RESUMEN
Kupffer cells (KCs) is the main macrophage in liver, and its inflammation is related to liver diseases. It has been shown that inflammatory macrophages are accompanied by changes in monounsaturated fatty acid (MUFA) content. However, the effect of gondoic acid (GA) on inflammation and its underlying mechanism have not been described. In the current study, we demonstrated that GA significantly inhibited the expression of pro-inflammatory factors in LPS-exposed KCs. Further research found that GA reduced lipopolysaccharide (LPS)-stimulated reactive oxygen species (ROS) levels and enhanced the expression of antioxidant genes. Meanwhile, GA obviously blocked the LPS-stimulated PKCθ/ERK/STAT3 signaling pathways to alleviate the inflammatory responses. These results demonstrated for the first time that GA improves KCs inflammation through the inhibition of ROS production and PKCθ/ERK/STAT3 signaling pathway, the results assist in the potential development of functional foods or prodrugs based on the GA rich plant oils.
Asunto(s)
Macrófagos del Hígado , Lipopolisacáridos , Ácidos Grasos Monoinsaturados/farmacología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Proteína Quinasa C-theta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de SeñalRESUMEN
The incurable Type 2 diabetes mellitus (T2DM) has now been considered a pandemic with only supportive care in existence. Due to the adverse effects of available anti-diabetic drugs, there arises a great urgency to develop new drug molecules. One of the alternatives that can be considered for the treatment of T2DM are natural compounds from traditionally used herbal medicine. The present study undertakes, an integrated multidisciplinary concept of Network Pharmacology to evaluate the efficacy of potent anti-diabetic compound from traditionally used anti-diabetic plants of north east India and followed by DFT analysis. In the course of the study, 22 plant species were selected on the basis of their use in traditional medicine for the treatment of T2DM by various ethnic groups of the north eastern region of India. Initially, a library of 1053 compounds derived from these plants was generated. This was followed by network preparation between compounds and targets based on the docking result. The compounds having the best network property were considered for DFT analysis. We have identified that auraptene, a monoterpene coumarin for its activity in the management of Type 2 diabetes mellitus and deciphered its unexplored probable mechanisms. Molecular dynamics simulation of the ligand-protein complexes also reveals the stable binding of auraptene with the target proteins namely, Protein Kinase C θ, Glucocorticoid receptor, 11-ß hydroxysteroid dehydrogenase 1 and Aldose Reductase, all of which form uniform interactions throughout the MD simulation trajectory. Therefore, this finding could provide new insights for the development of a new anti-diabetic drug.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Diabetes Mellitus Tipo 2 , Medicamentos Herbarios Chinos , Aldehído Reductasa , Cumarinas , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/uso terapéutico , Ensayos Analíticos de Alto Rendimiento , Humanos , Hidroxiesteroide Deshidrogenasas , Ligandos , Simulación del Acoplamiento Molecular , Monoterpenos , Farmacología en Red , Proteína Quinasa C-theta , Receptores de GlucocorticoidesRESUMEN
Ischemic stroke is a common cerebrovascular disease with the main cause considered to be cerebral ischemia and reperfusion (I/R), which exerts irreparable injury on nerve cells. Thus, the development of neuroprotective drugs is an urgent concern. Curcumin, a known antioxidant, has been found to have neuroprotective effects. To determine the protective mechanism of curcumin in ischemic stroke, oxygen and glucose deprivation/reoxygenation (OGD/R) was used to treat PC12 cells to mimic the cerebral I/R cell model. Curcumin (20 µM) was applied to OGD/R PC12 cells, followed by Ca2+ concentration, transepithelial electrical resistance (TEER), and cell permeability measurements. The results showed that OGD/R injury induced a decrease in TEER and increases in Ca2+ concentration and cell permeability. In contrast, curcumin alleviated these effects. The protein kinase C θ (PKC-θ) was associated with the protective function of curcumin in the OGD/R cell model. Moreover, the middle cerebral artery occlusion and reperfusion model (MCAO/R) was applied to simulate the I/R rat model. Our results demonstrated that curcumin could reverse the MCAO/R-induced increase in Ca2+ concentration and blood-brain barrier (BBB) disruption. Our study demonstrates the mechanisms by which curcumin exhibited a protective function against cerebral I/R through PKC-θ signaling by reducing BBB dysfunction.
