Buel D. Rodgers

Isolation and characterization of myostatin complementary deoxyribonucleic acid clones from two commercially important fish: Oreochromis mossambicus and Morone chrysops.

In mammals, skeletal muscle mass is negatively regulated by a muscle-derived growth/differentiating factor named myostatin (MSTN) that belongs to the transforming growth factor-β superfamily. Although putative MSTN homologs have been identified from several vertebrates, nonmammalian orthologs remained poorly defined. Thus, we isolated and characterized MSTN complementary DNA clones from the skeletal muscle of the tilapia Oreochromis mossambicus and the white bass Morone chrysops. The nucleic and amino acid sequences from both fish species are highly homologous to the previously identified mammalian and avian orthologs, and both possess conserved cysteine residues and putative RXXR proteolytic processing sites that are common to all transforming growth factor-β family members. Western blotting of conditioned medium from human embryonal kidney (HEK293) cells overexpressing a His-tagged tilapia MSTN indicates that the secreted fish protein is processed in a manner similar to mouse MSTN. However, in contrast ...

Resveratrol induces brown-like adipocyte formation in white fat through activation of AMP-activated protein kinase (AMPK) α1

Objective:Development of brown-like/beige adipocytes in white adipose tissue (WAT) helps to reduce obesity. Thus we investigated the effects of resveratrol, a dietary polyphenol capable of preventing obesity and related complications in humans and animal models, on brown-like adipocyte formation in inguinal WAT (iWAT).Methods:CD1 female mice (5-month old) were fed a high-fat diet with/without 0.1% resveratrol. In addition, primary stromal vascular cells separated from iWAT were subjected to resveratrol treatment. Markers of brown-like (beige) adipogenesis were measured and the involvement of AMP-activated protein kinase (AMPK) α1 was assessed using conditional knockout.Results:Resveratrol significantly increased mRNA and/or protein expression of brown adipocyte markers, including uncoupling protein 1 (UCP1), PR domain-containing 16, cell death-inducing DFFA-like effector A, elongation of very long-chain fatty acids protein 3, peroxisome proliferator-activated receptor-γ coactivator 1α, cytochrome c and pyruvate dehydrogenase, in differentiated iWAT stromal vascular cells (SVCs), suggesting that resveratrol induced brown-like adipocyte formation in vitro. Concomitantly, resveratrol markedly enhanced AMPKα1 phosphorylation and differentiated SVC oxygen consumption. Such changes were absent in cells lacking AMPKα1, showing that AMPKα1 is a critical mediator of resveratrol action. Resveratrol also induced beige adipogenesis in vivo along with the appearance of multiocular adipocytes, increased UCP1 expression and enhanced fatty acid oxidation.Conclusions:Resveratrol induces brown-like adipocyte formation in iWAT via AMPKα1 activation and suggest that its beneficial antiobesity effects may be partly due to the browning of WAT and, as a consequence, increased oxygen consumption.

Phylogenetic analysis of the myostatin gene sub-family and the differential expression of a novel member in zebrafish

Summary The myostatin (MSTN)‐null phenotype in mammals is characterized by extreme gains in skeletal muscle mass or “double muscling” as the cytokine negatively regulates skeletal muscle growth. Recent attempts, however, to reproduce a comparable phenotype in zebrafish have failed. Several aspects of MSTN biology in the fishes differ significantly from those in mammals and at least two distinct paralogs have been identified in some species, which possibly suggests functional divergence between the different vertebrate classes or between fish paralogs. We therefore conducted a phylogenetic analysis of the entire MSTN gene sub‐family. Maximum likelihood, Bayesian inference, and bootstrap analyses indicated a monophyletic distribution of all MSTN genes with two distinct fish clades: MSTN‐1 and ‐2. These analyses further indicated that all Salmonid genes described are actually MSTN‐1 orthologs and that additional MSTN‐2 paralogs may be present in most, if not all, teleosts. An additional zebrafish homolog was identified by BLAST searches of the zebrafish Hierarchical Tets Generation System database and was subsequently cloned. Comparative sequence analysis of both genes (zebrafish MSTN (zfMSTN)‐1 and ‐2) revealed many differences, primarily within the latency‐associated peptide regions, but also within the bioactive domains. The 2‐kb promoter region of zfMSTN‐2 contained many putative cis regulatory elements that are active during myogenesis, but are lacking in the zfMSTN‐1 promoter. In fact, zfMSTN‐2 expression was limited to the early stages of somitogenesis, whereas zfMSTN‐1 was expressed throughout embryogenesis. These data suggest that zfMSTN‐2 may be more closely associated with skeletal muscle growth and development. They also resolve the previous ambiguity in classification of fish MSTN genes.

Myostatin represses physiological hypertrophy of the heart and excitation–contraction coupling

Although myostatin negatively regulates skeletal muscle growth, its function in heart is virtually unknown. Herein we demonstrate that it inhibits basal and IGF‐stimulated proliferation and differentiation and also modulates cardiac excitation–contraction (EC) coupling. Loss of myostatin induced eccentric hypertrophy and enhanced cardiac responsiveness to β‐adrenergic stimulation in vivo. This was due to myostatin null ventricular myocytes having larger [Ca2+]i transients and contractions and responding more strongly to β‐adrenergic stimulation than wild‐type cells. Enhanced cardiac output and β‐adrenergic responsiveness of myostatin null mice was therefore due to increased SR Ca2+ release during EC coupling and to physiological hypertrophy, but not to enhanced myofilament function as determined by simultaneous measurement of force and ATPase activity. Our studies support the novel concept that myostatin is a repressor of physiological cardiac muscle growth and function. Thus, the controlled inhibition of myostatin action could potentially help repair damaged cardiac muscle by inducing physiological hypertrophy.

Reduced Circulating GDF11 Is Unlikely Responsible for Age-Dependent Changes in Mouse Heart, Muscle, and Brain

Recent high-profile studies report conflicting data on the age-related change in circulating growth/differentiation factor 11 (GDF11) and myostatin as well as the formers influence on muscle regeneration. Both ligands bind and activate ActRIIB receptors with similar affinities and should therefore have similar actions, yet these studies suggest that GDF11 activates muscle regeneration whereas myostatin is well known to inhibit it. They also suggest that circulating GDF11 levels, but not those of myostatin, decline with age. We performed a careful assessment of the ELISA used to quantify circulating myostatin in these studies and determined that assay reagents significantly cross react with each protein, each of which is highly homologous. Circulating myostatin levels decreased with age and estimates of GDF11 levels using myostatin null mice indicate that they were almost 500 times lower than those for myostatin. This suggests that circulating GDF11 has little physiological relevance as it could not outcompete myostatin for ActRIIB binding sites. Together, these results further suggest that the previously reported aging muscle, heart, and brain phenotypes attributed to reduced circulating GDF11 should be reconsidered.

Myostatin inhibits myosatellite cell proliferation and consequently activates differentiation: evidence for endocrine-regulated transcript processing.

Myostatin is a potent negative regulator of muscle growth in mammals. Despite high structural conservation, functional conservation in nonmammalian species is only assumed. This is particularly true for fish due to the presence of several myostatin paralogs: two in most species and four in salmonids (MSTN-1a, -1b, -2a, and -2b). Rainbow trout are a rich source of primary myosatellite cells as hyperplastic muscle growth occurs even in adult fish. These cells were therefore used to determine myostatins effects on proliferation whereas our earlier studies reported its effects on quiescent cells. As in mammals, recombinant myostatin suppressed proliferation with no changes in cell morphology. Expression of MSTN-1a was several fold higher than the other paralogs and was autoregulated by myostatin, which also upregulated the expression of key differentiation markers: Myf5, MyoD1, myogenin, and myosin light chain. Thus, myostatin-stimulated cellular growth inhibition activates rather than represses differentiation. IGF-1 stimulated proliferation but had minimal and delayed effects on differentiation and its actions were suppressed by myostatin. However, IGF-1 upregulated MSTN-2a expression and the processing of its transcript, which is normally unprocessed. Myostatin therefore appears to partly mediate IGF-stimulated myosatellite differentiation in rainbow trout. This also occurs in mammals, although the IGF-stimulated processing of MSTN-2a transcripts is highly unique and is indicative of subfunctionalization within the gene family. These studies also suggest that the myokines actions, including its antagonistic relationship with IGF-1, are conserved and that the salmonid gene family is functionally diverging.

AMPK/α-Ketoglutarate Axis Dynamically Mediates DNA Demethylation in the Prdm16 Promoter and Brown Adipogenesis

Promoting brown adipose tissue (BAT) development is an attractive strategy for the treatment of obesity, as activated BAT dissipates energy through thermogenesis; however, the mechanisms controlling BAT formation are not fully understood. We hypothesized that as a master regulator of energy metabolism, AMP-activated protein kinase (AMPK) may play a direct role in the process and found that AMPKα1 (PRKAA1) ablation reduced Prdm16 expression and impaired BAT development. During early brown adipogenesis, the cellular levels of α-ketoglutarate (αKG), a key metabolite required for TET-mediated DNA demethylation, were profoundly increased and required for active DNA demethylation of the Prdm16 promoter. AMPKα1 ablation reduced isocitrate dehydrogenase 2 activity and cellular αKG levels. Remarkably, postnatal AMPK activation with AICAR or metformin rescued obesity-induced suppression of brown adipogenesis and thermogenesis. In summary, AMPK is essential for the epigenetic control of BAT development through αKG, thus linking a metabolite to progenitor cell differentiation and thermogenesis.

Myostatin stimulates myosatellite cell differentiation in a novel model system: evidence for gene subfunctionalization

Myosatellite cells play an important role in mammalian muscle regeneration as they differentiate and fuse with mature fibers. In fish, they also contribute to postnatal growth and the formation of new fibers. The relative conservation of fish systems, however, is not well known nor are the underlying mechanisms that control myosatellite cell differentiation. We therefore characterized this process in primary cells from rainbow trout and determined the effects of two known regulators in mammalian systems: IGF-I and myostatin. Unlike mammalian cell lines, subconfluent and proliferating trout myosatellite cells differentiated spontaneously and at a rate proportional to serum concentration. The expression of key myogenic markers (Myf5, MyoD1, myogenin, and MLC) and of the different myostatin paralogs (MSTN-1a/1b/2a) increased with serum-stimulated differentiation, although MSTN-1a expression was consistently higher than that of the other paralogs. In addition, MSTN-2a was only expressed as an unprocessed transcript. In low serum, where differentiation is normally suppressed, recombinant myostatin stimulated myogenic marker expression over time. The opposite was true for IGF-I as it stimulated proliferation, not differentiation, and additionally antagonized myostatin. This includes myostatins effects on marker expression and on the autoregulation of MSTN-1a and -1b expression. These results conflict with studies using mammalian cell lines and suggest, alternatively, that myostatin is a positive, not negative, regulator of myosatellite cell differentiation. Mammalian myoblasts differentiate when confluent and with serum withdrawal, which differs considerably from how myosatellite cells differentiate in vivo. Thus the primary rainbow trout myosatellite cell culture system appears to be more physiologically relevant.

Myostatin stimulates, not inihibits, C2C12 myoblast proliferation.

The immortal C2C12 cell line originates from dystrophic mouse thigh muscle and has been used to study the endocrine control of muscle cell growth, development, and function, including those actions regulated by myostatin. Previous studies suggest that high concentrations of recombinant myostatin generated in bacteria inhibit C2C12 proliferation and differentiation. Recombinant myostatin generated in eukaryotic systems similarly inhibits the proliferation of primary myosatellite cells, but consequently initiates, rather than inhibits, their differentiation and is bioactive at far lower concentrations. Our studies indicate that 2 different sources of recombinant myostatin made in eukaryotes stimulate, not inhibit, C2C12 proliferation. This effect occurred at different cell densities and serum concentrations and in the presence of IGF-I, a potent myoblast mitogen. This stimulatory effect was comparable to that obtained with TGFβ1, a related factor that also inhibits primary myosatellite cell proliferation. Attenuating the myostatin/activin (ie, Acvr2b) and TGFβ1 receptor signaling pathways with the Alk4/5 and Alk5 inhibitors, SB431542 and SB505142, respectively, similarly attenuated proliferation induced by serum, myostatin or TGFβ1 and in a dose-dependent manner. In serum-free medium, both myostatin and TGFβ1 stimulated Smad2 phosphorylation, but not that of Smad3, and a Smad3 inhibitor (SIS3) only inhibited proliferation in cells cultured in high serum. Thus, myostatin and TGFβ1 stimulate C2C12 proliferation primarily via Smad2. These results together question the physiological relevance of the C2C12 model and previous studies using recombinant myostatin generated in bacteria. They also support the alternative use of primary myosatellite cells and recombinant myostatin generated in eukaryotes.

Maternal high-fat diet during lactation impairs thermogenic function of brown adipose tissue in offspring mice

Maternal obesity and high-fat diet (HFD) predisposes offspring to obesity and metabolic diseases. Due to uncoupling, brown adipose tissue (BAT) dissipates energy via heat generation, mitigating obesity and diabetes. The lactation stage is a manageable period for improving the health of offspring of obese mothers, but the impact of maternal HFD during lactation on offspring BAT function is unknown. To determine, female mice were fed either a control or HFD during lactation. At weaning, HFD offspring gained more body weight and had greater body fat mass compared to the control, and these differences maintained into adulthood, which correlated with glucose intolerance and insulin resistance in HFD offspring. Adaptive thermogenesis of BAT was impaired in HFD offspring at weaning. In adulthood, HFD offspring BAT had lower Ucp1 expression and thermogenic activity. Mechanistically, maternal HFD feeding during lactation elevated peripheral serotonin, which decreased the sensitivity of BAT to sympathetic β3-adrenergic signaling. Importantly, early postnatal metformin administration decreased serotonin concentration and ameliorated the impairment of offspring BAT due to maternal HFD. Our data suggest that attenuation of BAT thermogenic function may be a key mechanism linking maternal HFD during lactation to persisted metabolic disorder in the offspring.