RESUMEN
BACKGROUND: Inflammation in the central nervous system (CNS) is observed in many neurological disorders. Nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling plays an essential role in modulating neuroinflammation. CYR119 is a CNS-penetrant sGC stimulator that amplifies endogenous NO-sGC-cGMP signaling. We evaluated target engagement and the effects of CYR119 on markers of neuroinflammation in vitro in mouse microglial cells and in vivo in quinolinic acid (QA)-induced and high-fat diet-induced rodent neuroinflammation models. METHODS: Target engagement was verified in human embryonic kidney (HEK) cells, rat primary neurons, mouse SIM-A9 cells, and in rats by measuring changes in cGMP and downstream targets of sGC signaling [phosphorylated vasodilator-stimulated phosphoprotein (pVASP), phosphorylated cAMP-response element binding (pCREB)]. In SIM-A9 cells stimulated with lipopolysaccharides (LPS), markers of inflammation were measured when cells were treated with or without CYR119. In rats, microinjections of QA and vehicle were administered into the right and left hemispheres of striatum, respectively, and then rats were dosed daily with either CYR119 (10 mg/kg) or vehicle for 7 days. The activation of microglia [ionized calcium binding adaptor molecule 1 (Iba1)] and astrocytes [glial fibrillary acidic protein (GFAP)] was measured by immunohistochemistry. Diet-induced obese (DIO) mice were treated daily with CYR119 (10 mg/kg) for 6 weeks, after which inflammatory genetic markers were analyzed in the prefrontal cortex. RESULTS: In vitro, CYR119 synergized with exogenous NO to increase the production of cGMP in HEK cells and in primary rat neuronal cell cultures. In primary neurons, CYR119 stimulated sGC, resulting in accumulation of cGMP and phosphorylation of CREB, likely through the activation of protein kinase G (PKG). CYR119 attenuated LPS-induced elevation of interleukin 6 (IL-6) and tumor necrosis factor (TNF) in mouse microglial cells. Following oral dosing in rats, CYR119 crossed the blood-brain barrier (BBB) and stimulated an increase in cGMP levels in the cerebral spinal fluid (CSF). In addition, levels of proinflammatory markers associated with QA administration or high-fat diet feeding were lower in rodents treated with CYR119 than in those treated with vehicle. CONCLUSIONS: These data suggest that sGC stimulation could provide neuroprotective effects by attenuating inflammatory responses in nonclinical models of neuroinflammation.
Asunto(s)
Antiinflamatorios/metabolismo , Sistema Nervioso Central/metabolismo , GMP Cíclico/metabolismo , Mediadores de Inflamación/metabolismo , Neuronas/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Antiinflamatorios/farmacología , Biomarcadores/metabolismo , Células Cultivadas , Sistema Nervioso Central/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Mediadores de Inflamación/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND AND PURPOSE: Reduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso-occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP-amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα-induced inflammation in wild-type C57BL/6 mice and in 'humanised' mouse models of SCD. EXPERIMENTAL APPROACH: Effects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα-induced and systemic inflammation associated with SCD. KEY RESULTS: Acute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte-endothelial cell interactions in TNFα-challenged mice. Co-treatment with hydroxyurea, an FDA-approved SCD therapeutic agent, further augmented the anti-inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα-induced vaso-occlusive crisis, a single dose of olinciguat attenuated leukocyte-endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle-treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat- than in vehicle-treated mice. In addition, kidney mass, water consumption, 24-h urine excretion, plasma levels of cystatin C and urinary excretion of N-acetyl-ß-d-glucosaminidase and neutrophil gelatinase-associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle-treated mice. CONCLUSION AND IMPLICATIONS: Our results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso-occlusion and kidney injury in mouse models of SCD and systemic inflammation.
Asunto(s)
Anemia de Células Falciformes , Enfermedades Vasculares , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/tratamiento farmacológico , Animales , Humanos , Inflamación , Ratones , Ratones Endogámicos C57BL , Guanilil Ciclasa SolubleRESUMEN
Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-ß-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.
