Catabolic Signaling Pathways Research Articles

Abstract LB022: A novel first-in-class USP19 inhibitor for the treatment of cancer-induced muscle atrophy

Abstract Cancer cachexia is a metabolic wasting syndrome characterized by weight loss, anorexia and anemia as a result of tumor burden, and affects up to 80% of advanced cancer patients #1. Cachexia is particularly prevalent in pancreatic, lung, colorectal and gastro-intestinal cancers and can lead to reduced tolerance and responsiveness to chemotherapy, increased treatment-related toxicity and morbidity, and poor overall quality of life. There are currently no approved therapies for cancer cachexia. The development and maintenance of muscle tissue is dependent on the balance between protein synthesis and protein degradation, controlled through various anabolic and catabolic signaling pathways. Dysregulation of these pathways can result in muscle atrophy, which arises in many chronic illnesses. The ubiquitin proteasome system (UPS) has a central role in regulating skeletal muscle physiology. Previous work utilizing USP19 knock out mouse models has demonstrated that USP19 plays an important role in muscle wasting and can protect against denervation-induced muscle atrophy #2. We have previously demonstrated that inhibition of USP19 enzymatic activity spares the muscle wasting observed in limb-casted and denervated mouse models of muscle wasting. Here, we report the discovery of a novel, highly potent and selective inhibitor of USP19 (ADC-846) and demonstrate its utility in a cancer-induced muscle atrophy model in vivo. Pharmacological inhibition of USP19 by ADC-846 increased lean muscle and fat mass following oral dosing in a Lewis Lung Carcinoma-induced cachexia model and reduced the cachexic index by >60% compared to controls. This data, in combination with our previous work detailing the effect of USP19 inhibition on muscle force and function, provides a much-needed novel pharmacological strategy for therapeutic intervention in muscle wasting conditions. Citation Format: Natalie Page, Vignesh Karthikaisamy, Darren O'Hara, Aaron N. Cranston, Colin R. O'Dowd, Xavier Jacq, Richard Wilkinson, Stephanie Burton, Hayley Gibson, Joana Costa, Daniel Longley, Matthew Helm, Chris McGivern, Steven Shepherd, Christina Bell, Peter Hewitt, Mary McFarland, Hugues Miel, Steven Whitehead, Lauren Proctor, Shane J. Rountree, Mark Wappett, Mauricio Berriel Diaz, Stephan Herzig, Timothy Harrison. A novel first-in-class USP19 inhibitor for the treatment of cancer-induced muscle atrophy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB022.

Disruptive lysosomal-metabolic signaling and neurodevelopmental deficits that precede Purkinje cell loss in a mouse model of Niemann-Pick Type-C disease

Purkinje cell (PC) loss occurs at an early age in patients and animal models of Niemann-Pick Type C (NPC), a lysosomal storage disease caused by mutations in the Npc1 or Npc2 genes. Although degeneration of PCs occurs early in NPC, little is known about how NPC1 deficiency affects the postnatal development of PCs. Using the Npc1nmf164 mouse model, we found that NPC1 deficiency significantly affected the postnatal development of PC dendrites and synapses. The developing dendrites of Npc1nmf164 PCs were significantly deficient in mitochondria and lysosomes. Furthermore, anabolic (mTORC1) and catabolic (TFEB) signaling pathways were not only perturbed but simultaneously activated in NPC1-deficient PCs, suggesting a loss of metabolic balance. We also found that mice with conditional heterozygous deletion of the Phosphatase and Tensin Homolog Deleted on Chromosome 10 gene (Pten-cHet), an inhibitor of mTORC1, showed similar early dendritic alterations in PCs to those found in Npc1-deficient mice. However, in contrast to Npc1nmf164 mice, Pten-cHet mice exhibited the overactivation of the mTORC1 pathway but with a strong inhibition of TFEB signaling, along with no dendritic mitochondrial reductions by the end of their postnatal development. Our data suggest that disruption of the lysosomal-metabolic signaling in PCs causes dendritic and synaptic developmental deficits that precede and promote their early degeneration in NPC.

