Muscle oxidative capacity during IL-6-dependent cancer cachexia.
ABSTRACT: Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1? expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.
Project description:Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. Inhibiting the signaling of the transcription factor nuclear factor kappa B (NF-?B) largely prevents cancer-induced muscle wasting in murine models. We have previously shown the utility of Compound A, a highly selective novel NF-?B inhibitor that targets the I?B kinase complex, to provide clinical benefit in cancer-induced skeletal muscle and cardiac atrophy.Using a metabolomics approach, we describe the changes found between cachectic and noncachectic gastrocnemius muscles before and after Compound A treatment at various doses.Of the 234 metabolites in the gastrocnemius, cachexia-induced changes in gastrocnemius metabolism reset the steady-state abundances of 42 metabolites (p?<?0.05). These changes, not evenly distributed across biochemical categories, are concentrated in amino acids, peptides, carbohydrates and energetics intermediates, and lipids. The gastrocnemius glycolytic pathway is markedly altered-changes consistent with tumor Warburg physiology. This is the first account of a Warburg effect that is not exclusively restricted to cancer cells or rapidly proliferating nonmalignant cells. Cachectic gastrocnemius also displays tricarboxylic acid cycle disruptions, signs of oxidative stress, and impaired redox homeostasis. Compound A only partially rescues the phenotype of the cachectic gastrocnemius, failing to restore the gastrocnemius' baseline metabolic profile.The findings in the present manuscript enumerate the metabolic consequences of cachexia in the gastrocnemius and demonstrate that NF-kB targeted treatment only partly rescues the cachectic metabolic phenotype. These data strengthen the previous findings from metabolomic characterization of serum in cachectic animals, suggesting that many of the metabolic alterations observed in the blood originate in the diseased muscle. These findings provide significant insight into the complex pathophysiology of cancer cachexia and provide objective criteria for evaluating future therapeutics.
Project description:Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Therapeutic options are still scarce. We hypothesized that cachexia-induced muscle oxidative stress may be attenuated in response to treatment with beta2-adrenoceptor-selective agonist formoterol in rats. In diaphragm and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with formoterol (0.3 mg/kg body weight/day for seven days, daily subcutaneous injection), redox balance (protein oxidation and nitration and antioxidants) and muscle proteins (1-dimensional immunoblots), carbonylated proteins (2-dimensional immunoblots), inflammatory cells (immunohistochemistry), and mitochondrial respiratory chain (MRC) complex activities were explored. In the gastrocnemius, but not the diaphragm, of cancer cachectic rats compared to the controls, protein oxidation and nitration levels were increased, several functional and structural proteins were carbonylated, and in both study muscles, myosin content was reduced, inflammatory cell counts were greater, while no significant differences were seen in MRC complex activities (I, II, and IV). Treatment of cachectic rats with formoterol attenuated all the events in both respiratory and limb muscles. In this in vivo model of cancer-cachectic rats, the diaphragm is more resistant to oxidative stress. Formoterol treatment attenuated the rise in oxidative stress in the limb muscles, inflammatory cell infiltration, and the loss of myosin content seen in both study muscles, whereas no effects were observed in the MRC complex activities. These findings have therapeutic implications as they demonstrate beneficial effects of the beta2 agonist through decreased protein oxidation and inflammation in cachectic muscles, especially the gastrocnemius.
Project description:Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.
Project description:BACKGROUND:Cachexia is the direct cause of at least 20% of cancer-associated deaths. Muscle wasting in skeletal muscle results in weakness, immobility, and death secondary to impaired respiratory muscle function. Muscle proteins are massively degraded in cachexia; nevertheless, the molecular mechanisms related to this process are poorly understood. Previous studies have reported conflicting results regarding the amino acid abundances in cachectic skeletal muscle tissues. There is a clear need to identify the molecular processes of muscle metabolism in the context of cachexia, especially how different types of molecules are involved in the muscle wasting process. METHODS:New in situ -omics techniques were used to produce a more comprehensive picture of amino acid metabolism in cachectic muscles by determining the quantities of amino acids, proteins, and cellular metabolites. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, we determined the in situ concentrations of amino acids and proteins, as well as energy and other cellular metabolites, in skeletal muscle tissues from genetic mouse cancer models (n = 21) and from patients with cancer (n = 6). Combined results from three individual MALDI mass spectrometry imaging methods were obtained and interpreted. Immunohistochemistry staining for mitochondrial proteins and myosin heavy chain expression, digital image analysis, and transmission electron microscopy complemented the MALDI mass spectrometry imaging results. RESULTS:Metabolic derangements in cachectic mouse muscle tissues were detected, with significantly increased quantities of lysine, arginine, proline, and tyrosine (P = 0.0037, P = 0.0048, P = 0.0430, and P = 0.0357, respectively) and significantly reduced quantities of glutamate and aspartate (P = 0.0008 and P = 0.0124). Human skeletal muscle tissues revealed similar tendencies. A majority of altered amino acids were released by the breakdown of proteins involved in oxidative phosphorylation. Decreased energy charge was observed in cachectic muscle tissues (P = 0.0101), which was related to the breakdown of specific proteins. Additionally, expression of the cationic amino acid transporter CAT1 was significantly decreased in the mitochondria of cachectic mouse muscles (P = 0.0133); this decrease may play an important role in the alterations of cationic amino acid metabolism and decreased quantity of glutamate observed in cachexia. CONCLUSIONS:Our results suggest that mitochondrial dysfunction has a substantial influence on amino acid metabolism in cachectic skeletal muscles, which appears to be triggered by diminished CAT1 expression, as well as the degradation of mitochondrial proteins. These findings provide new insights into the pathobiochemistry of muscle wasting.