Asunto(s)
Isquemia Encefálica/metabolismo , Curcumina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Proteína Quinasa C-theta/metabolismo , Daño por Reperfusión/metabolismo , Animales , Isquemia Encefálica/prevención & control , Curcumina/farmacología , Masculino , Fármacos Neuroprotectores/farmacología , Células PC12 , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/prevención & controlRESUMEN
Excessive energy intake leads to fat overload and the formation of lipotoxic compounds mainly derived from the saturated fatty acid palmitate (PAL), thus promoting insulin resistance (IR) in skeletal muscle. N-3 polyunsaturated fatty acids (n-3PUFA) may prevent lipotoxicity and IR. The purpose of this study was to examine the differential effects of n-3PUFA on fatty acid metabolism and insulin sensitivity in muscle cells. C2C12 myotubes were treated with 500 µM of PAL without or with 50 µM of alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) for 16 h. PAL decreased insulin-dependent AKT activation and glucose uptake and increased the synthesis of ceramides and diglycerides (DG) derivatives, leading to protein kinase Cθ activation. EPA and DHA, but not ALA, prevented PAL-decreased AKT activation but glucose uptake was restored to control values by all n-3PUFA vs. PAL. Total DG and ceramide contents were decreased by all n-3PUFA, but only EPA and DHA increased PAL ß-oxidation, decreased PAL incorporation into DG and reduced protein kinase Cθ activation. EPA and DHA emerge as better candidates than ALA to improve fatty acid metabolism in skeletal muscle cells, notably via their ability to increase mitochondrial ß-oxidation.
Asunto(s)
Ácidos Grasos Omega-3/farmacología , Mioblastos Esqueléticos/efectos de los fármacos , Palmitatos/toxicidad , Animales , Células Cultivadas , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina , Isoenzimas/fisiología , Fluidez de la Membrana/efectos de los fármacos , Ratones , Mioblastos Esqueléticos/metabolismo , Fosforilación , Proteína Quinasa C/fisiología , Proteína Quinasa C-thetaRESUMEN
Skeletal muscle plays a major role in the control of whole body glucose disposal in response to insulin stimulus. Excessive supply of fatty acids to this tissue triggers cellular and molecular disturbances leading to lipotoxicity, inflammation, mitochondrial dysfunctions, impaired insulin response and decreased glucose uptake. This study was conducted to analyze the preventive effect of docosahexaenoic acid (DHA), a long-chain polyunsaturated n-3 fatty acid, against insulin resistance, lipotoxicity and inflammation in skeletal muscle at doses compatible with nutritional supplementation. DHA (30 µM) prevented insulin resistance in C2C12 myotubes exposed to palmitate (500 µM) by decreasing protein kinase C (PKC)-θ activation and restoring cellular acylcarnitine profile, insulin-dependent AKT phosphorylation and glucose uptake. Furthermore, DHA protected C2C12 myotubes from palmitate- or lipopolysaccharide-induced increase in Ptgs2, interleukin 6 and tumor necrosis factor-α mRNA level, probably through the inhibition of p38 MAP kinase and c-Jun amino-terminal kinase. In LDLR -/- mice fed a high-cholesterol-high-sucrose diet, supplementation with DHA reaching up to 2% of daily energy intake enhanced the insulin-dependent AKT phosphorylation and reduced the PKC-θ activation in skeletal muscle. Therefore, DHA used at physiological doses participates in the regulation of muscle lipid and glucose metabolisms by preventing lipotoxicity and inflammation.
