RESUMEN
Tongmai Yangxin (TMYX) is a complex compound of the Traditional Chinese Medicine (TCM) used to treat several cardiac rhythm disorders; however, no information regarding its mechanism of action is available. In this study we provide a detailed characterization of the effects of TMYX on the electrical activity of pacemaker cells and unravel its mechanism of action. Single-cell electrophysiology revealed that TMYX elicits a reversible and dose-dependent (2/6 mg/ml) slowing of spontaneous action potentials rate (-20.8/-50.2%) by a selective reduction of the diastolic phase (-50.1/-76.0%). This action is mediated by a negative shift of the If activation curve (-6.7/-11.9 mV) and is caused by a reduction of the cyclic adenosine monophosphate (cAMP)-induced stimulation of pacemaker channels. We provide evidence that TMYX acts by directly antagonizing the cAMP-induced allosteric modulation of the pacemaker channels. Noticeably, this mechanism functionally resembles the pharmacological actions of muscarinic stimulation or ß-blockers, but it does not require generalized changes in cytoplasmic cAMP levels thus ensuring a selective action on rate. In agreement with a competitive inhibition mechanism, TMYX exerts its maximal antagonistic action at submaximal cAMP concentrations and then progressively becomes less effective thus ensuring a full contribution of If to pacemaker rate during high metabolic demand and sympathetic stimulation.
Asunto(s)
AMP Cíclico , Sistemas de Mensajero Secundario , Potenciales de Acción , Animales , China , AMP Cíclico/metabolismo , Miocitos Cardíacos/metabolismo , ConejosRESUMEN
Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.
Asunto(s)
Síndrome de Bardet-Biedl/metabolismo , Chaperoninas/metabolismo , Hipotálamo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación Missense , Neuronas/metabolismo , Sistemas de Mensajero Secundario , Sustitución de Aminoácidos , Animales , Síndrome de Bardet-Biedl/genética , Chaperoninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Deciphering signal transduction processes is crucial for understanding how plants sense and respond to environmental changes. Various chemical compounds function as central messengers within deeply intertwined signaling networks. How such compounds act in concert remains to be elucidated. We have developed dual-reporting transcriptionally linked genetically encoded fluorescent indicators (2-in-1-GEFIs) for multiparametric in vivo analyses of the phytohormone abscisic acid (ABA), Ca2+, protons (H+), chloride (anions), the glutathione redox potential, and H2O2 Simultaneous analyses of two signaling compounds in Arabidopsis (Arabidopsis thaliana) roots revealed that ABA treatment and uptake did not trigger rapid cytosolic Ca2+ or H+ dynamics. Glutamate, ATP, Arabidopsis PLANT ELICITOR PEPTIDE, and glutathione disulfide (GSSG) treatments induced rapid spatiotemporally overlapping cytosolic Ca2+, H+, and anion dynamics, but except for GSSG, only weakly affected the cytosolic redox state. Overall, 2-in-1-GEFIs enable complementary, high-resolution in vivo analyses of signaling compound dynamics and facilitate an advanced understanding of the spatiotemporal coordination of signal transduction processes in Arabidopsis.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Citosol/metabolismo , Colorantes Fluorescentes/metabolismo , Sistemas de Mensajero Secundario , Transcripción Genética , Adenosina Trifosfato/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/metabolismo , Calcio/metabolismo , Cloruros/metabolismo , Citosol/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Ácido Glutámico/farmacología , Disulfuro de Glutatión/farmacología , Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Concentración de Iones de Hidrógeno , Ácidos Indolacéticos/farmacología , Oxidación-Reducción , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
We have previously reported that argan oil and argan press-cake from the kernels of Argania spinosa have an anti-melanogenesis effect. Here, the effect of argan fruit shell ethanol extract (AFSEE) on melanogenesis in B16F10 cells was determined, and the mechanism underlying its effect was elucidated. The proliferation of AFSEE-treated B16F10 cells was evaluated using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, while the melanin content was quantified using a spectrophotometric method. The expression of melanogenesis-related proteins was determined by Western blot and real-time PCR, while global gene expression was determined using a DNA microarray. In vitro analysis results showed that the melanin content of B16F10 cells was significantly increased by AFSEE, without cytotoxicity, by increasing the melanogenic enzyme tyrosinase (TRY), tyrosinase related-protein 1 (TRP1), and dopachrome tautomerase (DCT) protein and mRNA expression, as well as upregulating microphthalmia-associated transcription factor (MITF) expression through mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38, and the cyclic adenosine monophosphate (cAMP) signaling pathway, as indicated by the microarray analysis results. AFSEE's melanogenesis promotion effect is primarily attributed to its polyphenolic components. In conclusion, AFSEE promotes melanogenesis in B16F10 cells by upregulating the expression of the melanogenic enzymes through the cAMP-MITF signaling pathway.AFSEE may be used as a cosmetics product component to promote melanogenesis, or as a therapeutic against hypopigmentation disorders.