The use of physiological and transcriptional analyses to examine molting regulatory mechanisms in juvenile horseshoe crab Tachypleus tridentatus

Molting is a necessary developmental process in crustaceans, and is holistically regulated by a range of hormones and associated genes. In order to investigate physiological changes occurring at the molecular level during the molting cycle of juvenile Tachypleus tridentatus, chitinase and N-acetyl-β-D-glucosaminidase (β-NAGase) activities were firstly measured by absorbance spectrophotometry. In addition, ecdysone content was quantified by ELISA, and changes in expression levels of genes relevant to ecdysis were examined based on quantitative real-time PCR (qRT-PCR) and RNA-sequencing (RNA-seq). Results showed that chitinase, β-NAGase activity, and ecdysone content increased at the intermolt stages, peaked at premolt stages, and decreased to a lower level at molt and postmolt stages. Chitinase and β-NAGase were positively correlated with ecdysone. RNA-seq revealed that a total of 6482 differentially expressed genes (DEGs) in the four different molting stages, and 5763 DEGs had higher expression levels in the premolt stage than in the intermolt stage. A total of 59 shared DEGs were identified in all molting stages, and the highly expressed DEGs were mainly related to chitin binding, stratum corneum structural composition and neuropeptide hormone activity signaling pathways. KEGG pathway analysis showed that at the premolt stage, digestion, transport, and catabolic signaling pathways were significantly enriched and innate immune response pathways were significantly enriched as molting progressed. These results suggested that the molting of juvenile horseshoe crabs may be directly regulated by ecdysone, and chitinase and β-NAGase play a synergistic role with ecdysone during the molting cycle. The key biological processes and signaling pathways involved in hormone regulation, epidermal structural proteins, catabolism, apoptosis, and immune response potentially regulate the molting process of horseshoe crab juveniles. This study provides a theoretical basis for enhancing understanding of the molting and growth processes in marine arthropods, and a strong theoretical support for the developmenting of captive rearing technologies for Chinese horseshoe crabs.

AMPK: Thekey to ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) refers to a syndrome in which tissue damage is further aggravated and organ function further deteriorates when blood flow is restored after a period of tissue ischemia. Acute myocardial infarction, stress ulcer, pancreatitis, intestinal ischemia, intermittent claudication, acute tubular necrosis, postshock liver failure, and multisystem organ failure are all related to reperfusion injury. AMP-activated protein kinase (AMPK) has been identified in multiple catabolic and anabolic signaling pathways. The functions of AMPK during health and diseases are intriguing but still need further research. Except for its conventional roles as an intracellular energy switch, emerging evidence reveals the critical role of AMPK in IRI as an energy-sensing signal molecule by regulating metabolism, autophagy, oxidative stress, inflammation, and other progressions. At the same time, drugs based on AMPK for the treatment of IRI are constantly being researched and applied in clinics. In this review, we summarize the mechanisms underlying the effects of AMPK in IRI and describe the AMPK-targeting drugs in treatment, hoping to increase the understanding of AMPK in IRI and provide new insights into future clinical treatment.

Sex-specific changes in metabolism during the transition from chow to high-fat diet feeding are abolished in response to dieting in C57BL/6J mice

Background/ObjectiveFemale mice are often excluded from diet-induced obesity studies as they are more resistant to the obesifying effects of a high-fat diet (HFD). However, the underlying mechanisms behind this sex disparity may actually have important implications for the development and management of obesity in humans. Therefore, we systematically investigated the immediate sex-specific effects of transitioning to a HFD in C57BL/6J mice as well as monitored whether these effects are altered after sustained HFD feeding and whether sex affects the response to a return to chow, representative of dieting.MethodsDual X-ray absorptiometry (DXA) analysis of body composition, indirect calorimetry measurements, and qPCR analysis of hypothalamic and brainstem regions were performed on male and female C57BL/6J mice.ResultsHFD had immediate and dramatic effects in males, increasing fat mass by 58% in the first 3 days. The resistance to the obesifying effect of HFD in females was linked both to an ability to maintain activity levels as well as to an immediate and significantly enhanced reduction in respiratory quotient (RQ), suggesting a greater ability to utilise fat in the diet as a source of fuel. Mechanistically, this sex disparity may be at least partially due to inherent sex differences in the catabolic (POMC/CART) versus anabolic (NPY/AgRP) neurological signalling pathways. Interestingly, the reintroduction of chow following HFD had immediate and consistent responses between the sexes with body composition and most metabolic parameters normalised within 3 days. However, both sexes displayed elevated hypothalamic Npy levels reminiscent of starvation. The difference in RQ seen between the sexes on HFD was immediately abolished suggesting similar abilities to burn fat reserves for fuel.ConclusionsC57BL/6J mice have markedly different sex-specific behavioural and metabolic responses to the introduction as well as the sustained intake of a HFD, but consistent responses to a dieting situation.

