Comparison of Atrogin-1/MAFbx mRNA Expression in the Gizzards of Egg- and Meat-Type Chickens

We examined atrogin-1/MAFbx mRNA expression in the smooth muscle of gizzards from egg- and meat-type chickens. Gizzard weight relative to body weight was significantly lower in the meat-type chickens than in the egg-type at 14 d of age. In contrast, the level of atrogin-1/MAFbx mRNA in the gizzard was significantly higher in the meat-type chickens than in the egg-type chickens. Thus atrogin-1/MAFbx mRNA expression in the smooth muscle of the gizzard was higher in meat-type chickens than in egg-type chickens, in contrast to its expression in the skeletal muscles.     

Curcumin prevents lipopolysaccharide-induced atrogin-1/MAFbx upregulation and muscle mass loss

Because elevated ubiquitin ligase atrogin-1/MAFbx and MuRF1 mediate skeletal muscle wasting associated with various catabolic conditions, the signaling pathways involved in the upregulation of these genes under pathological conditions are considered therapeutic targets. AKT and NF-kappaB have been previously shown to regulate the expression of atrogin-1/MAFbx or MuRF1, respectively. In addition, we recently found that p38 MAPK mediates TNF-alpha upregulation of atrogin-1/MAFbx expression, suggesting that multiple signaling pathways mediate muscle wasting in inflammatory diseases. To date, however, these advances have not resulted in a practical clinical intervention for disease-induced muscle wasting. In the present study, we tested the effect of curcumin--a non-toxic anti-inflammatory reagent that inhibits p38 and NF-kappaB--on lipopolysaccharide (LPS)-induced muscle wasting in mice. Daily intraperitoneal (i.p.) injection of curcumin (10-60 micro g/kg) for 4 days inhibited, in a dose-dependent manner, the LPS-stimulated (1 mg/kg, i.p.) increase of atrogin-1/MAFbx expression in gastrocnemius and extensor digitorum longus (EDL) muscles, resulting in the attenuation of muscle protein loss. It should also be noted that curcumin administration did not alter the basal expression of atrogin-1/MAFbx, nor did it affect LPS-stimulated MuRF1 and polyubiquitin expression. LPS activated p38 and NF-kappaB, while inhibiting AKT; whereas, curcumin administration inhibited LPS-stimulated p38 activation, without altering the effect of LPS on NF-kappaB and AKT. These results indicate that curcumin is effective in blocking LPS-induced loss of muscle mass through the inhibition of p38-mediated upregulation of atrogin-1/MAFbx.     

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.     

Experimental arthritis inhibits the insulin-like growth factor-I axis and induces muscle wasting through cyclooxygenase-2 activation

Chronic arthritis induces cachexia associated with an inhibition of the growth hormone (GH)-insulin-like growth factor-I (IGF-I) system and an activation of the E3 ubiquitin-ligating enzymes muscle atrophy F-box (MAFbx) and muscle Ring finger 1 (MuRF1) in the skeletal muscle. The aim of this work was to study the role of cyclooxygenase (COX)-2 in chronic arthritis-induced cachexia. Arthritis was induced in rats by Freund's adjuvant injection, and the effects of two COX inhibitors (indomethacin, a nonspecific inhibitor, and meloxicam, a selective COX-2 inhibitor on pituitary GH and on liver and serum IGF-I levels) were tested. Arthritis decreased body weight gain and GH and liver IGF-I gene expression. In the arthritic rats, both inhibitors, indomethacin and meloxicam, prevented the inhibitory effect of arthritis on body weight gain. Indomethacin and meloxicam administration to arthritic rats increased pituitary GH and liver IGF-I mRNA as well as serum levels of IGF-I. These data suggest that induction of COX-2 during chronic inflammation is involved in the inhibition of the GH-IGF-I axis and in the body weight loss. In the gastrocnemius muscle, arthritis increased the gene expression of tumor necrosis factor (TNF)-alpha, the E3 ubiquitin-ligating enzymes MAFbx and MuRF1, as well as of IGF-I and IGF-binding protein-5 (IGFBP-5). Inhibition of COX-2 by meloxicam administration increased gastrocnemius weight and decreased MAFbx, MuRF1, TNF-alpha, and IGFBP-5 gene expression. In summary, our data indicate that chronic arthritis-induced cachexia and muscle wasting are mediated by the COX-2 pathway resulting in a decreased GH-IGF-I secretion and increased expression of MAFbx and MuRF1 mRNA.     