Asunto(s)
Suplementos Dietéticos , Ácidos Docosahexaenoicos/uso terapéutico , Aceites de Pescado/uso terapéutico , Resistencia a la Insulina , Metabolismo de los Lípidos , Músculo Esquelético/metabolismo , Miositis/prevención & control , Absorción Fisiológica , Animales , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/inmunología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevención & control , Dieta Occidental/efectos adversos , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/metabolismo , Aceites de Pescado/administración & dosificación , Glucosa/metabolismo , Miembro Posterior , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Músculo Esquelético/enzimología , Músculo Esquelético/inmunología , Miositis/sangre , Miositis/inmunología , Miositis/metabolismo , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/agonistas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , AtúnRESUMEN
HIV-1 infection cannot be cured due to reservoirs formed early after infection. Decreasing the massive CD4+ T cell activation that occurs at the beginning of the disease would delay reservoir seeding, providing a better prognosis for patients. CD4+ T cell activation is mediated by protein kinase C (PKC) theta (θ), which is involved in T-cell proliferation, as well as NF-κB, NF-AT, and AP-1 activation. We found that PKCθ activity increased viral replication, but also that HIV-1 induced higher activation of PKCθ in infected CD4+ T cells, creating a feedback loop. Therefore, specific inhibition of PKCθ activity could contribute to control HIV-1 replication. We tested the efficacy of seven PKCθ specific inhibitors to control HIV-1 replication in CD4+ T cells and selected two of the more potent and safer: CGX1079 and CGX0471. They reduced PKCθ phosphorylation at T538 and its translocation to the plasma membrane, which correlated with decreased HIV-1 retrotranscription through partial inhibition of SAMHD1 antiviral activity, rendering lower proviral integration. CGX1079 and CGX0471 also interfered with viral transcription, which would reduce the production of new virions, as well as the subsequent spread and infection of new targets that would increase the reservoir size. CGX1079 and CGX0471 did not completely abrogate T-cell functions such as proliferation and CD8-mediated release of IFN-γ in PBMCs from HIV-infected patients, thereby avoiding general immunosuppresion. Consequently, using PKCθ inhibitors as adjuvant of antiretroviral therapy in recently infected patients would decrease the pool of activated CD4+ T cells, thwarting proviral integration and reducing the reservoir size.
Asunto(s)
Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , VIH-1/efectos de los fármacos , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Retroelementos , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/virología , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , VIH-1/genética , VIH-1/fisiología , Humanos , Células Jurkat , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosforilación , Proteína Quinasa C-theta , Transporte de Proteínas , Proteína 1 que Contiene Dominios SAM y HD , Transcripción Genética , Integración Viral/efectos de los fármacos , Internalización del Virus , Replicación ViralRESUMEN
Protein kinase Cθ (PKCθ) regulates a key step in the activation of T cells. On the basis of its mechanism of action, inhibition of this kinase is hypothesized to serve as an effective therapy for autoimmune diseases such as rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis. Herein, the discovery of a small molecule PKCθ inhibitor is described, starting from a fragment hit 1 and advancing to compound 41 through the use of structure-based drug design. Compound 41 demonstrates excellent in vitro activity, good oral pharmacokinetics, and efficacy in both an acute in vivo mechanistic model and a chronic in vivo disease model but suffers from tolerability issues upon chronic dosing.
Asunto(s)
Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Área Bajo la Curva , Artritis Experimental/tratamiento farmacológico , Disponibilidad Biológica , Células Cultivadas , Cromatografía Liquida , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Descubrimiento de Drogas , Femenino , Humanos , Isoenzimas/metabolismo , Espectrometría de Masas , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Modelos Moleculares , Estructura Molecular , Unión Proteica , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Inhibidores de Proteínas Quinasas/farmacocinética , Estructura Terciaria de Proteína , Ratas , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T/efectos de los fármacosRESUMEN
We previously demonstrated that selective inhibition of protein kinase Cθ (PKCθ) with triazinone 1 resulted in dose-dependent reduction of paw swelling in a mouse model of arthritis.1,2 However, a high concentration was required for efficacy, thus providing only a minimal safety window. Herein we describe a strategy to deliver safer compounds based on the hypothesis that optimization of potency in concert with good oral pharmacokinetic (PK) properties would enable in vivo efficacy at reduced exposures, resulting in an improved safety window. Ultimately, transformation of 1 yielded analogues that demonstrated excellent potency and PK properties and fully inhibited IL-2 production in an acute model. In spite of good exposure, twice-a-day treatment with 17l in the glucose-6-phosphate isomerase chronic in vivo mouse model of arthritis yielded only moderate efficacy. On the basis of the exposure achieved, we conclude that PKCθ inhibition alone is insufficient for complete efficacy in this rodent arthritis model.