Modulation of Inflammation by Plant-Derived Nutraceuticals in Tendinitis

Tendinitis (tendinopathy) is a pro-inflammatory and painful tendon disease commonly linked with mechanical overuse and associated injuries, drug abuse, and lifestyle factors (including poor diet and physical inactivity) that causes significant healthcare expenditures due to its high incidence. Nuclear factor kappa B (NF-κB) is one of the major pro-inflammatory transcription factors, along with other inflammation signaling pathways, triggered by a variety of stimuli, including cytokines, endotoxins, physical and chemical stressors, hypoxia, and other pro-inflammatory factors. Their activation is known to regulate the expression of a multitude of genes involved in inflammation, degradation, and cell death. The pathogenesis of tendinitis is still poorly understood, whereas efficient and sustainable treatment is missing. Targeting drug suppression of the key inflammatory regulators represents an effective strategy for tendinitis therapy, but requires a comprehensive understanding of their principles of action. Conventional monotherapies are often ineffective and associated with severe side effects in patients. Therefore, agents that modulate multiple cellular targets represent therapeutic treatment potential. Plant-derived nutraceuticals have been shown to act as multi-targeting agents against tendinitis via various anti-oxidant and anti-inflammatory mechanisms, whereat they were able to specifically modulate numerous signaling pathways, including NF-κB, p38/MAPK, JNK/STAT3, and PI3K/Akt, thus down-regulating inflammatory processes. This review discusses the utility of herbal nutraceuticals that have demonstrated safety and tolerability as anti-inflammatory agents for the prevention and treatment of tendinitis through the suppression of catabolic signaling pathways. Limitations associated with the use of nutraceuticals are also described.

Nutraceuticals and Exercise against Muscle Wasting during Cancer Cachexia.

Cancer cachexia (CC) is a debilitating multifactorial syndrome, involving progressive deterioration and functional impairment of skeletal muscles. It affects about 80% of patients with advanced cancer and causes premature death. No causal therapy is available against CC. In the last few decades, our understanding of the mechanisms contributing to muscle wasting during cancer has markedly increased. Both inflammation and oxidative stress (OS) alter anabolic and catabolic signaling pathways mostly culminating with muscle depletion. Several preclinical studies have emphasized the beneficial roles of several classes of nutraceuticals and modes of physical exercise, but their efficacy in CC patients remains scant. The route of nutraceutical administration is critical to increase its bioavailability and achieve the desired anti-cachexia effects. Accumulating evidence suggests that a single therapy may not be enough, and a bimodal intervention (nutraceuticals plus exercise) may be a more effective treatment for CC. This review focuses on the current state of the field on the role of inflammation and OS in the pathogenesis of muscle atrophy during CC, and how nutraceuticals and physical activity may act synergistically to limit muscle wasting and dysfunction.

TAK1 preserves skeletal muscle mass and mitochondrial function through redox homeostasis.

Skeletal muscle atrophy is debilitating consequence of a large number of chronic disease states, aging, and disuse conditions. Skeletal muscle mass is regulated through coordinated activation of a number of signaling cascades. Transforming growth factor‐β activated kinase 1 (TAK1) is a central kinase that mediates the activation of multiple signaling pathways in response to various growth factors, cytokines, and microbial products. Accumulating evidence suggests that TAK1 promotes skeletal muscle growth and essential for the maintenance of muscle mass in adults. Targeted inactivation of TAK1 leads to severe muscle wasting and kyphosis in mice. However, the mechanisms by which TAK1 prevents loss of muscle mass remain poorly understood. Through generation of inducible skeletal muscle‐specific Tak1‐knockout mice, we demonstrate that targeted ablation of TAK1 disrupts redox signaling leading to the accumulation of reactive oxygen species and loss of skeletal muscle mass and contractile function. Suppression of oxidative stress using Trolox improves muscle contractile function and inhibits the activation of catabolic signaling pathways in Tak1‐deficient muscle. Moreover, Trolox inhibits the activation of ubiquitin‐proteasome system and autophagy markers in skeletal muscle of Tak1‐deficient mice. Furthermore, inhibition of oxidative stress using Trolox prevents the slow‐to‐fast type fiber transition and improves mitochondrial respiration in skeletal muscle of Tak1‐deficient mice. Overall, our results demonstrate that TAK1 maintains skeletal muscle mass and health through redox homeostasis.