Doxorubicin acts via mitochondrial ROS to stimulate catabolism in C2C12 myotubes

Doxorubicin, a commonly prescribed chemotherapeutic agent, causes skeletal muscle wasting in cancer patients undergoing treatment and increases mitochondrial reactive oxygen species (ROS) production. ROS stimulate protein degradation in muscle by activating proteolytic systems that include caspase-3 and the ubiquitin-proteasome pathway. We hypothesized that doxorubicin causes skeletal muscle catabolism through ROS, causing upregulation of E3 ubiquitin ligases and caspase-3. We tested this hypothesis by exposing differentiated C2C12 myotubes to doxorubicin (0.2 μM). Doxorubicin decreased myotube width 48 h following exposure, along with a 40-50% reduction in myosin and sarcomeric actin. Cytosolic oxidant activity was elevated in myotubes 2 h following doxorubicin exposure. This increase in oxidants was followed by an increase in the E3 ubiquitin ligase atrogin-1/muscle atrophy F-box (MAFbx) and caspase-3. Treating myotubes with SS31 (opposes mitochondrial ROS) inhibited expression of ROS-sensitive atrogin-1/MAFbx and protected against doxorubicin-stimulated catabolism. These findings suggest doxorubicin acts via mitochondrial ROS to stimulate myotube atrophy.     

Muscle oxidative capacity during IL-6-dependent cancer cachexia

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-1α 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.     

Differential effects of thyroid status on regional H2O2 production in slow- and fast-twitch muscle of ducklings

Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T₃ levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T₃. Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.     

Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle

Muscle wasting during sepsis reflects increased expression and activity of the ubiquitin-proteasome proteolytic pathway and is at least in part mediated by glucocorticoids. The ubiquitination of proteins destined to be degraded by the proteasome is regulated by multiple enzymes, including ubiquitin ligases. We tested the hypothesis that sepsis upregulates the gene expression of the newly described ubiquitin ligases, MuRF1 and atrogin-1/MAFbx. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. In some experiments, rats were treated with the glucocorticoid receptor antagonist RU 38486 before induction of sepsis. At various time points after induction of sepsis, mRNA levels for MuRF1 and atrogin-1/MAFbx were determined in extensor digitorum longus muscles by real-time PCR. Sepsis resulted in a 10-16-fold increase in gene expression of the ubiquitin ligases studied here. These changes were much greater than those observed previously for another ubiquitin ligase, E3alpha, in muscle during sepsis. Treatment of rats with RU 38486 prevented the sepsis-induced increase in mRNA levels for MuRF1 and atrogin-1/MAFbx, suggesting that glucocorticoids participate in the upregulation of these genes in muscle during sepsis. The present results lend further support to the concept that the ubiquitin-proteasome pathway plays an important role in sepsis-induced muscle proteolysis and suggest that multiple ubiquitin ligases may participate in the development of muscle wasting during sepsis.     

Mycobacterium ulcerans infections cause progressive muscle atrophy and dysfunction, and mycolactone impairs satellite cell proliferation

Clinical observations from Buruli ulcer (BU) patients in West Africa suggest that severe Mycobacterium ulcerans infections can cause skeletal muscle contracture and atrophy leading to significant impairment in function. In the present study, male mice C57BL/6 were subcutaneously injected with M. ulcerans in proximity to the right biceps muscle, avoiding direct physical contact between the infectious agent and the skeletal muscle. The histological, morphological, and functional properties of the muscles were assessed at different times after the injection. On day 42 postinjection, the isometric tetanic force and the cross-sectional area of the myofibers were reduced by 31% and 29%, respectively, in the proximate-infected muscles relative to the control muscles. The necrotic areas of the proximate-infected muscles had spread to 7% of the total area by day 42 postinjection. However, the number of central nucleated fibers and myogenic regulatory factors (MyoD and myogenin) remained stable and low. Furthermore, Pax-7 expression did not increase significantly in mycolactone-injected muscles, indicating that the satellite cell proliferation is abrogated by the toxin. In addition, the fibrotic area increased progressively during the infection. Lastly, muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (atrogin-1/MAFbx), two muscle-specific E3 ubiquitin ligases, were upregulated in the presence of M. ulcerans. These findings confirmed that skeletal muscle is affected in our model of subcutaneous infection with M. ulcerans and that a better understanding of muscle contractures and weakness is essential to develop a therapy to minimize loss of function and promote the autonomy of BU patients.     

