Muscle RING-finger Protein-1 Research Articles

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

Suppressive Effects of Arriheuk Wheat Sprout Extract on Muscle Atrophy in Dexamethasone-Induced C2C12 Myotubes and a Mouse Model.

Skeletal muscle atrophy refers to the loss of muscle strength and mass due to decreased protein synthesis or increased protein degradation. Various conditions can cause muscle atrophy, including aging, heart disease, chronic illness, obstructive pulmonary disease, kidney failure, diabetes, AIDS, cancer, sepsis, and steroid use. Various natural materials have been studied for the prevention of muscle atrophy. In this study, we found that extracts from the sprouts of purple wheat, Arriheuk, prevented muscle atrophy in vitro and invivo. Arriheuk wheat sprouts extract inhibited the expression of muscle protein breakdown factors, which were increased by dexamethasone, and improved muscle strength. In C2C12 myotubes, Arriheuk wheat sprout extract (ARE) protected against dexamethasone-induced muscle atrophy by potentiating Akt/mammalian target of rapamycin and AMP-activated protein kinase (AMPK)/forkhead box O3 (AMPK/Foxo3) signaling and inhibiting the expression of Atrogin-1, muscle RING-finger protein-1 (MuRF1), and Myostatin. In addition, the administration of ARE in an animal model of muscle atrophy induced by dexamethasone prevented myocardial and muscle strength loss by regulating the expression of muscle atrophy-related factors by affecting AMPK/Foxo3 signaling. Taken together, these results suggest that Arriheuk wheat sprouts extract effectively alleviates muscle atrophy by regulating the synthesis and breakdown of muscle proteins.

Changes in Skeletal Muscle Atrophy over Time in a Rat Model of Adenine-Induced Chronic Kidney Disease

This study evaluated changes over time in skeletal muscle atrophy, expressions of skeletal muscle anabolic and catabolic genes, and mitochondrial activity by skeletal muscle type in an adenine-induced chronic kidney disease (CKD) model. A CKD model was successfully established by feeding male Wistar rats a 0.75% adenine diet for 4 weeks starting at 8 weeks of age. Control and CKD groups were sacrificed at 12 and 20 weeks of age. The back muscles were analyzed histologically, and succinate dehydrogenase (SDH) staining was performed to evaluate mitochondrial activity. Gene expressions of myogenic determination gene number 1 and myogenin as indicators of muscle anabolism, atrogin-1 and muscle RING-finger protein-1 (MuRF1) as indicators of muscle catabolism, and peroxisome proliferator-activated receptor-γ coactivator-1-α as a marker of mitochondrial biogenesis were assessed. Type I and type II muscle cross-sectional areas (CSAs) were decreased at 12 weeks, but type I muscle CSA was recovered at 20 weeks. SDH staining was lower in CKD than in control rats at 12 weeks, but no significant difference was observed at 20 weeks. Increased expressions of myogenin, atrogin-1, and MuRF-1 were observed only at 12 weeks, but no differences were observed at 20 weeks. The adenine-induced CKD rat model appears to show changes in muscle atrophy over time.

6′-Sialyllactose Prevents Dexamethasone-Induced Muscle Atrophy by Controlling the Muscle Protein Degradation Pathway

Sarcopenia is associated with various geriatric diseases, such as gait disorders, falls, malnutrition, and osteoporosis. Accordingly, interest in the prevention and treatment of sarcopenia has grown over the years. The human milk oligosaccharide (HMO) 6′-sialyllactose (6′-SL) is known to improve exercise performance, reduce muscle fatigue, and improve GNE myopathy; however, its effect on sarcopenia has not yet been reported. In this study, we aimed to investigate the efficacy of 6′-SL in dexamethasone-induced muscle atrophy, which is a widely used model for the study of sarcopenia. The effects of 6′-SL on differentiated C2C12 skeletal muscle cells and on mice were examined by treatment with 6′-SL in the presence or absence of dexamethasone. 6′-SL was found to inhibit the dexamethasone-induced decrease of MHC expression, as well as to prevent reduction in the number, length, and width of myotubes. Furthermore, the dexamethasone-induced upregulation of myostatin, muscle RING-finger protein-1 (MuRF1), and atrogin-1 were also inhibited by 6′-SL treatment. In mice, intraperitoneal administration of dexamethasone caused decreases in muscle fiber diameter, muscle weight, and exercise performance, most of which were significantly inhibited by oral treatment with 6′-SL. Therefore, utilization of 6′-SL could contribute to the prevention and treatment of muscle atrophy and sarcopenia.