AMPK, Mitochondrial Function, and Cardiovascular Disease.

Adenosine monophosphate-activated protein kinase (AMPK) is in charge of numerous catabolic and anabolic signaling pathways to sustain appropriate intracellular adenosine triphosphate levels in response to energetic and/or cellular stress. In addition to its conventional roles as an intracellular energy switch or fuel gauge, emerging research has shown that AMPK is also a redox sensor and modulator, playing pivotal roles in maintaining cardiovascular processes and inhibiting disease progression. Pharmacological reagents, including statins, metformin, berberine, polyphenol, and resveratrol, all of which are widely used therapeutics for cardiovascular disorders, appear to deliver their protective/therapeutic effects partially via AMPK signaling modulation. The functions of AMPK during health and disease are far from clear. Accumulating studies have demonstrated crosstalk between AMPK and mitochondria, such as AMPK regulation of mitochondrial homeostasis and mitochondrial dysfunction causing abnormal AMPK activity. In this review, we begin with the description of AMPK structure and regulation, and then focus on the recent advances toward understanding how mitochondrial dysfunction controls AMPK and how AMPK, as a central mediator of the cellular response to energetic stress, maintains mitochondrial homeostasis. Finally, we systemically review how dysfunctional AMPK contributes to the initiation and progression of cardiovascular diseases via the impact on mitochondrial function.

Diurnal Regulation Of Exercise-induced Anabolic And Catabolic Signaling In White Adipose Tissue

PURPOSE: Autophagy is a degradation system where damaged cellular components can be recycled, resulting in higher cellular efficiency. White adipose tissue has been shown to have a catabolic response to exercise where autophagy will increase immediately after exercise. mTOR is an anabolic signaling pathway that inhibits autophagy, and plays a key role regulating cell size. Autophagy is sensitive to bioenergetic stressors such as exercise and has been shown to be regulated in a circadian fashion. However, it is currently unknown if exercise-induced autophagy is sensitive to the time-of-day at which exercise occurs. The purpose was to assess anabolic and catabolic signaling in white adipose tissue following exercise at two times of day; Zeitgeber time (ZT) 0 (light phase) and ZT12 (dark phase). METHODS: 21 week old male C57/BL6 mice (n=38) were habituated to treadmill exercise for 5 days under red light during the active phase, and allowed to recover for 2 days. Following a single 60-minute bout of treadmill exercise at 10 m/min, mice were sacrificed at 3 time points, pre exercise (SED), immediately post exercise (POST), and 1-hour post exercise (1HR). Tissue was analyzed for anabolic (p-mTOR (Ser2448) and p-S6 (Ser240/244)), and catabolic (LC3II/I) signaling pathways via western blotting. Results were analyzed with a 2x3 ANOVA and significance was accepted at p<0.05. RESULTS: Following exercise, there was a significant repression of mTOR (SED=0.990 ±0.17 vs POST=0.747 ± 0.14, p<0.05) and S6 (SED=0.702 ± 0.39 vs POST=0.186 ± 0.14, p<0.05) activation (Main Effect (ME) Exercise; p<0.05, both), which was significantly stronger following exercise performed at ZT12 (Interaction Effect for p-S6;; p<0.05). Autophagy signaling (LC3II/I) was increased at ZT12 (ZT0=1.22 ± 0.33 vs ZT12=2.28 ± 1.09, p<0.05), which appeared to be driven by changes in LC3I expression. LC3II (normalized to total protein) was increased following exercise (SED=0.81 ± 0.26 vs POST=1.28 ± 0.43 and 1HR=1.22 ± 0.28, p<0.05, both) and was higher at ZT0 (ZT0=1.33 ± 0.33 vs ZT12=0.91 ± 0.33, p<0.05), although no interaction was present. CONCLUSION: Anabolic and catabolic signaling in adipose tissue may be differentially impacted by exercise performed at different times of day.

