Inhibition of myostatin protects against diet-induced obesity by enhancing fatty acid oxidation and promoting a brown adipose phenotype in mice.

AIMS/HYPOTHESIS: Although myostatin-null (Mstn (-/-)) mice fail to accumulate fat in adipose tissue when fed a high-fat diet (HFD), little is known about the molecular mechanism(s) behind this phenomenon. We therefore sought to identify the signalling pathways through which myostatin regulates accumulation and/or utilisation of fat. METHODS: Wild-type, Mstn (-/-) and wild-type mice treated with soluble activin type IIB receptor (sActRIIB) were fed a control chow diet or an HFD for 12 weeks. Changes in gene expression were measured by microarray and quantitative PCR. Histological changes in white adipose tissue were assessed together with peripheral tissue fatty acid oxidation and changes in circulating hormones following HFD feeding. RESULTS: Our results demonstrate that inactivation of myostatin results in reduced fat accumulation in mice on an HFD. Molecular analysis revealed that metabolic benefits, due to lack of myostatin, are mediated through at least two independent mechanisms. First, lack of myostatin increased fatty acid oxidation in peripheral tissues through induction of enzymes involved in lipolysis and in fatty acid oxidation in mitochondria. Second, inactivation of myostatin also enhanced brown adipose formation in white adipose tissue of Mstn (-/-) mice. Consistent with the above, treatment of HFD-fed wild-type mice with the myostatin antagonist, sActRIIB, reduced the obesity phenotype. CONCLUSIONS/INTERPRETATION: We conclude that absence of myostatin results in enhanced peripheral tissue fatty acid oxidation and increased thermogenesis, culminating in increased fat utilisation and reduced adipose tissue mass. Taken together, our data suggest that anti-myostatin therapeutics could be beneficial in alleviating obesity.

Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway.

AIMS/HYPOTHESIS: Myostatin-null mice (Mstn(-/-)) have reduced body fat and increased tolerance to glucose. To date the molecular mechanisms through which myostatin regulates body fat content and insulin sensitivity are not known. Therefore, the aim of the current study was to identify signalling pathways through which myostatin regulates insulin sensitivity. METHODS: Wild-type (WT) mice and Mstn(-/-) mice were fed either a control chow diet or a high fat diet (HFD) for 12 weeks. Glucose tolerance testing and insulin stimulated glucose uptake by M. extensor digitorum longus (EDL) were used as variables to determine insulin sensitivity. Quantitative PCR, Western blotting and enzyme assays were used to monitor AMP-activated protein kinase (AMPK) levels and activity. RESULTS: Mstn(-/-) mice exhibited reduced fat accumulation and peripheral insulin resistance when compared with WT mice, even when they were fed an HFD. Furthermore, treatment with a myostatin antagonist also increased insulin sensitivity during HFD. Consistent with increased insulin sensitivity, we also detected elevated levels of GLUT4, AKT, p-AKT and insulin receptor substrate-1 in Mstn(-/-) muscles. Molecular analysis showed that there is increased expression and activity of AMPK in Mstn(-/-) muscles. Furthermore, we also observed an increase in the AMPK downstream target genes, Sirt1 and Pgc-1α (also known as Ppargc1a), in skeletal muscle of Mstn(-/-) mice. CONCLUSIONS/INTERPRETATION: We conclude that myostatin inactivation leads to increased AMPK levels and activity resulting in increased insulin sensitivity of skeletal muscle. We propose that, by regulating AMPK in skeletal muscle and adipose tissues, myostatin plays a major role in regulating insulin signalling.

Prolonged underfeeding of sheep increases myostatin and myogenic regulatory factor Myf-5 in skeletal muscle while IGF-I and myogenin are repressed.