Asunto(s)
Apoptosis/efectos de los fármacos , Nefropatías Diabéticas/tratamiento farmacológico , Agonistas de la Guanilato Ciclasa C/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Nefritis/tratamiento farmacológico , Pirazoles/farmacología , Pirimidinas/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Línea Celular , Citocinas/metabolismo , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Enalapril/farmacología , Humanos , Mediadores de Inflamación/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Nefritis/metabolismo , Nefritis/patología , Fosforilación , Ratas Zucker , Transducción de Señal , Proteína smad3/metabolismoRESUMEN
The age-related effects of GDF11 have been a subject of controversy. Here, we find that elevated GDF11 causes signs of cachexia in mice: reduced food intake, body weight, and muscle mass. GDF11 also elicited a significant elevation in plasma Activin A, previously shown to contribute to the loss of skeletal muscle. The effects of GDF11 on skeletal muscle could be reversed by administration of antibodies to the Activin type II receptors. In addition to the effects on muscle, GDF11 increased plasma GDF15, an anorectic agent. The anorexia, but not the muscle loss, could be reversed with a GDF15-neutralizing antibody. GDF15 upregulation is due to GDF11-induced recruitment of SMAD2/3 to the GDF15 promoter. Inhibition of GDF15 can restore appetite but cannot restore the GDF11-induced loss of muscle mass, which requires blockade of ActRII signaling. These findings are relevant for treatment of cachexia.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Caquexia , Factor 15 de Diferenciación de Crecimiento/biosíntesis , Factores de Diferenciación de Crecimiento/metabolismo , Activinas/metabolismo , Animales , Proteínas Morfogenéticas Óseas/farmacología , Factores de Diferenciación de Crecimiento/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
The PI3K-Akt-FoxO1 pathway contributes to the actions of insulin and leptin in several cell types, including neurons in the CNS. However, identifying these actions in chemically identified neurons has proven difficult. To address this problem, we have developed a reporter mouse for monitoring PI3K-Akt signaling in specific populations of neurons, based on FoxO1 nucleocytoplasmic shuttling. The reporter, FoxO1 fused to green fluorescent protein (FoxO1GFP), is expressed under the control of a ubiquitous promoter that is silenced by a loxP flanked transcriptional blocker. Thus, the expression of the reporter in selected cells is dependent on the action of Cre recombinase. Using this model, we found that insulin treatment resulted in the nuclear exclusion of FoxO1GFP within POMC and AgRP neurons in a dose- and time-dependent manner. FoxO1GFP nuclear exclusion was also observed in POMC neurons following in vivo administration of insulin. In addition, leptin induced transient nuclear export of FoxO1GFP in POMC neurons in a dose dependent manner. Finally, insulin-induced nuclear export was impaired in POMC neurons by pretreatment with free fatty acids, a paradigm known to induce insulin resistance in peripheral insulin target tissues. Thus, our FoxO1GFP mouse provides a tool for monitoring the status of PI3K-Akt signaling in a cell-specific manner under physiological and pathophysiological conditions.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , Ácidos Grasos no Esterificados/metabolismo , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/genética , Hipotálamo/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Hibridación in Situ , Insulina/metabolismo , Leptina/metabolismo , Ratones , Técnicas de Cultivo de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Drugs activating 5-hydroxytryptamine 2C receptors (5-HT2CRs) potently suppress appetite, but the underlying mechanisms for these effects are not fully understood. To tackle this issue, we generated mice with global 5-HT2CR deficiency (2C null) and mice with 5-HT2CRs re-expression only in pro-opiomelanocortin (POMC) neurons (2C/POMC mice). We show that 2C null mice predictably developed hyperphagia, hyperactivity, and obesity and showed attenuated responses to anorexigenic 5-HT drugs. Remarkably, all these deficiencies were normalized in 2C/POMC mice. These results demonstrate that 5-HT2CR expression solely in POMC neurons is sufficient to mediate effects of serotoninergic compounds on food intake. The findings also highlight the physiological relevance of the 5-HT2CR-melanocortin circuitry in the long-term regulation of energy balance.
Asunto(s)
Metabolismo Energético/genética , Homeostasis/genética , Hipotálamo/metabolismo , Proopiomelanocortina/metabolismo , Receptor de Serotonina 5-HT2C/genética , Serotonina/metabolismo , Animales , Apetito/efectos de los fármacos , Apetito/genética , Depresores del Apetito/farmacología , Regulación del Apetito/genética , Resistencia a Medicamentos/genética , Hiperfagia/genética , Hiperfagia/metabolismo , Hiperfagia/fisiopatología , Hipotálamo/citología , Ratones , Ratones Noqueados , Actividad Motora/genética , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Obesidad/genética , Obesidad/metabolismo , Obesidad/fisiopatologíaRESUMEN
Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Since its discovery, accumulating evidence has suggested that ghrelin may play a role in signaling and reversing states of energy insufficiency. For example, ghrelin levels rise following food deprivation, and ghrelin administration stimulates feeding and increases body weight and adiposity. However, recent loss-of-function studies have raised questions regarding the physiological significance of ghrelin in regulating these processes. Here, we present results of a study using a novel GHSR-null mouse model, in which ghrelin administration fails to acutely stimulate food intake or activate arcuate nucleus neurons. We show that when fed a high-fat diet, both female and male GHSR-null mice eat less food, store less of their consumed calories, preferentially utilize fat as an energy substrate, and accumulate less body weight and adiposity than control mice. Similar effects on body weight and adiposity were also observed in female, but not male, GHSR-null mice fed standard chow. GHSR deletion also affected locomotor activity and levels of glycemia. These findings support the hypothesis that ghrelin-responsive pathways are an important component of coordinated body weight control. Moreover, our data suggest that ghrelin signaling is required for development of the full phenotype of diet-induced obesity.
Asunto(s)
Dieta , Obesidad/genética , Hormonas Peptídicas/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Tejido Adiposo/metabolismo , Alelos , Análisis de Varianza , Alimentación Animal , Animales , Glucemia/metabolismo , Southern Blotting , Western Blotting , Composición Corporal , Peso Corporal , Cruzamientos Genéticos , ADN/metabolismo , Femenino , Eliminación de Gen , Predisposición Genética a la Enfermedad , Genotipo , Ghrelina , Heterocigoto , Homeostasis , Hiperglucemia/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Leptina/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Neuronas/metabolismo , Obesidad/metabolismo , Hormonas Peptídicas/química , Fenotipo , ARN Mensajero/metabolismo , Receptores de Ghrelina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación Genética , Transducción de Señal , Tinción con Nitrato de Plata , Factores de TiempoRESUMEN
Of the various environmental factors influencing reproduction, food availability plays a particularly significant role, and an insufficient supply of oxidizable metabolic fuels inhibits reproduction in female mammals. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. However, the specific neuroendocrine alterations that mediate the suppression of estrous behavior are unknown. Several conditions that inhibit female sexual behavior are thought to be associated with altered neuropeptide Y (NPY) activity in the brain. Intracerebroventricular (ICV) infusion of NPY inhibits estrous behavior in ovariectomized steroid-primed rats and hamsters. Furthermore, food-deprived rats have an increase in NPY mRNA in the arcuate nucleus (ARC) of the hypothalamus. Unlike rats, studies in Syrian hamsters have failed to detect any alterations in ARC NPY mRNA following food deprivation. Here we show that ARC NPY immunoreactivity and mRNA is increased in food-deprived hamsters but not in hamsters given other metabolic challenges that inhibit estrous behavior. These findings support the hypothesis that NPY contribute to, but not be critical for, the nutritional inhibition of sexual receptivity.
Asunto(s)
Antimetabolitos/farmacología , Desoxiglucosa/farmacología , Estradiol/análogos & derivados , Estro/efectos de los fármacos , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Animales , Núcleo Arqueado del Hipotálamo/citología , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Conducta Animal , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Recuento de Células/métodos , Frío , Cricetinae , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Compuestos Epoxi/farmacología , Estradiol/administración & dosificación , Femenino , Privación de Alimentos , Hipoglucemiantes/farmacología , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Mesocricetus , Neuronas/efectos de los fármacos , Neuropéptido Y/genética , Ovariectomía/métodos , Postura , Progesterona/administración & dosificación , Propionatos/farmacología , ARN Mensajero/metabolismo , Conducta Sexual Animal/efectos de los fármacosRESUMEN
Food deprivation and similar metabolic challenges inhibit estrous behavior in female Syrian hamsters. The relevant metabolic cues appear to be detected in the hindbrain, and this information is then relayed synaptically to the forebrain circuits controlling estrous behavior. Neuropeptide Y (NPY) may be one of the neuropeptides/neurotransmitters serving this function. Infusion of NPY or the Y2/Y5 agonist, peptide YY3-36 (PYY3-36), into the lateral ventricles rapidly inhibits estrous behavior in ovariectomized, steroid-primed hamsters. This experiment sought to determine the neural loci where NPY acts to inhibit estrous behavior. Steroid-primed animals received infusions of artificial cerebrospinal fluid (aCSF) vehicle, 0.024 nmol PYY3-36 and 0.24 nmol PYY3-36 in separate tests 30 min prior to testing for sexual receptivity. Infusion of 0.24 nmol, but not 0.024 nmol, of PYY3-36 reduced lordosis duration when infused into the paraventricular nucleus of the hypothalamus (PVN), the caudal part of the medial preoptic area (MPO), the anterior hypothalamus (AH) or the lateral ventricles. Placements in the ventromedial hypothalamus (VMH), the arcuate nucleus (ARC) and the fourth ventricle were generally without effect. These data suggest that increased endogenous release of NPY into the caudal MPO-AH-PVN continuum during food deprivation could contribute to the observed inhibition of sexual receptivity. The possible contributions of other neuropeptides and neural estrogen receptors to this action of NPY are discussed.
Asunto(s)
Ciclo Estral/fisiología , Neuropéptido Y/fisiología , Prosencéfalo/fisiología , Animales , Cricetinae , Femenino , Privación de Alimentos/fisiología , Mesocricetus , Ovariectomía , Postura , Área Preóptica/fisiología , Conducta Sexual Animal/efectos de los fármacosRESUMEN
Acute food deprivation or chronic food restriction suppresses reproduction in female mammals. Although a link between undernutrition and ovarian function is well established in rats, a similar link with reproductive behavior in this species is yet to be described. Therefore, we compared the display of estrous behaviors induced by exogenous steroid hormone treatment in ovariectomized fed and fasted rats. In addition, estrogen receptor-alpha immunoreactivity (ERIR) was measured in fed and fasted animals to determine whether changes in behavior were associated with changes in the number of detectable ERIR-containing cells in several brain regions. Fasting for 74 h decreased lordosis quotients (LQ) and lordosis ratings (LR) in ovariectomized, steroid-primed rats. The number of detectable ERIR cells decreased after a 74-h fast in the mid-region of the arcuate (ARC), paraventricular (PVN) and ventromedial nuclei of the hypothalamus (VMH) and the ventral bed nucleus of the stria terminalis (BST) but did not change in a number of other areas examined. Taken together, these data demonstrate that, similar to the effect on the reproductive-endocrine axis, food deprivation for 74 h suppresses steroid-induced display of lordosis in adult, female rats. Furthermore, this suppression in sexual receptivity is associated with a decrease in ERIR in a number of areas, including the VMH, a region of the hypothalamus known to be critical for the display of reproductive behaviors in female rats.
Asunto(s)
Encéfalo/metabolismo , Receptores de Estrógenos/metabolismo , Caracteres Sexuales , Animales , Núcleo Arqueado del Hipotálamo/metabolismo , Estradiol/farmacología , Receptor alfa de Estrógeno , Ayuno , Femenino , Privación de Alimentos/fisiología , Ovariectomía , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas , Núcleos Septales/metabolismo , Conducta Sexual Animal , Núcleo Hipotalámico Ventromedial/metabolismoRESUMEN
Estradiol and progesterone (P) induce female mammalian reproductive behaviors, which are, in turn, sensitive to food availability. When ovariectomized, steroid-primed hamsters are food deprived for 48 h, estrous behavior is suppressed. While this suppression of estrous behavior may be due to alterations in neural steroid receptor levels, it is also possible that decreased levels of circulating estradiol could be involved in mediating this suppression. Ovariectomized Syrian hamsters given varying doses of estradiol benzoate (EB) and P were tested to determine whether increasing doses of sex steroids would overcome the suppressive effects of food deprivation on estrous behavior. As expected, lordosis duration decreased in food-deprived animals. Increasing the levels of EB, but not P, increased lordosis duration in the food-deprived animals so that animals who were given 20 microg of EB had lordosis durations significantly longer than food-deprived hamsters that received 1.5 microg and 2.5 microg EB. Following an injection of 2.5 microg of EB, food-deprived hamsters actually had higher circulating levels of estradiol than ad libitum-fed animals. Therefore, increasing circulating levels of estradiol can increase lordosis durations in fasted animals; however, the suppression of estrous behavior occurs despite increased circulating estradiol levels in ovariectomized, steroid-treated animals. The most parsimonious explanation for this phenomenon is a deprivation-induced reduction in neural responsiveness to estradiol.