Proteasome activity and expression of mammalian target of rapamycin signaling factors in skeletal muscle of dairy cows supplemented with conjugated linoleic acids during early lactation

The mammalian target of rapamycin (mTOR) is a major regulator of protein synthesis via its main downstream effectors, ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein (4EBP1). The ubiquitin-proteasome system (UPS) is the main proteolytic pathway in muscle, and the muscle-specific ligases tripartite motif containing 63 (TRIM63; also called muscle-specific ring-finger protein 1, MuRF-1) and F-box only protein 32 (FBXO32; also called atrogin-1) are important components of the UPS. We investigated 20S proteasome activity and mRNA expression of key components of mTOR signaling and UPS in skeletal muscle of dairy cows during late gestation and early lactation and tested the effects of dietary supplementation (from d 1 in milk) with conjugated linoleic acids (sCLA; 100 g/d; n = 11) compared with control fat-supplemented cows (CTR; n = 10). Blood and muscle tissue (semitendinosus) samples were collected on d -21, 1, 21, and 70 relative to parturition. Dry matter intake increased with time of lactation in both groups. It was lower in sCLA than in CTR on d 21, which resulted in a reduced calculated metabolizable protein balance. Most serum and muscle concentrations of AA followed time-related changes but were unaffected by CLA supplementation. In both groups, serum and muscle 3-methylhistidine (3-MH) concentrations and the ratio of 3-MH:creatinine increased from d -21 to d 1, followed by a decline on d 21. The mRNA abundance of MTOR on d 21 and 70 was greater in sCLA than in CTR. The abundance of 4EBP1 mRNA did not differ between groups but was upregulated in both on d 1. The mRNA abundance of S6K1 on d 70 was greater in CTR than in sCLA, but remained unchanged over time in both groups. The mRNA abundance of FBXO32 (encoding atrogin-1) on d 21 was greater in sCLA than in CTR. The mRNA abundance of TRIM63 (also known as MuRF1) showed a similar pattern as FBXO32 in both groups: an increase from d -21 to d 1, followed by a decline. The mRNA for the α (BCKDHA) and β (BCKDHB) polypeptide of branched-chain α-keto acid dehydrogenase was elevated in sCLA and CTR cows on d 21, respectively, suggesting a role of CLA in determining the metabolic fate of branched-chain AA. For the mTOR protein, no group differences were observed. The abundance of S6K1 protein was greater across all time points in sCLA versus CTR. The antepartum 20S proteasome activity in muscle was elevated in both groups compared with postpartum, probably reflecting the start of protein mobilization before parturition. Plasma insulin concentrations decreased in both groups postpartum but to a greater extent in CTR than in sCLA, resulting in greater insulin concentrations in sCLA than in CTR. Thus, the greater abundance of MTOR mRNA and S6K1 protein in sCLA compared with CTR might be mediated by the greater plasma insulin postpartum. The upregulation of MTOR mRNA in sCLA cows on d 21, despite greater FBXO32 mRNA abundance, may reflect a simultaneous activation of both anabolic and catabolic signaling pathways, likely resulting in greater protein turnover.

The Effect of β-GPA on the Markers of Anabolic and Catabolic Signaling Pathways in Rat Soleus Muscle at the Initial Stage of Hindlimb Unloading

