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Phosphodiesterase inhibitor and the control of proteolitical processes in muscular atrophy induced by diabetes mellitus

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Carlos Alberto Arcaro Filho
Total Authors: 1
Document type: Doctoral Thesis
Press: Araraquara. 2018-07-16.
Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
Defense date:
Advisor: Amanda Martins Baviera

Considering the advances in the knowledge of the mechanisms controlling the protein metabolism in skeletal muscles that allowed the discover of new options for the treatment of muscle atrophies, the present study aimed to understand the antiproteolytic potential of phosphodiesterase (PDE) inhibitors (pentoxifylline, a non-selective PDE inhibitor; rolipram, a selective PDE 4 inhibitor), in skeletal muscles of rats submitted to muscle atrophy due to insulin insufficiency (experimental diabetes mellitus), with emphasis on the elucidation of the participation of cyclic AMP (cAMP) signaling components. Normal and diabetic rats (60 mg/kg streptozotocin, intravenous administration) were treated intraperitoneally with saline (NS and DS) or with 2 mg/kg rolipram (NROL and DROL) or with 25 mg/kg pentoxifylline (NPTX and DPTX) for 3 days. After three days of treatments, soleus and extensor digitorum longus (EDL) muscles were removed, weighed, frozen and processed for several analyzes: (i) cAMP content; (ii) activities of proteasome, calpain and caspase-3 (use of specific fluorigenic substrates); (iii) protein levels and/or phosphorylation levels of components of proteolytic pathways, intracellular signaling effectors and transcription factors (Western blotting); (iv) determination of serum insulin and proinflammatory cytokines levels. Ex vivo experiments were performed to verify the direct action of the drugs in the control of muscle proteolysis and activation of intracellular effectors, via muscle incubations in the presence of rolipram or agonists of EPAC (Exchange protein directly activated by cAMP) and PKA (cAMP-dependent protein kinase), intracellular effectors activated by cAMP. Experiments were also carried out in the Laboratory of Prof. Dr. Marco Sandri at the Venetian Institute of Molecular Medicine, Padova, Italy, for the evaluation of the role of PDE4D in controlling the autophagic-lysosomal process in skeletal muscles of starved mice. Treatments of diabetic animals with rolipram (DROL) or pentoxifylline (DPTX) promoted a reduction in the activities of proteasome and calpain in soleus and EDL, as well as reduced the levels of key components of the ubiquitin-proteasome system (MuRF-1, atrogin-1, polyubiquitinated conjugates), and increased the levels of calpastatin (calpain inhibitor). Interestingly, DROL rats showed a reduction in the activity and in the protein levels of caspase-3 in both muscles, whereas DPTX rat had reductions only in EDL muscles. Contributing to the reduction in caspase-3 activity, it was observed a reduction in the content of Bax (pro-apoptotic protein) and an increase of Bcl-2 (anti-apoptotic protein) in both muscles of DROL rats. Diabetic animals treated with saline (DS) showed an increase in the activities of the three proteases, as well as increases in the levels of components belonging to these proteolytic processes. Normal and diabetic animals treated with saline (NS and DS) had basal and similar levels of cAMP in both soleus and EDL, whereas the treatments of normal and diabetic rats with pentoxifylline (NPTX and DPTX) or with rolipram (NROL and DROL) promoted increases in cAMP in both muscles. One of the mechanisms that may be involved in the muscle proteolysis inhibition after increases in cAMP involves the EPAC protein, responsible for integrating the cAMP and the insulin signaling pathways via AKT activation. Diabetic animals treated with pentoxifylline or with rolipram showed an increase in the protein levels of EPAC 1 and in the phosphorylation of AKT, when compared with the DS group. We also observed an increase in the phosphorylation (inhibitory) of FoxO 1 and 3a in both muscles of DROL rats. It can be suggested that part of the rolipram actions causing AKT activation and FoxO inhibition in skeletal muscles may be associated with the increases in the circulating levels of insulin observed in DROL animals. It was investigated, only in animals treated with rolipram, the possible involvement of PKA in the control of muscle proteolysis. DROL rats had activation of PKA, verified both by the increase in the phosphorylation of PKA substrates, as well as in the phophorylation of the transcription factor CREB, in soleus and EDL. DS rats had decreased levels of p-CREB and of the PKA substrates, in soleus and EDL. Diabetic animals treated with PDE inhibitors showed a decrease in serum proinflammatory cytokines (TNF-, PTX and ROL; IL-1, ROL) when compared with DS. In ex vivo studies, incubations of soleus and EDL with rolipram caused a reduction of the total proteolysis as well as an increase in the phosphorylation of PKA substrates and and of AKT. Soleus and EDL muscles incubated with EPAC agonist showed increased in the AKT phosphorylation, whereas incubation with PKA agonist promoted an increase in the phosphorylation of PKA substrates (in both muscles) and and increase in the AKT phosphorylation (EDL), when compared with muscles incubated in the absence of the drugs. In studies to understand the role of PDE4D in the control of the autophagic-lysosomal process, it was observed that the PDE4D gene silencing in anterior tibialis muscles caused a preservation of the muscle mass and fiber area in fasted animals when compared with control muscle. Flexor digitorium brevis muscles, silenced for PDE4D, showed a decreased expression of key proteins of the autophagic-lysosomal process, such as LC3 and p62. These results suggested the mechanisms that may be involved in the direct action of PDE inhibitors in the control of skeletal muscle protein metabolism, through activation of two cAMP-dependent pathways: (i) the PKA/CREB pathway, which may participate in transcriptional control of Bcl-2 and calpastatin, as well as causing direct inactivation of caspases, thus inhibiting the proteolytic processes dependent on caspase-3 and calpains, (ii) the EPAC/AKT pathway, via phosphorylation and inhibition of FoxO 1 and 3a factors, regulating the expression of atrogenes (MuRF-1 and atrogin-1) and promoting a decrease in activity of ubiquitin-proteasome system. Treatments with PDE inhibitors also decreased the inflammatory process and increased the circulating linsulin levels, which may be contributing to the antiproteolytic responses. Initial evidence also suggests that PDE4D participates in the control of the autophagy-lysosomal system in skeletal muscles. All these results indicate that PDE participate in the control of proteolytic processes, therefore PDE inhibitors emerge as an interesting option to activate the cAMP signaling in the skeletal muscles, which may be used in the future in treatments muscle mass loss during atrophy situations. (AU)

FAPESP's process: 14/12202-0 - Role of cyclic AMP-dependent effectors, PKA and EPAC, in the control of the Calcium-dependent proteolysis, caspase-mediated proteolysis and ubiquitin-proteasome system in diabetes muscle atrophy
Grantee:Carlos Alberto Arcaro Filho
Support Opportunities: Scholarships in Brazil - Doctorate