Muscle Atrophy F-box Expression Research Articles

SAT004 A Novel Therapeutic Target Of Mitochondrial Protein To Prevent Steroid-induced Muscle Atrophy Through Regulation Of Proteolysis And Myogenesis

Abstract Disclosure: M. Kim: None. I.S. Sinam: None. S. Kim: None. Z. Siddique: None. S. Kwon: None. J. Jeon: None. I. Lee: None. Objectives: Muscle atrophy, defined as a progressive loss of muscle mass and function induced by various etiology. Steroids is well-known muscle atrophy-inducible drug by enhancing of muscle catabolism and proteolysis. Pyruvate dehydrogenase kinase (PDK), a mitochondrial protein that inhibits the pyruvate dehydrogenase complex, is highly upregulated in metabolic diseases, especially pathologic muscle conditions associated with enhanced muscle proteolysis and aberrant myogenesis. Here, we investigated the role of PDK on proteolysis and myogenesis in steroid-induced muscle atrophy. Methods & Materials: Human skeletal (gluteus-maximus) muscles obtained from patients who underwent hip replacement surgery according to steroid administration were used for evaluated at the molecular level by expression of key genes and proteins involved in myogenesis. Steroid-induced muscle atrophy models were developed administered with glucocorticoid (GC) 25 mg/kg, intraperitoneally, for 10 days. Muscle dysfunction was studied in vivo in wild-type and PDK4 knockout mice treated with GC. We used C2C12 myotubes treated with GC for 24 h for in vitro study. Results: The expression of PDK4, as well as muscle-specific E3 ligases: muscle RING finger 1(MuRF1) and muscle atrophy F box (MAFbx), are significantly increased in skeletal muscle of steroid-user. In mouse model of steroid-induced muscle atrophy, expression of PDK4 and MAFbx are also upregulated, whereas knockdown of PDK4 reduces MAFbx expression and ameliorated steroid-induced muscle atrophy; increased muscle strength and muscle fibers. In C2C12 myotubes, knockdown of PDK4 was also associated with reduction of myogenin induction and MAFbx expression. To find out the mechanism, we used coimmunoprecipitation and liquid chromatography-mass spectrometry analysis and found that myogenin is novel substrate of PDK4. Mechanistically, this result suggests that PDK4 promotes MAFbx recruitment to catalyze muscle atrophy-induced MYOG ubiquitination and degradation. Conclusion: Taken together, the results suggest that PDK4-dependent activation of the ubiquitin degradation pathway appears to underly the promotion of muscle atrophy by directly targeting the muscle-specific MAFbx. Therefore, the inhibition of PDK4 represents as a novel therapeutic target to alleviate steroid-induced muscle atrophy. Presentation: Saturday, June 17, 2023

Attenuation of skeletal muscle atrophy via acupuncture, electro-acupuncture, and electrical stimulation

BackgroundAccelerated skeletal muscle wasting is a shared trait among many pathologies and aging. Acupuncture has been used as a therapeutic intervention to control pain; however, little is known about its effects on skeletal muscle atrophy and function. The study's purpose was to compare the effects of acupuncture, electro-acupuncture, and electrical stimulation on cast-induced skeletal muscle atrophy. MethodsForty female Sprague Dawley rats were randomly divided into groups: Control, casted (CAST), CAST+Acupuncture (CAST-A), 4) CAST+Electro-acupuncture (CAST-EA), and CAST+Electrical stimulation (CAST-ES) (n = 8). Plaster casting material was wrapped around the left hind limb. Acupuncture and electro-acupuncture (10 Hz, 6.4 mA) treatments were applied by needling acupoints (stomach-36 and gallbladder-34). Electrical stimulation (10 Hz, 6.4 mA) was conducted by needling the lateral and medial gastrocnemius muscles. Treatments were conducted for 15 min, three times/week for 14 days. Muscle atrophy F-box (MAFbx), muscle RING finger 1 (MuRF1), and contractile properties were assessed. ResultsFourteen days of cast-immobilization decreased muscle fiber CSA by 56% in the CAST group (p = 0.00); whereas, all treatment groups demonstrated greater muscle fiber CSA than the CAST group (p = 0.00). Cast-immobilization increased MAFbx and MuRF1 protein expression in the CAST group (p<0.01) while the CAST-A, CAST-EA, and CAST-ES groups demonstrated lower levels of MAFbx and MuRF1 protein expression (p<0.02) compared to the CAST group. Following fourteen days of cast-immobilization, peak twitch tension did not differ between the CAST-A and CON groups (p = 0.12). ConclusionSkeletal muscle atrophy, induced by 14 days of cast-immobilization, was significantly attenuated by acupuncture, electro-acupuncture, or electrical stimulation.

Effects on Goat Meat Extracts on α-Glucosidase Inhibitory Activity, Expression of Bcl-2-Associated X (BAX), p53, and p21 in Cell Line and Expression of Atrogin-1, Muscle Atrophy F-Box (MAFbx), Muscle RING-Finger Protein-1 (MuRF-1), and Myosin Heavy Chain-7 (MYH-7) in C2C12 Myoblsts.

This study examined the α-glucosidase inhibitory, and apoptosis- and anti-muscular-related factors of goat meat extracts from forelegs, hind legs, loin, and ribs. The goat meat extracts were evaluated for their α-glucosidase inhibitory activity. The gene and protein expression levels of Bcl-2-associated X (bax), p53, and p21 were examined by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting in AGS and HT-29 cells. The expression levels of Atrogin-1 and MHC1b were examined by RT-PCR in C2C12 myoblasts, and the expression levels of Atrogin-1, muscle atrophy F-box (MAFbx), muscle RING-finger protein-1 (MuRF-1), and myosin heavy chain-7 were investigated by immunoblotting. α-Glucosidase inhibitory activity was higher in ethanol extract than in hydrous and hot water extracts. BAX and p53 expression levels were higher (p<0.05) in AGS cells treated with goat meat extract than those of cells treated with no goat meat extract. In HT-29 cells, the protein expression levels of BAX, p53, and p21 were higher (p<0.05) in the cells treated with goat meat extract than those of cells not treated with goat meat extract. In dexamethasone-treated C2C12 cells, goat meat extract treatment lower (p<0.05) the expression of Atrogin-1 and lower (p<0.05) the expression of MAFbx and MuRF-1. The results of the present study indicate that goat meat extracts have α-glucosidase inhibitory activity in vitro. In addition, apoptosis was induced in AGS cells and HT-29 cells treated with goat meat extract, and anti-muscular atrophy activity was also observed in C2C12 cells treated with goat meat extract.

β-Sitosterol Attenuates Dexamethasone-Induced Muscle Atrophy via Regulating FoxO1-Dependent Signaling in C2C12 Cell and Mice Model.

Sarcopenia refers to a decline in muscle mass and strength with age, causing significant impairment in the ability to carry out normal daily functions and increased risk of falls and fractures, eventually leading to loss of independence. Maintaining protein homeostasis is an important factor in preventing muscle loss, and the decrease in muscle mass is caused by an imbalance between anabolism and catabolism of muscle proteins. Although β-sitosterol has various effects such as anti-inflammatory, protective effect against nonalcoholic fatty liver disease (NAFLD), antioxidant, and antidiabetic activity, the mechanism of β-sitosterol effect on the catabolic pathway was not well known. β-sitosterol was assessed in vitro and in vivo using a dexamethasone-induced muscle atrophy mice model and C2C12 myoblasts. β-sitosterol protected mice from dexamethasone-induced muscle mass loss. The thickness of gastrocnemius muscle myofibers was increased in dexamethasone with the β-sitosterol treatment group (DS). Grip strength and creatine kinase (CK) activity were also recovered when β-sitosterol was treated. The muscle loss inhibitory efficacy of β-sitosterol in dexamethasone-induced muscle atrophy in C2C12 myotube was also verified in C2C12 myoblast. β-sitosterol also recovered the width of myotubes. The protein expression of muscle atrophy F-box (MAFbx) was increased in dexamethasone-treated animal models and C2C12 myoblast, but it was reduced when β-sitosterol was treated. MuRF1 also showed similar results to MAFbx in the mRNA level of C2C12 myotubes. In addition, in the gastrocnemius and tibialis anterior muscles of mouse models, Forkhead Box O1 (FoxO1) protein was increased in the dexamethasone-treated group (Dexa) compared with the control group and reduced in the DS group. Therefore, β-sitosterol would be a potential treatment agent for aging sarcopenia.

Fucoxanthin rescues dexamethasone induced C2C12 myotubes atrophy

Muscle atrophy and weakness are the adverse effects of long-term or high dose usage of glucocorticoids. In the present study, we explored the effects of fucoxanthin (10 μM) on dexamethasone (10 μM)-induced atrophy in C2C12 myotubes and investigated its underlying mechanisms. The diameter of myotubes was observed under a light microscope, and the expression of myosin heavy chain (MyHC), proteolysis-related, autophagy-related, apoptosis-related, and mitochondria-related proteins was analyzed by western blots or immunoprecipitation. Fucoxanthin alleviates dexamethasone-induced muscle atrophy in C2C12 myotubes, indicated by increased myotubes diameter and expression of MyHC, decreased expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1). Through activating SIRT1, fucoxanthin inhibits forkhead box O (FoxO) transcriptional activity to reduce protein degradation, induces autophagy to enhance degraded protein clearance, promotes mitochondrial function and diminishes apoptosis. In conclusion, we identified fucoxanthin ameliorates dexamethasone induced C2C12 myotubes atrophy through SIRT1 activation.

Effects of electroacupuncture on proangiogenesis process and protein turnover in a mouse model of sarcopenia

To observe the effect of electroacupuncture(EA) on proangiogenesis process and protein turn-over in a mouse model of sarcopenia, so as to explore its potential molecular mechanism anti-aging. Fourteen 30-week-old male SAMP8 mice were randomly divided into a model group (n=7) and an EA group (n=7). Seven anti-rapidly aging SAMR1 mice of the same age were used as the control group (n=7). EA (1 mA, 4 Hz) was applied to bilateral "Zusanli"(ST36) and "Yanglingquan"(GB34) for 20 minutes each time once a day, 6 times a week for 4 weeks. The exhausted running platform was used to test the sports function. Gastrocnemius muscle mass and relative ratio of gastrocnemius muscle mass to body mass were measured. HE staining and transmission electron microscope were used to observe the morphology, and the cross-sectional area of gastrocnemius muscle was calculated. Relative protein expressions of protein kinase B (AKT) , phosphorylated (p) -AKT, mammalian target of rapamycin (mTOR) , p-mTOR, p70 ribosomal protein S6 kinase (p70S6K) , p-p70S6K,hypoxia inducible factor-1α (HIF-1α) and relative mRNA expressions of HIF-1α, vascular endothelial growth factor A (VEGF-A) , muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx) were detected by Western blot and real-time fluorescence quantitative PCR, seperatively. Compared with the control group, the running time and distance, body mass and gastrocnemius mass, and the ratio of gastrocnemius mass to body mass decreased(P<0.01, P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A decreased (P<0.01), while mRNA expression of MuRF1 and MAFbx increased (P<0.01) in the model group. Following EA intervention, the running time and distance, body mass and gastrocnemius mass and the ratio of gastrocnemius mass to body mass increased (P<0.05), cross-sectional area of gastrocnemius, related protein expression of p-AKT,p-mTOR, p-p70S6K and HIF-1α, mRNA expression of HIF-1α and VEGF-A were significantly up-regulated (P<0.01), mRNA expression of MuRF1 and MAFbx down-regulated (P<0.01, P<0.05) in the EA group compared with the model group. EA may delay the aging muscle atrophy in mice by regulating the gastrocnemius muscle's proangiogenesis process and protein turnover.

Water Extract of Lotus Leaf Alleviates Dexamethasone-Induced Muscle Atrophy via Regulating Protein Metabolism-Related Pathways in Mice.

Muscle atrophy is an abnormal condition characterized by loss of skeletal muscle mass and function and is primarily caused by injury, malnutrition, various diseases, and aging. Leaf of lotus (Nelumbo nucifera Gaertn), which has been used for medicinal purposes, contains various active ingredients, including polyphenols, and is reported to exert an antioxidant effect. In this study, we investigated the effect of water extract of lotus leaf (LL) on muscle atrophy and the underlying molecular mechanisms of action. Amounts of 100, 200, or 300 mg/kg/day LL were administered to dexamethasone (DEX)-induced muscle atrophy mice for 4 weeks. Micro-computed tomography (CT) analysis revealed that the intake of LL significantly increased calf muscle volume, surface area, and density in DEX-induced muscle atrophy mice. Administration of LL recovered moving distance, grip strength, ATP production, and body weight, which were decreased by DEX. In addition, muscle damage caused by DEX was also improved by LL. LL reduced the protein catabolic pathway by suppressing gene expression of muscle atrophy F-Box (MAFbx; atrogin-1), muscle RING finger 1 (MuRF1), and forkhead box O (FoxO)3a, as well as phosphorylation of AMP-activated kinase (AMPK). The AKT-mammalian target of the rapamycin (mTOR) signal pathway, which is important for muscle protein synthesis, was increased in LL-administered groups. The HPLC analysis and pharmacological test revealed that quercetin 3-O-beta-glucuronide (Q3G) is a major active component in LL. Thus, Q3G decreased the gene expression of atrogin-1 and MuRF1 and phosphorylation of AMPK. This compound also increased phosphorylation levels of mTOR and its upstream enzyme AKT in DEX-treated C2C12 cells. We identified that LL improves muscle wasting through regulation of muscle protein metabolism in DEX-induced muscle atrophy mice. Q3G is predicted to be one of the major active phenolic components in LL. Therefore, we propose LL as a supplement or therapeutic agent to prevent or treat muscle wasting, such as sarcopenia.

Fermented Feed Modulates Meat Quality and Promotes the Growth of Longissimus Thoracis of Late-Finishing Pigs.

Simple SummaryLittle is known about the effect of fermented feed on lean mass production of finishing pigs and the underlying mechanism. This study aimed to investigate the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. Our results found that fermented diet significantly increased loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. Fermented diet also increased the abundance of insulin, insulin receptor, myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of protein kinase B (AKT), mammalian target of rapamycin (mTORC1), initiation factor 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1) in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Our results indicated that fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy. This study investigated the effect of fermented diet on growth performance, carcass traits, meat quality and growth of longissimus thoracis (LT) of finishing pigs. A total of 48 finishing pigs [Duroc × (Landrace × Large White), male, 126 ± 5-d-old] weighing 98.76 ± 1.27 kg were randomly assigned to two treatments (eight pens per treatment and three pigs per pen) for a 28-d feeding trial, including control diet and fermented diet. Fermented diet significantly increased the loin eye area and lean mass percentage, decreased backfat thickness and improved meat quality of LT by decreasing the shear force and drip loss at 48 h post slaughter and improving meat sensory characteristics compared with control diet. A fermented diet also significantly increased the abundance of insulin, insulin receptor (IR), myoblast determination protein (MyoD) and myosin heavy chain-I (MyHC-I) transcripts, and the phosphorylation levels of AKT, mTORC1, 4EBP1 and S6K1 in LT, while decreasing the expression of muscle atrophy F-box (MAFbx) and forkhead Box O1 (Foxo1) mRNA transcripts. Moreover, proteomic analysis revealed that differentially expressed proteins predominantly involved in protein synthesis and muscle development were modulated by fermented diet. Our results indicated that a fermented diet improved meat quality and enhanced LT growth of finishing pigs by increasing insulin/AKT/mTORC1 protein synthesis cascade and activating the Foxo1/MAFbx pathway, along with the regulation of ribosomal protein and proteins involved in muscle contraction and muscle hypertrophy.

Effects of electroacupuncture on skeletal muscle and blood glucose in rats with diabetic amyotrophy

To explore the effects of electroacupuncture (EA) on skeletal muscle and blood glucose in rats with diabetic amyotrophy. Among 40 SD rats, 10 rats were randomly selected into the control group and received no treatment. The remaining 30 rats were treated with intraperitoneal injection of streptozotocin (STZ, 60 mg/kg) to establish diabetes mellitus (DM) model, and then the rats were treated with vascular ligation at right posterior limb to establish amyotrophy model. The rats with diabetic amyotrophy were randomly divided into a model group and an EA group, 10 rats in each group (10 rats were excluded due to unsuccessful model establishment and death). The rats in the EA group was treated with EA at right-side "Yishu (EX-B 3)" "Shenshu (BL 23)" "Zusanli (ST 36)" and "Sanyinjiao (SP 6)", disperse-dense wave, 2 Hz/ 15 Hz, 20 minutes each time, once a day for 3 weeks. Before and after EA treatment, the blood sample was collected from inner canthus and the "glucose oxidase-peroxidase" method was used to detect fasting blood glucose level; ELISA method was used to detect insulin content. At the end of the treatment, HE staining method was used to observe the morphology of ischemic skeletal muscle in the right hindlimb; the real-time PCR method was used to detect the mRNA expression of muscle atrophy F-box (MAFbx), muscle ring finger-1 (MuRF1) and forkhead box O3a (FOXO3a) in the ischemic skeletal muscle tissue of right hindlimb. Before the treatment, the body mass in the model group and EA group was lower than that in the control group (P<0.01); after the treatment, the body mass in the control group was increased, while the body mass in the model group and EA group was decreased (P<0.01). Compared with the control group, the fasting blood glucose was significantly increased and insulin content was significantly decreased in the model group (P<0.01); compared with the model group, the fasting blood glucose was significantly decreased and the insulin content was significantly increased in the EA group after treatment (P<0.01). The muscle fibers of the model group were obviously broken, the number of the nuclei decreased, and the nuclei shrinked or even dissolved; the morphology of the muscle tissue of the EA group after intervention was improved compared with the model group. Compared with the control group, the cross-sectional area of ischemic skeletal muscle cells in the right hindlimb in the model group was decreased (P<0.01); compared with the model group, the cross-sectional area of ischemic skeletal muscle cells in the right hindlimb was increased in EA group (P<0.05). Compared with the control group, the levels of MAFbx, MuRF1 and FOXO3a mRNA in the right hindlimb ischemic skeletal muscle in the model group were increased significantly (P<0.01, P<0.05); compared with the model group, the levels of MAFbx, MuRF1 and FOXO3a mRNA in the EA group were decreased significantly (P<0.05, P<0.01). EA may play a role in the treatment of diabetic amyotrophy by inducing FOXO3a to reduce the transcription of MAFbx and MuRF1.

Contralateral C7 transfer to axillary and median nerves in rats with total brachial plexus avulsion

BackgroundContralateral cervical 7 nerve (cC7) was used to repair two recipient nerves simultaneously for patients with total brachial plexus avulsion (TBPA).ObjectiveTo evaluate the effect of cC7 transfer to axillary and median nerves in rats with TBPA.MethodsEighty S-D rats were divided into 4 groups randomly on average. Group A: cC7-median nerve, Group B: cC7-axillary nerve, Group C: cC7-median and axillary nerves, Group D: TBPA without repair. The evaluation tools included behavioral tests, electromyogram (EMG), measurement of cross-sectional area of muscle fiber, nerve fiber count and gene expression assay.ResultsThe effective rates of EMG were 90 and 70% in Flexor Carpi Radialis (FCR) in Group A and C, while 70 and 60% in deltoid (DEL) in Group B and C, respectively. In behavioral test, the differences of effective rates between groups were not significant. The mean cross-sectional area of FCR in Group A or C was significantly larger than that in Group D. Either the number of median or axillary nerve fibers in Group A, B or C was statistically more than that in Group D. No matter for FCR or DEL, there were no significant differences in the ratios of relative expression of Muscle Atrophy F-box(MAFBOX)and Muscle RING Finger 1(MURF1)among these groups.ConclusionCompared with cC7 transfer to median nerve, cC7 transfer to both median and axillary nerves did not affect median nerve recovery. The deltoid muscle also could be restored. The recovery proportion of axillary nerve was less than that of median nerve.

High-intensity interval training changes the expression of muscle RING-finger protein-1 and muscle atrophy F-box proteins and proteins involved in the mechanistic target of rapamycin pathway and autophagy in rat skeletal muscle.

What is the central question of this study? What are the adaptations of protein synthesis and degradation that occur in skeletal muscle in response to high-intensity interval training (HIIT), and what are the magnitudes of the changes in response to HIIT, compared to moderate-intensity continuous training (MICT), and the mechanisms underlying these changes? What is the main finding and its importance? HIIT is more effective than MICT in altering the expression of muscle RING-finger protein-1 and muscle atrophy F-box, and enhancing the autophagic flux in rat soleus muscle. In addition, HIIT could activate the mechanistic target of rapamycin pathway. These findings suggest that HIIT might be an effective exercise strategy for health promotion in skeletal muscle. This study aimed to investigate the impact of high-intensity interval training (HIIT) on the proteins involved in protein synthesis, the ubiquitin-proteasome system (UPS) and autophagy in skeletal muscle of middle-aged rats. Nine-month-old male Wistar rats (n=56) were randomly divided into three groups: a control (C) group, a moderate-intensity continuous training (MICT) group and a HIIT group. Rats in the training groups ran on treadmills 5days per week for 8weeks. The MICT group ran for 50min at 60% , while the HIIT group ran for 3min at 80% of six times separated by 3-min periods at 40% . Aerobic endurance, number of autophagosomes and expression of proteins involved in protein synthesis and degradation in the soleus muscle were measured at three time points: before training, after 4weeks and after 8weeks of training. Compared to the C group, HIIT and MICT increased the expression of phosphorylated mechanistic target of rapamycin (mTOR) after 8weeks (P<0.05 and P<0.01, respectively). HIIT increased the expression of muscle RING-finger protein-1 (MuRF-1) after 4weeks (P<0.01), and decreased its expression after 8weeks (P<0.01). Both HIIT and MICT decreased the expression of muscle atrophy F-box (MAFbx) after 4weeks (P<0.05). HIIT improved the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II (P<0.05), and decreased the P62 content (P<0.01) after 4weeks. The LC3II/LC3I ratio was increased after 8weeks (P<0.01). This study demonstrated that HIIT could activate the mTOR pathway, alter the expression of MuRF-1 and MAFbx proteins, and enhance autophagic flux in soleus muscle of middle-aged rats.

Effects Of Intermittent Fasting And High Intensity Training On Regulators Of Muscle Mass And Metabolism

Diet-induced obesity can lead to higher intramuscular fat deposition and inflammatory cell accumulation, ultimately having a negative impact on skeletal muscle morphology and function leading to mitochondrial dysfunction and insulin resistance. Intermittent fasting (IF) and high intensity interval training (HIIT) are both effective strategies for losing weight, specifically fat mass. However, the effects on skeletal muscle, specifically genes that regulate mitochondrial function, energy homeostasis, and muscle atrophy are unknown. PURPOSE: To investigate the effects of IF and/or HIIT on molecular markers of skeletal muscle mass and metabolic function in diet-induced obesity. METHODS: Eight week old mice (C57BL/6, n=39) had ad libitum access to an obesogenic diet (60% fat, 30% sugar) for 12 weeks. They were then randomly allocated to three intervention groups: IF (fasting for 2 alternate days/week), HIIT (3 days/week), combined IF+HIIT (2 alternate fasting days and 3 days HIIT) or control (CON) for a further 12 weeks. Extensor digitorum longus (EDL) muscle weight and expression of PGC1α, AMPK, citrate synthase (CS), muscle atrophy F-box (MAFbx), and muscle RING Finger-1 (MuRF1) genes were measured at the end of the intervention period. Data was analysed using ANOVA. RESULTS: Muscle weights were similar between groups at the end of the intervention period (CON: 9.5±1.3mg, HIIT: 9.2±0.8mg, IF: 9.2±0.4mg, IF+HIIT: 9.7±0.8mg, p>0.05). PGC1α and CS gene expression was significantly lower in the IF group compared to the CON (PGC1α: 0.8±0.1 vs 1.0±0.2, CS: 0.8±0.2 vs 1.0±0.4, p<0.05). AMPK gene expression was also significantly lower in the IF group, but only compared to the IF+HIIT group (0.9±0.1 vs 1.0±0.1, p<0.05). MAFbx and MuRF1 gene expression was significantly higher in the HIIT group (1.7±0.8 & 2.2±0.9) group compared to CON (1.0±0.4 & 1.0±0.2), IF (0.9±0.2 & 0.8±0.2), and IF+HIIT (1.4±0.3 & 0.9±0.2, p<0.01).groups. CONCLUSIONS: Intermittent fasting reduced gene expression markers of mitochondrial biogenesis and energy homeostasis, while HIIT appeared to increase markers of atrophy at the end of the intervention period. The combination of IF and HIIT did not show any synergistic effects within the muscle.

Oral Treatment with the Ghrelin Receptor Agonist HM01 Attenuates Cachexia in Mice Bearing Colon-26 (C26) Tumors.

The gastrointestinal hormone ghrelin reduces energy expenditure and stimulates food intake. Ghrelin analogs are a possible treatment against cancer anorexia-cachexia syndrome (CACS). This study aimed to investigate whether oral treatment with the non-peptidergic ghrelin receptor agonist HM01 counteracts CACS in colon-26 (C26) tumor-bearing mice. The C26 tumor model is characterized by pronounced body weight (BW) loss and muscle wasting in the absence of severe anorexia. We analyzed the time course of BW loss, body composition, muscle mass, bone mineral density, and the cytokines interleukin-6 (IL-6) and macrophage-inhibitory cytokine-1 (MIC-1). Moreover, we measured the expression of the muscle degradation markers muscle RING-finger-protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx). After tumor inoculation, MIC-1 levels increased earlier than IL-6 and both cytokines were elevated before MuRF-1/MAFbx expression increased. Oral HM01 treatment increased BW, fat mass, and neuronal hypothalamic activity in healthy mice. In tumor-bearing mice, HM01 increased food intake, BW, fat mass, muscle mass, and bone mineral density while it decreased energy expenditure. These effects appeared to be independent of IL-6, MIC-1, MuRF-1 or MAFbx, which were not affected by HM01. Therefore, HM01 counteracts cachectic body weight loss under inflammatory conditions and is a promising compound for the treatment of cancer cachexia in the absence of severe anorexia.

Aspartate inhibits LPS-induced MAFbx and MuRF1 expression in skeletal muscle in weaned pigs by regulating Akt, AMPKα and FOXO1.

Infection and inflammation can result in the rapid loss of muscle mass and myofibrillar proteins (muscle atrophy). In addition, aspartate (Asp) is necessary for protein synthesis in mammalian cells. We hypothesized that Asp could attenuate LPS-induced muscle atrophy in a piglet model. Twenty-four weaning piglets were allotted to four treatments, including non-challenged control, LPS challenged control, LPS+0.5% Asp and LPS+1.0% Asp. On d 21, the piglets were injected with i.p. injection of LPS (100 ug/kg BM) or saline. At 4 h post-injection, blood, gastrocnemius and longissimus dorsi muscles samples were collected for biochemical and molecular analyses. Asp decreased the concentrations of cortisol and glucagon in plasma. In addition, Asp increased protein and RNA contents in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). Moreover, Asp decreased phosphorylation of AMPKα but increased phosphorylation of Akt and Forkhead Box O (FOXO) 1 in the muscles. Our results indicate that Asp suppresses LPS-induced MAFbx and MuRF1 expression via activation of Akt signaling, and inhibition of AMPKα and FOXO1 signaling.

Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

Glycine enhances muscle protein mass associated with maintaining Akt-mTOR-FOXO1 signaling and suppressing TLR4 and NOD2 signaling in piglets challenged with LPS.

Pro-inflammatory cytokines play a critical role in the pathophysiology of muscle atrophy. We hypothesized that glycine exerted an anti-inflammatory effect and alleviated lipopolysaccharide (LPS)-induced muscle atrophy in piglets. Pigs were assigned to four treatments including the following: 1) nonchallenged control, 2) LPS-challenged control, 3) LPS+1.0% glycine, and 4) LPS+2.0% glycine. After receiving the control, 1.0 or 2.0% glycine-supplemented diets, piglets were treated with either saline or LPS. At 4 h after treatment with saline or LPS, blood and muscle samples were harvested. We found that 1.0 or 2.0% glycine increased protein/DNA ratio, protein content, and RNA/DNA ratio in gastrocnemius or longissimus dorsi (LD) muscles. Glycine also resulted in decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1) in gastrocnemius muscle. In addition, glycine restored the phosphorylation of Akt, mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and Forkhead Box O 1 (FOXO1) in gastrocnemius or LD muscles. Furthermore, glycine resulted in decreased plasma tumor necrosis factor-α (TNF-α) concentration and muscle TNF-α mRNA abundance. Moreover, glycine resulted in decreased mRNA expresson of Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain protein 2 (NOD2), and their respective downstream molecules in gastrocnemius or LD muscles. These results indicate glycine enhances muscle protein mass under an inflammatory condition. The beneficial roles of glycine on the muscle are closely associated with maintaining Akt-mTOR-FOXO1 signaling and suppressing the activation of TLR4 and/or NOD2 signaling pathways.

Effects of GHRP-2 and Cysteamine Administration on Growth Performance, Somatotropic Axis Hormone and Muscle Protein Deposition in Yaks (Bos grunniens) with Growth Retardation.

The objective of this study was to investigate the effects of growth hormone-releasing peptide-2 (GHRP-2) and cysteamine (CS) administration on growth performance in yaks with growth retardation and try to elucidate its regulatory mechanisms. Trial 1, thirty-six 1-year-old Qinghai high plateau yaks (body weight 38–83.2 kg) were randomly chosen for body weight and jugular blood samples collection. The relationship between body weight and serum GHRH (P < 0.05, R = 0.45), GH (P < 0.05, R = 0.47), IGF-1 (P < 0.05, R = 0.62) was significantly correlated in yaks colonies with lighter body weights. Trial 2, fifteen 1-year-old Qinghai high plateau yaks with growth retardation (average body weight 54.8 ± 8.24 kg) were randomly selected and assigned to negative control group (NG), GHRP-2 injection group (GG) and cysteamine feeding group (CG), with 5 yaks per group. Another five 1-year-old Qinghai high plateau yaks with normal growth performance (average body weight 75.3 ± 2.43 kg) were selected as positive control group (PG). The average daily gain (ADG) of the GG and CG were significantly higher than those in the PG and NG (P < 0.05). Both GHRP-2 and CS administration significantly enhanced the myofiber diameter and area of skeletal muscle (P<0.05). GHRP-2 significantly enhanced the serum GH and IGF-1 levels (P < 0.05), and up-regulated GHR, IGF-1 and IGF-1R mRNA expression in the liver and skeletal muscle (P < 0.05), enhanced the mRNA expression of PI3K, AKt and mTOR in the skeletal muscle (P<0.05). CS significantly reduced the serum SS levels and the hypothalamus SS mRNA expression (P < 0.05), and enhanced GHR and IGF-1 mRNA expression in the liver (P < 0.05), decreased the mRNA expression of muscle atrophy F-box (Atrogin-1) and muscle ring finger 1 (MuRF1) mRNA (P < 0.05). Conclusions: Growth retardation in yaks was primarily due to somatotropic axis hormones secretion deficiency. Both GHRP-2 and CS administration can accelerate growth performance and GH, IGF-1 secretion in yaks with growth retardation. GHRP-2 enhanced muscle protein deposition mainly by up-regulated the protein synthesis pathways, whereas CS worked mainly by down-regulated the ubiquitin-proteasome pathway.

The role of E3 ubiquitin-ligases MuRF-1 and MAFbx in loss of skeletal muscle mass

The ubiquitin–proteasome system (UPS) is the main regulatory mechanism of protein degradation in skeletal muscle. The ubiquitin-ligase enzymes (E3s) have a central role in determining the selectivity and specificity of the UPS. Since their identification in 2001, the muscle specific E3s, muscle RING finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx), have been shown to be implicated in the regulation of skeletal muscle atrophy in various pathological and physiological conditions. This review aims to explore the involvement of MuRF-1 and MAFbx in catabolism of skeletal muscle during various pathologies, such as cancer cachexia, sarcopenia of aging, chronic kidney disease (CKD), diabetes, and chronic obstructive pulmonary disease (COPD). In addition, the effects of various lifestyle and modifiable factors (e.g. nutrition, exercise, cigarette smoking, and alcohol) on MuRF-1 and MAFbx regulation will be discussed. Finally, evidence of potential strategies to protect against skeletal muscle wasting through inhibition of MuRF-1 and MAFbx expression will be explored.

1α,25(OH)2D3 downregulates gene expression levels of muscle ubiquitin ligases MAFbx and MuRF1 in human myotubes.

Clinical trials involving in patients with osteoporosis have reported that activated vitamin D3 (1α,25(OH)2D3, calcitriol) can prevent falling by acting on the skeletal muscles. However, pharmacological mechanisms of 1α,25(OH)2D3 with respect to skeletal muscle hypertrophy or atrophy are still poorly understood. Therefore, we examined changes in the expression of several related genes in human myotubes to test whether 1α,25(OH)2D3 influences hypertrophy and atrophy of skeletal muscle. Myotubes treated with 1α,25(OH)2D3 increased interleukin-6 (IL-6) expression and inhibited expression of tumor necrosis factor alpha (TNF-α), whereas the expression of insulin-like growth factor-1 (IGF-1) that is involved in muscle hypertrophy was not affected. However, 1α,25(OH)2D3 treatment significantly inhibited the expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), ubiquitin ligases involved in muscle atrophy. The analysis of pathways using microarray data suggested that 1α,25(OH)2D3 upregulates AKT-1 by inhibiting the expression of protein phosphatase 2 (PP2A), a phosphatase acting on AKT-1, in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, thereby inhibiting the expression of ubiquitin ligases. Thus, this study showed that 1α,25(OH)2D3 might have an inhibitory effect on the expression of MAFbx and MuRF1 in skeletal muscle and a suppressive effect on muscle degradation in patients with osteoporosis.

Endotoxemia-induced muscle wasting is associated with the change of hypothalamic neuropeptides in rats

In critical patients, sepsis-induced muscle wasting is considered to be an important contributor to complications and mortality. Previous work mainly focuses on the peripheral molecular mechanism of muscle degradation, however little evidence exists for the role of central nervous system in the process. In the present study, we, for the first time, characterized the relationship between muscle wasting and central neuropeptide changes in a septic model. Thirty-six adult male Sprague–Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) or saline. Twelve, 24 and 48 hrs after injection, skeletal muscle and hypothalamus tissues were harvested. Muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle ring finger 1 (MuRF-1) and muscle atrophy F-box (MAFbx), as well as 3-methyl-histidine (3-MH) and tyrosine release. Hypothalamic neuropeptides and inflammatory marker expressions were also measured in three time points. LPS injection caused an increase expression of MuRF-1 and MAFbx, and a significant higher release of 3-MH and tyrosine. Hypothalamic neuropeptides, proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), agouti-related protein (AgRP) and neuropeptide Y (NPY) presented a dynamic change after LPS injection. Also, hypothalamic inflammatory markers, interleukin-1 β (IL-1β) and tumor necrosis factor α (TNF-α) increased substantially after LPS administration. Importantly, the expressions of POMC, AgRP and CART were well correlated with muscle atrophy gene, MuRF-1 expression. These findings suggest hypothalamic peptides and inflammation may participate in the sepsis-induced muscle wasting, but the exact mechanism needs further study.