Molecular mechanisms initiating the development of atrophic changes in a mammalian postural muscle remain unclear. We have suggested that AMP-activated protein kinase (AMPK) may play an important role in the regulation of anabolic and catabolic signaling pathways in postural muscle at the initial stage of mechanical unloading, since skeletal muscle inactivation can lead to changes in the balance of intramuscular high-energy phosphates. Beta-guanidinopropionic acid (β-GPA) can act as a modulator of the balance of high-energy phosphates, application of which can reduce the content of intracellular high-energy phosphates. The purpose of this study was to assess signaling pathways of protein synthesis and degradation in rat soleus muscle after preliminary treatment with β-GPA followed by 1-day hindlimb unloading. The experiments were performed on male Wistar rats, which were divided into the following groups: (1) vivarium control (C); (2) vivarium control + β-GPA (C + GPA); (3) 1-day hindlimb unloading (HU); and (4) 1-day hindlimb unloading + β-GPA (HU + GPA). β-GPA was administered daily by intraperitoneal injections (400 mg/kg). The content of the key signaling proteins (AMPK, ACC, p70S6K, 4E-BP1, and FOXO3) was determined by gel electrophoresis followed by immunoblotting. The expression of mRNA of ubiquitin ligases (MuRF1 and MAFbx) was evaluated by real-time PCR. A prevention of reduction in phosphorylation of AMPK, ACC and 4E-BP1 and the return to control values of the increased level of p70S6K phosphorylation were observed in the group with 1-day unloading and β-GPA pretreatment. The phosphorylation of AKT and FOXO3, as well as the expression levels of MuRF1 and MAFbx in the HU + GPA group did not differ from the HU group. The obtained data indicate that the AMPK activity may play an important role in the modulation of anabolic mTORC1-signaling in rat soleus muscle at the early stage of simulated microgravity.

Effects of bathing in hot water after resistance exercise on muscle anabolic and catabolic signaling pathways in rat skeletal muscle

Effects of bathing in hot water after resistance exercise on muscle anabolic and catabolic signaling pathways in rat skeletal muscle

Interactions of the super complexes: When mTORC1 meets the proteasome.

Homeostatic regulation of energy and metabolic status requires that anabolic and catabolic signaling pathways be precisely regulated and coordinated. Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is a mega protein complex that promotes energy-consuming anabolic processes of protein and nucleic acid synthesis as well lipogenesis in times of energy and nutrient abundance. However, it is best characterized as the regulator of steps leading to protein synthesis. The ubiquitin-proteasome proteolytic system (UPS) is a major intracellular proteolytic system whose activity is increased during periods of nutrient scarcity and in muscle wasting conditions such as cachexia. Recent studies have examined the impact of mTORC1 on levels and functions of the 26S proteasome, the mega protease complex of the UPS. Here we first briefly review current understanding of the regulation of mTORC1, the UPS, and the 26S proteasome complex. We then review evidence of the effect of each complex on the abundance and functions of the other. Given the fact that drugs that inhibit either complex are either in clinical trials or are approved for treatment of cancer, a muscle wasting condition, we identify studying the effect of combinatory mTORC1-proteasome inhibition on skeletal muscle mass and health as a critical area requiring investigation.

Effects of Plantar Mechanical Stimulation on Anabolic and Catabolic Signaling in Rat Postural Muscle Under Short-Term Simulated Gravitational Unloading.

It is known that plantar mechanical stimulation (PMS) is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. However, molecular mechanisms underlying the effect of PMS on skeletal muscle during unloading remain undefined. The aim of the study was to evaluate the effects of PMS on anabolic and catabolic signaling pathways in rat soleus at the early stages of mechanical unloading. Wistar rats were randomly assigned to ambulatory control, hindlimb suspension (HS) for 1 or 3 days, and HS for 1 or 3 days with PMS. The key anabolic and catabolic markers were assessed by western blotting and RT-PCR. Protein synthesis (PS) rate was estimated using SUnSET technique. PMS attenuated a 1-day HS-induced decrease in 4E-BP1, GSK-3β, and AMPK phosphorylation. PMS also partially prevented a decrease in PS, phosphorylation of GSK-3β, nNOS, and an increase in eEF2 phosphorylation after 3-day HS. PMS during 1- and 3-day HS prevented MuRF-1, but not MAFbx, upregulation but did not affect markers of ribosome biogenesis (18S + 28S rRNA, c-myc) as well as AKT phosphorylation. Thus, PMS during 3-day HS partially prevented a decrease in the global rate of PS in rat soleus muscle, which was accompanied by attenuation of MuRF-1 mRNA expression as well as changes in GSK-3β, nNOS, and eEF2 phosphorylation.

