BACKGROUND:Adiponectin (ApN) is a hormone known to exhibit insulin-sensitizing, fat-burning, and anti-inflammatory properties in several tissues, including the skeletal muscle. Duchenne muscular dystrophy (DMD) is a devastating disease characterized by dystrophin deficiency with subsequent chronic inflammation, myofiber necrosis, and impaired regeneration. Previously, we showed that transgenic up-regulation of ApN could significantly attenuate the dystrophic phenotype in mdx mice (model of DMD). Recently, an orally active ApN receptor agonist, AdipoRon, has been identified. This synthetic small molecule has the advantage of being more easily produced and administrable than ApN. The aim of this study was to investigate the potential effects of AdipoRon on the dystrophic muscle.METHODS:Four-week-old mdx mice (n = 6-9 per group) were orally treated with AdipoRon (mdx-AR) for 8 weeks and compared with untreated (mdx) mice and to control (wild-type) mice. In vivo functional tests were carried out to measure the global force and endurance of mice. Ex vivo biochemical and molecular analyses were performed to evaluate the pathophysiology of the skeletal muscle. Finally, in vitro tests were conducted on primary cultures of healthy and DMD human myotubes.RESULTS:AdipoRon treatment mitigated oxidative stress (-30% to 45% for 4-hydroxy-2-nonenal and peroxiredoxin 3, P < 0.0001) as well as inflammation in muscles of mdx mice (-35% to 65% for interleukin 1 beta, tumour necrosis factor alpha, and cluster of differentiation 68, a macrophage maker, P < 0.0001) while increasing the anti-inflammatory cytokine, interleukin 10 (~5-fold, P < 0.0001). AdipoRon also improved the myogenic programme as assessed by a ~2-fold rise in markers of muscle proliferation and differentiation (P < 0.01 or less vs. untreated mdx). Plasma lactate dehydrogenase and creatine kinase were reduced by 30-40% in mdx-AR mice, reflecting less sarcolemmal damage (P < 0.0001). When compared with untreated mdx mice, mdx-AR mice exhibited enhanced physical performance with an increase in both muscle force and endurance and a striking restoration of the running capacity during eccentric exercise. AdipoRon mainly acted through ApN receptor 1 by increasing AMP-activated protein kinase signalling, which led to repression of nuclear factor-kappa B, up-regulation of utrophin (a dystrophin analogue), and a switch towards an oxidative and more resistant fibre phenotype. The effects of AdipoRon were then recapitulated in human DMD myotubes.CONCLUSIONS:These results demonstrate that AdipoRon exerts several beneficial effects on the dystrophic muscle. This molecule could offer promising therapeutic prospect for managing DMD or other muscle and inflammatory disorders.

Although the high prevalence of anxiety in obesity increasingly emerges as significant risk factor for related severe health complications, the underlying pathophysiological mechanisms remain poorly understood. Considering that chronic inflammation is a key component of obesity and is well known to impact brain function and emotional behavior, we hypothesized that it may similarly contribute to the development of obesity-related anxiety. This hypothesis was experimentally tested by measuring whether chronic food restriction, a procedure known to reduce inflammation, or chronic anti-inflammatory treatment with ibuprofen improved anxiety-like behavior and concomitantly decreased peripheral and/or hippocampal inflammation characterizing a model of severe obesity, the db/db mice. In both experiments, reduced anxiety-like behaviors in the open-field and/or elevated plus-maze were selectively associated with decreased hippocampal tumor necrosis factor-α (TNF-α) mRNA expression. Highlighting the causality of both events, chronic central infusion of the TNF-α blocker etanercept was then shown to be sufficient to improve anxiety-like behavior in db/db mice. Lastly, by measuring the impact of ex-vivo etanercept on hippocampal synaptic processes underlying anxiety-like behaviors, we showed that the anxiolytic effect of central TNF-α blockade likely involved modulation of synaptic transmission within the ventral hippocampus. Altogether, these results uphold the role of brain TNF-α in mediating obesity-related anxiety and provide important clues about how it may modulate brain function and behavior. They may therefore help to introduce novel therapeutic strategies to reduce anxiety associated with inflammatory conditions.

Background: The hormone adiponectin (ApN) exerts powerful anti-inflammatory effects on skeletal muscle and can reverse devastating myopathies, like Duchenne muscular dystrophy (DMD), where inflammation exacerbates disease progression. The NLRP3 inflammasome plays a key role in the inflammation process, and its aberrant activation leads to several inflammatory or immune diseases. Here we investigated the expression of the NLRP inflammasome in skeletal muscle and its contribution to DMD. Results: We find that NLRP3 is expressed in skeletal muscle and show that ApN downregulates NLRP3 via its antiinflammatory mediator, miR-711. This repression occurs both in vitro in C2C12 myotubes and in vivo after either local (via muscle electrotransfer) or systemic (by using transgenic mice) ApN supplementation. To explore the role of the NLRP3 inflammasome in a murine model of DMD, we crossed mdx mice with Nlrp3-knockout mice. In mdx mice, all components of the inflammasome were upregulated in muscle, and the complex was overactivated. By contrast, in mdx mice lacking Nlrp3, there was a reduction in caspase-1 activation, inflammation and oxidative stress in dystrophic muscle, and these mice showed higher global muscle force/endurance than regular mdx mice as well as decreased muscle damage. To investigate the relevance of NLPR3 regulation in a human disease context, we characterized NLRP3 expression in primary cultures of myotubes from DMD subjects and found a threefold increase compared to control subjects. This overexpression was attenuated by ApN or miR-711 mimic treatments. Conclusions: The NLRP3 inflammasome plays a key pathogenic role in DMD and muscle inflammation, thereby opening new therapeutic perspectives for these and other related disorders.

Individual data values for all experiments are available in an Excel file. Data sets are sorted by figure, and an additional section is provided in order to recap n < 6 experiments. (XLSX 45 kb)

BACKGROUND:Persistent inflammation exacerbates the progression of Duchenne muscular dystrophy (DMD). The hormone, adiponectin (ApN), which is decreased in the metabolic syndrome, exhibits anti-inflammatory properties on skeletal muscle and alleviates the dystrophic phenotype of mdx mice. Here, we investigate whether ApN retains its anti-inflammatory action in myotubes obtained from DMD patients. We unravel the underlying mechanisms by studying the secretome and the early events of ApN.METHODS:Primary cultures of myotubes from DMD and control patients were treated or not by ApN after an inflammatory challenge. Myokines secreted in medium were identified by cytokine antibody-arrays and ELISAs. The early events of ApN signaling were assessed by abrogating selected genes.RESULTS:ApN retained its anti-inflammatory properties in both dystrophic and control myotubes. Profiling of secretory products revealed that ApN downregulated the secretion of two pro-inflammatory factors (TNFα and IL-17A), one soluble receptor (sTNFRII), and one chemokine (CCL28) in DMD myotubes, while upregulating IL-6 that exerts some anti-inflammatory effects. These changes were explained by pretranslational mechanisms. Earlier events of the ApN cascade involved AdipoR1, the main receptor for muscle, and the AMPK-SIRT1-PGC-1α axis leading, besides alteration of the myokine profile, to the upregulation of utrophin A (a dystrophin analog).CONCLUSION:ApN retains its beneficial properties in dystrophic muscles by activating the AdipoR1-AMPK-SIRT1-PGC-1α pathway, thereby inducing a shift in the secretion of downstream myokines toward a less inflammatory profile while upregulating utrophin. ApN, the early events of the cascade and downstream myokines may be therapeutic targets for the management of DMD.

Assembly of the NLRP3 inflammasome leads to caspase-1 activation and mediates the cleaving and release of several inflammatory cytokines. The NLRP3 inflammasome can amplify inflammatory responses and thus worsen several diseases. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle disease and it is known to harbour a severe inflammation. We have recently shown that NLRP3 was more expressed in skeletal muscle fibers of mdx mice (a murine model of DMD) than in Wild-Type (WT) mice. Adiponectine (ApN) is a hormone known to possess powerful anti-inflammatory effects on skeletal muscle. Interestingly, transgenic mdx mice that overexpress ApN exhibited lower muscle inflammation/damage as well as higher globular muscle force/endurance when compared to regular mdx mice. These beneficial effects of ApN were associated with a reduction in NLRP3 expression in skeletal muscle. In this study, we investigated the effects of the absence of NLRP3 on the dystrophic phenotype by crossing mdx mice with NLRP3-knockout (NLRP3-KO) mice. First, functional in vivo studies (grip test, wire test and treadmill exercise) were performed on 4 groups of mice: WT, NLRP3-KO, mdx and NLRP3-KO-mdx. Compared to WT, mdx mice presented a strong decrease of global force and endurance that was partially restored in NLRP3-KO-mdx mice. In addition, NLRP3-KO-mdx mice also exhibited a significant decrease in muscle damage, oxidative stress and inflammation as well as a reduction in caspase-1 activation, when compared to regular mdx mice. Furthermore, satellite cells obtained from control and DMD subjects were cultured and differentiated into myotubes. We found that NLRP3 basal expression was 3.5-fold higher in DMD myotubes than in control myotubes. This expression was then reduced after ApN treatment. These novel data show that NLRP3 is implicated in DMD where it plays a key pathogenic role, thus opening new therapeutic perspectives to control muscle inflammation and damage.

Adiponectin (ApN) is a hormone that exhibits anti-inflammatory effects on skeletal muscle exposed to acute and chronic inflammation. We have previously tested the implication of ApN in Duchenne muscular dystrophy (DMD) using mdx mice, a model of DMD, and by generating transgenic mdx mice overexpressing ApN. We showed that ApN can act as a preventive agent and delay disease progression by reducing muscle inflammation/injury and improving force/myogenesis. Herein, we took an opposite approach and crossed mdx mice with ApN knockout mice, to obtain mdx mice with ApN depletion. The aims were to test whether ApN deficiency could worsen the mdx phenotype and whether ApN supplementation can reverse several muscle abnormalities once the disease is settled. mdx-knockout mice exhibited Lower muscle force/endurance as well as increased muscle damage when compared to regular mdx mice. Local administration of the ApN gene significantly reduced the expression of several oxidative stress/inflammatory markers and increased the expression of myogenic markers in the skeletal muscle. Finally, the presence of ApN markedly reduced the activity of NF-kappa B, a key player in muscle inflammation and myogenesis. ApN proves to be a powerful protector of the skeletal muscle capable of reversing the disease progression, thus making it a potential therapeutic agent for DMD.

NLRP3 inflammasome activation cleaves several pro-inflammatory cytokines including pro-IL-1β, resulting in their activation and secretion. Several myopathies result in excessive inflammation that plays a worsening pathogenic role. IL-1β is overexpressed in skeletal muscle of mdx mice, a murine model of Duchenne muscular dystrophy. However, NLRP3 expression/activity has not yet been investigated in skeletal muscle. Adiponectin (ApN) is a hormone mainly secreted by adipose tissue, which exerts anti-inflammatory properties on skeletal muscle. Recently, transgenic mdx mice overexpressing ApN exhibited higher global force/endurance as well as decreased muscle damage/inflammation. We have shown that ApN upregulated miR-711 in skeletal muscle, thereby contributing to its anti-inflammatory effects via the inhibition of NF-κB activity. In this study, we investigated the presence of NLRP3 in myofibers and whether its expression is potentially regulated by ApN and miR-711 in vivo and in vitro. One tibialis anterior of ApN-KO mice was electroporated with a plasmid coding for the ApN gene or the miR-711, while the contralateral one received a respective control plasmid. Mice were next challenged by lipopolysaccharide (LPS) to induce inflammation. Muscle electrotransfer of either the ApN gene or miR-711 induced anti-inflammatory effects: downregulation (~50%) of inflammation (TNFα, IL-1β) and oxidative stress (peroxiredoxin-3) stress markers. NLRP3 labeling found as cluster stains in sarcoplasm was also reduced (40%), while being undetectable in NLRP3-KO mice. In C2C12 myotubes, both ApN treatment and transfection of miR-711 mimic reduced gene expression of TNFα, IL-1β and NLRP3 after LPS challenge. In mdx mice, the expression of NLRP3 was 4-fold higher than in WT mice, while mdx mice overexpressing ApN presented a 30% reduction. These novel data show that NLRP3 is present within myofibers and may open new therapeutic perspectives to control muscle inflammation.

Muscle inflammation worsens metabolic disorders as well as devastating myopathies. The hormone adiponectin (ApN) has emerged has a master regulator of inflammation/immunity in several tissues including the skeletal muscle. In this work, we explore whether microRNAs regulated by ApN may represent novel mechanisms for controlling muscle inflammation. By screening arrays, we found miR-711 as a strong candidate for mediating ApN action. Thus, ApN-knockout mice showed decreased muscular expression of miR-711 together with enhanced inflammation/oxidative stress markers, while mice overexpressing ApN showed increased miR-711 levels. Likewise, electrotransfer of the ApN gene in muscle of ApN-knockout mice upregulated miR-711 while reducing inflammation and oxidative stress. Similar data were obtained in murine C2C12 cells or in human primary myotubes treated with ApN. MiR-711 overexpression downregulated several components of the Toll-like receptor-4 (TLR4) pathway, which led to repression of NF-κB activity and downstream pro-inflammatory cytokines. MiR-711 blockade had opposite effects. Moreover, muscle electrotransfer of pre-miR-711 recapitulated in vivo the anti-inflammatory effects observed in vitro . Thus, miR-711, which is upregulated by ApN represses TLR4 signaling, acting therefore as a major mediator of the anti-inflammatory action of ApN. This novel miRNA and its related target genes may open new therapeutic perspectives for controlling muscle inflammation.

L'adiponectine (ApN) est une hormone diminuée dans le syndrome métabolique qui possède des propriétés antidiabétiques et anti-inflammatoires. Dans le muscle squelettique, l'ApN prévient l'inflammation et le stress oxydatif causés par l'obésité. Les microARNs contrôlent l'expression génique en induisant notamment la dégradation des ARNm. Dans ce travail, nous avons caractérisé les microARNs qui pourraient contribuer à l'action anti-inflammatoire de l'ApN sur le muscle squelettique. Le muscle tibialis anterior gauche des souris déficientes en ApN (ApN-KO) a été injecté, puis électroporé, avec un plasmide codant pour le gène de l'ApN tandis que la patte controlatérale a reçu un plasmide contrôle. Les souris ont ensuite été soumises à une injection ip de lipopolysaccharides (LPS) afin d'induire un état inflammatoire. L'expression de marqueurs de l'inflammation (TNFα, IL-1β) et du stress oxydatif (péroxiredonine-3 ; PRDX3) a été quantifiée. Un microRNA array (Exiqon) a été réalisé. Des myotubes murins (C2C12) ont été traités avec de l'ApN recombinante ou transfectés par un miR-mimic, avant une inflammation causée par le LPS. L'ApN exerçait, comme attendu, son action anti-inflammatoire: l'expression du TNFα, de l'IL-1β et du PRDX3 étaient diminués (– 50 %) dans le muscle électroporé avec l'ApN comparé au controlatéral. Le microRNA array a révélé que l'ApN augmentait (~ +150 %) l'expression du miR-711, données validées par RT-qPCR. L'analyse in silico a montré que ce microARN peut réprimer l'expression de gènes impliqués dans différentes voies inflammatoires et notamment la voie Toll-like receptor 4, activée par le LPS. Dans les cellules C2C12, l'ApN atténuait également l'induction du TNFα causée par le LPS. La transfection du miR-711 mimic reproduisait l'effet de l'ApN. Le miR-711 semble être impliqué dans l'action anti-inflammatoire de l'ApN, ce qui peut ouvrir de nouvelles perspectives thérapeutiques pour contrôler l'inflammation musculaire. Les auteurs déclarent ne pas avoir d'intérêt direct ou indirect (financier ou en nature) avec un organisme privé, industriel ou commercial en relation avec le sujet présenté.