RESUMO
The potential role of liver kinase B1 (LKB1) in the altered activation of the master metabolic and epigenetic regulator adenosine monophosphate-activated protein kinase (AMPK) in Duchenne muscular dystrophy has not been investigated so far. Hence, we analyzed both gene and protein levels of LKB1 and its related targets in gastrocnemius muscles of adult C57BL/10 mdx mice and D2 mdx mice, a model with a more severe dystrophic phenotype, as well as the sensitivity of the LKB1-AMPK pathway to AMPK activators, such as chronic exercise. Our data show, for the first time, a reduction in the levels of LKB1 and accessory proteins, MO25 and STRADα, in both mdx strains versus the respective wild type, which was further impaired by exercise, in parallel with a lack of further phosphorylation of AMPK. The AMPK-like kinase salt-inducible kinase (SIK) and class II histone deacetylases, along with expression of the HDAC target gene Mef2c, were also altered, supporting an impairment of LKB1-SIK-class II histone deacetylase signaling. Our results demonstrate that LKB1 may be involved in dystrophic progression, paving the way for future preclinical studies.
Assuntos
Proteínas Quinases Ativadas por AMP , Distrofia Muscular de Duchenne , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
In age-related sarcopenia, the gradual loss of skeletal muscle mass, function and strength is underpinned by an imbalanced rate of protein synthesis/breakdown. Hence, an adequate protein intake is considered a valuable strategy to mitigate sarcopenia. Here, we investigated the effects of a 12-week oral supplementation with branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) with recognized anabolic properties, in 17-month-old (AGED) C57BL/6J male mice. BCAAs (2:1:1) were formulated in drinking water, alone or plus two L-Alanine equivalents (2ALA) or dipeptide L-Alanyl-L-Alanine (Di-ALA) to boost BCAAs bioavailability. Outcomes were evaluated on in/ex vivo readouts vs. 6-month-old (ADULT) mice. In vivo hind limb plantar flexor torque was improved in AGED mice treated with BCAAs + Di-ALA or 2ALA (recovery score, R.S., towards ADULT: ≥20%), and all mixtures significantly increased hind limb volume. Ex vivo, myofiber cross-sectional areas were higher in gastrocnemius (GC) and soleus (SOL) muscles from treated mice (R.S. ≥ 69%). Contractile indices of isolated muscles were improved by the mixtures, especially in SOL muscle (R.S. ≥ 20%). The latter displayed higher mTOR protein levels in mice supplemented with 2ALA/Di-ALA-enriched mixtures (R.S. ≥ 65%). Overall, these findings support the usefulness of BCAAs-based supplements in sarcopenia, particularly as innovative formulations potentiating BCAAs bioavailability and effects.
Assuntos
Aminoácidos de Cadeia Ramificada , Sarcopenia , Masculino , Camundongos , Animais , Aminoácidos de Cadeia Ramificada/metabolismo , Sarcopenia/metabolismo , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Suplementos NutricionaisRESUMO
Introduction: Growth hormone secretagogues (GHSs) exert multiple actions, being able to activate GHS-receptor 1a, control inflammation and metabolism, to enhance GH/insulin-like growth factor-1 (IGF-1)-mediated myogenesis, and to inhibit angiotensin-converting enzyme. These mechanisms are of interest for potentially targeting multiple steps of pathogenic cascade in Duchenne muscular dystrophy (DMD). Methods: Here, we aimed to provide preclinical evidence for potential benefits of GHSs in DMD, via a multidisciplinary in vivo and ex vivo comparison in mdx mice, of two ad hoc synthesized compounds (EP80317 and JMV2894), with a wide but different profile. 4-week-old mdx mice were treated for 8 weeks with EP80317 or JMV2894 (320 µg/kg/d, s.c.). Results: In vivo, both GHSs increased mice forelimb force (recovery score, RS towards WT: 20% for EP80317 and 32% for JMV2894 at week 8). In parallel, GHSs also reduced diaphragm (DIA) and gastrocnemius (GC) ultrasound echodensity, a fibrosis-related parameter (RS: ranging between 26% and 75%). Ex vivo, both drugs ameliorated DIA isometric force and calcium-related indices (e.g., RS: 40% for tetanic force). Histological analysis highlighted a relevant reduction of fibrosis in GC and DIA muscles of treated mice, paralleled by a decrease in gene expression of TGF-ß1 and Col1a1. Also, decreased levels of pro-inflammatory genes (IL-6, CD68), accompanied by an increment in Sirt-1, PGC-1α and MEF2c expression, were observed in response to treatments, suggesting an overall improvement of myofiber metabolism. No detectable transcript levels of GHS receptor-1a, nor an increase of circulating IGF-1 were found, suggesting the presence of a novel receptor-independent mechanism in skeletal muscle. Preliminary docking studies revealed a potential binding capability of JMV2894 on metalloproteases involved in extracellular matrix remodeling and cytokine production, such as ADAMTS-5 and MMP-9, overactivated in DMD. Discussion: Our results support the interest of GHSs as modulators of pathology progression in mdx mice, disclosing a direct anti-fibrotic action that may prove beneficial to contrast pathological remodeling.