The loss of skeletal muscle mass during aging is a significant health concern linked to adverse outcomes in older individuals. Understanding the molecular basis of age-related muscle loss is crucial for developing strategies to combat this debilitating condition. Long noncoding RNAs (lncRNAs) are a largely uncharacterized class of biomolecules that have been implicated in cellular homeostasis and dysfunction across a many tissues and cell types. To identify lncRNAs that might contribute to skeletal muscle aging, we screened for lncRNAs whose expression was altered in vastus lateralis muscle from older compared to young adults. We identified FRAIL1 as an aging-induced lncRNA with high abundance in human skeletal muscle. In healthy young and older adults, skeletal muscle FRAIL1 was increased with age in conjunction with lower muscle function. Forced expression of FRAIL1 in mouse tibialis anterior muscle elicits a dose-dependent reduction in skeletal muscle fiber size that is independent of changes in muscle fiber type. Furthermore, this reduction in muscle size is dependent on an intact region of FRAIL1 that is highly conserved across non-human primates. Unbiased transcriptional and proteomic profiling of the effects of FRAIL1 expression in mouse skeletal muscle revealed widespread changes in mRNA and protein abundance that recapitulate age-related changes in pathways and processes that are known to be altered in aging skeletal muscle. Taken together, these findings shed light on the intricate molecular mechanisms underlying skeletal muscle aging and implicate FRAIL1 in age-related skeletal muscle phenotypes.

BACKGROUND:The favorable health-promoting adaptations to exercise result from cumulative responses to individual bouts of physical activity. Older adults often exhibit anabolic resistance; a phenomenon whereby the anabolic responses to exercise and nutrition are attenuated in skeletal muscle. The mechanisms contributing to age-related anabolic resistance are emerging, but our understanding of how chronological age influences responsiveness to exercise is incomplete. The objective was to determine the effects of healthy aging on peripheral blood metabolomic response to a single bout of resistance exercise and whether any metabolites in circulation are predictive of anabolic response in skeletal muscle.METHODS:Thirty young (20-35 years) and 49 older (65-85 years) men and women were studied in a cross-sectional manner. Participants completed a single bout of resistance exercise consisting of eight sets of 10 repetitions of unilateral knee extension at 70% of one-repetition maximum. Blood samples were collected before exercise, immediately post exercise, and 30-, 90-, and 180-minutes into recovery. Proton nuclear magnetic resonance spectroscopy was used to profile circulating metabolites at all timepoints. Serial muscle biopsies were collected for measuring muscle protein synthesis rates.RESULTS:Our analysis revealed that one bout of resistance exercise elicits significant changes in 26 of 33 measured plasma metabolites, reflecting alterations in several biological processes. Furthermore, 12 metabolites demonstrated significant interactions between exercise and age, including organic acids, amino acids, ketones, and keto-acids, which exhibited distinct responses to exercise in young and older adults. Pre-exercise histidine and sarcosine were negatively associated with muscle protein synthesis, as was the pre/post-exercise fold change in plasma histidine.CONCLUSIONS:This study demonstrates that while many exercise-responsive metabolites change similarly in young and older adults, several demonstrate age-dependent changes even in the absence of evidence of sarcopenia or frailty.TRIAL REGISTRATION:Clinical trial registry: ClinicalTrials.gov NCT03350906.