Asunto(s)
AMP Cíclico/metabolismo , Frutas/química , Melaninas/biosíntesis , Extractos Vegetales/química , Extractos Vegetales/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Sapotaceae/química , Sistemas de Mensajero Secundario/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Melanoma Experimental , Ratones , Fosforilación , Fitoquímicos/química , Fitoquímicos/farmacologíaRESUMEN
FGF2 is a tumor cell survival factor that is exported from cells by an ER/Golgi-independent secretory pathway. This unconventional mechanism of protein secretion is based on direct translocation of FGF2 across the plasma membrane. The Na,K-ATPase has previously been shown to play a role in this process, however, the underlying mechanism has remained elusive. Here, we define structural elements that are critical for a direct physical interaction between FGF2 and the α1 subunit of the Na,K-ATPase. In intact cells, corresponding FGF2 mutant forms were impaired regarding both recruitment at the inner plasma membrane leaflet and secretion. Ouabain, a drug that inhibits both the Na,K-ATPase and FGF2 secretion, was found to impair the interaction of FGF2 with the Na,K-ATPase in cells. Our findings reveal the Na,K-ATPase as the initial recruitment factor for FGF2 at the inner plasma membrane leaflet being required for efficient membrane translocation of FGF2 to cell surfaces.
Asunto(s)
Membrana Celular/enzimología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Células CHO , Cricetulus , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Sistemas de Mensajero Secundario , Vías Secretoras , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Previously, we designed, synthesized, and evaluated a series of quinolone-benzofuran derivatives as multitargeted anti-Alzheimer's disease (anti-AD) compounds, and we discovered that WBQ5187 possesses superior anti-AD bioactivity. In this work, we investigated the pharmacokinetics of this new molecule, as well as its therapeutic efficacy in restoring cognition and neuropathology, in the APP/PS1 mouse model of AD. Pharmacokinetic analyses demonstrated that WBQ5187 possessed rational oral bioavailability, metabolic stability, and excellent blood-brain barrier (BBB) permeability. Pharmacodynamics studies indicated that a 12-week treatment with the lead compound at doses of 40 mg/kg or higher significantly enhanced the learning and memory performance of the APP/PS1 transgenic mice, and the effect was more potent than that of clioquinol (CQ). Furthermore, WBQ5187 notably reduced cerebral ß-amyloid pathology, gliosis, and neuronal cell loss and increased the levels of cAMP in the hippocampus of these mice. The surrogate measures of emesis indicated that WBQ5187 had no effect at its cognitive effective doses. Overall, our results demonstrated that this compound markedly improves cognitive and spatial memory functions in AD mice and represents a promising pharmaceutical agent with potential for the treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Benzofuranos/uso terapéutico , Química Encefálica/efectos de los fármacos , Clioquinol/análogos & derivados , Fármacos Neuroprotectores/uso terapéutico , Inhibidores de Fosfodiesterasa 4/uso terapéutico , Resorcinoles/uso terapéutico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Anestésicos Generales/toxicidad , Animales , Benzofuranos/química , Benzofuranos/farmacocinética , Disponibilidad Biológica , Barrera Hematoencefálica , Clioquinol/química , Clioquinol/farmacocinética , Clioquinol/uso terapéutico , AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Gliosis/tratamiento farmacológico , Gliosis/prevención & control , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Náusea/inducido químicamente , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacocinética , Inhibidores de Fosfodiesterasa 4/química , Inhibidores de Fosfodiesterasa 4/farmacocinética , Inhibidores de Fosfodiesterasa 4/toxicidad , Resorcinoles/química , Resorcinoles/farmacocinética , Sistemas de Mensajero Secundario/efectos de los fármacos , Vómitos/inducido químicamenteRESUMEN
