Burmese pythons exhibit a transient adaptation to nutrient overload that prevents liver damage.

As an opportunistic predator, the Burmese python (Python molurus bivittatus) consumes large and infrequent meals, fasting for up to a year. Upon consuming a large meal, the Burmese python exhibits extreme metabolic responses. To define the pathways that regulate these postprandial metabolic responses, we performed a comprehensive profile of plasma metabolites throughout the digestive process. Following ingestion of a meal equivalent to 25% of its body mass, plasma lipoproteins and metabolites, such as chylomicra and bile acids, reach levels observed only in mammalian models of extreme dyslipidemia. Here, we provide evidence for an adaptive response to postprandial nutrient overload by the python liver, a critical site of metabolic homeostasis. The python liver undergoes a substantial increase in mass through proliferative processes, exhibits hepatic steatosis, hyperlipidemia-induced insulin resistance indicated by PEPCK activation and pAKT deactivation, and de novo fatty acid synthesis via FASN activation. This postprandial state is completely reversible. We posit that Burmese pythons evade the permanent hepatic damage associated with these metabolic states in mammals using evolved protective measures to inactivate these pathways. These include a transient activation of hepatic nuclear receptors induced by fatty acids and bile acids, including PPAR and FXR, respectively. The stress-induced p38 MAPK pathway is also transiently activated during the early stages of digestion. Taken together, these data identify a reversible metabolic response to hyperlipidemia by the python liver, only achieved in mammals by pharmacologic intervention. The factors involved in these processes may be relevant to or leveraged for remediating human hepatic pathology.

Palmitic Acid Reduces the Autophagic Flux and Insulin Sensitivity Through the Activation of the Free Fatty Acid Receptor 1 (FFAR1) in the Hypothalamic Neuronal Cell Line N43/5.

Chronic consumption of high fat diets (HFDs), rich in saturated fatty acids (SatFAs) like palmitic acid (PA), is associated with the development of obesity and obesity-related metabolic diseases such as type II diabetes mellitus (T2DM). Previous studies indicate that PA accumulates in the hypothalamus following consumption of HFDs; in addition, HFDs consumption inhibits autophagy and reduces insulin sensitivity. Whether malfunction of autophagy specifically in hypothalamic neurons decreases insulin sensitivity remains unknown. PA does activate the Free Fatty Acid Receptor 1 (FFAR1), also known as G protein-coupled receptor 40 (GPR40); however, whether FFAR1 mediates the effects of PA on hypothalamic autophagy and insulin sensitivity has not been shown. Here, we demonstrate that exposure to PA inhibits the autophagic flux and reduces insulin sensitivity in a cellular model of hypothalamic neurons (N43/5 cells). Furthermore, we show that inhibition of autophagy and the autophagic flux reduces insulin sensitivity in hypothalamic neuronal cells. Interestingly, the inhibition of the autophagic flux, and the reduction in insulin sensitivity are prevented by pharmacological inhibition of FFAR1. Our findings show that dysregulation of autophagy reduces insulin sensitivity in hypothalamic neuronal cells. In addition, our data suggest FFAR1 mediates the ability of PA to inhibit autophagic flux and reduce insulin sensitivity in hypothalamic neuronal cells. These results reveal a novel cellular mechanism linking PA-rich diets to decreased insulin sensitivity in the hypothalamus and suggest that hypothalamic autophagy might represent a target for future T2DM therapies.

Transforming growth factor type-ß inhibits Mas receptor expression in fibroblasts but not in myoblasts or differentiated myotubes; Relevance to fibrosis associated to muscular dystrophies.

Duchenne muscular dystrophy is a genetic disorder characterized by myofiber degeneration, muscle weakness, and increased fibrosis. Transforming growth factor type-ß (TGF-ß), a central mediator of fibrosis, is upregulated in fibrotic diseases. Angiotensin-(1-7) [Ang-(1-7)] is a peptide with actions that oppose those of angiotensin-II (Ang II). Ang-(1-7) effects are mediated by the Mas receptor. Treatment with Ang-(1-7) produce positive effects in the mdx mouse, normalizing skeletal muscle architecture, decreasing local fibrosis, and fibroblasts, and improving muscle function. Mdx mice deficient for the Mas receptor showed the opposite effects. To identify the cell type(s) responsible for Mas receptor expression, and to characterize whether profibrotic effectors had any effect on its expression, we determined the effect of profibrotic agents on Mas expression. TGF-ß, but not connective tissue growth factor or Ang-II, reduced the expression of Mas receptor in fibroblasts isolated from skeletal muscle cells and fibroblasts from two established cell lines. In contrast, no effects were observed in myoblasts and differentiated myotubes. This inhibition was mediated by the Smad-dependent (canonical) and the PI3K and MEK1/2 (noncanonical) TGF-ß signaling pathways. When both canonical and noncanonical inhibitors of the TGF-ß-dependent pathways were added together, the inhibitory effect of TGF-ß on Mas expression was lost. The decrease in Mas receptor induced by TGF-ß in fibroblasts reduced the Ang-(1-7) mediated stimulation of phosphorylation of AKT pathway proteins. These results suggest that reduction of Mas receptor in fibroblasts, by TGF-ß, could increase the fibrotic phenotype observed in dystrophic skeletal muscle decreasing the beneficial effect of Ang-(1-7).

Novel and optimized strategies for inducing fibrosis in vivo: focus on Duchenne Muscular Dystrophy.

BACKGROUND: Fibrosis, an excessive collagen accumulation, results in scar formation, impairing function of vital organs and tissues. Fibrosis is a hallmark of muscular dystrophies, including the lethal Duchenne muscular dystrophy (DMD), which remains incurable. Substitution of muscle by fibrotic tissue also complicates gene/cell therapies for DMD. Yet, no optimal models to study muscle fibrosis are available. In the widely used mdx mouse model for DMD, extensive fibrosis develops in the diaphragm only at advanced adulthood, and at about two years of age in the 'easy-to-access' limb muscles, thus precluding fibrosis research and the testing of novel therapies. METHODS: We developed distinct experimental strategies, ranging from chronic exercise to increasing muscle damage on limb muscles of young mdx mice, by myotoxin injection, surgically induced trauma (laceration or denervation) or intramuscular delivery of profibrotic growth factors (such as TGFß). We also extended these approaches to muscle of normal non-dystrophic mice. RESULTS: These strategies resulted in advanced and enhanced muscle fibrosis in young mdx mice, which persisted over time, and correlated with reduced muscle force, thus mimicking the severe DMD phenotype. Furthermore, increased fibrosis was also obtained by combining these procedures in muscles of normal mice, mirroring aberrant repair after severe trauma. CONCLUSIONS: We have developed new and improved experimental strategies to accelerate and enhance muscle fibrosis in vivo. These strategies will allow rapidly assessing fibrosis in the easily accessible limb muscles of young mdx mice, without necessarily having to use old animals. The extension of these fibrogenic regimes to the muscle of non-dystrophic wild-type mice will allow fibrosis assessment in a wide array of pre-existing transgenic mouse lines, which in turn will facilitate understanding the mechanisms of fibrogenesis. These strategies should improve our ability to combat fibrosis-driven dystrophy progression and aberrant regeneration.

ACE2 is augmented in dystrophic skeletal muscle and plays a role in decreasing associated fibrosis.

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease and is characterized by absence of the cytoskeletal protein dystrophin, muscle wasting, and fibrosis. We previously demonstrated that systemic infusion or oral administration of angiotensin-(1-7) (Ang-(1-7)), a peptide with opposing effects to angiotensin II, normalized skeletal muscle architecture, decreased local fibrosis, and improved muscle function in mdx mice, a dystrophic model for DMD. In this study, we investigated the presence, activity, and localization of ACE2, the enzyme responsible for Ang-(1-7) production, in wild type (wt) and mdx skeletal muscle and in a model of induced chronic damage in wt mice. All dystrophic muscles studied showed higher ACE2 activity than wt muscle. Immunolocalization studies indicated that ACE2 was localized mainly at the sarcolemma and, to a lesser extent, associated with interstitial cells. Similar results were observed in the model of chronic damage in the tibialis anterior (TA) muscle. Furthermore, we evaluated the effect of ACE2 overexpression in mdx TA muscle using an adenovirus containing human ACE2 sequence and showed that expression of ACE2 reduced the fibrosis associated with TA dystrophic muscles. Moreover, we observed fewer inflammatory cells infiltrating the mdx muscle. Finally, mdx gastrocnemius muscles from mice infused with Ang-(1-7), which decreases fibrosis, contain less ACE2 associated with the muscle. This is the first evidence supporting ACE2 as an important therapeutic target to improve the dystrophic skeletal muscle phenotype.

Transforming growth factor type beta 1 increases the expression of angiotensin II receptor type 2 by a SMAD- and p38 MAPK-dependent mechanism in skeletal muscle.

Excessive deposition of extracellular matrix (ECM) proteins, a condition known as fibrosis, is a hallmark of Duchenne muscular dystrophy. Among the factors that trigger muscle fibrosis are transforming growth factor beta (TGF-ß) and angiotensin II (Ang-II). Ang-II belongs to the renin-angiotensin system, and its biological effects are exerted by Ang-II receptors type 1 and type 2 (AT-1 and AT-2, respectively). This study aims to determine the effect of TGF-ß1 on the expression of AT-1 and AT-2 receptor in skeletal muscle. C2 C12 myoblasts exposed to TGF-ß1 showed a dose-dependent increase in AT-2 expression but with no effect on AT-1 levels. Injection of TGF-ß1 in the skeletal muscle of mice increased the levels of AT-2 and ECM protein but unchanged AT-1 levels. We also detected higher expression levels of AT-2 receptor in dystrophic skeletal muscle of mdx mice than in normal mice. The induction of AT-2 was mediated by the canonical TGF-ß pathway because under the inhibitory conditions of the kinase activity of TGFß receptor I or the knockdown of Smad2/3 levels, TGF-ß-induced AT-2 receptor increase was strongly inhibited. Furthermore, we demonstrated that p38MAPK activity in response to TGF-ß is also required for AT-2 increase as evaluated by a p38MAPK inhibitor. Our results show that the levels of AT-2 but not AT-1 receptor are modulated by the pro-fibrotic factor TGF-ß1 in myoblasts and mouse skeletal muscle. This finding suggests that AT-2 might be involved in the physiopathology of fibrosis in dystrophic skeletal muscle.

Fatty acids identified in the Burmese python promote beneficial cardiac growth.

Burmese pythons display a marked increase in heart mass after a large meal. We investigated the molecular mechanisms of this physiological heart growth with the goal of applying this knowledge to the mammalian heart. We found that heart growth in pythons is characterized by myocyte hypertrophy in the absence of cell proliferation and by activation of physiological signal transduction pathways. Despite high levels of circulating lipids, the postprandial python heart does not accumulate triglycerides or fatty acids. Instead, there is robust activation of pathways of fatty acid transport and oxidation combined with increased expression and activity of superoxide dismutase, a cardioprotective enzyme. We also identified a combination of fatty acids in python plasma that promotes physiological heart growth when injected into either pythons or mice.

Structure of p300 bound to MEF2 on DNA reveals a mechanism of enhanceosome assembly.

Transcription co-activators CBP and p300 are recruited by sequence-specific transcription factors to specific genomic loci to control gene expression. A highly conserved domain in CBP/p300, the TAZ2 domain, mediates direct interaction with a variety of transcription factors including the myocyte enhancer factor 2 (MEF2). Here we report the crystal structure of a ternary complex of the p300 TAZ2 domain bound to MEF2 on DNA at 2.2Å resolution. The structure reveals three MEF2:DNA complexes binding to different sites of the TAZ2 domain. Using structure-guided mutations and a mammalian two-hybrid assay, we show that all three interfaces contribute to the binding of MEF2 to p300, suggesting that p300 may use one of the three interfaces to interact with MEF2 in different cellular contexts and that one p300 can bind three MEF2:DNA complexes simultaneously. These studies, together with previously characterized TAZ2 complexes bound to different transcription factors, demonstrate the potency and versatility of TAZ2 in protein-protein interactions. Our results also support a model wherein p300 promotes the assembly of a higher-order enhanceosome by simultaneous interactions with multiple DNA-bound transcription factors.

Whole transcriptome analysis of the fasting and fed Burmese python heart: insights into extreme physiological cardiac adaptation.

The infrequently feeding Burmese python (Python molurus) experiences significant and rapid postprandial cardiac hypertrophy followed by regression as digestion is completed. To begin to explore the molecular mechanisms of this response, we have sequenced and assembled the fasted and postfed Burmese python heart transcriptomes with Illumina technology using the chicken (Gallus gallus) genome as a reference. In addition, we have used RNA-seq analysis to identify differences in the expression of biological processes and signaling pathways between fasted, 1 day postfed (DPF), and 3 DPF hearts. Out of a combined transcriptome of ∼2,800 mRNAs, 464 genes were differentially expressed. Genes showing differential expression at 1 DPF compared with fasted were enriched for biological processes involved in metabolism and energetics, while genes showing differential expression at 3 DPF compared with fasted were enriched for processes involved in biogenesis, structural remodeling, and organization. Moreover, we present evidence for the activation of physiological and not pathological signaling pathways in this rapid, novel model of cardiac growth in pythons. Together, our data provide the first comprehensive gene expression profile for a reptile heart.

Extracellular proteoglycans modify TGF-beta bio-availability attenuating its signaling during skeletal muscle differentiation.

The onset and progression of skeletal muscle regeneration are controlled by a complex set of interactions between muscle precursor cells and their environment. Satellite cells constitute the main source of muscle precursor cells for growth and repair. After skeletal muscle injury, cell-derived signals induce their re-entry into the cell cycle and their migration into the damaged zone, where they proliferate and differentiate into mature myofibers. The surrounding extracellular matrix (ECM) together with inhibitory growth factors, such as transforming growth factor-beta (TGF-beta), also likely play an important role in growth control and muscle differentiation. Decorin, biglycan and betaglycan are proteoglycans that bind TGF-beta during skeletal muscle differentiation. In this paper, we show that the binding of TGF-beta to the receptors TGF-betaRI and-betaRII diminished in a satellite cell-derived cell line during differentiation, in spite of an increase expression of both receptors. In contrast, during the differentiation of decorin-null myoblasts (Dcn null), which lack decorin expression, the binding of TGF-beta to TGF-betaRI and -betaRII increased concomitantly with receptors levels. Both the addition and re-expression of decorin, in these myoblasts, diminished the binding of TGF-beta to its transducing receptors. Similar results were obtained when biglycan was added or over-expressed in Dcn null myoblasts. The binding of TGF-beta to TGF-betaRIII, alternatively known as betaglycan, was also augmented in Dcn null myoblasts and diminished by decorin, biglycan and betaglycan. These results suggest that decorin, biglycan and betaglycan compete for the binding of TGF-beta to its transducing receptors. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1, coupled with luciferase, revealed that the addition of each proteoglycan diminished TGF-beta-dependent activity, for both TGF-beta1 and -beta2. The modulation of TGF-beta signaling by ECM proteoglycans diminishing the bio-availability of TGF-beta for its transducing receptors appears to be a feasible mechanism for the attenuation of this inhibitory growth factor during skeletal muscle formation.

Ubc9 expression is essential for myotube formation in C2C12.

Myogenic differentiation is a fundamental biological process that involves a hierarchical series of events that ultimately leads to muscle-specific gene expression and myofiber formation. Posttranslational modifications of the myogenic regulatory factors have been implicated as important regulatory mechanisms in this process. Here we investigate whether covalent protein modification by a small ubiquitin-like modifier (SUMO) that is known to affect transcription factor activity can impact muscle differentiation. We show that the overall load of sumoylated proteins present in myoblasts diminishes progressively throughout myogenesis. Interestingly, knockdown of the SUMO-conjugating enzyme, Ubc9, severely compromises C2C12 muscle cell terminal differentiation. However, it does not affect the expression, the localization and the activation of MyoD and myogenin. These novel results suggest that protein sumoylation plays a pivotal role in myoblast differentiation and is required to regulate the activity of key targets downstream of MyoD and myogenin.

SUMO-1 modification of MEF2A regulates its transcriptional activity.

Myocyte enhancer factor 2 (MEF2) transcription factors are crucial regulators controlling muscle-specific and growth factor-inducible genes. Numerous studies have reported that the activity of these transcription factors is tightly modulated by posttranslational modifications such as activation by specific phosphorylation as well as repression by class II histone deacetylases (HDACs). We hypothesized that MEF2 could also be regulated by covalent modification by SUMO-1, a reversible posttranslational modification which has been shown to regulate key proteins involved in cell proliferation, differentiation and tumor suppression. In this study, we demonstrate that MEF2A undergoes sumoylation primarily at a single lysine residue (K395) both in vitro and in vivo. We also show that the nuclear E3 ligase, PIAS1, promotes sumoylation of MEF2A. Mutation of lysine 395 to arginine abolishes MEF2A sumoylation and the sumoylation incompetent mutant protein has enhanced transcriptional activity compared to the wild type protein. Our results suggest that protein sumoylation could play a pivotal role in controlling MEF2 transcriptional activity.

Betaglycan expression is transcriptionally up-regulated during skeletal muscle differentiation. Cloning of murine betaglycan gene promoter and its modulation by MyoD, retinoic acid, and transforming growth factor-beta.

Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C(2)C(12) skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C(2)C(12) cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C(2)C(12) myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.