BackgroundThe unfolded protein response (UPR) is a proteostatic process that is activated in response to endoplasmic reticulum stress. It is currently unclear how aging influences the chronic and adaptive UPR in human skeletal muscle. Here we determined the effect of aging on UPR activation at rest, in response to exercise, and the associations with muscle function.MethodsThirty young (20-35 yr) and 50 older (65-85 yr) individuals were enrolled. Vastus lateralis biopsies were performed at rest and 3 and 48 h after a single bout of resistance exercise. The abundance of UPR-related transcripts and proteins was measured by RNA sequencing and Western blotting, respectively. Fractional synthetic rates of muscle protein were determined by mass spectrometry after intravenous infusion of 13C6 phenylalanine.ResultsOlder adults demonstrated elevated transcriptional and proteomic markers of UPR activation in resting muscle. Resting UPR gene expression was negatively associated with muscle strength and power in older adults. The UPR is similarly activated by acute resistance exercise in young and older adults and positively associated with muscle function but not the anabolic response to exercise.ConclusionsSkeletal muscle from older adults exhibits chronically activated UPR, which accompanies functional decline. The adaptive UPR is a proteostatic mechanism that is upregulated in response to exercise in young and older adults and positively associated with muscle function.

Metabolic Syndrome and Related DisordersVol. 21, No. 1 EditorialFree AccessA Fresh Look at Fuel Selection in Working MuscleHawley E. Kunz and Ian R. LanzaHawley E. KunzEndocrine Research Unit, Division of Endocrinology, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.Search for more papers by this author and Ian R. LanzaAddress correspondence to: Ian R. Lanza, PhD, Endocrine Research Unit, Division of Endocrinology, Department of Internal Medicine, Mayo Clinic, 200 1st Street Southwest, Rochester, MN 55902, USA E-mail Address: lanza.ian@mayo.eduEndocrine Research Unit, Division of Endocrinology, Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota, USA.Search for more papers by this authorPublished Online:15 Feb 2023https://doi.org/10.1089/met.2022.0087AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Skeletal muscle is unique in its ability to rapidly increase energetic demand from rest to exercise. Even more impressive is that intracellular adenosine triphosphate (ATP) concentrations are well maintained even in the face of >25-fold increases in ATP demand during some types of vigorous exercise. This remarkable feat of bioenergetic homeostasis is achieved through the coordinated metabolic machinery that generates chemical energy from macronutrients. Skeletal muscle is designed to be nimble in its fuel preference depending on the prevailing conditions. For example, under resting postabsorptive conditions healthy muscle prefers to oxidize lipids for fuel, preserving glycogen for future needs and sparing glucose for other tissues (e.g., brain).However, under postprandial conditions where insulin levels are high, skeletal muscle quickly changes its appetite, switching to carbohydrate as a preferred fuel. In the situation of exercise, skeletal muscle has a menu to choose from that includes glycogen and triglyceride in muscle and glucose and fatty acids in blood. The intensity and duration of exercise dictate how skeletal muscle taps into these fuel sources to support the energetic demands of exercise.Flexibility in substrate preference is critical in sustaining physical activity. This concept of metabolic flexibility has become mainstream after the important limb balance studies that were done in the 1990s1,2 and more recent whole-body gas exchange measurements3–5 that demonstrated that people with obesity or type 2 diabetes lose the ability to respond or adapt appropriately to changing conditions. This phenomenon known as metabolic inflexibility has been implicated in insulin resistance and recognized as a central feature of metabolic derangements of obesity and diabetes.The article by Barakati and colleagues takes aim at this important topic from the standpoint of skeletal muscle fuel selection during exercise. The current gold-standard approach is arteriovenous catheterization, which may not be feasible in many situations. The authors describe a technique whereby whole-body gas exchange data are used to estimate fuel oxidation in working skeletal muscle. The technique goes beyond simply estimating muscle fuel oxidation from indirect calorimetry, which represents fuel oxidation of many tissues in addition to muscle. Instead, the increments in VO2 and VCO2 from rest to exercise are used to calculate a respiratory exchange ratio in working muscle (ΔRER) and estimate the fraction of fuel supplied by lipid.This approach is appealing because it represents an alternative to the invasive limb balance studies and is conceivably more representative of fuel oxidation in working muscle than the conventional use of whole-body indirect calorimetry. There are several noteworthy aspects of this study. First, Barakati et al provide data to support that ΔRER appropriately estimates fuel use in exercising muscle at very low intensities well below the lactate threshold that could be maintained at steady state. Whether this approach has utility at higher intensities that go beyond mild exercise and require energy from both oxidative and nonoxidative processes remains to be determined. The authors go on to evaluate the extent to which insulin sensitivity influences fuel preference of working muscle in young sedentary individuals. Surprisingly, they found that insulin-stimulated glucose disposal did not predict fuel selection during muscle activity, and that carbohydrate was the preferred substrate in the working muscle of very sedentary individuals.Furthermore, muscle citrate synthase activity, a proxy for mitochondrial content, was a significant predictor of fat oxidation during exercise. These findings suggest that metabolic inflexibility in response to exercise may arise from distinct mechanisms to those that drive metabolic inflexibility in response to a meal. The extent to which these findings go beyond the situation of very mild exercise are unclear, particularly given prior literature demonstrating distinct patterns of substrate utilization during moderate exercise in people with and without obesity.4 From this study, it is difficult to ignore the possibility that whole-body indirect calorimetry overestimates muscle lipid oxidation during exercise. Sedentary individuals appear to have greater reliance on carbohydrate as a fuel for skeletal muscle at very low intensities than previously believed.References1. Kelley DE, Mokan M, Simoneau JA, et al. Interaction between glucose and free fatty acid metabolism in human skeletal muscle. J Clin Invest 1993;92:91–98. Crossref, Medline, Google Scholar2. Kelley DE, Simoneau JA. Impaired free fatty acid utilization by skeletal muscle in non-insulin-dependent diabetes mellitus. J Clin Invest 1994;94:2349–2356. Crossref, Medline, Google Scholar3. Horowitz JF, Klein S. Oxidation of nonplasma fatty acids during exercise is increased in women with abdominal obesity. J Appl Physiol 2000;89:2276–2282. Crossref, Medline, Google Scholar4. Goodpaster BH, Wolfe RR, Kelley DE. Effects of obesity on substrate utilization during exercise. Obes Res 2002;10:575–584. Crossref, Medline, Google Scholar5. Braun B, Sharoff C, Chipkin SR, et al. Effects of insulin resistance on substrate utilization during exercise in overweight women. J Appl Physiol 2004;97:991–997. Crossref, Medline, Google ScholarFiguresReferencesRelatedDetails Volume 21Issue 1Feb 2023 InformationCopyright 2023, Mary Ann Liebert, Inc., publishersTo cite this article:Hawley E. Kunz and Ian R. Lanza.A Fresh Look at Fuel Selection in Working Muscle.Metabolic Syndrome and Related Disorders.Feb 2023.1-2.http://doi.org/10.1089/met.2022.0087Published in Volume: 21 Issue 1: February 15, 2023Online Ahead of Print:November 1, 2022PDF download

Aging is associated with profound alterations in skeletal muscle, including loss of muscle mass and function, local inflammation, altered mitochondrial physiology, and attenuated anabolic responses to exercise termed anabolic resistance. "Inflammaging," the chronic, low-grade inflammation associated with aging, may contribute to many of the age-related derangements in skeletal muscle, including its ability to respond to exercise and nutritional stimuli. Inflammation and exercise are closely intertwined in numerous ways. A single bout of muscle-damaging exercise stimulates an acute inflammatory response in the skeletal muscle that is essential for muscle repair and regeneration; however, the chronic systemic and local inflammation associated with aging may impair acute inflammatory and anabolic responses to exercise. In contrast, exercise training is anti-inflammatory, targeting many of the potential root causes of inflammaging. In this review, we discuss the interplay between inflammation and exercise in aging and highlight potential therapeutic targets for improving adaptive responses to exercise in older adults.

Aging and many illnesses and injuries impair skeletal muscle mass and function, but the molecular mechanisms are not well understood. To better understand the mechanisms, we generated and studied transgenic mice with skeletal muscle–specific expression of growth arrest and DNA damage inducible α (GADD45A), a signaling protein whose expression in skeletal muscle rises during aging and a wide range of illnesses and injuries. We found that GADD45A induced several cellular changes that are characteristic of skeletal muscle atrophy, including a reduction in skeletal muscle mitochondria and oxidative capacity, selective atrophy of glycolytic muscle fibers, and paradoxical expression of oxidative myosin heavy chains despite mitochondrial loss. These cellular changes were at least partly mediated by MAP kinase kinase kinase 4, a protein kinase that is directly activated by GADD45A. By inducing these changes, GADD45A decreased the mass of muscles that are enriched in glycolytic fibers, and it impaired strength, specific force, and endurance exercise capacity. Furthermore, as predicted by data from mouse models, we found that GADD45A expression in skeletal muscle was associated with muscle weakness in humans. Collectively, these findings identify GADD45A as a mediator of mitochondrial loss, atrophy, and weakness in mouse skeletal muscle and a potential target for muscle weakness in humans.

Background Omega-3 (n-3) PUFAs are recognized for triglyceride-lowering effects in people with dyslipidemia, but it remains unclear if n-3-PUFA intake influences lipoprotein profiles in older adults without hypertriglyceridemia. Objectives The objective was to determine the effect of n-3-PUFA supplementation on plasma lipoprotein subfractions in healthy older men and women in the absence of cardiovascular disease (CVD) or hypertriglyceridemia. This was a secondary analysis and considered exploratory. Methods Thirty young (20-35 y old) and 54 older (65-85 y old) men and women were enrolled in the study. Fasting plasma samples were collected. After baseline sample collection, 44 older adults were randomly assigned to receive either n-3-PUFA ethyl esters (3.9 g/d) or placebo (corn oil) for 6 mo. Pre- and postintervention plasma samples were used for quantitative lipoprotein subclass analysis using high-resolution proton NMR spectroscopy. Results The number of large, least-dense LDL particles decreased 17%-18% with n-3 PUFAs compared with placebo (P < 0.01). The number of small, dense LDL particles increased 26%-44% with n-3 PUFAs compared with placebo (similar to 11% decrease; P < 0.01). The cholesterol content of large HDL particles increased by 32% with n-3 PUFAs and by 2% in placebo (P < 0.01). The cholesterol content of small HDL particles decreased by 23% with n-3 PUFAs and by 2% in placebo (P < 0.01). Conclusions Despite increasing abundance of small, dense LDL particles that are associated with CVD risk, n-3 PUFAs reduced total triglycerides, maintained HDL, reduced systolic blood pressure, and shifted the HDL particle distribution toward a favorable cardioprotective profile in healthy older adults without dyslipidemia. This study suggests potential benefits of n-3-PUFA supplementation to lipoprotein profiles in healthy older adults without dyslipidemia, which should be considered when weighing the potential health benefits against the cost and ecological impact of widespread use of n-3-PUFA supplements. This trial was registered at clinicaltrials.gov as NCT03350906.

Objective: The health benefits of exercise are well documented, but several exercise-response parameters are attenuated in individuals with obesity. The goal of this pilot study was to identify molecular mechanisms that may influence exercise response with obesity. Methods: A multi-omics comparison of the transcriptome, proteome, and phosphoproteome in muscle from a preliminary cohort of lean individuals (n = 4) and individuals with obesity (n = 4) was performed, before and after a single bout of 30 minutes of unilateral cycling at 70% maximal oxygen uptake (VO2 peak). Mass spectrometry and RNA sequencing were used to interrogate the proteome, phosphoproteome, and transcriptome from muscle biopsy tissue. Results: The main findings are that individuals with obesity exhibited transcriptional and proteomic signatures consistent with reduced mitochondria! function, protein synthesis, and glycogen synthesis. Furthermore, individuals with obesity demonstrated markedly different transcriptional, proteomic, and phosphoproteomic responses to exercise, particularly biosynthetic pathways of glycogen synthesis and protein synthesis. Casein kinase II subunit alpha and glycogen synthase kinase-3 beta signaling was identified as exercise-response pathways that were notably altered by obesity. Conclusions: Opportunities to enhance exercise responsiveness by targeting specific molecular pathways that are disrupted in skeletal muscle from individuals with obesity await a better understanding of the precise molecular mechanisms that may limit exercise-response pathways in obesity.