Because of climate change, heat stress (HS) causes more and more impacts on dairy animals to decrease lactation performance. The neuroendocrine system is key in regulating systemic physiological processes and milk synthesis. However, the hypothalamic-pituitary axis response to HS is still unclear. In this study, a group of lactating mice underwent a daily 2-h heat treatment (36°C) for 14 d to explore possible cross-talk between the hypothalamic-pituitary axis and mammary gland under HS. Transcriptome analyses by multitissue RNA-Seq indicated the possible mechanisms of reduced lactation performance in animals under HS. In the hypothalamus, the cAMP signaling pathway was activated to resist neuronal death, and the expression of downstream genes was increased to promote cell survival under HS. Reduced food intake might be caused by down-regulated appetite-related peptide, whereas up-regulated neuropeptide Y acted to attenuate reduced food intake. In pituitary, energy stress from lower food intake might result in reduced secretion of prolactin and growth hormone. Under HS, the mammary gland may undergo hypoxic stress, causing mammary epithelial cell apoptosis. Together, these data showed systemic changes in tissues to accommodate the effects of HS on lactation.-Han, J., Shao, J., Chen, Q., Sun, H., Guan, L., Li, Y., Liu, J., Liu, H. Transcriptional changes in the hypothalamus, pituitary, and mammary gland underlying decreased lactation performance in mice under heat stress.
Asunto(s)
Respuesta al Choque Térmico , Hipotálamo/metabolismo , Lactancia , Glándulas Mamarias Animales/metabolismo , Hipófisis/metabolismo , Transcripción Genética , Animales , AMP Cíclico/metabolismo , Femenino , Ratones , Sistemas de Mensajero SecundarioRESUMEN
Nutrient overload in combination with decreased energy dissipation promotes obesity and diabetes. Obesity results in a hormonal imbalance, which among others activates G protein-coupled receptors utilizing diacylglycerol (DAG) as secondary messenger. Protein kinase D1 (PKD1) is a DAG effector, which integrates multiple nutritional and hormonal inputs, but its physiological role in adipocytes is unknown. Here, we show that PKD1 promotes lipogenesis and suppresses mitochondrial fragmentation, biogenesis, respiration, and energy dissipation in an AMP-activated protein kinase (AMPK)-dependent manner. Moreover, mice lacking PKD1 in adipocytes are resistant to diet-induced obesity due to elevated energy expenditure. Beiging of adipocytes promotes energy expenditure and counteracts obesity. Consistently, deletion of PKD1 promotes expression of the ß3-adrenergic receptor (ADRB3) in a CCAAT/enhancer binding protein (C/EBP)-α- and δ-dependent manner, which leads to the elevated expression of beige markers in adipocytes and subcutaneous adipose tissue. Finally, deletion of PKD1 in adipocytes improves insulin sensitivity and ameliorates liver steatosis. Thus, depletion of PKD1 in adipocytes increases energy dissipation by several complementary mechanisms and might represent an attractive strategy to treat obesity and its related complications.