Asunto(s)
Antiinflamatorios/farmacología , Artritis Experimental/tratamiento farmacológico , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Artritis Experimental/metabolismo , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Interleucina-2/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , Ratones , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Resultado del TratamientoRESUMEN
We have demonstrated previously that n-3 PUFA endogenously produced by fat-1 transgenic mice regulate CD4+ T-cell function by affecting the formation of lipid rafts, liquid-ordered mesodomains in the plasma membrane. In the present study, we tested the effects of dietary sources of n-3 PUFA, i.e. fish oil (FO) or purified DHA, when compared with an n-6 PUFA-enriched maize oil control diet in DO11.10 T-cell receptor transgenic mice. Dietary n-3 PUFA were enriched in CD4+ T-cells, resulting in the increase of the n-3:n-6 ratio. Following antigen-specific CD4+ T-cell activation by B-lymphoma cells pulsed with the ovalbumin 323-339 peptide, the formation of liquid-ordered mesodomains at the immunological synapse relative to the whole CD4+ T-cell, as assessed by Laurdan labelling, was increased (P< 0·05) in the FO-fed group. The FO diet also suppressed (P< 0·05) the co-localisation of PKCθ with ganglioside GM1 (monosialotetrahexosylganglioside), a marker for lipid rafts, which is consistent with previous observations. In contrast, the DHA diet down-regulated (P< 0·05) PKCθ signalling by moderately affecting the membrane liquid order at the immunological synapse, suggesting the potential contribution of the other major n-3 PUFA components of FO, including EPA.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Ácidos Docosahexaenoicos/farmacología , Aceites de Pescado/farmacología , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Membrana Celular , Aceite de Maíz/química , Dieta/veterinaria , Ácidos Docosahexaenoicos/química , Regulación hacia Abajo , Ácidos Grasos Omega-6/química , Ácidos Grasos Omega-6/farmacología , Femenino , Gangliósido G(M1)/genética , Gangliósido G(M1)/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-thetaRESUMEN
In the present study, receptor induced 3D-QSAR model was developed for a set of 46 thieno[2,3-b]pyridine-5- carbonitrile PKC-θ inhibitors, to explore the structural requirements of the molecules necessary for PKC-θ inhibition. Since the chemical nature of the studied molecules was different from the crystal ligand of the selected protein, induced fit docking (IFD) protocol was employed to induce the conformational changes in the active site of the selected protein. Thereafter, all molecules were docked into the newly generated active site environment of the selected protein using glide docking program, and the 3D-QSAR analysis was performed in PHASE program utilizing the docking based alignment of the molecules. The best 3D-QSAR model was selected on the basis of the highest value of Q(2)test (0.600), and the selected model also showed high values of R(2)train , 0.915, Pearson-r, 0.801 and low value of SD, 0.241. The contour maps corresponding to the selected 3D-QSAR model, in combination with docking analysis, helped to explore the essential amino acid residues involved in binding, and structural requirements of the ligand molecules necessary for complementary fit with the active site of the protein. Therefore, the information revealed from the generated model can further be explored as a novel tool for the designing of new congener molecules that can serve as potential therapeutics for the treatment of various disease conditions associated with abnormal PKC-θ signalling.