Project description:Cachexia is a devastating muscle wasting syndrome that occurs in patients suffering from chronic diseases, most commonly observed in 80% of advanced cancer patients. One of the primary causes of cachexia-associated morbidity and mortality is involuntary muscle wasting. And while many cachexia patients show hypermetabolism, its causative role in muscles had remained unclear. To understand the molecular basis of this muscle wasting, accurate models of cachexia are necessary. Using transcriptomics and cytokine profiling of human muscle stem cell-based models and human cancer-induced cachexia models in mice, we found that cachectic cancer cells secreted many inflammatory factors which rapidly led to higher levels of fatty acid metabolism and the activation of a p38 stress response signature, before the cachectic muscle wasting is manifested. Metabolomics profiling revealed that factors secreted by cachectic cancer cells rapidly induce excessive fatty acid oxidation in human myotubes, leading to oxidative stress, p38 activation, and impaired muscle growth. Pharmacological blockade of fatty acid oxidation not only rescued human myotubes, but also significantly improved muscle mass and total weight in cancer cachexia models in vivo. Therefore, fatty acid-induced oxidative stress could be targeted to prevent cancer cachexia. Overall design: Three NOD-SCID mice were subcutaneously transplanted with RXF393 or SKRC39 cancer cells for 22 d. Quadriceps muscles were harvested and total RNA was extracted with the Trizol reagent (Ambion) for each mouse, processed with the Total Prep RNA for Illumina kit (Ambion), and hybridized to an Illumina MouseWG-6 v2.0 beadchip, then scanned on the Illumina Beadarray Reader. Array normalization was performed with Illumina Genome Studio Gene Expression module 1.9.0 using quantile normalisation. In total 6 RNA samples were prepared on the beadchip.
Project description:Cancer cachexia is observed in more than 50% of advanced cancer patients, and impairs quality of life and prognosis. A variety of pathways are likely to be dysregulated. Hence, a broad-spectrum understanding of the disease process is best achieved by a discovery based approach such as proteomics.More than 300 proteins were identified with > 95% confidence in correct sequence identification, of which 5-10% were significantly differentially expressed in cachectic tissues (p-value of 0.05; 27 proteins from gastrocnemius, 34 proteins from soleus and 24 proteins from heart). The two most pronounced functional groups being sarcomeric proteins (mostly upregulated across all three muscle types) and energy/metabolism proteins (mostly downregulated across all muscle types). Electron microscopy revealed disintegration of the sarcomere and morphological aberrations of mitochondria in the cardiac muscle of colon 26 (C26) carcinoma mice.The colon 26 (C26) carcinoma mouse model of cachexia was used to analyse soleus, gastrocnemius and cardiac muscles using two 8-plex iTRAQ proteomic experiments and tandem mass spectrometry (LCMSMS). Differentially expressed proteomic lists for protein clustering and enrichment of biological processes, molecular pathways, and disease related pathways were analysed using bioinformatics. Cardiac muscle ultrastructure was explored by electron microscopy.Morphological and proteomic analyses suggested molecular events associated with disintegrated sarcomeric structure with increased dissolution of Z-disc and M-line proteins. Altered mitochondrial morphology, in combination with the reduced expression of proteins regulating substrate and energy metabolism, suggest that muscle cells are likely to be undergoing a state of energy crisis which ultimately results in cancer-induced cachexia.
Project description:Cachexia is a devastating muscle wasting syndrome that occurs in patients suffering from chronic diseases, most commonly observed in 80% of advanced cancer patients. One of the primary causes of cachexia-associated morbidity and mortality is involuntary muscle wasting. And while many cachexia patients show hypermetabolism, its causative role in muscles had remained unclear. To understand the molecular basis of this muscle wasting, accurate models of cachexia are necessary. Using transcriptomics and cytokine profiling of human muscle stem cell-based models and human cancer-induced cachexia models in mice, we found that cachectic cancer cells secreted many inflammatory factors which rapidly led to higher levels of fatty acid metabolism and the activation of a p38 stress response signature, before the cachectic muscle wasting is manifested. Metabolomics profiling revealed that factors secreted by cachectic cancer cells rapidly induce excessive fatty acid oxidation in human myotubes, leading to oxidative stress, p38 activation, and impaired muscle growth. Pharmacological blockade of fatty acid oxidation not only rescued human myotubes, but also significantly improved muscle mass and total weight in cancer cachexia models in vivo. Therefore, fatty acid-induced oxidative stress could be targeted to prevent cancer cachexia. Overall design: Primary human myoblasts were exposed to RXF or SKR conditioned media for 6 d, by which time they grew and differentiated into early myotubes. Human early myotubes were harvested and total RNA was extracted with the Trizol reagent (Ambion) for each sample, processed with the Total Prep RNA for Illumina kit (Ambion), and hybridized to an Illumina HumanHT-12 v4.0 beadchip, then scanned on the Illumina Beadarray Reader. Array normalization was performed with Illumina Genome Studio Gene Expression module 1.9.0 using quantile normalisation.
Project description:Cachexia is a syndrome characterized by wasting of skeletal muscle and contributes to nearly one-third of all cancer deaths. Cytokines and tumor factors mediate wasting by suppressing muscle gene products, but exactly which products are targeted by these cachectic factors is not well understood. Because of their functional relevance to muscle architecture, such targets are presumed to represent myofibrillar proteins, but whether these proteins are regulated in a general or a selective manner is also unclear. Here we demonstrate, using in vitro and in vivo models of muscle wasting, that cachectic factors are remarkably selective in targeting myosin heavy chain. In myotubes and mouse muscles, TNF-alpha plus IFN-gamma strongly reduced myosin expression through an RNA-dependent mechanism. Likewise, colon-26 tumors in mice caused the selective reduction of this myofibrillar protein, and this reduction correlated with wasting. Under these conditions, however, loss of myosin was associated with the ubiquitin-dependent proteasome pathway, which suggests that mechanisms used to regulate the expression of muscle proteins may be cachectic factor specific. These results shed new light on cancer cachexia by revealing that wasting does not result from a general downregulation of muscle proteins but rather is highly selective as to which proteins are targeted during the wasting state.
Project description:Nearly 80% of advanced cancer patients are afflicted with cachexia, a debilitating syndrome characterized by extensive loss of muscle mass and function. Cachectic cancer patients have a reduced tolerance to antineoplastic therapies and often succumb to premature death from the wasting of respiratory and cardiac muscles. Since there are no available treatments for cachexia, it is imperative to understand the mechanisms that drive cachexia in order to devise effective strategies to treat it. Although 25% of metastatic breast cancer patients develop symptoms of muscle wasting, mechanistic studies of breast cancer cachexia have been hampered by a lack of experimental models. Using tumor cells deficient for BARD1, a subunit of the BRCA1/BARD1 tumor suppressor complex, we have developed a new orthotopic model of triple-negative breast cancer that spontaneously metastasizes to the lung and leads to systemic muscle deterioration. We show that expression of the metal-ion transporter, Zip14, is markedly upregulated in cachectic muscles from these mice and is associated with elevated intramuscular zinc and iron levels. Aberrant Zip14 expression and altered metal-ion homeostasis could therefore represent an underlying mechanism of cachexia development in human patients with triple-negative breast cancer. Our study provides a unique model for studying breast cancer cachexia and identifies a potential therapeutic target for its treatment.
Project description:BACKGROUND: Cancer cachexia is a complex syndrome related to a negative energy balance resulting in muscle wasting. Implication of muscle mitochondrial bioenergetics alterations during cancer cachexia was suggested. Therefore, the aim of this study was to explore the efficiency of oxidative phosphorylation in skeletal muscle mitochondria in a preclinical model of cancer cachexia. METHODS: Berlin-Druckrey IX rats with peritoneal carcinosis (PC) were used as a model of cancer cachexia with healthy pair-fed rats (PF) as control. Hindlimb muscle morphology and fibre type composition were analysed in parallel with ubiquitin ligases and UCP gene expression. Oxidative phosphorylation was investigated in isolated muscle mitochondria by measuring oxygen consumption and ATP synthesis rate. RESULTS: PC rats underwent significant muscle wasting affecting fast glycolytic muscles due to a reduction in fibre cross-sectional area. MuRF1 and MAFbx gene expression were significantly increased (9- and 3.5-fold, respectively) in the muscle of PC compared to PF rats. Oxygen consumption in non-phosphorylating state and the ATP/O were similar in both groups. Muscle UCP2 gene was overexpressed in PC rats. State III and the uncoupled state were significantly lower in muscle mitochondria from PC rats with a parallel reduction in complex IV activity (-30 %). CONCLUSION: This study demonstrated that there was neither alteration in ATP synthesis efficiency nor mitochondrial uncoupling in skeletal muscle of cachectic rats despite UCP2 gene overexpression. Muscle mitochondrial oxidative capacities were reduced due to a decrease in complex IV activity. This mitochondrial bioenergetics alteration could participate to insulin resistance, lipid droplet accumulation and lactate production.