Examinando por Autor "Salgado, Magdiel"

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    Glial hypothalamic inhibition of GLUT2 expression alters satiety, impacting eating behavior
    (John Wiley and Sons Inc., 2018-03) Barahona, María J.; Llanos, Paula; Recabal, Antonia; Escobar-Acuña, Kathleen; Elizondo-Vega, Roberto; Salgado, Magdiel; Ordenes, Patricio; Uribe, Elena; Sepúlveda, Fernando J.; Araneda, Ricardo C.; García-Robles, María A.
    Glucose is a key modulator of feeding behavior. By acting in peripheral tissues and in the central nervous system, it directly controls the secretion of hormones and neuropeptides and modulates the activity of the autonomic nervous system. GLUT2 is required for several glucoregulatory responses in the brain, including feeding behavior, and is localized in the hypothalamus and brainstem, which are the main centers that control this behavior. In the hypothalamus, GLUT2 has been detected in glial cells, known as tanycytes, which line the basal walls of the third ventricle (3V). This study aimed to clarify the role of GLUT2 expression in tanycytes in feeding behavior using 3V injections of an adenovirus encoding a shRNA against GLUT2 and the reporter EGFP (Ad-shGLUT2). Efficient in vivo GLUT2 knockdown in rat hypothalamic tissue was demonstrated by qPCR and Western blot analyses. Specificity of cell transduction in the hypothalamus and brainstem was evaluated by EGFP-fluorescence and immunohistochemistry, which showed EGFP expression specifically in ependymal cells, including tanycytes. The altered mRNA levels of both orexigenic and anorexigenic neuropeptides suggested a loss of response to increased glucose in the 3V. Feeding behavior analysis in the fasting-feeding transition revealed that GLUT2-knockdown rats had increased food intake and body weight, suggesting an inhibitory effect on satiety. Taken together, suppression of GLUT2 expression in tanycytes disrupted the hypothalamic glucosensing mechanism, which altered the feeding behavior. © 2017 The Authors GLIA Published by Wiley Periodicals, Inc.
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    Ítem
    Poly (I:C)-induced inflammation requires the activation of toll-like receptor 3/Ca2+/CaMKII/pannexin 1-dependent signaling
    (Ivyspring International Publisher, 2025) Salgado, Magdiel; Salgado M.; Sepúlveda-Arriagada, Vania; Konar-Nié, Macarena; García-Robles, María A.; Sáez, Juan C.
    Pannexin1 (Panx1) is a glycoprotein, ubiquitously expressed throughout vertebrate tissues. In the cell membrane, it forms non-selective hemichannels (Panx1 HCs) that allow the release of ATP. This extracellular ATP triggers purinergic signaling relevant to the immune responses to pathogens, including viruses. While the activity of Panx1 HCs is known to be elevated by some viruses, the underlying molecular mechanism remains elusive. Methods: In this study, we used Poly(I:C), a double-stranded RNA analog that constitutes a hallmark of viral infections. Peritoneal macrophages were obtained from wild-type and Panx1 knock-out mice. The mRNA levels of proinflammatory cytokines were quantified by RT-qPCR. We also evaluated hemichannel activity through dye uptake assays, whereas Ca2+ signals were studied using Fura-2 and GcamP6. Panx1-P2X7R interaction was studied by proximity ligation assays. Results: Panx1 expression and activity were crucial for the proinflammatory response induced by Poly(I:C) in RAW264.7 cells and peritoneal macrophages. In HeLa cells transfected with mPanx1 (HeLa-mPanx1) and RAW264.7 cells, Poly (I:C) increased Panx1 HC activity in a concentration-dependent manner, which was inhibited by 10Panx1, a peptide that selectively blocks Panx1 HCs. Furthermore, the Poly(I:C)-induced rise in Panx1 HC activity correlated with a rapid increase in intracellular Ca2+ signal, dependent on TLR3 and P2X7R activity. Interestingly, lasting exposure to Poly (I:C) promoted the interaction and internalization of the Panx1-P2X7R complex, which depended on CaMKII, Panx1 HC, and P2X7R activities. The Poly (I:C)-induced increase in Panx1 HC activity was entirely prevented by Ca2+ chelation with BAPTA-AM, CaMKII blockage with KN-62, or PKA activation with db-cAMP. These findings were consistent with data from Panx1 mutants that either avoid or mimic phosphorylation at kinase target sites. Supporting this finding, we demonstrated that CaMKII activity is essential for the inflammatory response triggered by Poly (I:C) in macrophages. Conclusion: A TLR3/Ca2+/CaMKII/Panx1 HC pathway is crucial in orchestrating the cellular response to viral patterns and presents a potential novel target for preventing infections and alleviating the harmful effects associated with RNA-based viral infections.
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    Skeletal Muscle Atrophy Induced by Diabetes Is Mediated by Non-Selective Channels and Prevented by Boldine
    (MDPI, 2023-04) Cea, Luis A.; Vásquez, Walter; Hernández-Salinas, Romina; Vielma, Alejandra Z.; Castillo-Ruiz, Mario; Velarde, Victoria; Salgado, Magdiel; Sáez, Juan C.
    Individuals with diabetes mellitus present a skeletal muscle myopathy characterized by atrophy. However, the mechanism underlying this muscular alteration remains elusive, which makes it difficult to design a rational treatment that could avoid the negative consequences in muscles due to diabetes. In the present work, the atrophy of skeletal myofibers from streptozotocin-induced diabetic rats was prevented with boldine, suggesting that non-selective channels inhibited by this alkaloid are involved in this process, as has previously shown for other muscular pathologies. Accordingly, we found a relevant increase in sarcolemma permeability of skeletal myofibers of diabetic animals in vivo and in vitro due to de novo expression of functional connexin hemichannels (Cx HCs) containing connexins (Cxs) 39, 43, and 45. These cells also expressed P2X7 receptors, and their inhibition in vitro drastically reduced sarcolemma permeability, suggesting their participation in the activation of Cx HCs. Notably, sarcolemma permeability of skeletal myofibers was prevented by boldine treatment that blocks Cx43 and Cx45 HCs, and now we demonstrated that it also blocks P2X7 receptors. In addition, the skeletal muscle alterations described above were not observed in diabetic mice with myofibers deficient in Cx43/Cx45 expression. Moreover, murine myofibers cultured for 24 h in high glucose presented a drastic increase in sarcolemma permeability and levels of NLRP3, a molecular member of the inflammasome, a response that was also prevented by boldine, suggesting that, in addition to the systemic inflammatory response found in diabetes, high glucose can promote the expression of functional Cx HCs and activation of the inflammasome in skeletal myofibers. Therefore, Cx43 and Cx45 HCs play a critical role in myofiber degeneration, and boldine could be considered a potential therapeutic agent to treat muscular complications due to diabetes. © 2023 by the authors.