A novel nutritional supplement prevents muscle loss and accelerates muscle mass recovery in caloric-restricted mice

BackgroundMuscle atrophy is defined as decreased muscle mass, associated with aging as well as with various chronic diseases and is a fundamental cause of frailty, functional decline and disability. Frailty represents a huge potential public health issue worldwide with high impact on healthcare costs. A major clinical issue is therefore to devise new strategies preventing muscle atrophy. In this study, we tested the efficacy of Vital01, a novel oral nutritional supplement (ONS), on body weight and muscle mass using a caloric restriction-induced mouse model for muscle atrophy. MethodsMice were calorically restricted for 2 weeks to induce muscle atrophy: one control group received 60% kcal of the normal chow diet and one intervention group received 30% kcal chow and 30 kcal% Vital01. The effects on body weight, lean body mass, muscle histology and transcriptome were assessed. In addition, the effects of Vital01, in mice with established muscle atrophy, were assessed and compared to a standard ONS. To this end, mice were first calorically restricted on a 60% kcal chow diet and then refed with either 100 kcal% chow, a mix of Vital01 (receiving 60% kcal chow and 40 kcal% Vital01) or with a mix of standard, widely prescribed ONS (receiving 60 kcal% chow and 40 kcal% Fortisip Compact). ResultsVital01 attenuated weight loss (−15% weight loss for Vital01 vs. −25% for control group, p < 0.01) and loss of muscle mass (Vital01 with −13%, −12% and −18%, respectively, for gastrocnemius, quadriceps and tibialis vs. 25%, −23% and −28%, respectively, for control group, all p < 0.05) and also restored body weight, fat and muscle mass more efficiently when compared to Fortisip Compact. As assessed by transcriptome analysis and Western blotting of key proteins (e.g. phospoAKT, mTOR and S6K), Vital01 attenuated the catabolic and anabolic signaling pathways induced by caloric restriction and modulated inflammatory and mitochondrial pathways. In addition, Vital01 affected pathways related to matrix proteins/collagens homeostasis and tended to reduce caloric restriction-induced collagen fiber density in the quadriceps (with −27%, p = 0.051). ConclusionsWe demonstrate that Vital01 preserves muscle mass in a calorically restricted mouse model for muscle atrophy. Vital01 had preventive effects when administered during development of muscle atrophy. Furthermore, when administered in a therapeutic setting to mice with established muscle atrophy, Vital01 rapidly restored body weight and accelerated the recurrence of fat and lean body mass more efficiently than Fortisip Compact. Bioinformatics analysis of gene expression data identified regulatory pathways that were specifically influenced by Vital01 in muscle.

Cellular and molecular signatures of alcohol-induced myopathy in women.

Alcoholic myopathy is characterized by the reduction in cross-sectional area (CSA) of muscle fibers and impaired anabolic signaling. The goal of the current study was to investigate the causes and compare the changes in CSA and fiber type composition with the modifications of anabolic and catabolic signaling pathways at the early stages of chronic alcohol consumption in women. Skeletal muscle samples from 5 female patients with alcohol abuse (AL; 43 ± 5 yr old; alcohol abuse duration 5,6 ± 0,6 yr) were compared with the muscle from the control group of 8 healthy women (C; 35 ± 4 yr old). The average daily dose of alcohol consumption was 110 ± 10 ml of pure ethanol. In women patients, a significant decrease in CSA of type I and II muscle fibers, titin and nebulin content, plasma IGF-1 level and total IRS-1, p-Akt and p-4E-BP1 in vastus lateralis was found in comparison with the control group. The p-AMPK level was found to be increased versus the control group. In women patients with chronic alcoholic myopathy 1) both fast and slow muscle fibers are subjected to atrophy; 2) impairments in IGF-I-dependent signaling and pathways controlling translation initiation (AMPK/mTOR/4E-BP1), but not translation elongation, are observed; 3) the level of calpain-1 and ubiquitinated proteins increases, unlike E3 ligases content.

Inhibition of Proteasomes by Bortezomib Decreases MuRF-1 and MAFbx mRNA Expression in Rat Soleus during Hindlimb Unloading

