Daniel J. Ham

Leucine as a treatment for muscle wasting: A critical review

Amino acids are potent modulators of protein turnover and skeletal muscle cells are highly sensitive to changes in amino acid availability. During amino acid abundance increased activity of mTORC1 drives protein synthesis and growth. In skeletal muscle, it has been clearly demonstrated that of all the amino acids, leucine is the most potent stimulator of mTORC1 and protein synthesis in vitro and in vivo. As such, leucine has received considerable attention as a potential pharmaconutrient for the treatment of numerous muscle wasting conditions. However, despite a multitude of studies showing enhanced acute protein synthesis with leucine or leucine-rich supplements in healthy individuals, additional leucine intake does not necessarily enhance protein synthesis during muscle wasting conditions. In addition, long-term, placebo controlled, iso-caloric studies in humans consistently show no beneficial effect of leucine supplementation on skeletal muscle mass or function. This review, critically evaluates the therapeutic potential of leucine to attenuate the skeletal muscle wasting associated with ageing, cancer and immobilization/bed rest. It also highlights the impact of inflammation on amino acid sensing, mTORC1 activation and stimulation of protein synthesis and challenges the underlying hypothesis that the acute activation of mTORC1 and stimulation of protein synthesis by leucine increases in muscle mass over time. We conclude that leucine, as a standalone nutritional intervention, is not effective in the prevention of muscle wasting. Future work should focus on identifying and utilizing other nutrients or treatments that sensitize skeletal muscle to leucine, thereby enhancing its therapeutic potential for muscle wasting conditions.

Glycine administration attenuates skeletal muscle wasting in a mouse model of cancer cachexia

BACKGROUND AND AIMS The non-essential amino acid, glycine, is often considered biologically neutral, but some studies indicate that it could be an effective anti-inflammatory agent. Since inflammation is central to the development of cancer cachexia, glycine supplementation represents a simple, safe and promising treatment. We tested the hypothesis that glycine supplementation reduces skeletal muscle inflammation and preserves muscle mass in tumor-bearing mice. METHODS To induce cachexia, CD2F1 mice received a subcutaneous injection of PBS (control, n = 12) or C26 tumor cells (n = 32) in accordance with the protocols developed by Murphy et al. [Murphy KT, Chee A, Trieu J, Naim T, Lynch GS. Importance of functional and metabolic impairments in the characterization of the C-26 murine model of cancer cachexia. Dis Models Mech 2012;5(4):533-545.]. Subcutaneous injections of glycine (n = 16) or PBS (n = 16) were administered daily for 21 days and at the conclusion of treatment, selected muscles, tumor and adipose tissue were collected and prepared for Real-Time RT-PCR or western blot analysis. RESULTS Glycine attenuated the loss of fat and muscle mass, blunted increases in markers of inflammation (F4/80, P = 0.01 & IL-6 mRNA, P = 0.01) and atrophic signaling (MuRF, P = 0.047; atrogin-1, P = 0.04; LC3B, P = 0.06 and; BNIP3, P = 0.10) and tended to attenuate the loss of body mass (P = 0.07), muscle function (P = 0.06), and oxidative stress (GSSG/GSH, P = 0.06 and DHE, P = 0.07) seen in tumor-bearing mice. Preliminary studies that compared the effect of glycine administration with isonitrogenous doses of alanine or citrulline showed that the observed protective effect was specific to glycine. CONCLUSIONS Glycine protects skeletal muscle from cancer-induced wasting and loss of function, reduces the oxidative and inflammatory burden, and reduces the expression of genes associated with muscle protein breakdown in cancer cachexia. Importantly, these effects were glycine specific.

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

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.

