Muscle Proteolysis Research Articles

Effects of thyroid hormones in skeletal muscle protein turnover.

Thyroid hormones (THs) are critical regulators of muscle metabolism in both healthy and unhealthy conditions. Acting concurrently as powerful anabolic and catabolic factors, THs are endowed with a vital role in muscle mass maintenance. As a result, thyroid dysfunctions are the leading cause of a wide range of muscle pathologies, globally identified as myopathies. Whether muscle wasting is a common feature in patients with hyperthyroidism and is mainly caused by THs-dependent stimulation of muscle proteolysis, also muscle growth is often associated with hyperthyroid conditions, linked to THs-dependent stimulation of muscle protein synthesis. Noteworthy, also hypothyroid status negatively impacts on muscle physiology, causing muscle weakness and fatigue. Most of these symptoms are due to altered balance between muscle protein synthesis andbreakdown. Thus, a comprehensive understanding of THs-dependent skeletal muscle protein turnover might facilitate the management of physical discomfort or weakness in conditions of thyroid disease. Herein, we describe the molecular mechanisms underlying the THs-dependent alteration of skeletal muscle structure and function associated with muscle atrophy and hypertrophy, thus providing new insights for targeted modulation of skeletal muscle dynamics.

Metabolic Effects of Ketogenic Diets: Exploring Whole-Body Metabolism in Connection with Adipose Tissue and Other Metabolic Organs.

The ketogenic diet (KD) is characterized by minimal carbohydrate, moderate protein, and high fat intake, leading to ketosis. It is recognized for its efficiency in weight loss, metabolic health improvement, and various therapeutic interventions. The KD enhances glucose and lipid metabolism, reducing triglycerides and total cholesterol while increasing high-density lipoprotein levels and alleviating dyslipidemia. It significantly influences adipose tissue hormones, key contributors to systemic metabolism. Brown adipose tissue, essential for thermogenesis and lipid combustion, encounters modified UCP1 levels due to dietary factors, including the KD. UCP1 generates heat by uncoupling electron transport during ATP synthesis. Browning of the white adipose tissue elevates UCP1 levels in both white and brown adipose tissues, a phenomenon encouraged by the KD. Ketone oxidation depletes intermediates in the Krebs cycle, requiring anaplerotic substances, including glucose, glycogen, or amino acids, for metabolic efficiency. Methylation is essential in adipogenesis and the body's dietary responses, with DNA methylation of several genes linked to weight loss and ketosis. The KD stimulates FGF21, influencing metabolic stability via the UCP1 pathways. The KD induces a reduction in muscle mass, potentially involving anti-lipolytic effects and attenuating proteolysis in skeletal muscles. Additionally, the KD contributes to neuroprotection, possesses anti-inflammatory properties, and alters epigenetics. This review encapsulates the metabolic effects and signaling induced by the KD in adipose tissue and major metabolic organs.

Myogenic microRNAs as Therapeutic Targets for Skeletal Muscle Mass Wasting in Breast Cancer Models.

Breast cancer is the type of cancer with the highest prevalence in women worldwide. Skeletal muscle atrophy is an important prognostic factor in women diagnosed with breast cancer. This atrophy stems from disrupted skeletal muscle homeostasis, triggered by diminished anabolic signalling and heightened inflammatory conditions, culminating in an upregulation of skeletal muscle proteolysis gene expression. The importance of delving into research on modulators of skeletal muscle atrophy, such as microRNAs (miRNAs), which play a crucial role in regulating cellular signalling pathways involved in skeletal muscle protein synthesis and degradation, has been recognised. This holds true for conditions of homeostasis as well as pathologies like cancer. However, the determination of specific miRNAs that modulate skeletal muscle atrophy in breast cancer conditions has not yet been explored. In this narrative review, we aim to identify miRNAs that could directly or indirectly influence skeletal muscle atrophy in breast cancer models to gain an updated perspective on potential therapeutic targets that could be modulated through resistance exercise training, aiming to mitigate the loss of skeletal muscle mass in breast cancer patients.