Asunto(s)
Adipocitos/metabolismo , Adiposidad , Metabolismo Energético , Hígado Graso/metabolismo , Obesidad/metabolismo , Proteína Quinasa C/metabolismo , Grasa Subcutánea/metabolismo , Células 3T3-L1 , Adipocitos/patología , Animales , Proteína delta de Unión al Potenciador CCAAT/genética , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Hígado Graso/genética , Hígado Graso/patología , Femenino , Humanos , Masculino , Ratones , Ratones Mutantes , Obesidad/genética , Obesidad/patología , Proteína Quinasa C/genética , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Sistemas de Mensajero Secundario/genética , Grasa Subcutánea/fisiologíaRESUMEN
Ginsenoside Re (GS-Re) is one of the main ingredients of ginseng, a widely known Chinese traditional medicine, and has a variety of beneficial effects, including vasorelaxation, antioxidative, anti-inflammatory, and anticancer properties. The aims of the present study were to observe the effect of GS-Re on balloon injury-induced neointimal hyperplasia in the arteries and to investigate the mechanisms underlying this effect. A rat vascular neointimal hyperplasia model was generated by rubbing the endothelium of the common carotid artery (CCA) with a balloon, and GS-Re (12.5, 25 or 50â¯mg/kg/d) were subsequently continuously administered to the rats by gavage for 14 days. After GS-Re treatment, the vessel lumen of injured vessels showed significant increases in the GS-Re 25.0 and 50.0â¯mg/kg/d (intermediate- and high-dose) groups according to H.E. staining. Additionally, a reduced percentage of proliferating cell nuclear antigen (PCNA)-positive cells and an increased number of SM α-actin-positive cells were detected, and the levels of NO, cyclic guanosine monophosphate (cGMP), and eNOS mRNA as well as the phos-eNOSser1177/eNOS protein ratio were obviously upregulated in the intermediate- and high-dose groups. Moreover, the promotive effects of GS-Re on NO and eNOS expression were blocked by L-NAME treatment to different degrees. These results suggested that GS-Re can suppress balloon injury-induced vascular neointimal hyperplasia by inhibiting VSMC proliferation, which is closely related to the activation of the eNOS/NO/cGMP pathway.
Asunto(s)
Angioplastia de Balón/instrumentación , Traumatismos de las Arterias Carótidas/prevención & control , Arteria Carótida Común/efectos de los fármacos , GMP Cíclico/metabolismo , Ginsenósidos/farmacología , Neointima , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Actinas/metabolismo , Animales , Traumatismos de las Arterias Carótidas/enzimología , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/patología , Arteria Carótida Común/enzimología , Arteria Carótida Común/patología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hiperplasia , Masculino , Óxido Nítrico Sintasa de Tipo III/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario/efectos de los fármacosRESUMEN
Dehydroepiandrosterone (DHEA) is commonly used as a nutritional supplement to control fat deposition, but the mechanism of this action is poorly understood. In this study, we demonstrated that DHEA increased phosphorylation of AMP-activated protein kinase (p-AMPK). Elevated p-AMPK levels resulted in reduced expression of sterol regulatory element binding protein-1c, acetyl CoA carboxylase, fatty acid synthase and enhanced expression of peroxisome proliferators-activated receptor α and carnitine palmitoyl transferase-I, ultimately leading to the reduction of lipid droplet accumulation in primary chicken hepatocytes. We found that DHEA activates the cyclic adenosine 3', 5'-monophosphate/protein kinase A - extracellular signal-regulated kinase 1/2 (cAMP/PKA-ERK1/2) signaling pathway, which regulates the conversion of DHEA into testosterone and estradiol by increasing the 17ß-hydroxysteroid dehydrogenase and aromatase protein expression. Importantly, the fat-reducing effects of DHEA are more closely associated with the conversion of DHEA into estradiol than with the action of DHEA itself as an active biomolecule, or to its alternative metabolite, testosterone. Taken together, our results indicate that DHEA is converted into active hormones through activation of the cAMP/PKA-ERK1/2 signaling pathway; the fat-reducing effects of DHEA are achieved through its conversion into estradiol, not testosterone, and not through direct action of DHEA itself, which led to the activation of the p-AMPK in primary chicken hepatocytes. These data provide novel insight into the mechanisms underlying the action of DHEA in preventing fat deposition, and suggest potential applications for DHEA treatment to control fat deposition or as an agent to treat disorders related to lipid metabolism in animals and humans.
