Ursolic acid increases skeletal muscle and brown fat and decreases diet-induced obesity, glucose intolerance and fatty liver disease

Skeletal muscle Akt activity stimulates muscle growth and imparts resistance to obesity, glucose intolerance and fatty liver disease. We recently found that ursolic acid increases skeletal muscle Akt activity and stimulates muscle growth in non-obese mice. Here, we tested the hypothesis that ursolic acid might increase skeletal muscle Akt activity in a mouse model of diet-induced obesity. We studied mice that consumed a high fat diet lacking or containing ursolic acid. In skeletal muscle, ursolic acid increased Akt activity, as well as downstream mRNAs that promote glucose utilization (hexokinase-II), blood vessel recruitment (Vegfa) and autocrine/paracrine IGF-I signaling (Igf1). As a result, ursolic acid increased skeletal muscle mass, fast and slow muscle fiber size, grip strength and exercise capacity. Interestingly, ursolic acid also increased brown fat, a tissue that shares developmental origins with skeletal muscle. Consistent with increased skeletal muscle and brown fat, ursolic acid increased energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis. These data support a model in which ursolic acid reduces obesity, glucose intolerance and fatty liver disease by increasing skeletal muscle and brown fat, and suggest ursolic acid as a potential therapeutic approach for obesity and obesity-related illness.     

Systems-based Discovery of Tomatidine as a Natural Small Molecule Inhibitor of Skeletal Muscle Atrophy

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.     

Developmentally regulated troponin C mRNAs of chicken skeletal muscle.

Fast and slow/cardiac troponin C (TnC) are the two different isoforms of TnC. Expression of these isoforms is developmentally regulated in vertebrate skeletal muscle. Therefore, in our studies, the pattern of their expression was analyzed by determining the steady-state levels of both TnC mRNAs. It was also examined if mRNAs for both isoforms of TnC were efficiently translated during chicken skeletal muscle development. We have used different methods to determine the steady-state levels of TnC mRNAs. First, probes specific for the fast and slow TnC mRNAs were developed using a 390 base pair (bp) and a 255 bp long fragment, of the full-length chicken fast and slow TnC cDNA clones, respectively. Our analyses using RNA-blot technique showed that fast TnC mRNA was the predominant isoform in embryonic chicken skeletal muscle. Following hatching, a significant amount of slow TnC mRNA began to accumulate in the skeletal (pectoralis) muscle. At 43 weeks posthatching, the slow TnC mRNA was nearly as abundant as the fast isoform. Furthermore, a majority of both slow and fast TnC mRNAs was found to be translationally active. A second method allowed a more reliable measure of the relative abundance of slow and fast TnC mRNAs in chicken skeletal muscle. We used a common highly conserved 18-nucleotide-long sequence towards the 5'-end of these mRNAs to perform primer extension analysis of both mRNAs in a single reaction. The result of these analyses confirmed the predominance of fast TnC mRNA in the embryonic skeletal muscle, while significant accumulation of slow TnC mRNA was observed in chicken breast (pectoralis) muscle following hatching. In addition to primer extension analysis, polymerase chain reaction was used to amplify the fast and slow TnC mRNAs from cardiac and skeletal muscle. Analysis of the amplified products demonstrated the presence of significant amounts of slow TnC mRNA in the adult skeletal muscle.     

Expression of ARPP-16/19 in rat denervated skeletal muscle

It is known that denervation of rat skeletal muscle causes atrophy and this is often adopted as a model for human muscle atrophy. To understand the molecular changes that occur, it is important to identify the profiles of differential gene expression. In the present study, we investigated differentially expressed genes in denervated muscle using DNA microarrays with printed genes preferentially expressed in skeletal muscle. We found that several genes are differentially expressed. Of these genes, ARPP-16/19 (cAMP-regulated phosphoprotein 16/19) is selectively enhanced after denervation. The expression of ARPP-16/19 in denervated muscles starts to increase from two days after denervation surgery. On the other hand, the expression of ARPP-16/19 does not change in hind-limb suspended muscles, such as EDL and soleus muscles. These results suggest that the increase in ARPP-16/19 mRNA expression is regulated by unknown factor(s) secreted from nerves, and not by electrical muscle activity.     

Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.     

Expression of perilipins in human skeletal muscle in vitro and in vivo in relation to diet, exercise and energy balance

The perilipin proteins enclose intracellular lipid droplets. We describe the mRNA expression of the five perilipins in human skeletal muscle in relation to fatty acid supply, exercise and energy balance. We observed that all perilipins were expressed in skeletal muscle biopsies with the highest mRNA levels of perilipin 2, 4 and 5. Cultured myotubes predominantly expressed perilipin 2 and 3. In vitro, incubation of myotubes with fatty acids enhanced mRNA expression of perilipin 1, 2 and 4. In vivo, low fat diet increased mRNA levels of perilipin 3 and 4. Endurance training, but not strength training, enhanced the expression of perilipin 2 and 3. Perilipin 1 mRNA correlated positively with body fat mass, whereas none of the perilipins were associated with insulin sensitivity. In conclusion, all perilipins mRNAs were expressed in human skeletal muscle. Diet as well as endurance exercise modulated the expression of perilipins.     

cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

eIF3-f function in skeletal muscles: To stand at the crossroads of atrophy and hypertrophy

The control of muscle cell size is a physiological process balanced by a fine tuning between protein synthesis and protein degradation. MAFbx/Atrogin-1 is a muscle specific E3 ubiquitin ligase up regulated during disuse, immobilization, and fasting or systemic diseases such as diabetes, cancer, SIDA and renal failure. This response is necessary to induce a rapid and functional atrophy. To date, the targets of MAFbx/Atrogin-1 in skeletal muscle remain to be identified. We have recently presented evidence that eIF3-f, a regulatory subunit of the eukaryotic translation factor eIF3 is a key target that accounts for MAFbx/Atrogin-1 function in muscle atrophy. More importantly, we showed that eIF3-f act as a “translational enhancer” that increases the efficiency of the structural muscle proteins synthesis leading to both in vitro and in vivo muscle hypertrophy. We propose that eIF3-f subunit, a mTOR/S6K1 scaffolding protein in the IGF-1/Akt/mTOR dependant control of protein translation, is a positive actor essential to the translation of specific mRNAs probably implicated in the muscle hypertrophy. The central role of eIF3-f in both the atrophic and hypertrophic pathways will be discussed in the light of its promising potential in muscle wasting therapy.     

Sepsis downregulates myostatin mRNA levels without altering myostatin protein levels in skeletal muscle

Myostatin is a negative regulator of muscle mass and has been reported to be upregulated in several conditions characterized by muscle atrophy. The influence of sepsis on myostatin expression and activity is poorly understood. Here, we tested the hypothesis that sepsis upregulates the expression and downstream signaling of myostatin in skeletal muscle. Because sepsis‐induced muscle wasting is at least in part regulated by glucocorticoids, we also determined the influence of glucocorticoids on myostatin expression. Sepsis was induced in rats by cecal ligation and puncture and control rats were sham‐operated. In other experiments, rats were injected intraperitoneally with dexamethasone (10 mg/kg) or corresponding volume of vehicle. Surprisingly, myostatin mRNA levels were reduced and myostatin protein levels were unchanged in muscles from septic rats. Muscle levels of activin A, follistatin, and total and phosphorylated Smad2 (p‐Smad2) were not influenced by sepsis, suggesting that myostatin downstream signaling was not altered during sepsis. Interestingly, total and p‐Smad3 levels were increased in septic muscle, possibly reflecting altered signaling through pathways other than myostatin. Similar to sepsis, treatment of rats with dexamethasone reduced myostatin mRNA levels and did not alter myostatin protein levels. Fasting, an additional condition characterized by muscle wasting, reduced myostatin mRNA and activin A protein levels, increased myostatin protein, and did not influence follistatin and p‐Smad2 levels. Of note, total and p‐Smad3 levels were reduced in muscle during fasting. The results suggest that sepsis and glucocorticoids do not upregulate the expression and activity of myostatin in skeletal muscle. The role of myostatin may vary between different conditions characterized by muscle wasting. Downstream signaling through Smad2 and 3 is probably regulated not only by myostatin but by other mechanisms as well. J. Cell. Biochem. 111: 1059–1073, 2010. © 2010 Wiley‐Liss, Inc.     

USP19 is a ubiquitin-specific protease regulated in rat skeletal muscle during catabolic states

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.     

mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.     

Interleukin-15 responses to aging and unloading-induced skeletal muscle atrophy

Interleukin-15 (IL-15) mRNA is constitutively expressed in skeletal muscle. Although IL-15 has proposed hypertrophic and anti-apoptotic roles in vitro, its role in skeletal muscle cells in vivo is less clear. The purpose of this study was to determine if skeletal muscle aging and unloading, two conditions known to promote muscle atrophy, would alter basal IL-15 expression in skeletal muscle. We hypothesized that IL-15 mRNA expression would increase as a result of both aging and muscle unloading and that muscle would express the mRNA for a functional trimeric IL-15 receptor (IL-15R). Two models of unloading were used in this study: hindlimb suspension (HS) in rats and wing unloading in quail. The absolute muscle wet weight of plantaris and soleus muscles from aged rats was significantly less when compared with muscles from young adult rats. Although 14 days of HS resulted in reduced muscle mass of plantaris and soleus muscles from young adult animals, this effect was not observed in muscles from aged animals. A significant aging times unloading interaction was observed for IL-15 mRNA in both rat soleus and plantaris muscles. Patagialis (PAT) muscles from aged quail retained a significant 12 and 6% of stretch-induced hypertrophy after 7 and 14 days of unloading, respectively. PAT muscles from young quail retained 15% hypertrophy at 7 days of unloading but regressed to control levels following 14 days of unloading. A main effect of age was observed on IL-15 mRNA expression in PAT muscles at 14 days of overload, 7 days of unloading, and 14 days of unloading. Skeletal muscle also expressed the mRNAs for a functional IL-15R composed of IL-15R, IL-2/15R-, and -c. Based on these data, we speculate that increases in IL-15 mRNA in response to atrophic stimuli may be an attempt to counteract muscle mass loss in skeletal muscles of old animals. Additional research is warranted to determine the importance of the IL-15/IL-15R system to counter muscle wasting. atrophy; interleukins; sarcopenia; gene signaling     

Myogenin and class II HDACs control neurogenic muscle atrophy by inducing E3 ubiquitin ligases

Maintenance of skeletal muscle structure and function requires innervation by motor neurons, such that denervation causes muscle atrophy. We show that myogenin, an essential regulator of muscle development, controls neurogenic atrophy. Myogenin is upregulated in skeletal muscle following denervation and regulates expression of the E3 ubiquitin ligases MuRF1 and atrogin-1, which promote muscle proteolysis and atrophy. Deletion of myogenin from adult mice diminishes expression of MuRF1 and atrogin-1 in denervated muscle and confers resistance to atrophy. Mice lacking histone deacetylases (HDACs) 4 and 5 in skeletal muscle fail to upregulate myogenin and also preserve muscle mass following denervation. Conversely, forced expression of myogenin in skeletal muscle of HDAC mutant mice restores muscle atrophy following denervation. Thus, myogenin plays a dual role as both a regulator of muscle development and an inducer of neurogenic atrophy. These findings reveal a specific pathway for muscle wasting and potential therapeutic targets for this disorder.