP1; Fig. 3) was lower in mice administered LPS compared with saline-treated CON. No difference was observed between ALA and GLY groups. Leucine-stimulated protein synthesis was 35% higher (P < 0.01) than basal conditions in control mice. Importantly, the relative increase in leucine-stimulated protein synthetic rate we observed utilizing SunSET methodology is quantitatively similar to that reported following leucine or protein administration in rats and humans using labeled tracers (1, 33). In contrast, leucine administration failed to increase protein synthesis in the ALA group treated with LPS. The failure of leucine to stimulate skeletal muscle protein synthesis, even at higher doses, has been well demonstrated in rodent models of sepsis and endotoxemia (36–38). The most important finding in this study was that the protein synthetic response to leucine was restored with glycine treatment. Compared with basal conditions, protein synthesis was markedly higher following leucine stimulation (+51%, P < 0.01) in GLY-treated mice, although relative rates still tended to be lower than in leucine-stimulated CON mice (−15%, P = 0.06). In addition, leucine failed to increase mean puromycin staining intensity in any fiber type in the ALA group, whereas in GLY-treated mice exposed to LPS, leucine-stimulated mean puromycin staining intensity was higher in type IIb/x (P < 0.05) but not type IIa fibers (Fig. 2). Despite the impaired protein synthetic response to leucine in the ALA group, LPS failed to measurably impair the leucine-induced increase in mTORC1 signaling, which was not different from CON. The physiological significance of the mismatch between the LPS-induced changes in leucine-stimulated protein synthesis and mTORC1 signaling is unclear. However, in line with higher relative rates of protein synthesis, the phosphorylation states of mTOR, S6, and 4E-BP1 were significantly higher under leucine-stimulated conditions in the GLY group than in the ALA group. The relative rate of protein synthesis can also be altered at the elongation level through phosphorylation and inhibition of eEF2. However, we did not observe alteration of eEF2 phosphorylation by either LPS or GLY. The exact molecular mechanisms responsible for the protective effects of GLY on leucine-stimulated mTORC1 signaling and protein synthesis are unclear. Interestingly, leucine administered to GLY-treated mice increased the phosphorylation status of the extracellular signal-regulated kinase (ERK) by ∼300% compared with all other groups. ERK activation has been linked to mTORC1 signaling, as pharmacological activation of ERK activates mTORC1 signaling in rat L6 myotubes (47) and may go some way toward explaining the positive effect of glycine observed here. Glycine reduces markers of muscle protein breakdown. Although increased protein breakdown has been observed 4 h after LPS administration in humans (52), we did not observe a pronounced alteration in the mRNA expression of genes involved in the ubiquitin-proteasomal (i.e., atrogin-1 and MuRF1) or autophagic (Bnip3) pathways of muscle protein breakdown (Fig. 4, A–C). The expression of these genes in skeletal muscle in response to LPS is highly temporal, with increased expression reported after 12 h (11, 12, 48) but not before 6 h in rodents (11). Despite the lack of difference in atrogin-1 mRNA expression, we observed a treatment main effect for the protein expression of the eukaryotic initiation factor 3f (eIF3f), an important component of the protein synthetic machinery and major target of atrogin-1 (35), with higher expression in GLY- than in ALA-treated mice (P < 0.05). We also observed a markedly higher LC3BII to LC3BI protein ratio, a reliable marker of autophagosome number (31), following LPS in both ALA and GLY groups compared with the CON group under basal conditions. Under leucine-stimulated conditions, the LC3BII to LC3BI protein ratio was significantly lower than under basal conditions for both CON and GLY mice but not ALA mice. In line with this, we observed a treatment main effect in the mRNA expression of Bnip3, which mediates the recruitment of the growing autophagosome to damaged mitochondria (29). Bnip3 mRNA was significantly lower for GLY than CON (P < 0.05) and tended to be lower than ALA (P = 0.10). Because the control of autophagy by mTORC1 is well documented (7, 8), the blunted formation of autophagosomes in leucine-stimulated GLY mice is entirely consistent with improvements in mTORC1 signaling. Glycine reduces markers of oxidative stress. Increases in ROS are commonly associated with muscle