Asunto(s)
AMP Cíclico/metabolismo , Deshidroepiandrosterona/metabolismo , Hepatocitos/metabolismo , Gotas Lipídicas/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Sistemas de Mensajero Secundario , Animales , Proteínas Aviares/metabolismo , Pollos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
TLR signaling is critical to innate immune system regulation; however, aberrant TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, and tumor metastasis. Moreover, a recent study found that TLR-4 signaling pathway inhibition might be a target for the suppression of chronic inflammatory disorders. In this article, we show that the green tea polyphenol epigallocatechin-3-O-gallate (EGCG) increases the expression of Toll interacting protein, a strong inhibitor of TLR4 signaling, by suppressing the expression of E74-like ETS transcription factor 1 (Elf-1). A mechanistic study revealed that EGCG suppressed Elf-1 expression via protein phosphatase 2A/cyclic GMP (cGMP)-dependent mechanisms. We also confirmed that orally administered EGCG and a cGMP inducer upregulated Toll interacting protein expression, increased intracellular levels of cGMP in macrophages, and suppressed Elf-1 expression. These data support EGCG and a cGMP inducer as potential candidate suppressors of TLR4 signaling.
Asunto(s)
Catequina/análogos & derivados , Proteínas de Unión al ADN/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas Nucleares/inmunología , Sistemas de Mensajero Secundario/inmunología , Té/química , Factores de Transcripción/inmunología , Regulación hacia Arriba/inmunología , Animales , Catequina/química , Catequina/farmacología , GMP Cíclico/genética , GMP Cíclico/inmunología , Proteínas de Unión al ADN/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Nucleares/genética , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/inmunología , Sistemas de Mensajero Secundario/genética , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Factores de Transcripción/genéticaRESUMEN
Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.
Asunto(s)
Neovascularización Coroidal/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Metabolismo de los Lípidos/fisiología , Sistemas de Mensajero Secundario/fisiología , Animales , Citocromo P-450 CYP2C8/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Epóxido Hidrolasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Leucocitos/metabolismo , Degeneración Macular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
PURPOSE OF REVIEW: The current article presents recent findings on the metabolic effects of fructose. RECENT FINDINGS: Fructose has always been considered as a simple 'caloric' hexose only metabolized by splanchnic tissues. Nevertheless, there is growing evidence that fructose acts as a second messenger and induces effects throughout the human body. SUMMARY: Recent discoveries made possible with the evolution of technology have highlighted that fructose induces pleiotropic effects on different tissues. The fact that all these tissues express the specific fructose carrier GLUT5 let us reconsider that fructose is not only a caloric hexose, but could also be a potential actor of some behaviors and metabolic pathways. The physiological relevance of fructose as a metabolic driver is pertinent regarding recent scientific literature.
