RESUMEN
Peptides, polymers of amino acids, comprise a vital and expanding therapeutic approach. Their rapid degradation by proteases, however, represents a major limitation to their therapeutic utility and chemical modifications to native peptides have been employed to mitigate this weakness. Herein, we describe functionalized thiocarbazate scaffolds as precursors of aza-amino acids, that, upon activation, can be integrated in a peptide sequence to generate azapeptides using conventional peptide synthetic methods. This methodology facilitates peptide editing-replacing targeted amino acid(s) with aza-amino acid(s) within a peptide-to form azapeptides with preferred therapeutic characteristics (extending half-life/bioavailability, while at the same time typically preserving structural features and biological activities). We demonstrate the convenience of this azapeptide synthesis platform in two well-studied peptides with short half-lives: FSSE/P5779, a tetrapeptide inhibitor of HMGB1/MD-2/TLR4 complex formation, and bradykinin, a nine-residue vasoactive peptide. This bench-stable thiocarbazate platform offers a robust and universal approach to optimize peptide-based therapeutics.
Asunto(s)
Aminoácidos , Bradiquinina , Semivida , Péptido Hidrolasas , EndopeptidasasRESUMEN
Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1-60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.
Asunto(s)
Adenilil Ciclasas/metabolismo , Inflamación/metabolismo , Reflejo/fisiología , Factores de Necrosis Tumoral/metabolismo , Animales , Proteína de Unión a CREB/metabolismo , Línea Celular , Endotoxinas/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7 , Ratas , Ratas Sprague-Dawley , Bazo/metabolismo , Nervio Vago/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterized by beta cell destruction, insulin deficiency and hyperglycemia. Activated macrophages and autoimmune T cells play a crucial role in the pathogenesis of hyperglycemia in NOD murine diabetes models, but the molecular mechanisms of macrophage activation are unknown. We recently identified pigment epithelium-derived factor (PEDF) as an adipocyte-derived factor that activates macrophages and mediates insulin resistance. Reasoning that PEDF might participate as a proinflammatory mediator in murine diabetes, we measured PEDF levels in NOD mice. PEDF levels are significantly elevated in pancreas, in correlation with pancreatic TNF levels in NOD mice. To identify experimental therapeutics, we screened 2,327 compounds in two chemical libraries (the NIH Clinical Collection and Pharmakon-1600a) for leads that inhibit PEDF mediated TNF release in macrophage cultures. The lead molecule selected, "emetine" is a widely used emetic. It inhibited PEDF-mediated macrophage activation with an EC50 or 146 nM. Administration of emetine to NOD mice and to C57Bl6 mice subjected to streptozotocin significantly attenuated hyperglycemia, reduced TNF levels in pancreas, and attenuated insulitis. Together, these results suggest that targeting PEDF with emetine may attenuate TNF release and hyperglycemia in murine diabetes models. This suggests that further investigation of PEDF and emetine in the pathogenesis of human diabetes is warranted.
RESUMEN
Type 1 diabetes in mice is characterized by autoimmune destruction of insulin-producing pancreatic ß-cells. Disease pathogenesis involves invasion of pancreatic islets by immune cells, including macrophages and T cells, and production of antibodies to self-antigens, including insulin. Activation of the inflammatory reflex, the neural circuit that inhibits inflammation, culminates on cholinergic receptor signals on immune cells to attenuate cytokine release and inhibit B-cell antibody production. Here, we show that galantamine, a centrally acting acetylcholinesterase inhibitor and an activator of the inflammatory reflex, attenuates murine experimental type 1 diabetes. Administration of galantamine to animals immunized with keyhole limpet hemocyanin (KLH) significantly suppressed splenocyte release of immunoglobulin G (IgG) and interleukin (IL)-4 and IL-6 during KLH challenge ex vivo. Administration of galantamine beginning at 1 month of age in nonobese diabetic (NOD) mice significantly delayed the onset of hyperglycemia, attenuated immune cell infiltration in pancreatic islets and decreased anti-insulin antibodies in serum. These observations indicate that galantamine attenuates experimental type 1 diabetes in mice and suggest that activation of the inflammatory reflex should be further studied as a potential therapeutic approach.
RESUMEN
Obesity is a major risk factor for insulin resistance, type 2 diabetes mellitus and cardiovascular disease. The pathophysiology of obesity is associated with chronic low-grade inflammation. Adipose tissue in obesity is significantly infiltrated by macrophages that secrete cytokines. The mechanisms of interaction between macrophages and adipocytes, leading to macrophage activation and increased cytokine release, remain to be elucidated. We reasoned that an adipocyte-derived factor might stimulate activation of macrophages. We have identified pigment epithelium-derived factor (PEDF) as a mediator of inflammation that is secreted by adipocytes and mediates macrophage activation. Recombinant PEDF activates macrophages to release tumor necrosis factor (TNF) and interleukin-1 (IL-1). The PEDF receptor adipose triglyceride lipase (ATGL) is required for PEDF-mediated macrophage activation. Selective inhibition of ATGL on macrophages attenuates PEDF-induced TNF production, and PEDF enhances the phosphorylation of p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases. PEDF administration to rats results in increased serum TNF levels, and insulin resistance. Together, these findings suggest that PEDF secreted by adipocytes contributes to the onset and maintenance of chronic inflammation in obesity, and may be a therapeutic target in ameliorating insulin resistance.
Asunto(s)
Adipocitos/metabolismo , Proteínas del Ojo/metabolismo , Mediadores de Inflamación/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Serpinas/metabolismo , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Animales , Proteínas del Ojo/administración & dosificación , Proteínas del Ojo/farmacología , Humanos , Inflamación/patología , Resistencia a la Insulina , Lipasa/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Factores de Crecimiento Nervioso/administración & dosificación , Factores de Crecimiento Nervioso/farmacología , Ratas , Ratas Sprague-Dawley , Serpinas/administración & dosificación , Serpinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Obesity, a serious and growing health threat, is associated with low-grade inflammation that plays a role in mediating its adverse consequences. Previously, we have discovered a role for neural cholinergic signaling in controlling inflammation, and demonstrated that the cholinergic agent galantamine suppresses excessive proinflammatory cytokine release. The main objective of this study was to examine the efficacy of galantamine, a clinically-approved drug, in alleviating obesity-related inflammation and associated complications. After 8 wks on a high-fat diet, C57BL/6J mice were treated with either galantamine (4 mg/kg, intraperitoneally [i.p.]) or saline for 4 wks in parallel with mice on a low-fat diet and treated with saline. Galantamine treatment of obese mice significantly reduced body weight, food intake, abdominal adiposity, plasma cytokine and adipokine levels, and significantly improved blood glucose, insulin resistance and hepatic steatosis. In addition, galantamine alleviated impaired insulin sensitivity and glucose intolerance significantly. These results indicate a previously unrecognized potential of galantamine in alleviating obesity, inflammation and other obesity-related complications in mice. These findings are of interest for studying the efficacy of this clinically-approved drug in the context of human obesity and metabolic syndrome.