Citrulline Does Not Prevent Skeletal Muscle Wasting or Weakness in Limb-Casted Mice

BACKGROUND Increasing arginine (Arg) availability reduces atrophy in cultured skeletal muscle cells. Supplementation with its metabolic precursor citrulline (Cit) is more effective at improving skeletal muscle Arg concentrations. OBJECTIVE We tested the hypothesis that Cit supplementation would attenuate skeletal muscle atrophy and loss of function during hindlimb immobilization in mice. METHODS Male C57BL/6JArc mice underwent 14 d of unilateral hindlimb immobilization/plaster casting and were supplemented with ~0.81 g Cit · kg⁻¹ · d⁻¹ (CIT group) or Ala (ALA group) mixed into their food. The uncasted contralateral limb (internal control) and an uncasted group (CON) served as controls. Muscle atrophy was evaluated with mass, fiber area, and in situ muscle function. RESULTS Tibialis anterior (TA) muscle mass [ALA: 37.6 ± 0.92 mg; CIT: 38.3 ± 1.25 mg] and peak tetanic force (ALA: 1150 ± 38.5 mN; CIT: 1150 ± 52.0 mN) were lower (P < 0.001) in the ALA (53.9 ± 0.42 mg) and CIT (1760 ± 28.5 mN) groups than in the CON group. No difference was found between ALA and CIT groups for TA mass, fiber area, or peak force. The mRNA expression of the nitric oxide synthase 2, inducible (Nos2; ~15-fold) and B-cell chronic lymphoid leukemia/lymphoma 2/adenovirus E1B 19 kDa interacting protein 3 (Bnip3; ~17-fold) genes and the ratio of microtubule-associated protein light chain 3BII to 3BI (LC3BII:LC3BI) (50.5% ± 17.7%) were higher (P < 0.05) in the ALA group than in the CON group, suggesting increased autophagy. In the CIT group, Bnip3 mRNA was lower (-70%; P < 0.05) and Nos2 mRNA tended to be lower (-45%; P = 0.05) than in the ALA group, whereas LC3BII:LC3BI was not different from the CON group. CONCLUSIONS Cit treatment of male mice did not affect therapeutically relevant outcome measures such as skeletal muscle mass and peak muscle force after 14 d of hindlimb immobilization.

Amino acid sensing and activation of mechanistic target of rapamycin complex 1: implications for skeletal muscle.

Purpose of reviewThis article evaluates recent studies on the mechanisms involved in sensing changes in amino acid availability and activation of the mechanistic target of rapamycin complex 1 (mTORC1). Recent findingsmTORC1 is sensitive to changes in amino acid availability and a well known regulator of protein turnover. The mechanisms of amino acid sensing and mTORC1 signaling are emerging with multiple potential sensors (e.g., solute carrier family 38, member 9, lysosomal protein transmembrane 4 beta/solute carrier family 7, member 5-solute carrier family 3, member 2) and signal transducers (e.g., Sestrins, ADP-ribosylation factor 1, and microspherule protein 1) identified. Studies in various cell lines have unveiled the importance of the lysosome in amino acid sensing and signal transmission. SummaryRecent discoveries in amino acid sensing highlight a complex scenario, whereby mTORC1 is not merely sensitive to some amino acids and not others, but where specific amino acids are sensed by specific pathways under specific conditions. The physiological purpose of such an arrangement remains to be unraveled, but it would allow mTORC1 to precisely regulate growth during different metabolic conditions. Understanding the mechanisms responsible for sensing amino acid availability and regulating mTORC1 activity is an important prerequisite for the development of nutritional strategies to combat skeletal muscle wasting disorders.

Glycine restores the anabolic response to leucine in a mouse model of acute inflammation

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 C2C12 myotubes. Our observations in male C57BL/6 mice suggest that glycine may represent a promising nutritional intervention for the attenuation of skeletal muscle wasting.