Asunto(s)
Fructosa/administración & dosificación , Fructosa/efectos adversos , Metabolismo/efectos de los fármacos , Terapia Nutricional/métodos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Fructosa/metabolismo , Microbioma Gastrointestinal/efectos de los fármacos , Transportador de Glucosa de Tipo 5 , Humanos , Absorción Intestinal/efectos de los fármacos , Mesenterio , Nutrición Parenteral , Factores de Riesgo , Sistemas de Mensajero SecundarioRESUMEN
Rheumatoid arthritis is an autoimmune disorder characterized by T cell dysregulation. We have shown that an altered peptide ligand (A9) activates T cells to use an alternate signaling pathway that is dependent on FcRγ and spleen tyrosine kinase, resulting in downregulation of inflammation. In the experiments described in this study, we have attempted to determine the molecular basis of this paradox. Three major Src family kinases found in T cells (Lck, Fyn, and Lyn) were tested for activation following stimulation by A9/I-Aq Unexpectedly we found they are not required for T cell functions induced by A9/I-Aq, nor are they required for APL stimulation of cytokines. On the other hand, the induction of the second messenger inositol trisphosphate and the mobilization of calcium are clearly triggered by the APL A9/I-Aq stimulation and are required for cytokine production, albeit the cytokines induced are different from those produced after activation of the canonical pathway. DBA/1 mice doubly deficient in IL-4 and IL-10 were used to confirm that these two cytokines are important for the APL-induced attenuation of arthritis. These studies provide a basis for exploring the effectiveness of analog peptides and the inhibitory T cells they induce as therapeutic tools for autoimmune arthritis.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Colágeno Tipo II/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de IgG/metabolismo , Quinasa Syk/metabolismo , Linfocitos T/inmunología , Animales , Señalización del Calcio , Colágeno Tipo II/genética , Colágeno Tipo II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Interleucina-10/genética , Interleucina-4/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de IgG/genética , Sistemas de Mensajero SecundarioRESUMEN
BACKGROUND: Kai-xin-san (KXS), composed of Ginseng Radix et Rhizoma, Polygalae Radix, Acori Tatarinowii Rhizoma and Poria, is a famous Chinese medicinal formula applied for treating stress-related psychiatric disease with the symptoms such as depression, forgetfulness and dizziness. Dependent on the symptom differentiation of patients, the composition ratio of KXS was varied and one ratio of 3:2:2:3 was widely applied. However, its molecular mechanism has seldom been investigated. PURPOSE: We aimed to reveal the action mechanism of KXS on anti-depression on synaptic protein regulation in both in vivo and in vitro models. STUDY DESIGN/METHODS: Firstly, the anti-depression effect of KXS was evaluated on a chronic mild stress induced depressive animal model and the mRNA expressions of various synaptic proteins in hippocampus of the depressive rat brains were determined. Then, KXS with different ratios as well as single herb were further evaluated on rat primary cultured hippocampus neurons and the possible signaling pathway was explored. RESULTS: Intra-gastric administration of a chemically standardized KXS for only 6h significantly alleviated the CUMS-induced depressive symptoms displayed by enhanced sucrose consumption and this effect was maintained after daily treatment for seven days. Simultaneously, the mRNA expressions of various synaptic proteins in hippocampus were regulated. Among these synaptic proteins, synaptotagmin (pre-synaptic marker) and post synaptic density protein (post-synaptic marker), with the higher altered magnitude on animal model, were further evaluated on rat primary cultured hippocampus neurons. After neuronal cultures treated with three ratios of KXS at the early and late stages of its life episode, the expression levels of synaptotagmin and PSD95 were both enhanced dramatically via stimulating cAMP dependent pathway. However, different ratio exerted different efficacy. The ratio with higher amounts of Ginseng Radix et Rhizoma and Polygalae Radix showed better effect in early life episode while higher amounts of Acori Tatarinowii Rhizoma and Poria behaved better in late life episode. The contribution of single herb on expressions of synaptic proteins was also evaluated. CONCLUSIONS: KXS was beneficial for synaptogenesis by inducing synaptic protein expressions, which might account for its anti-depression effect.
Asunto(s)
Antidepresivos/farmacología , Depresión/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Hipocampo/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Estrés Psicológico/complicaciones , Sinapsis/efectos de los fármacos , Animales , Células Cultivadas , Enfermedad Crónica , AMP Cíclico/metabolismo , Depresión/metabolismo , Depresión/fisiopatología , Depresión/psicología , Sacarosa en la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Conducta Alimentaria/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Masculino , Proteínas del Tejido Nervioso/genética , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Sistemas de Mensajero Secundario/efectos de los fármacos , Sinapsis/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. METHODS: GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. RESULTS: L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in â¼70% of colonic L-cells and 50% of small intestinal L-cells. CONCLUSIONS/INTERPRETATION: GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.
Asunto(s)
Aceite de Maíz/farmacología , Células Enteroendocrinas/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Aceite de Oliva/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Células Enteroendocrinas/efectos de los fármacos , Femenino , Masculino , Ratones Transgénicos , Aceite de Oliva/administración & dosificación , Cultivo Primario de Células , Sistemas de Mensajero SecundarioRESUMEN
Bombesin receptor subtype-3 (BRS-3) is an orphan G-protein coupled receptor which is classified in the bombesin receptor (BnR) family with which it shares high homology. It is present widely in the central nervous system and peripheral tissues and primarily receptor-knockout studies suggest it is involved in metabolic-glucose-insulin homeostasis, feeding and other CNS behaviors, gastrointestinal motility and cancer growth. However, the role of BRS-3 physiologically or in pathologic disorders has been not well defined because the natural ligand is unknown. Until recently, no selective agonists/antagonists were available; however, recently synthetic high-affinity agonists, chiral-diazepines nonpeptide-analogs (3F, 9D, 9F, 9G) with low CNS penetrance, were described, but are not well-categorized pharmacologically or in different labarotory species. The present study characterizes the affinities, potencies, selectivities of the chiral-diazepine BRS-3 agonists in human and rodents (mice,rat). In human BRS-3 receptors, the relative affinities of the chiral-diazepines was 9G>9D>9F>3F; each was selective for BRS-3. For stimulating PLC activity, in h-BRS-3 each of the four chiral diazepine analogs was fully efficacious and their relative potencies were: 9G (EC50: 9 nM)>9D (EC50: 9.4 nM)>9F (EC50: 39 nM)>3F (EC50: 48 nM). None of the four chiral diazepine analogs activated r,m,h-GRPR/NMBR. The nonpeptide agonists showed marked differences from each other and a peptide agonist in receptor-coupling-stiochiometry and in affinities/potencies in different species. These results demonstrate that chiral diazepine analogs (9G, 9D, 9F, 3F) have high/affinity/potency for the BRS-3 receptor in human and rodent cells, but different coupling-relationships and species differences from a peptide agonist.
Asunto(s)
Azepinas/farmacología , Receptores de Bombesina/agonistas , Animales , Células 3T3 BALB , Evaluación Preclínica de Medicamentos , Péptido Liberador de Gastrina/farmacología , Humanos , Concentración 50 Inhibidora , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Ratas , Sistemas de Mensajero Secundario , Especificidad de la EspecieRESUMEN
The phosphodiesterase inhibitor (PDEI)/eNOS enhancer KMUP-1, targeting G-protein coupled receptors (GPCRs), improves dyslipidemia. We compared its lipid-lowering effects with simvastatin and explored hormone-sensitive lipase (HSL) translocation in hepatic fat loss. KMUP-1 HCl (1, 2.5, and 5 mg/kg/day) and simvastatin (5 mg/kg/day) were administered in C57BL/6J male mice fed a high-fat diet (HFD) by gavage for 8 weeks. KMUP-1 inhibited HFD-induced plasma/liver TG, total cholesterol, and LDL; increased HDL/3-hydroxy-3-methylglutaryl-CoA reductase (HMGR)/Rho kinase II (ROCK II)/PPARγ/ABCA1; and decreased liver and body weight. KMUP-1 HCl in drinking water (2.5 mg/200 ml tap water) for 1-14 or 8-14 weeks decreased HFD-induced liver and body weight and scavenger receptor class B type I expression and increased protein kinase A (PKA)/PKG/LDLRs/HSL expression and immunoreactivity. In HepG2 cells incubated with serum or exogenous mevalonate, KMUP-1 (10(-7)â¼10(-5) M) reversed HMGR expression by feedback regulation, colocalized expression of ABCA1/apolipoprotein A-I/LXRα/PPARγ, and reduced exogenous geranylgeranyl pyrophosphate/farnesyl pyrophosphate (FPP)-induced RhoA/ROCK II expression. A guanosine 3',5'-cyclic monophosphate (cGMP) antagonist reversed KMUP-1-induced ROCK II reduction, indicating cGMP/eNOS involvement. KMUP-1 inceased PKG and LDLRs surrounded by LDL and restored oxidized LDL-induced PKA expresion. Unlike simvastatin, KMUP-1 could not inhibit (14)C mevalonate formation. KMUP-1 could, but simvastatin could not, decrease ROCK II expression by exogenous FPP/CGPP. KMUP-1 improves HDL via PPARγ/LXRα/ABCA1/Apo-I expression and increases LDLRs/PKA/PKG/HSL expression and immunoreactivity, leading to TG hydrolysis to lower hepatic fat and body weight.
Asunto(s)
Hiperlipoproteinemias/tratamiento farmacológico , Hipolipemiantes/farmacología , Piperidinas/farmacología , Xantinas/farmacología , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Dieta Alta en Grasa/efectos adversos , Evaluación Preclínica de Medicamentos , Células Hep G2 , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Hiperlipoproteinemias/etiología , Hipolipemiantes/uso terapéutico , Grasa Intraabdominal/efectos de los fármacos , Grasa Intraabdominal/fisiología , Lipoproteínas HDL/sangre , Lipoproteínas LDL/metabolismo , Hígado/patología , Masculino , Ácido Mevalónico/metabolismo , Ratones Endogámicos C57BL , PPAR gamma/metabolismo , Piperidinas/uso terapéutico , Receptores de LDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Sistemas de Mensajero Secundario , Esterol Esterasa/metabolismo , Xantinas/uso terapéuticoRESUMEN
Diabetes is a global disease, in which pancreatic dysfunction is an important pathological process. In previous years, interest in the biological activities of seaweed has increased. Fucoidan is an extract of the seaweed Fucus vesiculosus, which has been widely investigated. The present study aimed to determine the effects of fucoidan on insulin stimulation and pancreatic protection in vivo and in vitro. GotoKakizaki (GK) rats were provided with free access to standard food, with or without fucoidan, for 13 weeks, following which the body weights, and blood glucose and serum insulin levels of the rats were measured. Wistar rats were used as a control. In addition, the RIN5F rat insulinsecreting cell line was treated with fucoidan in high glucose conditions, following which the dosedependent and timedependent effects of fucoidan were determined, and the concentration of insulin was measured. Glybenclamide was used as a positive control. In vivo, the body weight and serum insulin levels decreased, whereas blood glucose levels increased significantly in the GK rats, compared with the Wistar control rats. Although, fucoidan did not improve changes in body weight, the increased blood glucose levels were reduced and the decreased serum insulin levels were increased in the GK rats following oral administration of fucoidan. In vitro, fucoidan did not exhibit significant cytotoxicity towards the RIN5F cells, and the insulin secretion increased significantly in a dose and timedependent manner. Treatment with amylin, an islet amyloid polypeptide and glybenclamide inhibitor, did not inhibit the stimulatory activity of fucoidan. The results of the present study also demonstrated that the concentration of cyclic adenosine monophosphate (cAMP) was significantly increased in the fucoidantreated RIN5F cells, and this increase was dose and timedependent. In addition, treatment with a phosphodiesterase inhibitor, which decreases the degradation of cAMP, significantly increased fucoidaninduced insulin secretion, whereas treatment with an adenylyl cyclase inhibitor, which decreases the generation of cAMP, significantly decreased fucoidaninduced insulin secretion. In conclusion, these data indicated that fucoidan may stimulate insulin secretion and provide pancreatic protection via the cAMP signaling pathway, in vivo and in vitro.
Asunto(s)
Hipoglucemiantes/farmacología , Insulina/fisiología , Páncreas/efectos de los fármacos , Polisacáridos/farmacología , Sistemas de Mensajero Secundario , Animales , Glucemia , Línea Celular , AMP Cíclico/metabolismo , Evaluación Preclínica de Medicamentos , Gliburida/farmacología , Masculino , Páncreas/metabolismo , Páncreas/patología , Ratas WistarRESUMEN
SCOPE: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis. METHODS AND RESULTS: Via Oil red O staining, triglyceride, and ß-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid ß-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis. CONCLUSION: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment.