Muscle Proteolysis Research Articles

Calpain activation and proteolysis in postmortem goose muscles.

Meat tenderness is considered as the most important criterion for meat quality by consumers and can be improved by the actions of endogenous proteases, mainly calpains, during postmortem storage at 0-5°C. The purpose of this study, therefore, was to examine the postmortem calpain activation and proteolysis in breast (BM) and leg and thigh (LM) muscles of White Roman goose. BM and LM were taken from goose carcasses (n=15) at 0 (10-15min postmortem), 1, 3, and 7days of storage at 5°C. The decrease in postmortem pH, calpain-1 and -11 activities, and contents of the calpain-1 80kDa subunit and desmin was more rapid (p<.05) in BM than in LM. Our results show that postmortem proteolysis was more extensive in BM than in LM of White Roman goose, not only because the difference in fiber type composition between two muscles, but because the rate and extent of calpain activation were greater in BM as well. These results may provide useful information to optimize meat processing for different muscles in goose industry.

Meat quality traits of European quails reared under different conditions of temperature and air velocity

This study’s objective was to evaluate the influence of thermal environment and air velocity during the rearing phase on European quail meat quality traits. A total of 1,152 one-day-old European quail chicks were placed inside floor pens within environmental chambers. Each experimental period was approximately 5 wks, with birds slaughtered at 37 d of age. The experimental design consisted of a 2 × 4 factorial arrangement of treatments in completely randomized design with 2 air velocities (0 and 2 m/s) × 4 air temperatures (severe cold [SC], moderate cold, thermal comfort, and moderate heat [MH]). ANOVA, with air velocity and thermal environment as fixed effects, was performed to evaluate the effect of main factors and their interaction on meat quality traits, using the GLM procedure (SAS 9.4). Least square means of treatments effects were compared using Tukey’s test (α = 0.05). Lightness (L∗), redness (a∗), and yellowness (b∗), of quail meat were affected by thermal environment and air velocity (P < 0.05). Initial and final L∗ values were greater for MH (P < 0.05). Meat from birds subjected to 2 m/s air velocity had lower final L∗, but no velocity effect was noted for initial L∗. Quail meat from SC presented higher initial and final a∗ values compared with the other thermal environment groups (P ≤ 0.001). Final a∗ was affected by air velocity (P < 0.05). Initial and final b∗ values for meat from MH were greater, 13.8 and 15.2, respectively, differing from the other treatment environments (P < 0.05). However, air velocity did not influence b∗ values (P > 0.05). Interactions were not significant for pHu (P = 0.993). Thawing loss and shear force were affected by treatments (P < 0.05) but not ultimate pH, drip loss, or sarcomere length. This study demonstrates that thermal environments and air velocity affect quail meat quality traits. Further investigation is recommended to explore effects of air velocity and thermal environment on muscle proteolysis of quail meat quality.

Postmortem role of calpain in Chinese and Wuzong goose muscles

The purpose of this study was to compare postmortem proteolysis and tenderization between Chinese and Wuzong goose breast muscles. Four months old Chinese (CG, n = 15) and Wuzong (WZ, n = 15) goose carcasses were vacuum-packaged 10 to 15 min postmortem and stored at 5°C. Breast (Pectoralis major) samples from each carcass were sampled at 0 (∼10 min postmortem), 1, 3, and 7 D of storage. Our results showed that the decrease in pH and calpain-1 activity was not different in CG and WG samples. However, the decrease in calpain-11 activity, desmin content, and shear force were more rapid (P < 0.05) in WZ than in CG samples. Our results indicate that postmortem proteolysis and tenderization of goose breast muscle were more extensive in WZ than in CG goose muscle.

Endogenous Glucocorticoid Signaling in the Regulation of Bone and Marrow Adiposity: Lessons from Metabolism and Cross Talk in Other Tissues.

The development of adiposity in the bone marrow, known as marrow adipose tissue (MAT), is often associated with musculoskeletal frailty. Glucocorticoids, which are a key component of the biological response to stress, affect both bone and MAT. These molecules signal through receptors such as the glucocorticoid receptor (GR), but the role of the GR in regulation of MAT is not yet clear from previous studies. The purpose of this review is to establish and determine the role of GR-mediated signaling in marrow adiposity by comparing and contrasting what is known against other energy-storing tissues like adipose tissue, liver, and muscle, to provide better insight into the regulation of MAT during times of metabolic stress (e.g., dietary challenges, aging). GR-mediated glucocorticoid signaling is critical for proper storage and utilization of lipids in cells such as adipocytes and hepatocytes and proteolysis in muscle, impacting whole-body composition, energy utilization, and homeostasis through a complex network of tissue cross talk between these systems. Loss of GR signaling in bone promotes increased MAT and decreased bone mass. GR-mediated signaling in the liver, adipose tissue, and muscle is critical for whole-body energy and metabolic homeostasis, and both similarities and differences in GR-mediated GC signaling in MAT as compared with these tissues are readily apparent. It is clear that GC-induced pathways work together through these tissues to affect systemic biology, and understanding the role of bone in these patterns of tissue cross talk may lead to a better understanding of MAT-bone biology that improves treatment strategies for frailty-associated diseases.

Effect of age on calpain changes in postmortem goose muscle

Meat tenderization can be affected by a variety of factors including animal age. However, results obtained from goose, chicken, turkey, and ostrich studies have shown that the effects of age on meat tenderization were insignificant, which may be due to a very limited age difference in birds used in those studies. Therefore, the purpose of this study was to investigate the effects of animal age on postmortem proteolysis and tenderization of breast muscle from developing and mature White Roman geese. Goose carcasses from mature (50 mo old, n = 10) and young (3 mo old, n = 10) geese were vacuum-packaged and stored at 5°C within 10min postmortem. Breast (pectoralis major) samples were taken at 0 (∼10min postmortem), 1, 3, and 7 D of storage. Our results showed that the decrease in pH, calpain-1 and -11 activities, desmin content and shear force, as well as the increase in myofibrillar fragmentation index were more rapid (P<0.05) in young than in mature goose breast. These results suggest that postmortem proteolysis and tenderization of goose muscle are extensively affected by age.

Revision of the cold processing phases to obtain a targeted salt reduction in typical Italian dry-cured ham

The objective of the study was to reduce the salt content of typical Italian dry-cured ham by at least 25%, to meet the “reduced salt” claim. Salt reduction needs process adjustments to maintain product safety and quality in the absence of nitrites. A way was to reduce the salt input and to shorten the salting period compared to the conventional process. The cold drying period of reduced salt (RS) hams was extended, to decrease aw below 0.97 in inner parts and obtain the same safety conditions of control hams (CS). In RS dry-cured hams the salt reduction was accomplished, the generation of biogenic amines was lower than the threshold values generating toxic effects, and color was the same as in the CS ones. However, in RS proteolysis increased, contributing to texture softening. A strengthened salt diffusion from backside rind could contribute to counteract the rise in proteolysis of biceps femoris muscle, depleted of salt during the process of RS hams.

Genome-Wide Identification and Transcriptomic Analysis of MicroRNAs Across Various Amphioxus Organs Using Deep Sequencing.

Amphioxus is the closest living invertebrate proxy of the vertebrate ancestor. Systematic gene identification and expression profile analysis of amphioxus organs are thus important for clarifying the molecular mechanisms of organ function formation and further understanding the evolutionary origin of organs and genes in vertebrates. The precise regulation of microRNAs (miRNAs) is crucial for the functional specification and differentiation of organs. In particular, those miRNAs that are expressed specifically in organs (OSMs) play key roles in organ identity, differentiation, and function. In this study, the genome-wide miRNA transcriptome was analyzed in eight organs of adult amphioxus Branchiostoma belcheri using deep sequencing. A total of 167 known miRNAs and 23 novel miRNAs (named novel_mir), including 139 conserved miRNAs, were discovered, and 79 of these were identified as OSMs. Additionally, analyses of the expression patterns of eight randomly selected known miRNAs demonstrated the accuracy of the miRNA deep sequencing that was used in this study. Furthermore, potentially OSM-regulated genes were predicted for each organ type. Functional enrichment of these predicted targets, as well as further functional analyses of known OSMs, was conducted. We found that the OSMs were potentially to be involved in organ-specific functions, such as epidermis development, gonad development, muscle cell development, proteolysis, lipid metabolism, and generation of neurons. Moreover, OSMs with non–organ-specific functions were detected and primarily include those related to innate immunity and response to stimuli. These findings provide insights into the regulatory roles of OSMs in various amphioxus organs.

Hungry for your alanine: when liver depends on muscle proteolysis.

Fasting requires complex endocrine and metabolic interorgan crosstalk, which involves shifting from glucose to fatty acid oxidation, derived from adipose tissue lipolysis, in order to preserve glucose for the brain. The glucose-alanine (Cahill) cycle is critical for regenerating glucose. In this issue of JCI, Petersen et al. report on their use of an innovative stable isotope tracer method to show that skeletal muscle-derived alanine becomes rate controlling for hepatic mitochondrial oxidation and, in turn, for glucose production during prolonged fasting. These results provide new insight into skeletal muscle-liver metabolic crosstalk during the fed-to-fasting transition in humans.

Aging- and obesity-related peri-muscular adipose tissue accelerates muscle atrophy.

Sarcopenia due to loss of skeletal muscle mass and strength leads to physical inactivity and decreased quality of life. The number of individuals with sarcopenia is rapidly increasing as the number of older people increases worldwide, making this condition a medical and social problem. Some patients with sarcopenia exhibit accumulation of peri-muscular adipose tissue (PMAT) as ectopic fat deposition surrounding atrophied muscle. However, an association of PMAT with muscle atrophy has not been demonstrated. Here, we show that PMAT is associated with muscle atrophy in aged mice and that atrophy severity increases in parallel with cumulative doses of PMAT. We observed severe muscle atrophy in two different obese model mice harboring significant PMAT relative to respective control non-obese mice. We also report that denervation-induced muscle atrophy was accelerated in non-obese young mice transplanted around skeletal muscle with obese adipose tissue relative to controls transplanted with non-obese adipose tissue. Notably, transplantation of obese adipose tissue into peri-muscular regions increased nuclear translocation of FoxO transcription factors and upregulated expression FoxO targets associated with proteolysis (Atrogin1 and MuRF1) and cellular senescence (p19 and p21) in muscle. Conversely, in obese mice, PMAT removal attenuated denervation-induced muscle atrophy and suppressed upregulation of genes related to proteolysis and cellular senescence in muscle. We conclude that PMAT accumulation accelerates age- and obesity-induced muscle atrophy by increasing proteolysis and cellular senescence in muscle.

Transcriptome profiling of muscle in Nelore cattle phenotypically divergent for the ribeye muscle area

This study aimed to use RNA-Seq to identify differentially expressed genes (DEGs) in muscle of uncastrated Nelore males phenotypically divergent for ribeye muscle area (REA). A total of 80 animals were phenotyped for REA, and 15 animals each with the highest REA and the lowest REA were selected for analyses. DEGs found (N = 288) belonging to families related to muscle cell growth, development, motility and proteolysis, such as actin, myosin, collagen, integrin, solute carrier, ubiquitin and kelch-like. Functional analysis showed that many of the significantly enriched gene ontology terms were closely associated with muscle development, growth, and degradation. Through co-expression network analysis, we predicted three hub genes (PPP3R1, FAM129B and UBE2G1), these genes are involved in muscle growth, proteolysis and immune system. The genes expression levels and its biological process found this study may result in differences in muscle deposition, and therefore, Nelore animals with different REA proportions.

α−Calcitonin gene-related peptide inhibits autophagy and calpain systems and maintains the stability of neuromuscular junction in denervated muscles

ObjectiveAlthough it is well established that a-calcitonin gene-related peptide (CGRP) stabilizes muscle-type cholinergic receptors nicotinic subunits (AChR), the underlying mechanism by which this neuropeptide regulates muscle protein metabolism and neuromuscular junction (NMJ) morphology is unclear. MethodsTo elucidate the mechanisms how CGRP controls NMJ stability in denervated mice skeletal muscles, we carried out physiological, pharmacological, and molecular analyses of atrophic muscles induced by sciatic nerve transection. ResultsHere, we report that CGRP treatment in vivo abrogated the deleterious effects on NMJ upon denervation (DEN), an effect that was associated with suppression of skeletal muscle proteolysis, but not stimulation of protein synthesis. CGRP also blocked the DEN-induced increase in endocytic AChR vesicles and the elevation of autophagosomes per NMJ area. The treatment of denervated animals with rapamycin blocked the stimulatory effects of CGRP on mTORC1 and its inhibitory actions on autophagic flux and NMJ degeneration. Furthermore, CGRP inhibited the DEN-induced hyperactivation of Ca2+-dependent proteolysis, a degradative system that has been shown to destabilize NMJ. Consistently, calpain was found to be activated by cholinergic stimulation in myotubes leading to the dispersal of AChR clusters, an effect that was abolished by CGRP. ConclusionTaken together, these data suggest that the inhibitory effect of CGRP on autophagy and calpain may represent an important mechanism for the preservation of synapse morphology when degradative machinery is exacerbated upon denervation conditions.

Differential structural features in soleus and gastrocnemius of carnitine-treated cancer cachectic rats.

Muscle wasting is associated with chronic diseases and cancer. Elucidation of the biological mechanism involved in the process of muscle mass loss and cachexia may help identify therapeutic targets. We hypothesized that l-carnitine treatment may differentially revert muscle fiber atrophy and other structural alterations in slow- and fast-twitch limb muscles of rats bearing the Yoshida ascites hepatoma. In soleus and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with l-carnitine (1 g/kg body weight for 7 days, intragastric), food intake, body and muscle weights, fiber typing and morphometry, morphological features, redox balance, autophagy and proteolytic, and signaling markers were explored. Levels of carnitine palmitoyl transferase were also measured in all the study muscles. l-Carnitine treatment ameliorated the atrophy of both slow- and fast-twitch fibers (gastrocnemius particularly), muscle structural alterations (both muscles), and attenuated oxidative stress, proteolytic and signaling markers (gastrocnemius). Despite that carnitine palmitoyl transferase-1 levels increased in both muscle types in a similar fashion, l-carnitine ameliorated muscle atrophy and proteolysis in a muscle-specific manner in cancer-induced cachexia. These data reveal the need to study muscles of different fiber type composition and function to better understand whereby l-carnitine exerts its beneficial effects on the myofibers in muscle wasting processes. These findings also have potential clinical implications, since combinations of various exercise and muscle training modalities with l-carnitine should be specifically targeted for the muscle groups to be trained.

Salidroside Attenuates Denervation-Induced Skeletal Muscle Atrophy Through Negative Regulation of Pro-inflammatory Cytokine

Skeletal muscle atrophy is associated with pro-inflammatory cytokines. Salidroside is a biologically active ingredient of Rhodiola rosea, which exhibits anti-inflammatory property. However, there is little known about the effect of salidroside on denervation-induced muscle atrophy. Therefore, the present study aimed to determine whether salidroside could protect against denervation-induced muscle atrophy and to clarify potential molecular mechanisms. Denervation caused progressive accumulation of inflammatory factors in skeletal muscle, especially interleukin 6 (IL6) and its receptor, and recombinant murine IL6 (rmIL6) local infusion could induce target muscle atrophy, suggesting that denervation induced inflammation in target muscles and the inflammation may trigger muscle wasting. Salidroside alleviated denervation-induced muscle atrophy and inhibited the production of IL6. Furthermore, the inhibition of phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the decreased levels of suppressor of cytokine signaling (SOCS3), muscle RING finger protein-1 (MuRF1), atrophy F-box (atrogin-1), microtubule-associated protein light chain 3 beta (LC3B) and PTEN-induced putative kinase (PINK1) were observed in denervated muscles that were treated with salidroside. Finally, all of these responses to salidroside were replicated in neutralizing antibody against IL6. Taken together, these results suggest that salidroside alleviates denervation-induced inflammation response, thereby inhibits muscle proteolysis and muscle atrophy. Therefore, it was assumed that salidroside might be a potential therapeutic candidate to prevent muscle wasting.

Impact of porcine reproductive and respiratory syndrome virus on muscle metabolism of growing pigs1.

Porcine reproductive and respiratory syndrome (PRRS) virus is one of the most economically significant pig pathogens worldwide. However, the metabolic explanation for reductions in tissue accretion observed in growing pigs remains poorly defined. Additionally, PRRS virus challenge is often accompanied by reduced feed intake, making it difficult to discern which effects are virus vs. feed intake driven. To account for this, a pair-fed model was employed to examine the effects of PRRS challenge and nutrient restriction on skeletal muscle and liver metabolism. Forty-eight pigs were randomly selected (13.1 ± 1.97 kg BW) and allotted to 1 of 3 treatments (n = 16 pigs/treatment): 1) PRRS naïve, ad libitum fed (Ad), 2) PRRS-inoculated, ad libitum fed (PRRS+), and 3) PRRS naïve, pair-fed to the PRRS-inoculated pigs' daily feed intake (PF). At days postinoculation (dpi) 10 and 17, 8 pigs per treatment were euthanized and tissues collected. Tissues were assayed for markers of proteolysis (LM only), protein synthesis (LM only), oxidative stress (LM only), gluconeogenesis (liver), and glycogen concentrations (LM and liver). Growth performance, feed intake, and feed efficiency were all reduced in both PRRS+ and PF pigs compared with Ad pigs (P < 0.001). Furthermore, growth performance and feed efficiency were additionally reduced in PRRS+ pigs compared with PF pigs (P < 0.05). Activity of most markers of LM proteolysis (μ-calpain, 20S proteasome, and caspase 3/7) was not increased (P > 0.10) in PRRS+ pigs compared with Ad pigs, although activity of m-calpain was increased in PRRS+ pigs compared with Ad pigs (P = 0.025) at dpi 17. Muscle reactive oxygen species production was not increased (P > 0.10) in PRRS+ pigs compared with Ad pigs. However, phosphorylation of protein synthesis markers was decreased in PRRS+ pigs compared with both Ad (P < 0.05) and PF (P < 0.05) pigs. Liver gluconeogenesis was not increased as a result of PRRS; however, liver glycogen was decreased (P < 0.01) in PRRS+ pigs compared with Ad and PF pigs at both time points. Taken together, this work demonstrates the differential impact a viral challenge and nutrient restriction have on metabolism of growing pigs. Although markers of skeletal muscle proteolysis showed limited evidence of increase, markers of skeletal muscle synthesis were reduced during PRRS viral challenge. Furthermore, liver glycogenolysis seems to provide PRRS+ pigs with glucose needed to fuel the immune response during viral challenge.

Effects of electrical stimulation and pre-rigor conditioning temperature on aging potential of hot-boned beef M. longissimus lumborum.

The objective of this study was to create various pH/temp decline rates in hot-boned bull beef M.longissimus lumborum (LL) through a combination of electrical stimulation (ES) and pre-rigor holding temperature. The relationship between the pre-rigor interventions, the activities of µ-calpain and small heat shock proteins (sHSP), and the impacts on meat product quality were determined. Paired LL loins from 13 bulls were hot-boned within 40min of slaughter, immediately ES and subjected to various holding temperatures (5, 15, 25, and 35°C) for 3hr. The rate of muscle pH decline, sarcomere length, shear force, and proteolysis of muscle proteins were measured. ES-25°C had a longer sarcomere length compared to non-electrical stimulation samples. ES-25°C and ES-35°C samples had lower shear force values, higher µ-calpain activity and higher desmin, troponin-T, and sHSP degradation. The above findings suggest that pH/temp decline rates created in hot-boned muscle impacted muscle protein proteolysis by increasing the activity of proteases and degradation of sHSP.

Expressions and characterization of MuRFs, Atrogin-1, F-box25 genes in tilapia, Oreochromis niloticus, in response to starvation.

Muscle accretion is affected by the difference between protein synthesis and its degradation. Studies on different species revealed that muscle proteolysis is mediated by different pathways including the ubiquitin-proteasome pathway in which the ubiquitin protein ligases play an important role. These muscle atrophy associated ligases were not well studied in tilapia. In this study, we characterized the ubiquitin protein ligases MuRF1/2/3, Atrogin-1 and F-box25, members of the ubiquitin-proteasome pathway in tilapia, Oreochromis niloticus, and their expressions in the muscle of starved, fed, refed, and control fish. Sequences of these genes revealed presence of Ring finger, B-box, and Cos domains in all MuRF genes, as well as F-box domain in Atrogin-1 and F-box25 genes. Real-time qPCR data analysis showed that expression of MuRF1/2/3, Atrogin-1, F-box25, and proteasome complex genes was significantly upregulated in starved fish compared to fed fish. Concurrently, the proteasome activity was 1.7-folds elevated in the starved fish compared to fed fish. These results confirm the important role of these genes in muscle degradation and suggest potential usage as markers of muscle accretion in tilapia.

1979-P: GLP-1 Recruits Skeletal Muscle Microvasculature without Impacting Glucose Uptake or Protein Metabolism in Older Men during Postabsorptive Insulin Clamps

Introduction: Ageing muscle is associated with impaired blood flow, “anabolic resistance” to dietary protein, and impaired glucose uptake - culminating in muscle atrophy and insulin resistance. Since GLP-1 has been shown to recruit muscle microvasculature, and due to the presence of its receptors in local tissue(s) (e.g., endothelium, myocytes) we hypothesized that GLP-1 infusions in older people, would positively impact muscle microcirculation, protein and/or glucose metabolism - in an insulin-independent fashion. Methods: We studied seven older men (∼70±1 y, mean±SEM) in a cross-over design. Baseline measures were taken (first 3 h) prior to insulin and glucose clamps where participants received co-infusions of octreotide, Vamin 14-EF (containing essential amino acids and dextrose) - with or without GLP-1, over the next 3 h. Four muscle biopsies were taken over 90-120 minute intervals. Muscle protein turnover was quantified via A-V balance, and direct incorporation of 13C6 phenylalanine via mass spectrometric techniques. Plasma GLP-1 and insulin were quantified via Milliplex Map, ELISA. Muscle microvascular flow was assessed via contrast enhanced ultrasound. Leg glucose uptake was determined via A-V balance. Results: GLP-1 increased microvascular flow in +GLP-1 vs. -GLP-1 conditions (increase vs. without GLP-1 infusion: 7.2±4.7 vs. 0.40±0.26-fold, p=0.009) while leg glucose uptake was unchanged. Muscle proteolysis (nb. no effects on synthesis) was equally suppressed vs. basal with GLP-1 (32.1±5.3 vs. 67.4±3.2 nmol.100 g leg-1.min-1, p&amp;lt;0.05) and without GLP-1 (32.77±6.1 vs. 57.9±5.1 nmol.100 g leg-1.min-1 p&amp;lt;0.05, respectively). Conclusion: GLP-1 recruits muscle microcirculation independent of insulin. A lack of impact of GLP-1 on muscle glucose uptake or protein metabolism, could reflect a requirement for fed-state insulin levels. GLP-1 may hold therapeutic value for muscle microvascular health. Disclosure H. Abdulla: None. B.E. Phillips: None. M.C. Limb: None. D.J. Wilkinson: None. T. Jandova: None. J. Cegielski: None. J.J. Bass: None. D. Rankin: None. J.S. Lewis: None. J. Williams: None. K. Smith: None. P.J. Atherton: None. I.R. Idris: None. Funding Medical Research Council UK; Arthritis Research UK

Low-Intensity Exercise Suppresses CCAAT/Enhancer-Binding Protein δ/Myostatin Pathway Through Androgen Receptor in Muscle Cells

Background: Androgen production following exercise has been suggested to contribute anabolic actions of muscle. However, the underlying mechanisms of the androgen receptor (AR) in androgen’s action are still unclear. Objective: In the present study, we examined androgen/AR-mediated action in exercise, especially for the suppression of myostatin, a potent negative regulator of muscle mass. Methods: To examine the effects of exercise, we employed low-intensity exercise in mice and electric pulse stimulation (EPS) in C2C12 myotubes. Androgen production by C2C12 myotubes was measured by enzyme-linked immunosorbent assay. To block the action of AR, we pretreated C2C12 myotubes with flutamide. Quantitative real-time polymerase chain reaction was used to determine the expression levels of proteolytic genes including CCAAT/enhancer-binding protein delta (C/EBPδ), myostatin and muscle E3 ubiquitin ligases, as well as myogenic genes such as myogenin and PGC1α. The activation of 5′-adenosine-activated protein kinase and STAT3 was determined by Western blot analysis. Results: Both mRNA and protein levels of AR significantly increased in skeletal muscle of low-intensity exercised mice and C2C12 myotubes exposed to EPS. Production of testosterone and dihydrotestosterone from EPS-treated C2C12 myotubes was markedly increased. Of interest, we found that myostatin was clearly inhibited by EPS, and its inhibition was significantly abrogated when AR was blocked by flutamide. To test how AR suppresses myostatin, we examined the effects of EPS on C/EBPδ because the promoter region of myostatin has several C/EBP recognition sites. C/EBPδ expression was decreased by EPS, and this decrease was negated by flutamide. IL-6 and phospho-STAT3 (pSTAT3) expression, the downstream pathway of myostatin, were decreased by EPS and this was also reversed by flutamide. Similar downregulation of C/EBPδ, myostatin, and IL-6 was seen in skeletal muscle of low-intensity exercised mice. Conclusions: Muscle AR expression and androgen production were increased by exercise and EPS treatment. As a mechanistical insight, it is suggested that AR inhibited myostatin expression transcriptionally by C/EBPδ suppression, which negatively influences IL-6/pSTAT3 expression and consequently contributes to the prevention of muscle proteolysis during exercise.

OR20-6 Ketones and Sepsis-Induced Muscle Weakness: Signal or Fuel for Protection?

Background: Septic patients often develop muscle weakness, contributing to ICU morbidity and mortality. We recently observed that obese septic mice and patients do not develop such weakness, explained by increased lipolysis and hepatic conversion of fatty acids into the ketone bodies. Furthermore, in lean septic mice, supplementing nutrition with 3-hydroxybutyrate (3HB) reproduced the protection against muscle weakness seen with obesity. The aim of the current study was to investigate underlying mechanisms of the 3HB-protection. Methods: We performed a mouse study (n=49) in a centrally-catheterized fluid-resuscitated model of prolonged (5 days) abdominal sepsis (cecal ligation and puncture). Lean septic mice received standard parenteral nutrition (PN) supplemented with either D,L-3HB (5 mg/kg/day) or isocaloric glucose (gluc). Healthy pair-fed mice served as controls. The use of 3HB as energy substrate in the muscle was assessed by quantifiying protein, triglyceride and glycogen content and markers of muscle ketolysis, proteolysis and fatty acid oxidation (FAO). Signaling function of 3HB was assessed by markers of muscle autophagy activation, inflammation and regeneration. Results: Although 3HB infusion abated sepsis-induced upregulation of ubiquitin-proteasome system marker Fxbo32 (p=0.002), loss of muscle mass was not prevented (p=0.8). Sepsis reduced triglyceride content (p≤0.01) equally in PN+gluc and PN+3HB mice (p=0.9), but did not affect markers of muscle FAO (Cd36, Cpt1b, Acadl, Hadha mRNA) in PN+gluc and PN+3HB mice (p≥0.2). Muscle glycogen content was unaltered by sepsis (p=0.1), and the illness-induced decrease in plasma glucose concentrations at day 5 (p≤0.004) was similar in PN+gluc and PN+3HB mice (p=1). Gene expression of the ketolysis enzyme Oxct1 was lower in PN+3HB compared to PN+gluc septic mice and healthy controls (p≤0.05). Also, ketone body transporter Mct1 mRNA was elevated by sepsis (p≤0.01) but attenuated by 3HB (p=0.05) compared to glucose. Sepsis-induced elevation of inflammation markers (Tnfα, IL-1β, Nlrp3 mRNA levels; p≥0.4) and autophagy (Atg5, Atg7 mRNA; p≥0.4; LC3 II/I, p-ULK1/ULK1 protein; p≥0.4) were not countered by 3HB infusion compared to glucose. However, PN+3HB septic mice did show enhanced markers of muscle regeneration (Myod1, Myf5 and Myog mRNA; p≤0.04) compared to PN+gluc septic mice and healthy controls. Also, sepsis-induced elevation of expression of Hdac4, an inhibitor of myoblast differentiation stimulator Mef2c, was reduced by 3HB (p=0.03) and resulted in increased Mef2c mRNA levels (p=0.01) compared to glucose. Conclusion: In conclusion, the observed protection against sepsis-induced muscle weakness with 3HB infusion coincided with enhanced markers of muscle regeneration, but not with changes in substrate handling. Supplemented 3HB during sepsis thus acted as a signaling molecule rather than as an energy source.

Citrulline and muscle protein homeostasis in three different models of hypercatabolism

Supplementation of enteral nutrition (EN) by specific amino acids (AAs) has been proposed to prevent muscle protein loss in intensive care unit (ICU) patients. Citrulline (Cit), which has been shown to stimulate muscle protein synthesis in other situations, may be of interest in this setting. Our aim was to assess the effect of Cit in three catabolic models relevant to critical illness: endotoxemia (LPS), traumatic brain injury (TBI), and TBI with infectious complications (TBI-Ec), which are characterized by different alterations in protein homeostasis. Fifty-eight male Sprague-Dawley rats (200-220g) were randomized to receive a standard diet ad libitum (CON, n=9) or to undergo catabolic injuries on day 0 (D0, n=49), and EN (Sondalis HP energy® 290kcal/kg/d) from day 1 (D1) combined with Cit (2g/kg/d) or isonitrogenous non-essential AAs (NEAAs) until day 3 (D3). Endotoxemia was induced by IP injection of LPS from E.coli (3mg/kg), TBI by hydraulic percussion, and infectious complications (TBI-Ec) by administration of luminescent E.coli on D1. Nitrogen balance (ΔN) and 3-methylhistidine (3-MHis) were measured daily. Muscle protein synthesis (MPS, measured by the SUnSET method) and mTORC1 activation (S6K-1 and 4E-BP1 phosphorylation) were measured on D3 2h after the arrest of enteral nutrition in soleus, extensor digitorum longus (EDL), gastrocnemius and tibialis muscles. ΔN was lower (p<0.001) in all three models of injury compared with basal and CON from D1 to D3, and more negative in the LPS-CIT (p<0.05) than in the LPS group. The 3-MHis/creatinine ratio was significantly increased on D1 in all groups compared with CON, and on D2 only in the LPS and TBI groups (p<0.0001, LPS and TBI vs. CON). MPS was similar in all groups in soleus and tibialis but significantly higher in EDL in LPS-CIT [LPS-CIT: 4.5±1.7 (mean±SD) vs. CON: 2.3±1.2; and vs. LPS-NEAA: 3.1±2.3] and in gastrocnemius (LPS-CIT vs. CON; p=0.05). S6K-1 phosphorylation in the EDL was also higher in LPS-CIT vs. CON (LPS-CIT: 0.94±0.51 CON: 0.42±0.28), but not in gastrocnemius. IL-6 plasma level was significantly higher in all the catabolic groups vs. CON (p<0.005) with no difference between treatments (Cit or NEAAs). In conclusion, the TBI model showed only a rise in muscle proteolysis, whereas the LPS model displayed a rise in both protein synthesis and proteolysis. Secondly, our results show that the Cit effect varies according to the type of injury and to the muscle under study. The stimulation of MPS rate and the mTOR pathway in LPS-treated rats contrasts with degraded ΔN, suggesting that the Cit effect on protein metabolism in critically ill rats is limited at the whole-body level.