wasting. ROS can damage lipids, DNA, and proteins and activate muscle catabolism (20, 42, 50). LPS increased DHE staining intensity, a marker of superoxide radical concentration, in the TA muscle (Fig. 5, A and B) but did not alter lipid damage as determined via Western blot quantification of 4HNE-bound proteins. DHE staining intensity was significantly lower in GLY compared with both ALA (P < 0.001) and CON groups (P < 0.05). Glycine has been shown to inhibit ROS formation and protect skeletal muscles from ischemia-reperfusion injury (2), and we recently reported reduced DHE staining intensity and markers of oxidative stress with glycine treatment in a C26 mouse model of cancer cachexia (28). Interestingly, the phosphorylation status of p38 MAPK was higher in the ALA group under leucine-stimulated conditions compared with all other groups (Fig. 6D). It has been reported that p38 MAPK represents a bridge between oxidative stress and autophagic muscle wasting, and its inhibition can block oxidative stress-induced activation of catabolic signaling and muscle wasting in skeletal muscle cells (42). Glycine does not alter the inflammatory response to LPS in skeletal muscle cells. LPS administration induced a pronounced skeletal muscle inflammatory response, with higher mRNA expression of cytokines (Tnfα, Il-6, and Socs3) and chemokines (Ccl2 and Ccl5) and a threefold higher phosphorylation status of the inflammatory response gene STAT3 (Fig. 5). However, contrary to our hypothesis, GLY did not alter the skeletal muscle inflammatory response. Although glycine administration has been clearly demonstrated to inhibit inflammatory cell activation and reduce tissue damage in numerous organs (56), significant inflammatory cell infiltration in skeletal muscle is not seen until 18–24 h after LPS administration in rodents (5). Although neutrophils usually migrate rapidly in extravascular tissue in response to invading pathogens, it has been demonstrated in pulmonary tissue that LPS can make neutrophils hyperadhesive to endothelial tissue, thereby restricting their migration into extravascular tissue (16, 44, 53). Unfortunately, the time-sensitive nature of tissue collection for our primary outcome measure of protein synthesis precluded plasma collection. As such, we cannot rule out the contribution of any glycine-induced reductions in systemic inflammation to the observed effects. Indeed, there was a tendency for a lower IL-6 protein concentration in GLY- compared with ALA-treated mice. However, the acute inflammatory response to LPS in skeletal muscle, which is increasingly recognized as a significant contributor to whole body inflammatory signaling (14), does not appear to be altered by glycine treatment and suggests that other (e.g., cytoprotective) properties of glycine may be responsible for the observed effects in skeletal muscle. To explore the potential for glycine to exert protective effects in skeletal muscle independent of its inhibitory effects on inflammatory cells, we stimulated inflammation and induced muscle wasting in mature C 2 C 12 myotubes by incubating cells in DMEM containing 1 μg/ml LPS. As observed previously (18), LPS stimulated a robust inflammatory response in C 2 C 12 muscle cells, and glycine did not blunt this response (Fig. 7). However, in line with the reported cytoprotective properties of glycine (56), we observed a glycine-induced protection of myotube diameter in LPS-treated skeletal muscle cells (Fig. 7, F and G), suggesting that glycine possesses cytoprotective but not anti-inflammatory properties in skeletal muscle cells.

CONCLUSIONS Our data show that glycine can restore the anabolic response to leucine during acute inflammation in skeletal muscle. These observations support the view that attempting to stimulate protein synthesis by increasing leucine availability alone constitutes an ineffective strategy to counteract anabolic resistance in muscle-wasting conditions (25). Further work is required to elucidate the key signaling pathways responsible for the beneficial effect of glycine but may include mTORC1 activation, ERK, p38 MAPK, or signaling pathways not investigated in this study, e.g., p53 and ATF4 (4, 13, 15). The identification of glycine as a novel nutritional intervention to restore the anabolic sensitivity of skeletal muscle to leucine has potential implications for a range of muscle-wasting conditions.

GRANTS D.J. Ham was supported by a postdoctoral fellowship from the European Society for Clinical Nutrition and Metabolism .

DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors.