Background: The hormone adiponectin (ApN) is decreased in the metabolic syndrome, where it plays a key pathogenic role. ApN also exerts some anti-inflammatory effects on skeletal muscles in mice exposed to acute or chronic inflammation. Here, we investigate whether ApN could be sufficiently potent to counteract a severe degenerative muscle disease, with an inflammatory component such as Duchenne muscular dystrophy (DMD). Methods: Mdx mice (a DMD model caused by dystrophin mutation) were crossed with mice overexpressing ApN in order to generate mdx-ApN mice; only littermates were used. Different markers of inflammation/oxidative stress and components of signaling pathways were studied. Global force was assessed by in vivo functional tests, and muscle injury with Evans Blue Dye (EBD). Eventually, primary cultures of human myotubes were used.

Adiponectin (ApN) exhibits metabolic and antiinflammatory properties. This hormone is exclusively secreted by adipocytes under normal conditions. We have shown that ApN was induced in tibialis anterior muscle of mice injected with lipopolysaccharide (LPS) and in C2C12 myotubes cultured with proinflammatory cytokines. We hypothesized that muscle ApN could be a local protective mechanism to counteract excessive inflammatory reaction and oxidative damage. To test this paradigm, we examined whether muscles of ApN-knockout (KO) mice exhibit a higher degree of oxidative stress and apoptosis than wild-type mice when challenged by ip LPS and whether these abnormalities may be corrected by local administration of ApN. Eventually we investigated the effects of ApN in vitro. When compared with wild-type mice, ApN-KO mice exhibited myocyte degenerescence, especially after LPS. Myocytes of ApN-KO mice also displayed much stronger immunolabeling for markers of oxidative stress (peroxiredoxin-3/5 and heme oxygenase-1) as well as for a lipid peroxidation product (hydroxynonenal). Expression of TNF-α, caspase-6, a marker of apoptosis, and nuclear factor-κB was enhanced as well. Eventually muscle electrotransfer of the ApN gene, which did not induce any rise of systemic ApN, corrected all these abnormalities in LPS-injected ApN-KO mice. Likewise, ApN attenuated LPS-induced production of proinflammatory cytokines and activation of nuclear factor-κB in C2C12 cells. Thus, induction of ApN into skeletal muscle in response to an inflammatory aggression appears to be a crucial mechanism to counteract in an autocrine or paracrine fashion excessive inflammatory damage, oxidative stress, and subsequent apoptosis.

BackgroundThe 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.ResultsWe find that NLRP3 is expressed in skeletal muscle and show that ApN downregulates NLRP3 via its anti-inflammatory 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.ConclusionsThe NLRP3 inflammasome plays a key pathogenic role in DMD and muscle inflammation, thereby opening new therapeutic perspectives for these and other related disorders.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0501-z) contains supplementary material, which is available to authorized users.

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 (À30% to 45% for 4-hydroxy-2-nonenal and peroxiredoxin 3, P < 0.0001) as well as inflammation in muscles of mdx mice (À35% 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, upregulation 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.

BackgroundPersistent 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.MethodsPrimary 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.ResultsApN 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).ConclusionApN 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.

A low-grade proinflammatory state contributes to the metabolic syndrome (MS). Adiponectin (ApN), which is reduced in the MS, has emerged as a master regulator of inflammation/immunity. We wanted to identify whether microRNAs (miRNAs) may mediate the antiinflammatory action of ApN on adipose tissue (AT). miRNA expression profiling was performed in mice overexpressing ApN specifically in AT and in wild-type controls. The role of specific miRNAs was analyzed by gain- or loss-of function approaches in 3T3-F442A (pre)-adipocytes and in de novo AT formed from engineered 3T3-F442A preadipocytes transplanted in nude mice. miRNA expression was compared in the omental AT of lean and obese subjects. The expression of miR532-5p and miR1983 was down-regulated, whereas that of miR883b-5p and miR1934 was up-regulated in AT of mice overexpressing ApN specifically in AT. We focused on miR883b-5p identified by computational analysis as being involved in inflammatory pathways. miR883b-5p overexpression down-regulated the lipopolysaccharide-binding protein (LBP) in 3T3-F442A cells, whereas miR883b-5p blockade had reverse effects. LBP aids in lipopolysaccharide binding to Toll-like receptor-4. miR883b-5p blockade also abolished the protective effects of ApN on proinflammatory adipokine induction. These data were recapitulated in the de novo AT in which miR883b-5p silencing induced LBP production and tissue inflammation. Eventually miR883b-5p expression was down-regulated in AT of obese subjects. We identified several novel miRNAs that are regulated by ApN in AT in vivo. miR883b-5p, which is up-regulated by ApN represses LBP and Toll-like receptor-4 signaling, acting therefore as a major mediator of the antiinflammatory action of ApN. These novel miRNAs may open new therapeutic perspectives for the MS.

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Upregulation of muscular adiponectin could act as a local protective mechanism to counteract cellular damage in obesity by weakening inflammation, oxidative stress, and apoptosis. To test this hypothesis, adiponectin-knockout (KO) and wild-type (WT) mice were fed a Western diet (WD). WT mice under WD conditions displayed 63% higher adiponectin expression in myocytes than those under standard laboratory diet (SLD) conditions (P = 0.011). WD-fed KO mice exhibited approximately threefold larger myocyte degeneration than WT mice (P = 0.003). Even under SLD conditions, myotubes of KO mice displayed already moderate immunolabeling for markers of oxidative stress (peroxiredoxin-3/5) and for a lipid peroxidation product (hydroxynonenal). Expression of tumor necrosis factor-α (TNF-α) and caspase-6, a marker of apoptosis, was also present. After WD challenge, immunoreactivity for these markers was strong in muscle of KO mice, although it was detected to a lesser extent in WT mice. Activation of NF-κB and caspase-6 doubled in myocytes of WD-fed KO mice when compared to WT mice (P < 0.001). Furthermore, muscle electrotransfer of the adiponectin gene prevented these abnormalities in WD-fed KO mice. Finally, gene abrogation of the adiponectin receptor 1 (AdipoR1) by siRNA recapitulated a pro-inflammatory state in C2C12 myotubes. Thus, upregulation of muscular adiponectin may be triggered by obesity and be crucial locally to counteract oxidative stress, inflammation, and apoptosis. These effects operate in an autocrine/paracrine manner via AdipoR1 and down-regulation of NF-κB signaling.