The modulatory effects of Lonicera caerulea L. extract and omega-3 on sarcopenia via regulating PI3K and FOXO signaling pathways in rats

Introduction: Sarcopenia is an inflammatory disease caused by a disruption of muscle homeostasis. Lonicera caerulea L. (Haskap berry) (HB) extract and omega-3 (ω−3) possess antioxidant and anti-inflammatory activities. This study assesses the potential ameliorating effects of HB extract and ω−3 supplementation on dexamethasone (DEXA)-induced sarcopenia. Methods: Rats were divided into five groups; the negative control group was injected with saline (i.p.); groups 2, 3, 4, and 5 were injected with DEXA (2 mg/kg/d, i.p.); groups 3 and 4 also received 400 mg/kg/d and 100 mg/kg/d of HB extract and ω−3, respectively, while group 5 received both treatments daily for 21 days. The ameliorative effects of treatments were investigated by measuring lysosomal proteolytic enzyme cathepsin activity, phosphatidylinositol 3 kinase (PI3K) activity, nuclear factor kappa beta (NF-κB) level, and heme oxygenase-1 (HO-1) activity. The gene expression levels of muscle ring-finger protein (MuRF) and forkhead box O (FOXO) transcription factor were also evaluated. Biochemical and histological examinations were done on muscle tissues. Results: DEXA caused a significant elevation (P<0.05) in NF-κβ level and cathepsin activity in muscle tissue. Also, it significantly upregulated the muscle atrophy markers. Meanwhile, PI3K and HO-1 activities were significantly reduced. HB extract and ω−3 administration significantly (P≤0.05) reversed these effects and downregulated the mRNA expression levels of MuRF and FOXO. Also, the histopathological examination confirmed these results. Conclusion: The current study proved the ameliorative effects of HB extract and ω−3 on sarcopenia parameters. Therefore, they might be potential therapeutic agents for myopathy/sarcopenia.

Melatonin protects against sarcopenia in middle-aged mice.

Sarcopenia is a common age-related disease. Melatonin (MEL) is an age-related endocrine hormone, which displays a crucial role in resisting oxidative stress during aging. Importantly, the antioxidant properties of MEL can be mediated by mitochondria. Therefore, we wondered whether MEL could mitigate oxidative stress caused by mitochondria in sarcopenia. The middle-aged mice were administered 5 mg/kg/d and 10 mg/kg/d of MEL for 2 months. Young mice were used as the control group. After treatment with MEL, the grip strength of the fore/hind limbs, running time, and distance were elevated, and the weights of the gastrocnemius (GA), tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL) were enhanced in middle-aged mice. Additionally, MEL was observed to alleviate histological damage and increase the cross-sectional area of muscle fibers in GA tissues of middle-aged mice. Furthermore, following MEL treatment, there was an increase in the percentage and size of normal mitochondria as well as mtDNA copy number but a reduction in the levels of malondialdehyde (MDA), protein carbonyl, and reactive oxygen species (ROS) in the GA tissues of middle-aged mice. At the molecular level, MEL repressed the levels of ATROGIN-1, muscle RING-finger protein-1 (MURF-1), and the ratio of p-P38/P38, but elevated the expression of cytochrome c oxidase subunit 4 (COX4), cystatin C (CYTC), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in the GA tissues of middle-aged mice. Importantly, 10 mg/kg MEL was more efficacious in the treatment of sarcopenia than 5 mg/kg MEL. MEL attenuates sarcopenia in middle-aged mice, and the mechanism may relate to mitochondria-induced oxidative stress and the PGC-1α/TFAM pathway.

Lactobacillus delbrueckii alleviates lipopolysaccharide-induced muscle inflammation and atrophy in weaned piglets associated with inhibition of endoplasmic reticulum stress and protein degradation.

Pro-inflammatory cytokines in muscle play a pivotal role in physiological responses and in the pathophysiology of inflammatory disease and muscle atrophy. Lactobacillus delbrueckii (LD), as a kind of probiotics, has inhibitory effects on pro-inflammatory cytokines associated with various inflammatory diseases. This study was conducted to explore the effect of dietary LD on the lipopolysaccharide (LPS)-induced muscle inflammation and atrophy in piglets and to elucidate the underlying mechanism. A total of 36 weaned piglets (Duroc × Landrace × Large Yorkshire) were allotted into three groups with six replicates (pens) of two piglets: (1) Nonchallenged control; (2) LPS-challenged (LPS); (3) 0.2% LD diet and LPS-challenged (LD+LPS). On d 29, the piglets were injected intraperitoneally with LPS or sterilized saline, respectively. All piglets were slaughtered at 4 h after LPS or saline injection, the blood and muscle samples were collected for further analysis. Our results showed that dietary supplementation of LD significantly attenuated LPS-induced production of pro-inflammatory cytokines IL-6 and TNF-α in both serum and muscle of the piglets. Concomitantly, pretreating the piglets with LD also clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits in the muscle, which correlated with the anti-inflammatory effects of LD on the muscle of piglets. Meanwhile, LPS-induced muscle atrophy, indicated by a higher expression of muscle atrophy F-box, muscle RING finger protein (MuRF1), forkhead box O 1, and autophagy-related protein 5 (ATG5) at the transcriptional level, whereas pretreatment with LD led to inhibition of these upregulations, particularly genes for MuRF1 and ATG5. Moreover, LPS-induced mRNA expression of endoplasmic reticulum stress markers, such as eukaryotic translational initiation factor 2α (eIF-2α) was suppressed by pretreatment with LD, which was accompanied by a decrease in the protein expression levels of IRE1α and GRP78. Additionally, LD significantly prevented muscle cell apoptotic death induced by LPS. Taken together, our data indicate that the anti-inflammatory effect of LD supply on muscle atrophy of piglets could be likely regulated by inhibiting the secretion of pro-inflammatory cytokines through the inactivation of the ER stress/NF-κB singling pathway, along with the reduction in protein degradation.

Glucosamine inhibits myoblast proliferation and differentiation, and stimulates myotube atrophy through distinct signal pathways

Glucosamine (GlcN) is one of the dietary supplements used in the treatment of osteoarthritis. Endogenously, GlcN is synthesized from glucose through the hexosamine pathway. In addition to ameliorating arthritis, several biological functions of GlcN have been reported, including insulin resistance in skeletal muscle. However, the regulatory role of GlcN in skeletal muscle development is not clear. We therefore investigated the effect of GlcN on myoblast proliferation, differentiation, and myotube development and their underlying mechanisms in C2C12 cells. Myoblast proliferation was measured by MTT assay. The expressions of MyoD, myogenin (MyoG), and myosin heavy chain (MyHC) were identified as determinants of myoblast differentiation. Expressions of atrogin-1 and muscle RING-finger protein-1 (MuRF-1) were identified as markers of myotube atrophy. The results show that treatment with GlcN significantly reduced myoblast proliferation and phosphorylation of Stat3 and S6K. These findings suggest that GlcN can inhibit growth of myoblasts through inhibiting phosphorylation of Stat3 and S6K. In addition, GlcN significantly suppressed the expression of MyoD, MyoG, and MyHC, as well as myotube formation. Pretreatment of C2C12 myoblast cells with ER stress inhibitors significantly blocked GlcN-inhibited MyHC expression and myotube formation. It can be concluded that GlcN suppressed myogenic differentiation via a pathway that involved ER stress. Moreover, GlcN decreased myotube diameter and expression of MyHC, as well as increased MuRF-1 in C2C12 myotubes. Meanwhile, GlcN also reduced the expressions of phosphorylated Akt and mTOR were stimulated after GlcN treatment in C2C12 myotubes. Thus, GlcN induced skeletal muscle atrophy by inhibiting the protein synthesis pathway. Chronic GlcN infusion also caused skeletal muscle atrophy in mice. In conclusion, GlcN regulated important stages of skeletal muscle development through different signaling pathways.

Murf1 alters myosin replacement rates in cultured myotubes in a myosin isoform-dependent manner.

Skeletal muscle tissue increases or decreases its volume by synthesizing or degrading myofibrillar proteins. The ubiquitin-proteasome system plays a pivotal role during muscle atrophy, where muscle ring finger proteins (Murf) function as E3 ubiquitin ligases responsible for identifying and targeting substrates for degradation. Our previous study demonstrated that overexpression of Ozz, an E3 specific to embryonic myosin heavy chain (Myh3), precisely reduced the Myh3 replacement rate in the thick filaments of myotubes (E. Ichimura et al., Physiol Rep. 9:e15003, 2021). These findings strongly suggest that E3 plays a critical role in regulating myosin replacement. Here, we hypothesized that the Murf isoforms, which recognize Myhs as substrates, reduced the myosin replacement rates through the enhanced Myh degradation by Murfs. First, fluorescence recovery after a photobleaching experiment was conducted to assess whether Murf isoforms affected the GFP-Myh3 replacement. In contrast to Murf2 or Murf3 overexpression, Murf1 overexpression selectively facilitated the GFP-Myh3 myosin replacement. Next, to examine the effects of Murf1 overexpression on the replacement of myosin isoforms, Cherry-Murf1 was coexpressed with GFP-Myh1, GFP-Myh4, or GFP-Myh7 in myotubes. Intriguingly, Murf1 overexpression enhanced the myosin replacement of GFP-Myh4 but did not affect those of GFP-Myh1 or GFP-Myh7. Surprisingly, overexpression of Murf1 did not enhance the ubiquitination of proteins. These results indicate that Murf1 selectively regulated myosin replacement in a Myh isoform-dependent fashion, independent of enhanced ubiquitination. This suggests that Murf1 may have a role beyond functioning as a ubiquitin ligase E3 in thick filament myosin replacement.

Effects of Ulmus macrocarpa Extract and Catechin 7-O-β-D-apiofuranoside on Muscle Loss and Muscle Atrophy in C2C12 Murine Skeletal Muscle Cells.

Muscle atrophy is known to be one of the symptoms leading to sarcopenia, which significantly impacts the quality of life, mortality, and morbidity. Therefore, the development of therapeutics for muscle atrophy is essential. This study focuses on addressing muscle loss and atrophy using Ulmus macrocarpa extract and its marker compound, catechin 7-O-β-D-apiofuranoside, by investigating their effects on biomarkers associated with muscle cell apoptosis. Additionally, protein and gene expression in a muscle atrophy model were examined using Western blotting and RT-PCR. Ulmus macrocarpa has been used as food or medicine due to its safety, including its roots, barks, and fruit. Catechin 7-O-β-D apiofuranoside is an indicator substance of plants of the Ulmus genus and has been reported to have various effects such as antioxidant and anti-inflammatory effects. The experimental results demonstrated that catechin glycoside and Ulmus macrocarpa extract decreased the expression of the muscle-degradation-related proteins Atrogin-1 and Muscle RING-Finger protein-1 (MuRF1) while increasing the expression of the muscle-synthesis-related proteins Myoblast determination (MyoD) and Myogenin. Gene expression confirmation experiments validated a decrease in the expression of Atrogin and MuRF1 mRNA and an increase in the expression of MyoD and Myogenin mRNA. Furthermore, an examination of muscle protein expression associated with the protein kinase B (Akt)/forkhead box O (FoxO) signaling pathway confirmed a decrease in the expression of FoxO, a regulator of muscle protein degradation. These results confirm the potential of Ulmus macrocarpa extract to inhibit muscle apoptosis, prevent muscle decomposition, and promote the development of functional materials for muscle synthesis, health-functional foods, and natural-product-derived medicines.

Pharmacological activation of pyruvate dehydrogenase by dichloroacetate protects against obesity-induced muscle atrophy in vitro and in vivo

Obesity-induced muscle atrophy leads to physical impairment and metabolic dysfunction, which are risky for older adults. The activity of pyruvate dehydrogenase (PDH), a critical regulator of glucose metabolism, is reduced in obesity. Additionally, PDH activator dichloroacetate (DCA) improves metabolic dysfunction. However, the effects of PDH activation on skeletal muscles in obesity remain unclear. Thus, this study aimed to evaluate the effects of PDH activation by DCA treatment on obesity-induced muscle atrophy in vitro and in vivo and elucidate the possible underlying mechanisms. Results showed that PDH activation by DCA treatment ameliorated muscle loss, decreased the cross-sectional area, and reduced grip strength in C57BL/6 mice fed a high-fat diet (HFD). Elevation of muscle atrophic factors atrogin-1 and muscle RING-finger protein-1 (MuRF-1) and autophagy factors LC3BII and p62 were abrogated by DCA treatment in palmitate-treated C2C12 myotubes and in the skeletal muscles of HFD-fed mice. Moreover, p-Akt, p-FoxO1, and p-FoxO3 protein levels were reduced and p-NF-κB p65 and p-p38 MAPK protein levels were elevated in palmitate-treated C2C12 myotubes, which were restored by DCA treatment. However, the protective effects of DCA treatment against myotube atrophy were reversed by treatment with Akt inhibitor MK2206. Taken together, our study demonstrated that PDH activation by DCA treatment can alleviate obesity-induced muscle atrophy. It may serve as a basis for developing novel strategies to prevent obesity-associated muscle loss.

Korean Mint (Agastache rugosa) Extract and Its Bioactive Compound Tilianin Alleviate Muscle Atrophy via the PI3K/Akt/FoxO3 Pathway in C2C12 Myotubes.

Skeletal muscle atrophy, which is characterized by diminished muscle mass, strength, and function, is caused by malnutrition, physical inactivity, aging, and diseases. Korean mint (Agastache rugosa Kuntze) possesses various biological functions, including anti-inflammatory, antioxidant, anticancer, and antiosteoporosis activities. Moreover, it contains tilianin, which is a glycosylated flavone that exerts antioxidant, anti-inflammatory, antidiabetic, and neuroprotective activities. However, no studies have analyzed the inhibitory activity of A. rugosa extract (ARE) and tilianin on muscle atrophy. Thus, the present study investigated the potential of ARE and tilianin on muscle atrophy and their underlying mechanisms of action in C2C12 myotubes treated with tumor necrosis factor-α (TNF-α). The results showed that ARE and tilianin promoted the phosphatidylinositol 3-kinase/protein kinase B pathway, thereby activating mammalian target of rapamycin (a protein anabolism-related factor) and its downstream factors. Moreover, ARE and tilianin inhibited the mRNA expression of muscle RING-finger protein-1 and atrogin-1 (protein catabolism-related factors) by blocking Forkhead box class O3 translocation. ARE and tilianin also mitigated inflammatory responses by downregulating nuclear factor-kappa B expression levels, thereby diminishing the expression levels of inflammatory cytokines, including TNF-α and interleukin-6. Additionally, ARE and tilianin enhanced the expression levels of antioxidant enzymes, including catalase, superoxide dismutase, and glutathione peroxidase. Overall, these results suggest that ARE and tilianin are potential functional ingredients for preventing or improving muscle atrophy.

Renin angiotensin system-induced muscle wasting: putative mechanisms and implications for clinicians.

Renin angiotensin system (RAS) alters various mechanisms related to muscle wasting. The RAS system consists of classical and non-classical pathways, which mostly function differently. Classical RAS pathway, operates through angiotensin II (AngII) and angiotensin type 1 receptors, is associated with muscle wasting and sarcopenia. On the other hand, the non-classical RAS pathway, which operates through angiotensin 1-7 and Mas receptor, is protective against sarcopenia. The classical RAS pathway might induce muscle wasting by variety of mechanisms. AngII reduces body weight, via reduction in food intake, possibly by decreasing hypothalamic expression of orexin and neuropeptide Y, insulin like growth factor-1 (IGF-1) and mammalian target of rapamycin (mTOR), signaling, AngII increases skeletal muscle proteolysis by forkhead box transcription factors (FOXO), caspase activation and muscle RING-finger protein-1 transcription. Furthermore, AngII infusion in skeletal muscle reduces phospho-Bad (Ser136) expression and induces apoptosis through increased cytochrome c release and DNA fragmentation. Additionally, Renin angiotensin system activation through AT1R and AngII stimulates tumor necrosis factor-α, and interleukin-6 which induces muscle wasting, Last but not least classical RAS pathway, induce oxidative stress, disturb mitochondrial energy metabolism, and muscle satellite cells which all lead to muscle wasting and decrease muscle regeneration. On the contrary, the non-classical RAS pathway functions oppositely to mitigate these mechanisms and protects against muscle wasting. In this review, we summarize the mechanisms of RAS-induced muscle wasting and putative implications for clinical practice. We also emphasize the areas of uncertainties and suggest potential research areas.

Brassinin alleviates cancer cachexia by suppressing diverse inflammatory mechanisms in mice.

Cancer cachexia is a multifactorial condition that contributes to the death of about 20% of cancer patients. It has the potential to cause weight loss, reduction in muscle mass, and loss of fat tissue, significantly lowering the quality of life. Currently, there are no approved drugs for cancer cachexia. Here, we have explored the possible impact of brassinin (BSN) on cancer cachexia under in vitro and in vivo settings. After differentiation, C2C12 and 3T3-L1 cells were incubated with colorectal carcinoma cells conditioned media or BSN. For preclinical studies, mice were injected with HT-29 cells followed by intraperitoneal administration of BSN, and muscle and adipose tissues were evaluated by Western blotting and hematoxylin and eosin staining. BSN effectively suppressed muscle atrophy by down-regulating the levels of Muscle RING-finger protein-1 and Atrogin-1, while also increasing the expression of myosin heavy chain in cachexia-induced-C2C12 myotubes. The induction of adipogenesis by BSN prevented adipocyte atrophy in cachexia-induced 3T3-L1 adipocytes. We also noted that BSN disrupted the interaction between COX-2 and signaling transducer and activator of transcription 3 (STAT3) promoter, leading to down-regulation of STAT3 activation. Moreover, it was found that BSN inhibited weight loss in mice and demonstrated anti-cachexic effects. Overall, our observations indicate that BSN can attenuate cancer cachexia through diverse mechanisms.

Epigallocatechin gallate protects against fat and muscle atrophy in B16BL6 melanoma-bearing mice on a high-fat diet

AimsEpidemiological evidence indicates that there is a substantial association between body mass index (BMI) and at least ten forms of cancer, including melanoma, and BMI imbalance contributes to the poor survival rate of cancer patients before and after therapy. Nevertheless, few pharmacological studies on models of obesity and cancer have been reported. In this study, we administered epigallocatechin gallate (EGCG) to B16BL6 tumor-bearing mice that received a high-fat diet (HFD) to examine its impact. MethodsB16BL6 tumor-bearing mice were fed a HFD. Body weight and food intake were documented every week. We conducted a Western blot analysis to examine the protein levels in the tumor, gastrocnemius (GAS), and tibialis anterior (TA) muscles, as well as the inguinal and epididymal white adipose tissues (iWAT and eWAT). Key findingsEGCG has been shown to have anti-cancer effects equivalent to those of cisplatin, a chemotherapy drug. Furthermore, EGCG protected against the loss of epidydimal white adipose tissue by regulating protein levels of lipolysis factors of adipose triglyceride lipase and hormone-sensitive lipase as well as WAT browning factors of uncoupling protein 1, as opposed to cisplatin. EGCG was shown to reduce the protein levels of muscular atrophy factors of muscle RING-finger protein-1, whereas cisplatin did not contribute to rescuing the atrophy of TA and GAS muscles. ConclusionTaken together, our findings indicate that EGCG has a preventive effect against cachexia symptoms and has anti-cancer effects similar to those of cisplatin in tumor-bearing mice fed a high-fat diet.

Myricanol improves metabolic profiles in dexamethasone induced lipid and protein metabolism disorders in mice

Myricanol (MY) is one of the main active components from bark of Myrica Rubra. It is demonstrated that MY rescues dexamethasone (DEX)-induced muscle dysfunction via activating silent information regulator 1 (SIRT1) and increasing adenosine 5’-monophosphate-activated protein kinase (AMPK) phosphorylation. Since SIRT1 and AMPK are widely involved in the metabolism of nutrients, we speculated that MY may exert beneficial effects on DEX-induced metabolic disorders. This study for the first time applied widely targeted metabolomics to investigate the beneficial effects of MY on glucose, lipids, and protein metabolism in DEX-induced metabolic abnormality in mice. The results showed that MY significantly reversed DEX-induced soleus and gastrocnemius muscle weight loss, muscle fiber damage, and muscle strength loss. MY alleviated DEX-induced metabolic disorders by increasing SIRT1 and glucose transporter type 4 (GLUT4) expressions. Additionally, myricanol prevented muscle cell apoptosis and atrophy by inhibiting caspase 3 cleavages and muscle ring-finger protein-1 (MuRF1) expression. Metabolomics showed that MY treatment reversed the serum content of carnitine ph-C1, palmitoleic acid, PS (16:0_17:0), PC (14:0_20:5), PE (P-18:1_16:1), Cer (t18:2/38:1(2OH)), four amino acids and their metabolites, and 16 glycerolipids in DEX mice. Kyoto encyclopedia of genes and genomes (KEGG) and metabolic set enrichment analysis (MSEA) analysis revealed that MY mainly affected metabolic pathways, glycerolipid metabolism, lipolysis, fat digestion and absorption, lipid and atherosclerosis, and cholesterol metabolism pathways through regulation of metabolites involved in glutathione, butanoate, vitamin B6, glycine, serine and threonine, arachidonic acid, and riboflavin metabolism. Collectively, MY can be used as an attractive therapeutic agent for DEX-induced metabolic abnormalities.

Lactate administration induces skeletal muscle synthesis by influencing Akt/mTOR and MuRF1 in non-trained mice but not in trained mice.

The perception regarding lactate has changed over the past decades, and some of its physiological roles have gradually been revealed. However, the effects of exogenous lactate on skeletal muscle synthesis remain unclear. This study aimed to confirm the effects of a 5-week lactate administration and post-exercise lactate administration on skeletal muscle synthesis. Thirty-two Institute of Cancer Research mice were randomly assigned to non-trained + placebo, non-trained + lactate, trained + placebo, and trained + lactate groups. Furthermore, 3 g/kg of lactate or an equivalent volume of saline was immediately administered after exercise training (maximum oxygen uptake: 70%). Lactate administration and/or exercise training was performed 5 days/week for 5 weeks. After the experimental period, it was observed that lactate administration tended to elevate skeletal muscle weight, increased protein kinase B (p < 0.05) and mammalian target of rapamycin (p < 0.05) mRNA levels, and decreased muscle ring-finger protein-1 expression (p < 0.05). Lactate administration after exercise training significantly enhanced plantaris muscle weight; however, it had no additional effects on most signaling factors. This study demonstrated that a 5-week lactate administration could stimulate skeletal muscle synthesis, and lactate administration after exercise training may provide additional effects, such as increasing skeletal muscle.

Unveiling the Potential of Nelumbo nucifera-Derived Liensinine to Target The Myostatin Protein and to Counteract Muscle Atrophy.

Muscle atrophy refers to a decline in muscle mass and function, which has become a global concern due to the aging population. Various clinical trials have investigated the inhibitors of myostatin (MSTN). They have shown promising improvements in muscle function and quality of life. However, there are no drugs specifically targeting MSTN that have been approved for clinical use. In this study, we virtually screened liensinine (LIE), a food (Nelumbo nucifera)-derived compound, with low toxicity, from over 1.1 million compounds. We subsequently identified it as a potential candidate that targets MSTN by a cellular thermal shift assay (CETSA) and drug affinity response target stability (DARTS) assay. Further validation through cellular and in vivo studies demonstrated its promising potential in combating muscle atrophy. The mechanism of action may involve hindering the interaction between MSTN and the activin receptor type IIB (ActRIIB) and downregulating the expression of downstream proteins, including the muscle RING-finger protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx)/Atrogin-1, ultimately promoting muscle regeneration. These results provide a strong foundation for future studies to explore the therapeutic potential of LIE in clinical settings.