Guava leaf extract attenuated muscle proteolysis in dexamethasone induced muscle atrophic mice via ubiquitin proteasome system, mTOR-autophagy, and apoptosis pathway

Muscle atrophy is the waste or loss of muscle mass and is caused by physical inactivity, aging, or diseases such as diabetes, cancer, and heart failure. The number of patients suffering from musculoskeletal disorders is expected to increase in the future. However, intervention for muscle atrophy is limited, so research on treatment for muscle wasting is needed. This study hypothesized that guava leaf (Psidium guajava L. [GL]) would have ameliorative effects on muscle atrophy by regulation of protein degradation pathways in a dexamethasone (DEX)-induced muscle atrophy mice model. Muscle atrophy was induced by DEX injection for 28 days in 7 week-old-male ICR mice. Then, low-dose GL (LGL, 200 mg/kg) or high-dose GL (HGL, 500 mg/kg) extract (GLE) was supplemented by oral gavage for 21 days. Muscle strength, calf thickness, and body composition were analyzed. Histopathological changes in the gastrocnemius muscle were examined using hematoxylin and eosin staining, and molecular pathways related to muscle degradation were analyzed by western blots. GLE treatment regardless of dose increased muscle strength in mice with muscle atrophy accompanied by attenuating autophagy related pathway in the DEX-induced muscle atrophy mice. Moreover, a high dose of GLE treatment ameliorated ubiquitin proteasome system and apoptosis in the DEX-induced muscle atrophy mice. This study suggested that GLE could be helpful to improve muscle health and alleviate proteolysis by regulation of the ubiquitin-proteasome system, autophagy, and apoptosis, which are involved in muscle degradation. In conclusion, GLE could be a potential nutraceutical to prevent 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.

Diabetic ketoacidosis in type 1 diabetes mellitus in children and adolescents

Background: Diabetic ketoacidosis (DKA) is the leading cause of morbidity and mortality in children with type 1 diabetes mellitus (T1DM). Although T1DM can occur in children and adults, DKA caused by T1DM is more severe in children and adolescents and metabolic changes occur more rapidly. This review discusses clinical factors associated with DKA assessed during diagnosis in children and adolescents with T1DM.Current Concepts: DKA is a severe complication that can occur in children with T1DM because of insulin deficiency, leading to high levels of catabolism with increased gluconeogenesis, glycogenolysis, lipolysis, muscle proteolysis, hyperglycemia, and osmotic diuresis. High levels of counterregulatory hormones that enhance ketogenesis have also been noted. High blood sugar levels, dehydration, and the release of ketone bodies into the circulation cause high occurrence of acidosis. DKA can lead to life-threatening complications, such as cerebral edema, which is more common in children than in adults. Other potential complications include kidney failure, respiratory distress, and cardiovascular collapse. Children with T1DM are at a higher risk of developing DKA, particularly during times of illness or stress, or when insulin doses are missed or insufficient. In addition, undiagnosed T1DM in children can lead to DKA when the body enters a state of severe insulin deficiency.Discussion and Conclusion: The risks should be detected earlier and the pathogenesis of T1DM should be understood and assessed by physicians in children and adolescents during check-ups. Furthermore, efforts to increase public awareness are required to reduce the cognitive delays associated with DKA.

Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective β2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a β2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

LSD1 inhibition circumvents glucocorticoid-induced muscle wasting of male mice

Synthetic glucocorticoids (GC), such as dexamethasone, are extensively used to treat chronic inflammation and autoimmune disorders. However, long-term treatments are limited by various side effects, including muscle atrophy. GC activities are mediated by the glucocorticoid receptor (GR), that regulates target gene expression in various tissues in association with cell-specific co-regulators. Here we show that GR and the lysine-specific demethylase 1 (LSD1) interact in myofibers of male mice, and that LSD1 connects GR-bound enhancers with NRF1-associated promoters to stimulate target gene expression. In addition, we unravel that LSD1 demethylase activity is required for triggering starvation- and dexamethasone-induced skeletal muscle proteolysis in collaboration with GR. Importantly, inhibition of LSD1 circumvents muscle wasting induced by pharmacological levels of dexamethasone, without affecting their anti-inflammatory activities. Thus, our findings provide mechanistic insights into the muscle-specific GC activities, and highlight the therapeutic potential of targeting GR co-regulators to limit corticotherapy-induced side effects.

Resistance training-induced changes in muscle proteolysis and extracellular matrix remodeling biomarkers in the untrained and trained states.

Resistance training (RT) induces muscle growth at varying rates across RT phases, and evidence suggests that the muscle-molecular responses to training bouts become refined or attenuated in the trained state. This study examined how proteolysis-related biomarkers and extracellular matrix (ECM) remodeling factors respond to a bout of RT in the untrained (UT) and trained (T) state. Participants (19 women and 19 men) underwent 10weeks of RT. Biopsies of vastus lateralis were collected before and after (24h) the first (UT) and last (T) sessions. Vastus lateralis cross-sectional area (CSA) was assessed before and after the experimental period. There were increases in muscle and type II fiber CSAs. In both the UT and T states, calpain activity was upregulated and calpain-1/-2 protein expression was downregulated from Pre to 24h. Calpain-2 was higher in the T state. Proteasome activity and 20S proteasome protein expression were upregulated from Pre to 24h in both the UT and T. However, proteasome activity levels were lower in the T state. The expression of poly-ubiquitinated proteins was unchanged. MMP activity was downregulated, and MMP-9 protein expression was elevated from Pre to 24h in UT and T. Although MMP-14 protein expression was acutely unchanged, this marker was lower in T state. TIMP-1 protein levels were reduced Pre to 24h in UT and T, while TIMP-2 protein levels were unchanged. Our results are the first to show that RT does not attenuate the acute-induced response of proteolysis and ECM remodeling-related biomarkers.

Differential effects of cholecalciferol and calcitriol on muscle proteolysis and oxidative stress in angiotensin II-induced C2C12 myotube atrophy.

Renin-angiotensin system activation contributes to skeletal muscle atrophy in aging individuals with chronic diseases. We aimed to explore the effects of cholecalciferol (VD3) and calcitriol (1,25VD3) on signaling of muscle proteolysis and oxidative stress in myotubes challenged with angiotensin II (AII). The mouse C2C12 myotubes were assigned to vehicle, AII, AII + VD3, AII + 1,25VD3, and AII + losartan groups. The expression levels of muscle-specific E3 ubiquitin ligase proteins, autophagy-related proteins, and oxidative stress markers were investigated. We demonstrated the diverse effects of VD3 and 1,25VD3 on AII-induced myotube atrophy. The myotube diameter was preserved by treatment with 100 nM VD3 and losartan, while 1 and 10 nM 1,25VD3 increased levels of FoxO3a, MuRF1, and atrogin-1 protein expression in myotubes exposed to AII. Treatment with AII + 10 nM 1,25VD3 resulted in the upregulation of LC3B-II, LC3B-II/LC3B-I, and mature cathepsin L, which are autophagic marker proteins. The p62/SQSTM1 protein was downregulated and vitamin D receptor was upregulated after treatment with AII + 10 nM 1,25VD3. A cellular redox imbalance was observed as AII + 10 nM 1,25VD3-induced reactive oxygen species and NADPH oxidase-2 overproduction, and these changes were associated with an inadequate response of antioxidant superoxide dismutase-1 and catalase proteins. Collectively, these findings provide a translational perspective on the role of vitamin D3 in alleviating muscle atrophy related to high levels of AII.

Glucocorticoid-Induced Hyperglycemia: A Neglected Problem.

Glucocorticoids provide a potent therapeutic response and are widely used to treat a variety of diseases, including coronavirus disease 2019 (COVID-19) infection. However, the issue of glucocorticoid-induced hyperglycemia (GIH), which is observed in over one-third of patients treated with glucocorticoids, is often neglected. To improve the clinical course and prognosis of diseases that necessitate glucocorticoid therapy, proper management of GIH is essential. The key pathophysiology of GIH includes systemic insulin resistance, which exacerbates hepatic steatosis and visceral obesity, as well as proteolysis and lipolysis of muscle and adipose tissue, coupled with β-cell dysfunction. For patients on glucocorticoid therapy, risk stratification should be conducted through a detailed baseline evaluation, and frequent glucose monitoring is recommended to detect the onset of GIH, particularly in high-risk individuals. Patients with confirmed GIH who require treatment should follow an insulin-centered regimen that varies depending on whether they are inpatients or outpatients, as well as the type and dosage of glucocorticoid used. The ideal strategy to maintain normoglycemia while preventing hypoglycemia is to combine basal-bolus insulin and correction doses with a continuous glucose monitoring system. This review focuses on the current understanding and latest evidence concerning GIH, incorporating insights gained from the COVID-19 pandemic.

Celecoxib attenuates hindlimb unloading-induced muscle atrophy via suppressing inflammation, oxidative stress and ER stress by inhibiting STAT3.

Improving inflammation may serve as useful therapeutic interventions for the hindlimb unloading-induced disuse muscle atrophy. Celecoxib is a selective non-steroidal anti-inflammatory drug. We aimed to determine the role and mechanism of celecoxib in hindlimb unloading-induced disuse muscle atrophy. Celecoxib significantly attenuated the decrease in soleus muscle mass, hindlimb muscle function and the shift from slow- to fast-twitch muscle fibers caused by hindlimb unloading in rats. Importantly, celecoxib inhibited the increased expression of inflammatory factors, macrophage infiltration in damaged soleus muscle. Mechanistically, Celecoxib could significantly reduce oxidative stress and endoplasmic reticulum stress in soleus muscle of unloaded rats. Furthermore, celecoxib inhibited muscle proteolysis by reducing the levels of MAFbx, MuRF1, and autophagy related proteins maybe by inhibiting the activation of pro-inflammatory STAT3 pathway in vivo and in vitro. This study is the first to demonstrate that celecoxib can attenuate disuse muscle atrophy caused by hindlimb unloading via suppressing inflammation, oxidative stress and endoplasmic reticulum stress probably, improving target muscle function and reversing the shift of muscle fiber types by inhibiting STAT3 pathways-mediated inflammatory cascade. This study not only enriches the potential molecular regulatory mechanisms, but also provides new potential therapeutic targets for disuse muscle atrophy.

Moderating AKT signaling with baicalein protects against weight loss by preventing muscle atrophy in a cachexia model caused by CT26 colon cancer

Cancer cachexia is a type of energy-wasting syndrome characterized by fatigue, anorexia, muscle weakness, fat loss, and systemic inflammation. Baicalein, a flavonoid with bioactive properties, has demonstrated the ability to mitigate cardiac and skeletal muscle atrophy in different experimental settings. This effect is achieved through the inhibition of muscle proteolysis, suggesting its potential in preserving skeletal muscle homeostasis. In this study, we investigated the anti-cancer cachexia effects of baicalein in the regulation of muscle and fat wasting, both in vivo and in vitro. Baicalein attenuated body weight loss, including skeletal muscle and white adipose tissue (WAT), in CT26-induced cachectic mice. Moreover, baicalein increased muscle fiber thickness and suppressed the muscle-specific ubiquitin-protease system, including F-box only protein 32 and muscle RING-finger protein-1, by activating AKT phosphorylation both in vivo and in vitro. The use of LY294002, a particular inhibitor of AKT, eliminated the observed impact of baicalein on the improvement of muscle atrophy. In conclusion, baicalein inhibits muscle proteolysis and enhances AKT phosphorylation, indicating its potential role in cancer cachexia-associated muscle atrophy.

Decreased Neuromuscular Function and Muscle Quality along with Increased Systemic Inflammation and Muscle Proteolysis Occurring in the Presence of Decreased Estradiol and Protein Intake in Early to Intermediate Post-Menopausal Women.

Menopause causes a reduction in estradiol (E2) and may be associated with neuromuscular degeneration. Compared to pre-menopausal (PRE-M) women, this study sought to determine dietary protein intake and whether lower levels of circulating E2 in post-menopausal women (POST-M) were occurring alongside increased levels of biomarkers of axonal and neuromuscular junction degeneration (NMJ), inflammation, muscle protein degradation, and reduced indices of muscle quality and performance. Employing a cross-sectional design, PRE-M (n = 6) and POST-M (n = 6) dietary analysis data were collected and participants then donated a blood and urine sample followed by assessments for body composition, motor unit activation, and muscle performance. Independent group t-tests were performed to determine differences between groups (p ≤ 0.05). In POST-M women, E2, motor unit activity, muscle quality, and muscle performance were significantly less than those for PRE-M women; however, the levels of c-terminal fragment of agrin, tumor necrosis factor-α, and urinary titin were significantly greater (p < 0.05). POST-M women were also shown to be ingesting fewer total calories and less protein than PRE-M (p < 0.05). Reduced E2 and dietary protein intake in POST-M women occurs in conjunction with increased levels of biomarkers of NMJ degradation, inflammation, and muscle proteolysis, which may be associated with reduced motor unit activation and muscle quality.

Muscle proteolysis is differentially influenced by mitochondrial intactness

This study examined the potential influence of mitochondrial calcium sequestering ability on calpain-1 autolysis and proteolysis in vitro. We first tested whether mitochondria can sequester calcium in an in vitro setting. Isolated bovine mitochondria (0, 0.5, or 2 mg/mL) were incubated in a buffer containing varying calcium levels (0, 50, or 100 μM). An inverse relationship between mitochondrial content and measured free calcium was observed (P < 0.05), confirming that mitochondria can sequester calcium within the concentration range tested. In the first in vitro experiment, intact mitochondria (0, 0.5, or 2 mg/mL) were incorporated into an in vitro model simulating postmortem muscle conditions, and calpain-1 autolysis and proteolysis were evaluated over a 168-h period. Adding intact mitochondria to the in vitro model decreased calpain-1 autolysis and proteolysis during the first 4 h of incubation (P < 0.05), likely through reducing calcium availability. However, accentuated calpain-1 autolysis and proteolysis were observed at 24 h. To further explore these effects, mitochondrial integrity was evaluated at varying pH and calcium levels. Mitochondrial integrity decreased as pH declined (P < 0.05), especially in the presence of calcium. Based on these results, we conducted a second in vitro experiment involving disrupted mitochondria. Unlike intact mitochondria, which exerted a suppressive effect on calpain-1 autolysis and proteolysis early on, disrupted mitochondria increased both parameters at most time points (P < 0.05). Overall, it appears that intact mitochondria initially cause a delay in calpain-1 autolysis and proteolysis, but as their integrity diminishes, both processes are enhanced.

Non-canonical Ca2+- Akt signaling pathway mediates the antiproteolytic effects induced by oxytocin receptor stimulation in skeletal muscle

Although it has been previously demonstrated that oxytocin (OXT) receptor stimulation can control skeletal muscle mass in vivo, the intracellular mechanisms that mediate this effect are still poorly understood. Thus, rat oxidative skeletal muscles were isolated and incubated with OXT or WAY-267,464, a non-peptide selective OXT receptor (OXTR) agonist, in the presence or absence of atosiban (ATB), an OXTR antagonist, and overall proteolysis was evaluated. The results indicated that both OXT and WAY-267,464 suppressed muscle proteolysis, and this effect was blocked by the addition of ATB. Furthermore, the WAY-induced anti-catabolic action on protein metabolism did not involve the coupling between OXTR and Gαi since it was insensitive to pertussis toxin (PTX). The decrease in overall proteolysis induced by WAY was probably due to the inhibition of the autophagic/lysosomal system, as estimated by the decrease in LC3 (an autophagic/lysosomal marker), and was accompanied by an increase in the content of Ca2+-dependent protein kinase (PKC)-phosphorylated substrates, pSer473-Akt, and pSer256-FoxO1. Most of these effects were blocked by the inhibition of inositol triphosphate receptors (IP3R), which mediate Ca2+ release from the sarcoplasmic reticulum to the cytoplasm, and triciribine, an Akt inhibitor. Taken together, these findings indicate that the stimulation of OXTR directly induces skeletal muscle protein-sparing effects through a Gαq/IP3R/Ca2+-dependent pathway and crosstalk with Akt/FoxO1 signaling, which consequently decreases the expression of genes related to atrophy, such as LC3, as well as muscle proteolysis.

Integrative profiling of metabolome and transcriptome of skeletal muscle after acute exercise intervention in mice.

This study aims to explore the molecular regulatory mechanisms of acute exercise in the skeletal muscle of mice. Male C57BL/6 mice were randomly assigned to the control group, and the exercise group, which were sacrificed immediately after an acute bout of exercise. The study was conducted to investigate the metabolic and transcriptional profiling in the quadriceps muscles of mice. The results demonstrated the identification of 34 differentially expressed metabolites (DEMs), with 28 upregulated and 6 downregulated, between the two groups. Metabolic pathway analysis revealed that these DEMs were primarily enriched in several, including the citrate cycle, propanoate metabolism, and lysine degradation pathways. In addition, the results showed a total of 245 differentially expressed genes (DEGs), with 155 genes upregulated and 90 genes downregulated. KEGG analysis indicated that these DEGs were mainly enriched in various pathways such as ubiquitin mediated proteolysis and FoxO signaling pathway. Furthermore, the analysis revealed significant enrichment of DEMs and DEGs in signaling pathways such as protein digestion and absorption, ferroptosis signaling pathway. In summary, the identified multiple metabolic pathways and signaling pathways were involved in the exercise-induced physiological regulation of skeletal muscle, such as the TCA cycle, oxidative phosphorylation, protein digestion and absorption, the FoxO signaling pathway, ubiquitin mediated proteolysis, ferroptosis signaling pathway, and the upregulation of KLF-15, FoxO1, MAFbx, and MuRF1 expression could play a critical role in enhancing skeletal muscle proteolysis.

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 &amp; 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

Jianpi Decoction Combined with Medroxyprogesterone Acetate Alleviates Cancer Cachexia and Prevents Muscle Atrophy by Directly Inhibiting E3 Ubiquitin Ligase.

To provide comprehensive evidence for the anti-cancer cachexia effect of Jianpi Decoction (JP) and to explore its mechanism of anti-cancer cachexia. A mouse model of colon cancer (CT26)-induced cancer cachexia (CC) was used to investigate the anti-CC effect of JP combined with medroxyprogesterone acetate (MPA). Thirty-six mice were equally divided into 6 groups: normal control, CC, MPA (100 mg•kg-1•d-1), MPA + low-dose (20 mg•kg-1•d-1) JP (L-JP), MPA + medium-dose (30 mg•kg-1•d-1) JP (M-JP), and MPA + high-dose (40 mg•kg-1•d-1) JP (H-JP) groups. After successful modeling, the mice were administered by gavage for 11 d. The body weight and tumor volume were measured and recorded every 2 d starting on the 8th day after implantation. The liver, heart, spleen, lung, kidney, tumor and gastrocnemius muscle of mice were collected and weighed. The pathological changes of the tumor was observed, and the cross-sectional area of the gastrocnemius muscle was calculated. The protein expressions of STAT3 and E3 ubiquitinase in the gastrocnemius muscle were measured by Western blot. In addition, an in vitro C2C12 myotube formation model was established to investigate the role of JP in hindering dexamethasone-induced muscle atrophy. In vitro experiments were divided into control, model, and JP serum groups. After 2-d administration, microscopic photographs were taken and myotube diameters were calculated. Western blot was performed to measure the protein expressions of STAT3 and E3 ubiquitinase. JP combined with MPA restored tumor-induced weight loss (P<0.05, vs. CC) and muscle fiber size (P<0.01, vs. CC). Mechanistically, JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx in tumor-induced muscle atrophy in vivo (P<0.05, vs. CC). In addition, JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx and p-STAT3 phosphorylation (P<0.05 or P<0.01 vs. model group) in C2C12 myotubes treated with dexamethasone in vitro. Administration of JP combined with MPA restores tumor-induced cachexia conditions. In addition, the profound effect of JP combined with MPA on tumor-induced cachexia may be due to its inhibition of muscle proteolysis (E3 ubiquitinase system).

Proteolytic markers associated with a gain and loss of leg muscle mass with resistance training followed by high-intensity interval training.

We recently reported that vastus lateralis (VL) cross-sectional area (CSA) increases after 7weeks of resistance training (RT, 2days/week), with declines occurring following 7weeks of subsequent treadmill high-intensity interval training (HIIT) (3days/week). Herein, we examined the effects of this training paradigm on skeletal muscle proteolytic markers. VL biopsies were obtained from 11 untrained college-aged males at baseline (PRE), after 7weeks of RT (MID), and after 7weeks of HIIT (POST). Tissues were analysed for proteolysis markers, and in vitro experiments were performed to provide additional insights. Atrogene mRNAs (TRIM63, FBXO32, FOXO3A) were upregulated at POST versus both PRE and MID (P<0.05). 20S proteasome core protein abundance increased at POST versus PRE (P=0.031) and MID (P=0.049). 20S proteasome activity, and protein levels for calpain-2 and Beclin-1 increased at MID and POST versus PRE (P<0.05). Ubiquitinated proteins showed model significance (P=0.019) with non-significant increases at MID and POST (P>0.05). in vitro experiments recapitulated the training phenotype when stimulated with a hypertrophic stimulus (insulin-like growth factor 1; IGF1) followed by a subsequent AMP-activated protein kinase activator (5-aminoimidazole-4-carboxamide ribonucleotide; AICAR), as demonstrated by larger myotube diameter in IGF1-treated cells versus IGF1 followed by AICAR treatments (I+A; P=0.017). Muscle protein synthesis (MPS) levels were also greater in IGF1-treated versus I+A myotubes (P<0.001). In summary, the loss in RT-induced VL CSA with HIIT coincided with increases in several proteolytic markers, and sustained proteolysis may have driven this response. Moreover, while not measured in humans, we interpret our in vitro data to suggest that (unlike RT) HIIT does not stimulate MPS. NEW FINDINGS: What is the central question of this study? Determining if HIIT-induced reductions in muscle hypertrophy following a period of resistance training coincided with increases in proteolytic markers. What is the main finding and its importance? Several proteolytic markers were elevated during the HIIT training period implying that increases in muscle proteolysis may have played a role in HIIT-induced reductions in muscle hypertrophy.