Parvalbumin Gene Transfer Impairs Skeletal Muscle Contractility in Old Mice

Sarcopenia is the progressive age-related loss of skeletal muscle mass associated with functional impairments that reduce mobility and quality of life. Overt muscle wasting with sarcopenia is usually preceded by a slowing of the rate of relaxation and a reduction in maximum force production. Parvalbumin (PV) is a cytosolic Ca(2+) buffer thought to facilitate relaxation in muscle. We tested the hypothesis that restoration of PV levels in muscles of old mice would increase the magnitude and hasten relaxation of submaximal and maximal force responses. The tibialis anterior (TA) muscles of young (6 month), adult (13 month), and old (26 month) C57BL/6 mice received electroporation-assisted gene transfer of plasmid encoding PV or empty plasmid (pcDNA3.1). Contractile properties of TA muscles were assessed in situ 14 days after transfer. In old mice, muscles with increased PV expression had a 40% slower rate of tetanic force development (p<0.01), and maximum twitch and tetanic force were 22% and 16% lower than control values, respectively (p<0.05). Muscles with increased PV expression from old mice had an 18% lower maximum specific (normalized) force than controls, and absolute force was `26% lower at higher stimulation frequencies (150-300 Hz, p<0.05). In contrast, there was no effect of increased PV expression on TA muscle contractile properties in young and adult mice. The impairments in skeletal muscle function in old mice argue against PV overexpression as a therapeutic strategy for ameliorating aspects of contractile dysfunction with sarcopenia and help clarify directions for therapeutic interventions for age-related changes in skeletal muscle structure and function.

Glycine metabolism in skeletal muscle: implications for metabolic homeostasis

Purpose of review The review summarizes the recent literature on the role of glycine in skeletal muscle during times of stress. Recent findings Supplemental glycine protects muscle mass and function under pathological conditions. In addition, mitochondrial dysfunction in skeletal muscle leads to increased cellular serine and glycine production and activation of NADPH-generating pathways and glutathione metabolism. These studies highlight how glycine availability modulates cellular homeostasis and redox status. Summary Recent studies demonstrate that supplemental glycine effectively protects muscles in a variety of wasting models, including cancer cachexia, sepsis, and reduced caloric intake. The underlying mechanisms responsible for the effects of glycine remain unclear but likely involve receptor-mediated responses and modulation of intracellular metabolism. Future research to understand these mechanisms will provide insight into glycines therapeutic potential. Our view is that glycine holds considerable promise for improving health by protecting muscles during different wasting conditions.

Muscle-specific deletion of SOCS3 does not reduce the anabolic response to leucine in a mouse model of acute inflammation

HighlightsSOCS3 deletion in skeletal muscle does not alter LPS‐induced inflammation.SOCS3 does not mediate the anabolic response to leucine in skeletal muscle.Leucine augments the LPS‐induced expression of IL‐6 and STAT3 phosphorylation. Abstract Excessive inflammation reduces skeletal muscle protein synthesis leading to wasting and weakness. The janus kinase/signal transducers and activators of transcription‐3 (JAK/STAT3) pathway is important for the regulation of inflammatory signaling. As such, suppressor of cytokine signaling‐3 (SOCS3), the negative regulator of JAK/STAT signaling, is thought to be important in the control of muscle homeostasis. We hypothesized that muscle‐specific deletion of SOCS3 would impair the anabolic response to leucine during an inflammatory insult. Twelve week old (n = 8 per group) SOCS3 muscle‐specific knockout mice (SOCS3‐MKO) and littermate controls (WT) were injected with lipopolysaccharide (LPS, 1 mg/kg) or saline and were studied during fasted conditions or after receiving 0.5 g/kg leucine 3 h after the injection of LPS. Markers of inflammation, anabolic signaling, and protein synthesis were measured 4 h after LPS injection. LPS injection robustly increased mRNA expression of inflammatory molecules (Socs3, Socs1, Il‐6, Ccl2, Tnf&agr; and Cd68). In muscles from SOCS3‐MKO mice, the Socs3 mRNA response to LPS was significantly blunted (˜6‐fold) while STAT3 Tyr705 phosphorylation was exacerbated (18‐fold). Leucine administration increased protein synthesis in both WT (˜1.6‐fold) and SOCS3‐MKO mice (˜1.5‐fold) compared to basal levels. LPS administration blunted this effect, but there were no differences between WT and SOCS3‐MKO mice. Muscle‐specific SOCS3 deletion did not alter the response of AKT, mTOR, S6 or 4EBP1 under any treatment conditions. Therefore, SOCS3 does not appear to mediate the early inflammatory or leucine‐induced changes in protein synthesis in skeletal muscle.