Skeletal muscle atrophy caused by unloading is accompanied by increased proteolysis and decreased protein synthesis. It is established that in conditions of muscle unloading, concentration of free amino acids is increased. We hypothesized that proteasome inhibition may decrease amino acid accumulation in skeletal muscle and prevent the atrophy. To test this hypothesis, we treated rats with bortezomib (a proteasome inhibitor) during 7-day hindlimb suspension. Content of key signaling proteins of various signaling pathways was measured by Western-blotting; mRNA level of E3 ligases by RT-PCR, rate of protein synthesis by SUnSET technique. Soleus muscle weight and intensity of the protein synthesis in the groups of “hindlimb suspension” (HS) and “HS + bortezomib” (HSB) were equally reduced as compared to control. Levels of MuRF-1 and MAFbx mRNAs, content of MuRF-1 and calpain-1 proteins, and level of the protein ubiquitination were increased only in the HS group and remained unchanged in the HSB group as compared to the control group. We conclude that inhibition of proteasomes during m. soleus unloading prevents increase in activity of some components of catabolic signaling pathways. However, this is not sufficient to reduce rate of the atrophic processes in skeletal muscle.

The metabolic and molecular mechanisms of hyperammonaemia- and hyperethanolaemia-induced protein catabolism in skeletal muscle cells.

Hyperammonaemia and hyperethanolaemia are thought to be driving factors behind skeletal muscle myopathy in liver disease, that is, cirrhosis. Despite this, the singular and combined impacts of ethanol‐ and ammonia‐induced protein catabolism are poorly defined. As such, we aimed to dissect out the effects of ammonia and ethanol on muscle catabolism. Murine C2C12 myotubes were treated with ammonium acetate (10 mM) and ethanol (100 mM) either alone or in combination for 4 hr and/or 24 hr. Myotube diameter, muscle protein synthesis and anabolic and catabolic signalling pathways were assessed. In separate experiments, cells were cotreated with selected inhibitors of protein breakdown to assess the importance of proteolytic pathways in protein loss with ammonia and ethanol. Ammonia and ethanol in combination resulted in a reduction in myotube width and total protein content, which was greater than the reduction observed with ammonia alone. Both ammonia and ethanol caused reductions in protein synthesis, as assessed by puromycin incorporation. There was also evidence of impairments in regulation of protein translation, and increased protein expression of markers of muscle protein breakdown. Myotube protein loss with ammonia plus ethanol was not affected by autophagy inhibition, but was completely prevented by proteasome inhibition. Thus, combined ammonia and ethanol incubation of C2C12 myotubes exacerbated myotube atrophy and dysregulation of anabolic and catabolic signalling pathways associated with either component individually. Ubiquitin proteasome‐mediated protein breakdown appears to play an important role in myotube protein loss with ethanol and ammonia.

Comparison of botulinum toxin type A and Rho-inhibitor on ATDC5 chondrocyte

Knee osteoarthritis (OA) has an incidence rate of 25% per year and its responses to treatment are variable. The analgesic effect of intra-articular injection of botulinum toxin type A (BTX-A) has been observed in patients with knee OA. BTX-A might inhibit Rho GTPase by ADP-ribosylation of amino acid asparagine-41. Rho activity has a role in mediation of chondrocyte catabolic signaling pathways, and Rho/ROCK (Rho-associated coiled-coil containing kinase) activation induces articular cartilage degradation. In our unpublished data, chondroprotection of BTX-A was observed based on pathological findings in rat knee OA. ROCK I & II were found to be altered in chondrocytes treated with BTXA, with dose-dependent effect. This study aimed to compare the effect on chondrocyte of BTX-A and Rho-inhibitor. ATDC5 chondrocytes were compared based on cell phenotype and markers for chondrocyte differentiation and growth after administration of BTX-A, Rho-inhibitor (Y27632), and normal saline (control) to clarify the downstream signaling pathway. Reverse transcription-PCR (RT-PCR) showed that in ATDC5 chondrocyte cell culture, Y27632 and BTX-A elevated aggrecan on culture day 4 and day 7. On day 21, the level of collagen type X and MMP13 were lowered in Y27632 and BTX-A. ROCK I and II level were decreased on day 4 and 7 in all three groups, but were remarkably higher on day 14 and 21 in Y27632. On day 8, in ATDC5 cells not pretreated with insulin, decreased cell density, small and flat chondrocyte morphology were observed after addition of Y27632 and BTX-A, in comparison to control. BTX-A and Rho-inhibitor decreased ROCK I & II and increased aggrecan level during early stage, and decreased collagen type X and MMP13 during late stage. Potential dedifferentiation effect in BTX-A and Rho-inhibitor were observed as well. Rho-signaling pathway might play different roles during early and late stage in chondrocyte growth.