Asunto(s)
Isoenzimas/antagonistas & inhibidores , Nitrilos/química , Nitrilos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/química , Piridinas/farmacología , Diseño de Fármacos , Humanos , Isoenzimas/metabolismo , Simulación del Acoplamiento Molecular , Proteína Quinasa C/metabolismo , Proteína Quinasa C-theta , Relación Estructura-Actividad CuantitativaRESUMEN
OBJECTIVE: To investigate the signaling mechanisms that might underlie the loss of anorectic response to insulin injections into the central nucleus of the amygdala (CeA) within 3 days of feeding a high fat diet. DESIGN AND METHODS: Protein samples from amygdala and hypothalamus of rats fed high or low fat diets were subjected to a phosphorylation screening assay. The effects of dietary fat intake on the expression and activation of protein kinase C theta (PKCθ) in brain regions was studied. Finally, lentiviral vectors were used to overexpress rat PKCθ unilaterally or bilaterally into the CeA of rats and the effects on food intake, body weight and insulin stimulation of Akt phosphorylation were studied. RESULTS: The level of pMARCKS (Myristoylated alanine-rich C-kinase substrate), a major substrate of PKCθ, was increased 116% in amygdala of high fat diet fed rats but reduced in the hypothalamus. High fat diets increased the level of PKCθ in a region specific manner in the brain and this PKCθ was activated by membrane association. Overexpressing rat PKCθ either unilaterally or bilaterally into the CeA inhibited insulin stimulation of Akt signaling and blocked the anorectic response to insulin injected into the amygdala. Bilaterally injected PKCθ rats gained more weight and body fat and had increased food intake when fed a high fat diet compared to the control rats that received a lentiviral-Green Fluorescent Protein construct. CONCLUSION: The data suggest that insulin may have a physiological role within the amygdala to regulate energy balance.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Peso Corporal , Ingestión de Energía , Insulina/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Tejido Adiposo/metabolismo , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/efectos de los fármacos , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dieta con Restricción de Grasas , Dieta Alta en Grasa , Grasas de la Dieta/administración & dosificación , Vectores Genéticos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Inmunohistoquímica , Resistencia a la Insulina , Isoenzimas/genética , Lentivirus/genética , Masculino , Fenotipo , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de SeñalRESUMEN
Protein kinase C-theta (PKCθ) is a protein kinase C (PKC) family member expressed predominantly in T lymphocytes, and extensive studies addressing its function have been conducted. PKCθ is the only T cell-expressed PKC that localizes selectively to the center of the immunological synapse (IS) following conventional T cell antigen stimulation, and this unique localization is essential for PKCθ-mediated downstream signaling. While playing a minor role in T cell development, early in vitro studies relying, among others, on the use of PKCθ-deficient (Prkcq(-/-)) T cells revealed that PKCθ is required for the activation and proliferation of mature T cells, reflecting its importance in activating the transcription factors nuclear factor kappa B, activator protein-1, and nuclear factor of activated T cells, as well as for the survival of activated T cells. Upon subsequent analysis of in vivo immune responses in Prkcq(-/-) mice, it became clear that PKCθ has a selective role in the immune system: it is required for experimental Th2- and Th17-mediated allergic and autoimmune diseases, respectively, and for alloimmune responses, but is dispensable for protective responses against pathogens and for graft-versus-leukemia responses. Surprisingly, PKCθ was recently found to be excluded from the IS of regulatory T cells and to negatively regulate their suppressive function. These attributes of PKCθ make it an attractive target for catalytic or allosteric inhibitors that are expected to selectively suppress harmful inflammatory and alloimmune responses without interfering with beneficial immunity to infections. Early progress in developing such drugs is being made, but additional studies on the role of PKCθ in the human immune system are urgently needed.
Asunto(s)
Enfermedades del Sistema Inmune/inmunología , Tolerancia Inmunológica , Sinapsis Inmunológicas/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/metabolismo , Tolerancia Inmunológica/efectos de los fármacos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/inmunología , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Terapia Molecular Dirigida , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C-theta , Transducción de Señal/efectos de los fármacosRESUMEN
The increase in PKCθ expression in the amygdala of rats fed high fat diet (HFD) has been related to the loss of the anorectic response to insulin injections into the central nucleus of the amygdala (CeA) in these animals. PKCθ overexpression in the CeA increases food intake, body weight and body fat and inhibits insulin stimulation of Akt signaling. To study the effects of bilateral overexpression of PKCθ in the CeA of rats on peripheral metabolism, rats were injected into the CeA or 3rd ventricle with a lentiviral (LV)-PKCθ construct or LV-Green fluorescent protein (GFP) construct as a control and fed either LFD or HFD. Insulin and glucose tolerance tests were undertaken and hepatic AMPK activation, Pepck, Srebp1c gene expression and lipid levels assayed. CeA LV-PKCθ injected rats increased food intake, body weight and body fat and increased hepatic, but not serum, triglyceride levels compared to control rats that received a CeA-LV-GFP construct. Hepatic AMP-kinase activity was reduced but expression of Pepck increased while serum insulin decreased, glucose tolerance improved and the hypoglycemic response to insulin was enhanced in CeA LV-PKCθ injected rats. In contrast, rats that received LV-PKCθ injections into the 3rd Ventricle did not show any changes in food intake or body weight although serum, but not hepatic, triglyceride levels were increased and glucose tolerance was impaired. The data suggest that activation of PKCθ in the CeA and hypothalamus have different effects on energy balance and peripheral metabolism and that insulin signaling in the amygdala regulates peripheral metabolism.
Asunto(s)
Núcleo Amigdalino Central/enzimología , Glucosa/metabolismo , Homeostasis/fisiología , Hipotálamo/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Adenilato Quinasa/metabolismo , Tejido Adiposo/fisiología , Animales , Peso Corporal/fisiología , Dieta con Restricción de Grasas , Dieta Alta en Grasa , Ingestión de Alimentos/fisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Isoenzimas/genética , Lentivirus/genética , Hígado/metabolismo , Masculino , Fosfoenolpiruvato Carboxiquinasa (GTP)/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Ratas Sprague-Dawley , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismoRESUMEN
Activation of delta-opioid receptors (DOR) is neuroprotective against hypoxic/ischemic injury in the cortex, which is at least partially related to its action against hypoxic/ischemic disruption of ionic homeostasis that triggers neuronal injury. Na(+) influx through TTX-sensitive voltage-gated Na(+) channels may be a main mechanism for hypoxia-induced disruption of K(+) homeostasis, with DOR activation attenuating the disruption of ionic homeostasis by targeting voltage-gated Na(+) channels. In the present study we examined the role of DOR in the regulation of Na(+) influx in anoxia and simulated ischemia (oxygen-glucose deprivation) as well as the effect of DOR activation on the Na(+) influx induced by a Na(+) channel opener without anoxic/ischemic stress and explored a potential PKC mechanism underlying the DOR action. We directly measured extracellular Na(+) activity in mouse cortical slices with Na(+) selective electrodes and found that (1) anoxia-induced Na(+) influx occurred mainly through TTX-sensitive Na(+) channels; (2) DOR activation inhibited the anoxia/ischemia-induced Na(+) influx; (3) veratridine, a Na(+) channel opener, enhanced the anoxia-induced Na(+) influx; this could be attenuated by DOR activation; (4) DOR activation did not reduce the anoxia-induced Na(+) influx in the presence of chelerythrine, a broad-spectrum PKC blocker; and (5) DOR effects were blocked by PKCßII peptide inhibitor, and PKCθ pseudosubstrate inhibitor, respectively. We conclude that DOR activation inhibits anoxia-induced Na(+) influx through Na(+) channels via PKC (especially PKCßII and PKCθ isoforms) dependent mechanisms in the cortex.
Asunto(s)
Lóbulo Frontal/metabolismo , Isoenzimas/fisiología , Lóbulo Parietal/metabolismo , Proteína Quinasa C/fisiología , Receptores Opioides delta/metabolismo , Canales de Sodio/metabolismo , Animales , Hipoxia de la Célula/fisiología , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Corteza Cerebral/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Proteína Quinasa C beta , Proteína Quinasa C-theta , Receptores Opioides delta/fisiología , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
PKC-theta (PKC-θ), a member of the novel protein kinase C family (nPKC), regulates a wide variety of functions in the periphery. However, its presence and role in the CNS has remained largely unknown. Recently, we demonstrated the presence of PKC-θ in the arcuate hypothalamic nucleus (ARC) and knockdown of PKC-θ from the ARC protected mice from developing diet-induced obesity. Another isoform of the nPKC group, PKC-delta (PKC-δ), is expressed in several non-hypothalamic brain sites including the thalamus and hippocampus. Although PKC-δ has been implicated in regulating hypothalamic glucose homeostasis, its distribution in the hypothalamus has not previously been described. In the current study, we used immunohistochemistry to examine the distribution of PKC-θ and -δ immunoreactivity in rat and mouse hypothalamus. We found PKC-θ immunoreactive neurons in several hypothalamic nuclei including the ARC, lateral hypothalamic area, perifornical area and tuberomammillary nucleus. PKC-δ immunoreactive neurons were found in the paraventricular and supraoptic nuclei. Double-label immunohistochemisty in mice expressing green fluorescent protein either with the long form of leptin receptor (LepR-b) or in orexin (ORX) neurons indicated that PKC-θ is highly colocalized in lateral hypothalamic ORX neurons but not in lateral hypothalamic LepR-b neurons. Double-label immunohistochemistry in oxytocin-enhanced yellow fluorescent protein mice or arginine vasopressin-enhanced green fluorescent protein (AVP-EGFP) transgenic rats revealed a high degree of colocalization of PKC-δ within paraventricular and supraoptic oxytocin neurons but not the vasopressinergic neurons. We conclude that PKC-θ and -δ are expressed in different hypothalamic neuronal populations.