Abstract C062: Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting

Abstract Background: Patients with pancreatic cancer (PC) often experience cachexia, a multifactorial syndrome that includes weight loss, malnutrition, appetite suppression, and tissue wasting. Unfortunately, the factors secreted from the pancreas or other tissues that drive cachexia are still poorly defined. Lipocalin 2 (LCN2) is a cytokine involved in innate immunity that we have previously shown to be increased in the pancreas and circulation of patients with PC and mouse models of PC. Recent studies suggest that LCN2 contributes to appetite suppression in cancer cachexia in orthotopic models of PC. However, a reduction in muscle regeneration is also commonly seen in cachexia that is not repaired by increased nutrient intake. In a non-cancer setting, LCN2 has been implicated as an important factor in mediating skeletal muscle repair. Therefore, we hypothesized that elevated LCN2 in PC contributes to cachexia by inhibiting muscle progenitor cell proliferation and differentiation into new muscle fibers. Methods: Muscles from a syngeneic orthotopic mouse model of PC were assessed for muscle RING-finger protein-1 (MuRF1, an E3 ubiquitin ligase highly expressed in muscle during wasting) and LCN2 by qPCR. C2C12 mouse muscle myoblasts were cultured in proliferation or differentiation media with recombinant mouse LCN2. Proliferation across 3 days was assessed by MTS assay. Differentiation was assessed by imaging myotubes and western blotting for myosin heavy chain (MyHC) expression which increases during differentiation. PC genetically engineered mice ± a whole body LCN2 were assessed for pancreatic tumor and gastroc muscle size. One-way and Two-way ANOVAs were used to determine statistical significance. Results: LCN2 and MuRF1 expression are elevated in muscles of mice with PC. C2C12 cells treated with LCN2 decreased proliferation in a dose-dependent manner up to concentrations of LCN2 commonly observed in PC. LCN2 treatments at a physiologic concentration observed in PC delayed differentiation of C2C12 cells into myotubes by reducing myotube diameter and delaying MyHC expression. Deleting LCN2 in a PC genetic mouse model prevented body weight and gastroc weight loss but did not affect pancreas weight. Conclusions: Here, we show a relationship between muscle wasting and LCN2 expression during PC. Elevated circulating LCN2 may be contributing to decreased muscle regeneration observed in PC-associated cachexia by directly inhibiting muscle progenitor cell proliferation and differentiation. Targeting LCN2 in PC may be a viable method of slowing or preventing muscle wasting in PC to improve patient outcomes. Citation Format: Kristyn Gumpper-Fedus, Valentina Pita Grisanti, Ericka Velez Bonet, Martha A. Belury, Christopher Coss, Zobeida Cruz-Monserrate. Lipocalin 2 slows myoblast growth and contributes to pancreatic cancer-associated muscle wasting [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C062.

Remedial effects of tilapia skin peptides against dexamethasone-induced muscle atrophy in mice by modulation of AKT/FOXO3a and Sirt1/PGC-1α signaling pathways

Tilapia skin peptides (TSP) possess a range of physiological activities. This study aimed to explore the effects of TSP on Dexamethasone (DEX)-induced muscle atrophy. In vitro, C2C12 myotube myotube diameter and expression levels of the muscle-specific E3 ubiquitin ligases F-box only protein 32 (Atrogin-1) and muscle ring finger protein 1 (MuRF1) challenged with DEX were reversed by TSP. In vivo, DEX was injected subcutaneously to build muscle atrophy model mice. TSP enhanced grip strength, running distance, body lean muscle content, cross-sectional area of the gastrocnemius and tibialis anterior muscles of DEX-induced mice. Moreover, TSP inhibited the expression levels of Atrogin-1 and MuRF1. Mechanically, TSP improved DEX-induced muscle atrophy by regulating the Protein Kinase B α (AKT)/Forkhead box O3 protein (FOXO3a), Sirtuin 1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway, and downstream factors such as nuclear respiratory factor (NRF)1/2 and mitochondrial transcription factor A (TFAM).