The IGF axis is nutritionally sensitive in vivo and IGFs stimulate myoblast proliferation and differentiation in vitro, while myostatin inhibits these processes in vitro. We hypothesised that underfeeding would reversibly inhibit the myogenic activity of satellite cells in vivo together with decreased IGF-I and increased myostatin in muscle. Satellite cell activity was measured indirectly from the expression of proliferating cell nuclear antigen (PCNA) and the myogenic regulatory factors (MRFs), MyoD, Myf-5 and myogenin. Young sheep were underfed (30% of maintenance) and some killed after 1, 4, 12, 17, 21 and 22 weeks. Remaining underfed animals were then re-fed a control ration of pellets and killed after 2 days, and 1, 6 and 30 weeks. Expression of PCNA and MRFs decreased during the first week of underfeeding. This coincided with reduced IGF-I and myostatin mRNA, and processed myostatin. Subsequently, Myf-5, MyoD, myostatin mRNA and processed myostatin increased, suggesting that satellite cells may have become progressively quiescent. Long-term underfeeding caused muscle necrosis in some animals and IGF-I and MRF expression was increased in these, indicating the activation of satellite cells for muscle repair. Re-feeding initiated rapid muscle growth and increased expression of PCNA, IGF-I and the MRFs concurrently with decreased myostatin proteins. In conclusion, these data indicate that IGF-I and myostatin may work in a coordinated manner to regulate the proliferation, differentiation and quiescence of satellite cells in vivo.

Growth factors controlling muscle development.

The enlarged muscles of certain breeds of cattle, such as the Belgian Blue, have been shown to result from a marked increase in the number of normal sized muscle fibers. Originally insulin-like growth factors (IGFs) were implicated in this myofiber hyperplasia, as IGFs have been shown to stimulate myoblast proliferation as well as maintain fiber differentiation. Recently it has been reported that mice lacking a myostatin gene, a member of the TGFbeta superfamily, have enhanced skeletal mass resulting from increased muscle fiber number and size. Mutations in this gene have been found in double-muscled cattle, indicating that myostatin is an inhibitor of muscle growth. Myostatin is expressed early in gestation and then maintained to adulthood in certain muscles. Myostatin expression in bovine muscle is highest during gestation when muscle fibers are forming and some of the myogenic regulatory factors have elevated expression over the same period as myostatin. Molecular expression of the IGF axis does not differ between Belgian Blue and normal muscled cattle, and IGF-II mRNA is increased throughout formation of secondary fibers in both breeds. However, myostatin and MyoD expression in muscle differ between normal and hypertrophied muscle cattle breeds. This evidence strongly suggests that lack of myostatin is associated with an increase in fiber number which then results in a marked increase in potential muscle mass in double-muscled cattle.

Actions of short-term fasting on human skeletal muscle myogenic and atrogenic gene expression.

BACKGROUND: Skeletal muscle mass is governed by multiple IGF-1-sensitive positive regulators of muscle-specific protein synthesis (myogenic regulatory factors which includes myoD, myogenin and Myf5) and negative regulators, including the atrogenic proteins myostatin, atrogin-1 and muscle ring finger 1 (MuRF-1). The coordinated control of these myogenic and atrogenic factors in human skeletal muscle following short-term fasting is currently unknown. METHOD: Healthy adults (n = 6, age 27.6 years) undertook a 40-hour fast. Skeletal muscle biopsy (vastus lateralis) and venous blood samples were taken 3, 15 and 40 h into the fast after an initial standard high-carbohydrate meal. Gene expression of the myogenic regulator factors (myoD, myogenin and Myf5) and the atrogenic factors (myostatin, atrogin-1 and MuRF-1) were determined by real-time PCR analysis. Plasma myostatin and IGF-1 were determined by ELISA. RESULTS: There were no significant alterations in either the positive or negative regulators of muscle mass at either 15 or 40 h, when compared to gene expression measured 3 h after a meal. Similarly, plasma myostatin and IGF-1 were also unaltered at these times. CONCLUSIONS: Unlike previous observations in catabolic and cachexic diseased states, short-term fasting (40 h) fails to elicit marked alteration of the genes regulating both muscle-specific protein synthesis or atrophy. Greater periods of fasting may be required to initiate coordinated inhibition of myogenic and atrogenic gene expression.

Cloned yeast and mammalian transcription factor TFIID gene products support basal but not activated metallothionein gene transcription.

Transcription factor IID (TFIID), the "TATA binding factor," is thought to play a key role in the regulation of eukaryotic transcriptional initiation. We have studied the role of TFIID in the transcription of the yeast metallothionein gene, which is regulated by the copper-dependent activator protein ACE1. Both basal and induced transcription of the metallothionein gene require TFIID and a functional TATA binding site. Crude human and mouse TFIID fractions, prepared from mammalian cells, respond to stimulation by ACE1. In contrast, human and yeast TFIID proteins expressed from the cloned genes do not respond to ACE1, except in the presence of wheat germ or yeast total cell extracts. These results indicate that the cloned TFIID gene products lack a component(s) or modification(s) that is required for regulated as compared to basal transcription.

Effect of protein calorie malnutrition and calcium deficiency on hepatic iso-metallothioneins in Cd-exposed monkeys.

Oral administration of cadmium results in the induction of metallothionein in liver of rhesus monkeys subjected to protein calorie malnutrition and calcium deficiency. Metallothionein was resolved into three iso-proteins viz. MTa, MTb & MTc which varied in their quantities and metal composition in these nutritional stress conditions. 'MTc' was the major iso-protein in protein calorie malnourished monkeys, while 'MTb' predominated in the calcium deficient group.

Role of metallothionein in metal detoxification and metal tolerance in protein calorie malnutrition and calcium deficient monkeys (Macaca mulatta).

A monkey model has been set up for protein calorie malnutrition and calcium deficiency. Oral exposure of 5ppm Cd/kg body wt./day for 24 weeks led to increased excretion of Cd, metallothionein (MT) and zinc. Rehabilitation of PCM monkeys for one year resulted in gradual reduction and finally complete disappearance of urinary metallothionein. During Cd exposure, the accumulation of Cd and induction of MT was significantly higher in liver, kidney and intestine. MT was also induced in heart, lung and testis of Cd exposed PCM and calcium deficient monkeys. Metallothionein from liver has been resolved into three isoforms, viz MTa, MTb and MTc on DEAE-Sephadex A 25 ion exchange column. MTc is the major isoform in Cd-treated, normal and protein calorie malnourished monkeys whereas MTb is the major isoprotein in the cadmium treated calcium deficient monkeys. The iso-metallothioneins varied in their metal composition in the nutritional stress conditions and showed different capacities to reactivate apo-enzymes viz. alkaline phosphatase, ceruloplasmin, superoxide dismutase and glutathione peroxidase. Thus, metallothionein plays a key role in metal metabolism during cadmium toxicity under nutritional stress conditions.

Myostatin in muscle growth and repair.

Myostatin, a member of the TGF beta superfamily, regulates skeletal muscle size by controlling embryonic myoblast proliferation. Recent results show that myostatin may also have a role in muscle regeneration and muscle wasting of adult animals. This review summarizes the recent developments in the regulation of myostatin gene expression and mechanism of its function.

Immunocytochemical studies with antibodies to three proteins prominent in the isolated microtubule cytoskeleton of a trypanosomatid.

The cytoskeleton of Crithidia fasciculata consists of a corset of parallel microtubules enclosing the cell body and closely underlying the plasma membrane. Distinct sets of crosslinks appear to connect tubules to each other and to membrane. Our objective is to determine the composition of these crosslinks and to elucidate the basis of this spectacular example of membrane-microtubule interaction. We purified three proteins (designated COP-33, -41, and -61 by their subunit Mr), which were consistently abundant in highly purified cytoskeletons. All three bound strongly to microtubules in vitro, and the first two induced bundles through periodic crosslinking. Polyclonal antibodies against each have been used to try to localize these proteins in thin sections of cells or whole mounts of cytoskeletons. Antibodies to COP-41 bound specifically to glycosomes, organelles that encapsulate many glycolytic enzymes in these protozoa, and COP-41 has been identified as glyceraldehyde 3-P dehydrogenase.

Characterization of putative cytoskeletal proteins from a trypanosomatid and their comparative binding to microtubules and soluble tubulin.

The corset of microtubules which encloses the cell body of Crithidia fasiculata displays cross-links joining tubules to each other and to plasma membrane. Two proteins, designated COP-33 and COP-61 on the basis of their subunit Mr values, have been considered putative components of this apparatus because of their abundance in isolated cytoskeleton and their ability to cross-link brain microtubules in vitro. The oligomeric structures of the native proteins have now been characterized, and they have been shown to be basic, rather symmetrical, and to require detergent for solubilization. Using monospecific antibodies, enzyme-linked immunoassays of subcellular fractions have shown that each is 5-fold enriched in the microtubule plasma membrane fraction and absent from flagellar and some internal membranes. The binding of each to soluble tubulin and to microtubules has been systematically studied and compared with that of two noncytoskeletal protein ligands (glycolytic enzymes). The observed positive cooperatively was unexpected for binding of these large ligands to microtubules. In each case the maximum number of mol of ligand bound per mol of tubulin (0.5-1.0) was identical whether the latter was dissociated or assembled and so were the dissociation constants (1.3-6.0 x 10(-7) M) for three of the ligand proteins.

Molecular aspects, physiological function, and clinical significance of metallothioneins.

Metallothioneins (MTs) are well-characterized low molecular weight, heat-stable cytosolic proteins with exceptional high content of cysteinyl sulfur and are known to bind heavy metals like cadmium (Cd), zinc (Zn), and copper (Cu). Since these proteins are induced on exposure to heavy metals, it is now accepted that they have a detoxifying role during heavy metal toxicity. It has also been suggested that the primary function of Mt is in the homeostasis of the essential metals Zn and Cu. Recently, a role MT in selenium metabolism in primates has been established. Further, MT has gained considerable importance in the clinical disorders related to trace metal metabolism.

Antiviral determinants of rat Mx GTPases map to the carboxy-terminal half.

Rat Mx2 and rat Mx3 are two alpha/beta interferon-inducible cytoplasmic GTPases that differ in three residues in the amino-terminal third, which also contains the tripartite GTP-binding domain, and that differ in five residues in the carboxy-terminal quarter, which also contains a dimerization domain. While Mx2 is active against vesicular stomatitis virus (VSV), Mx3 lacks antiviral activity. We mapped the functional difference between Mx2 and Mx3 protein to two critical residues in the carboxy-terminal parts of the molecules. An exchange of either residue 588 or 630 of Mx2 with the corresponding residues of Mx3 abolished anti-VSV activity, and the introduction of the two Mx2 residues on an Mx3 background partially restored anti-VSV activity. These results are consistent with the facts that Mx2 and Mx3 have similar intrinsic GTPase activities and that the GTPase domain of Mx3 can fully substitute for the GTPase domain of Mx2. Nevertheless, the amino-terminal third containing the GTP-binding domain is necessary for antiviral activity, since an amino-terminally truncated Mx2 protein is devoid of anti-VSV activity. Furthermore, Fab fragments of a monoclonal antibody known to neutralize antiviral activity block GTPase activity by binding an epitope in the carboxy-terminal half of Mx2 or Mx3 protein. The results are consistent with a two-domain model in which both the conserved amino-terminal half and the less-well-conserved carboxy-terminal half of Mx proteins carry functionally important domains.

Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle.

A visibly distinct muscular hypertrophy (mh), commonly known as double muscling, occurs with high frequency in the Belgian Blue and Piedmontese cattle breeds. The autosomal recessive mh locus causing double-muscling condition in these cattle maps to bovine chromosome 2 within the same interval as myostatin, a member of the TGF-beta superfamily of genes. Because targeted disruption of myostatin in mice results in a muscular phenotype very similar to that seen in double-muscled cattle, we have evaluated this gene as a candidate gene for double-muscling condition by cloning the bovine myostatin cDNA and examining the expression pattern and sequence of the gene in normal and double-muscled cattle. The analysis demonstrates that the levels and timing of expression do not appear to differ between Belgian Blue and normal animals, as both classes show expression initiating during fetal development and being maintained in adult muscle. Moreover, sequence analysis reveals mutations in heavy-muscled cattle of both breeds. Belgian Blue cattle are homozygous for an 11-bp deletion in the coding region that is not detected in cDNA of any normal animals examined. This deletion results in a frame-shift mutation that removes the portion of the Myostatin protein that is most highly conserved among TGF-beta family members and that is the portion targeted for disruption in the mouse study. Piedmontese animals tested have a G-A transition in the same region that changes a cysteine residue to a tyrosine. This mutation alters one of the residues that are hallmarks of the TGF-beta family and are highly conserved during evolution and among members of the gene family. It therefore appears likely that the mh allele in these breeds involves mutation within the myostatin gene and that myostatin is a negative regulator of muscle growth in cattle as well as mice.

NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation.

The cloned epithelial cell-specific Na+/H+ exchanger (NHE) isoform NHE2 is stimulated by fibroblast growth factor (FGF), phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), and fetal bovine serum (FBS) through a change in maximal velocity of the transporter. In the present study, we used COOH-terminal truncation mutants to delineate specific domains in the COOH terminus of NHE2 that are responsible for growth factor and/or protein kinase regulation. Five truncation mutants (designated by the amino acid number at the truncation site) were stably expressed in NHE-deficient PS120 fibroblasts. The effects of PMA, FGF, OA, FBS, and W-13 [a Ca2+/calmodulin (CaM) inhibitor] were studied. Truncation mutant E2/660, but not E2/573, was stimulated by PMA. OA stimulated E2/573 but not E2/540. FGF stimulated E2/540 but not E2/499. The most truncated mutant, E2/499, was stimulated by FBS. W-13 stimulated the basal activity of the wild-type NHE2. However, W-13 had no effect on E2/755. By monitoring the emission spectra of dansylated CaM fluorescence, we showed that dansylated CaM bound directly to a purified fusion protein of glutathione S-transferase and the last 87 amino acids of NHE2 in a Ca2+-dependent manner, with a stoichiometry of 1:1 and a dissociation constant of 300 nM. Our results showed that the COOH terminus of NHE2 is organized into separate stimulatory and inhibitory growth factor/protein kinase regulatory subdomains. This organization of growth factor/protein kinase regulatory subdomains is very similar to that of NHE3, suggesting that the tertiary structures of the putative COOH termini of NHE2 and NHE3 are very similar despite the minimal amino acid identity in this part of the two proteins.

Genomic organization and neonatal expression of the bovine myostatin gene.

Myostatin belongs to the Transforming Growth Factor-beta (TGF-beta) superfamily and is expressed in developing and mature skeletal muscle. Biologically, the role of myostatin seems to be extremely well conserved during evolution since inactivating mutations in myostatin gene cause similar phenotype of heavy muscling in both mice and cattle. In this report we have analysed the genomic structure and neonatal expression of the bovine myostatin gene. The molecular analysis shows that the bovine myostatin gene consists of three exons and two introns. The sizes of the first and second exons are 506 and 374 base pairs (bp) respectively. The size of the third exon was found to be variable in length (1701 or 1812 or 1887 nucleotides), whereas the size of the two introns is 1840 and 2033 bps. In the first exon of bovine myostatin, a single transcription initiation site is found at 133 bps from the translation start codon ATG. Sequencing the 3' untranslated region indicated that there are multiple polyadenylation signals at 1301, 1401 and 1477 bp downstream from the translation stop codon (TGA). Furthermore, 3' RACE analysis confirmed that all three polyadenylation sites are used in vivo. Using quantitative RT-PCR we have analysed neonatal expression of myostatin gene. In both the M. biceps femoris and M. semitendinosus, the highest level of myostatin expression was observed on day 1 postnatally, then gradually reduced on days 8 and 14 postnatally. In contrast, in the M. gastrocnemius, myostatin expression was highest on day 14 and lowest on day 8. These results indicate that myostatin gene structure and function is well conserved during evolution and that neonatal expression of myostatin in a number of predominantly fast twitch muscles is differentially regulated.

Myostatin regulation during skeletal muscle regeneration.

Myostatin, a member of the TGF-beta superfamily, is a key negative regulator of skeletal muscle growth. The role of myostatin during skeletal muscle regeneration has not previously been reported. In the present studies, normal Sprague-Dawley and growth hormone (GH)-deficient (dw/dw) rats were administered the myotoxin, notexin, in the right M. biceps femoris on day 0. The dw/dw rats then received either saline or human-N-methionyl GH (200microg/100g body weight/day) during the ensuing regeneration. Normal and dw/dw M. biceps femoris were dissected on days 1, 2, 3, 5, 9 and 13, formalin-fixed, then immunostained for myostatin protein. Immunostaining for myostatin revealed high levels of protein within necrotic fibres and connective tissue of normal and dw/dw damaged muscles. Regenerating myotubes contained no myostatin at the time of fusion (peak fusion on day 5), and only low levels of myostatin were observed during subsequent myotube enlargement. Fibres which survived assault by notexin (survivor fibres) contained moderate to high myostatin immunostaining initially. The levels in both normal and dw/dw rat survivor fibres decreased on days 2-3, then increased on days 9-13. In dw/dw rats, there was no observed effect of GH administration on the levels of myostatin protein in damaged muscle. The low level of myostatin observed in regenerating myotubes in these studies suggests a negative regulatory role for myostatin in muscle regeneration.