TNF-alpha is a potent pro-inflammatory cytokine. Previous studies have proved that biallelic polymorphisms in the TNF-alpha (-308, TNFA) and TNF-beta genes (intron 1, TNFB) influence TNF-alpha production. In sarcoidosis, a chronic granulomatous disease, as a result of an unknown in vivo activation bronchoalveolar lavage (BAL) cells release high amounts of TNF-alpha, spontaneously and after in vitro stimulation. Thus, sarcoidosis could serve as a model to test the in vivo effect of TNF gene polymorphisms. We determined the TNFA and TNFB polymorphisms of 44 patients with sarcoidosis and found the following allele frequencies: 0.80, 0.20, 0.38 and 0.62 for TNFA1, TNFA2, TNFB1 and TNFB2, respectively. To examine the in vivo effect of the named polymorphisms on the TNF-alpha production, the spontaneous and LPS-induced TNF-alpha release of BAL cells and peripheral blood mononuclear cells were also determined in patients with sarcoidosis. Statistical analysis did not reveal any significant difference between sarcoidosis patients with different genotypes. The results show that TNFA and TNFB polymorphisms do not determine the level of TNF-alpha release of mononuclear cells activated during the course of sarcoid inflammation.

To our knowledge, this is the first study to suggest that skeletal muscle mitochondrial deficits are associated with cancer-associated weight loss in humans. Mitochondrial deficits and alterations in the phospholipid metabolome were concurrent with reductions in whole body and skeletal muscle functional capacity. Whether mitochondrial deficits are causal or secondary to cancer-associated weight loss and functional deficits remains to be determined, but this study supports further exploration of mitochondria as a driver of cancer-associated losses in muscle mass and function.

These data highlight several unique pathways in individuals with obesity that result in a blunted exercise response.

PURPOSE: This study was conducted to examine the biological effects of dietary omega-3 polyunsaturated fatty acid (n3-PUFA) on plasma lipoprotein content of 44 healthy, older individuals METHODS: Following the baseline studies, the older adults were randomly assigned into placebo (n = 22) and treatment (n = 22) groups. They began a 26-week supplementation with placebo (corn oil) or n3-PUFAs (675 mg EPA, 300 mg DHA, 4 soft gels per day). Outcome measures were obtained before and after the intervention, with fasting blood samples collected following 3 days of a weight-maintaining diet and overnight stay in the hospital. In addition, 30 young participants were studied as a control group who did not receive an intervention. RESULTS: 1H-NMR post intervention analysis revealed that n3-PUFA supplementation did not change the total triglyceride and cholesterol levels in the treatment group, but appreciably lowered the total apo-AII content (p = .023). The particle numbers significantly decreased in LDL-1 and LDL-2 and increased in LDL-5 and LDL-6. Similar patterns were observed in apo-B contents. Other changes are remarkable increase in LDL triglyceride (p = .006) and HDL free cholesterol (p = .001), and a profound shift in the composition of LDL and HDL subclasses. CONCLUSIONS: Our findings suggest that 26 weeks of n3-PUFA supplementation does not change the total triglyceride and cholesterol levels, but modifies the HDL composition and function in healthy older adults. Changes in the composition of HDL subclasses, particularly an increase in the cholesterol and Apo-AI contents in the larger, less dense HDL particles, and a drop in the cholesterol and Apo-AI contents in the smaller, denser particles may be associated with the atheroprotective function of HDL. HDL particle is believed to participate in Reverse Cholesterol Transport process in which excess cholesterol in cells in the periphery is transported to the liver and ultimately excreted from the body

Skeletal muscle is critical for maintaining mobility, independence, and metabolic health in older adults. However, a common feature of aging is the progressive loss of skeletal muscle mass and function, which is often accompanied by mitochondrial impairments, oxidative stress, and insulin resistance. Exercise improves muscle strength, mitochondrial health, and cardiorespiratory fitness, but older adults often exhibit attenuated anabolic responses to acute exercise. Chronic inflammation associated with aging may contribute to this “anabolic resistance” and therapeutic interventions that target inflammation may improve exercise responsiveness. To this end, we conducted a randomized controlled trial to determine the effect of 6 months of dietary omega-3 polyunsaturated fatty acids (n3-PUFA) supplementation on skeletal muscle function (mass, strength), mitochondrial physiology (respiration, ATP production, ROS generation), and acute exercise responsiveness at the level of the muscle (fractional synthesis rate) and the whole-body (amino acid kinetics) in healthy older adults. When compared with a corn oil placebo (n = 33; 71.5 ± 4.8 years), older adults treated with 4 g/day n3-PUFA (n = 30; 71.4 ± 4.5 years) exhibited modest but significant increases in muscle strength (3.1 ± 14.7% increase in placebo vs. 7.5 ± 14.1% increase in n3-PUFA; p = 0.039). These improvements in muscle strength with n3-PUFA supplementation occurred in the absence of any effects on mitochondrial function and a minor attenuation of the acute response to exercise compared to placebo. Together, these data suggest modest benefits of dietary n3-PUFAs to muscle function in healthy older adults. Future studies may elucidate whether n3-PUFA supplementation improves the exercise response in elderly individuals with co-morbidities, such as chronic inflammatory disease or sarcopenia.

Habitual endurance exercise results in increased erythropoiesis, which is primarily controlled by erythropoietin (EPO), yet studies demonstrating upregulation of EPO via a single bout of endurance exercise have been equivocal. This study compares the acute EPO response to 30 min of high versus 90 min of moderate-intensity endurance exercise and whether that response can be upregulated via selective adrenergic receptor blockade. Using a counterbalanced, cross-over design, fifteen participants (age 28 ± 8) completed two bouts of running (30-min, high intensity vs 90-min, moderate intensity) matched for overall training stress. A separate cohort of fourteen participants (age 31 ± 6) completed three bouts of 30-min high-intensity cycling after ingesting the preferential β1-adrenergic receptor (AR) antagonist bisoprolol, the non-preferential β1 + β2 antagonist nadolol or placebo. Venous blood was collected before, during, and after exercise, and serum EPO levels were determined by ELISA. No detectable EPO response was observed during or after high intensity running, however, in the moderate-intensity trial EPO was significantly elevated at both during-exercise timepoints (+ 6.8% ± 2.3% at 15 min and + 8.7% ± 2.2% at 60 min). No significant change in EPO was observed post-cycling or between the trials involving βAR blockade. Neither training mode (running or cycling), nor beta-blockade significantly influenced the EPO response to 30 min of high-intensity exercise, however, 90 min of moderate-intensity running elevated EPO during exercise, returning to baseline immediately post-exercise. Identifying the optimal mode, duration and intensity required to evoke an EPO response to exercise may help tailor exercise prescriptions designed to maximize EPO response for both performance and clinical applications.

Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 nondiabetic adults across a range of body mass indexes (BMI 20.5-45.8 kg/m2). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (SI) and β cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, SI, and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced SI, and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with SI, and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements.NEW AND NOTEWORTHY Adipose inflammation is proposed to be at the nexus of the systemic inflammation and metabolic derangements associated with obesity. The present study provides evidence to support adipose inflammation as a central feature of the pathophysiology of obesity. Adipose inflammation is associated with systemic and peripheral metabolic derangements, including increased systemic inflammation, reduced insulin sensitivity, and reduced skeletal muscle mitochondrial respiration.