Inhibition of Xanthine Oxidase by Allopurinol Prevents Skeletal Muscle Atrophy: Role of p38 MAPKinase and E3 Ubiquitin Ligases

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.     

Deletion of UCP2 in iNOS deficient mice reduces the severity of the disease during experimental autoimmune encephalomyelitis

Uncoupling protein 2 is a member of the mitochondrial anion carrier family that is widely expressed in neurons and the immune cells of humans. Deletion of Ucp2 gene in mice pre-activates the immune system leading to higher resistance toward infection and to an increased susceptibility to develop chronic inflammatory diseases as previously exemplified with the Experimental Autoimmune Encephalomyelitis (EAE), a mouse model for multiple sclerosis. Given that oxidative stress is enhanced in Ucp2-/- mice and that nitric oxide (NO) also plays a critical function in redox balance and in chronic inflammation, we generated mice deficient for both Ucp2 and iNos genes and submitted them to EAE. Mice lacking iNos gene exhibited the highest clinical score (3.4+/-0.5 p<0.05). Surprisingly, mice deficient for both genes developed milder disease with reduced immune cell infiltration, cytokines and ROS production as compared to iNos-/- mice.     

Mitochondrial gene expression in mammalian striated muscle. Evidence that variation in gene dosage is the major regulatory event

The oxidative capacity of mammalian striated muscles can vary markedly over a nearly 10-fold range, reflecting major differences in the expression of genes that encode enzymes of oxidative metabolism, including genes located exclusively within mitochondrial DNA. To clarify the regulatory events that govern expression of mitochondrial genes in striated muscle, nucleic acid hybridization procedures employing cloned segments of mitochondrial DNA as probes were utilized to determine the concentrations of mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA (a mitochondrial gene product) in rabbit striated muscles of markedly different oxidative capacities. When cardiac muscle and Type I (red, oxidative) skeletal muscle were compared to Type II (white, glycolytic) skeletal muscle, mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA, each increased in direct proportion to increases in oxidative capacity. Furthermore, when the phenotypic characteristics of Type II skeletal muscle were altered by electrical stimulation in vivo, mitochondrial DNA, mitochondrial rRNA, and cytochrome b mRNA also increased proportionately with increases in oxidative capacity. These results indicate that the expression of mitochondrial genes in mammalian striated muscle is proportionate to their copy number, and support the hypothesis that amplification of the mitochondrial genome relative to chromosomal DNA is an important feature underlying enhanced expression of mitochondrial genes in highly oxidative tissues.     

Lack of skeletal muscle liver kinase B1 alters gene expression,mitochondrial content,inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice.KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling.KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.     

Effects of orally administered glycine on myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle in chicks

We examined the effects of orally administered glycine on myofibrillar proteolysis in food-deprived chicks. Food-deprived (24 h) chicks were orally administered 57, 113, and 225 mg glycine/100 g body weight and killed after 2 h. The plasma N(tau)-methylhistidine concentration, used as myofibrillar proteolysis, was decreased by glycine. We also examined the expression of proteolytic-related genes by real-time PCR of cDNA from chick skeletal muscles. The mRNA expression of atrogin-1/MAFbx, proteasome C2 subunit, m-calpain large subunit, and cathepsin B was decreased by glycine in a dose-dependent manner. The plasma corticosterone concentration was also decreased by glycine, but the plasma insulin concentration was unaffected. These results indicate that orally administered glycine suppresses myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle by decreasing the plasma corticosterone concentration in chicks.