Cancer cachexia is characterized by reductions in peripheral lean muscle mass. Prior studies have primarily focused on increased protein breakdown as the driver of cancer-associated muscle wasting. Therapeutic interventions targeting catabolic pathways have, however, largely failed to preserve muscle mass in cachexia, suggesting that other mechanisms might be involved. In pursuit of novel pathways, we used untargeted metabolomics to search for metabolite signatures that may be linked with muscle atrophy. We injected 7-week-old C57/BL6 mice with LLC1 tumor cells or vehicle. After 21 days, tumor-bearing mice exhibited reduced body and muscle mass and impaired grip strength compared with controls, which was accompanied by lower synthesis rates of mixed muscle protein and the myofibrillar and sarcoplasmic muscle fractions. Reductions in protein synthesis were accompanied by mitochondrial enlargement and reduced coupling efficiency in tumor-bearing mice. To generate mechanistic insights into impaired protein synthesis, we performed untargeted metabolomic analyses of plasma and muscle and found increased concentrations of two methylarginines, asymmetric dimethylarginine (ADMA) and NG-monomethyl-l-arginine, in tumor-bearing mice compared with control mice. Compared with healthy controls, human cancer patients were also found to have higher levels of ADMA in the skeletal muscle. Treatment of C2C12 myotubes with ADMA impaired protein synthesis and reduced mitochondrial protein quality. These results suggest that increased levels of ADMA and mitochondrial changes may contribute to impaired muscle protein synthesis in cancer cachexia and could point to novel therapeutic targets by which to mitigate cancer cachexia.

Cachexia, an illness-associated syndrome characterized by muscle wasting that cannot be reversed with nutritional support, is a significant contributor to cancer-associated morbidity and mortality. The mechanisms driving the loss of muscle mass are not well defined, and predictive or early diagnostic biomarkers have not been identified. PURPOSE: To determine factors that may contribute to cancer-associated losses in muscle mass and to identify potential biomarkers indicative or predictive of the severity of muscle wasting. METHODS: Lewis lung carcinoma (LLC1) cells or vehicle (CON) were injected subcutaneously into the left flank of seven week-old C57BL/J6 male and female mice. After 21 days, skeletal muscle mass and function were assessed. Mitochondrial energetics were assessed in permeabilized muscle fibers using high-resolution respirometry, and fractional protein synthesis rates following the administration of 13C6-phenylalanine were measured by mass spectrometry. To explore potential mechanisms and biomarkers of cachexia, untargeted metabolomics was performed using plasma and skeletal muscle from LLC1 and CON mice. RESULTS: Tumor-bearing mice showed evidence of cachexia, with 6.8% lower body mass (p<0.001), 10.0% lower quadriceps mass (p=0.010), 9.7% lower gastrocnemius mass (p=0.001), and 9.6% lower grip strength (p=0.004) at day 21. Mixed muscle protein synthesis was impaired in LLC1 mice (-18.6%, p=0.0279). Synthesis of both the sarcoplasmic and myofibrillar proteins was lower in LLC1 mice (-34.4%, p<0.0001 and -24.5%, p=0.0039, respectively). Mitochondrial protein synthesis was not significantly affected, and no differences in mitochondrial energetics were observed between LLC1 and CON mice. Untargeted metabolomics revealed significant increases in asymmetric dimethylarginine (ADMA) and N-monomethyl L-arginine (L-NMMA) in both the skeletal muscle and plasma of LLC1 mice. CONCLUSION: The synthesis of contractile and sarcoplasmic proteins was inhibited in cachectic, tumor-bearing mice. Elevations in ADMA and L-NMMA, endogenous nitric oxide synthase inhibitors formed during proteolysis, may both serve as biomarkers of cachexia and play a mechanistic role in the loss of muscle mass. Project supported by the Andersen Corporate Foundation and T32AR056950.