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Hipotálamo/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C/metabolismo , Animales , Arginina Vasopresina/metabolismo , Histidina Descarboxilasa/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Oxitocina/metabolismo , Proteína Quinasa C-theta , Ratas , Ratas Long-Evans , Receptores de Leptina/metabolismoRESUMEN
When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform theta (PKCtheta), a key regulator of TCR signaling. In contrast, PKCtheta was required for alloreactivity and GVHD induction. Furthermore, absence of PKCtheta raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCtheta-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCtheta is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.
Asunto(s)
Enfermedad Injerto contra Huésped/enzimología , Efecto Injerto vs Leucemia/fisiología , Isoenzimas/inmunología , Leucemia Experimental/enzimología , Leucemia Experimental/inmunología , Proteína Quinasa C/inmunología , Infecciones por Retroviridae/enzimología , Infecciones por Retroviridae/inmunología , Animales , Trasplante de Médula Ósea/inmunología , Femenino , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Efecto Injerto vs Leucemia/inmunología , Técnicas In Vitro , Isoantígenos , Isoenzimas/deficiencia , Isoenzimas/genética , Listeria monocytogenes/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-theta, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-theta was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-theta to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-theta nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-theta attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-theta activation, resulting in reduced insulin activity.
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Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Resistencia a la Insulina/fisiología , Isoenzimas/metabolismo , Ácido Palmítico/toxicidad , Proteína Quinasa C/metabolismo , Animales , Secuencia de Bases , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/toxicidad , Gluconeogénesis/efectos de los fármacos , Isoenzimas/deficiencia , Isoenzimas/genética , Leptina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido Palmítico/administración & dosificación , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Proteína Quinasa C-theta , ARN Interferente Pequeño/genética , Ratas , Ratas Long-Evans , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimologíaRESUMEN
GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKC(theta) (protein kinase C theta). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Evaluación Preclínica de Medicamentos/métodos , Transportador de Glucosa de Tipo 4/metabolismo , Hipoglucemiantes/uso terapéutico , Resistencia a la Insulina , Células Cultivadas , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/genética , Humanos , Hipoglucemiantes/aislamiento & purificación , Insulina/metabolismo , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Isoenzimas/genética , Proteína Quinasa 8 Activada por Mitógenos/genética , Fosfohidrolasa PTEN/genética , Ácido Palmítico/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C-theta , Transporte de Proteínas , Proteínas Quinasas S6 Ribosómicas/genética , TransfecciónRESUMEN
The protein kinase C (PKC) family of serine/threonine kinases is implicated in a wide variety of cellular processes. The PKC theta (PKCtheta) isoform is involved in TCR signal transduction and T cell activation and regulates T cell mediated diseases, including lung inflammation and airway hyperresponsiveness. Thus inhibition of PKCtheta enzyme activity by a small molecule represents an attractive strategy for the treatment of asthma. A PKCtheta high-throughput screening (HTS) campaign led to the identification of 4-(3-bromophenylamino)-5-(3,4-dimethoxyphenyl)-3-pyridinecarbonitrile 4a, a low microM ATP competitive PKCtheta inhibitor. Structure based hit-to-lead optimization led to the identification of 5-(3,4-dimethoxyphenyl)-4-(1H-indol-5-ylamino)-3-pyridinecarbonitrile 4p, a 70 nM PKCtheta inhibitor. Compound 4p was selective for inhibition of novel PKC isoforms over a panel of 21 serine/threonine, tyrosine, and phosphoinositol kinases, in addition to the conventional and atypical PKCs, PKCbeta, and PKCzeta, respectively. Compound 4p also inhibited IL-2 production in antiCD3/anti-CD28 activated T cells enriched from splenocytes.
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Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Nitrilos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Indoles/síntesis química , Indoles/química , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Isoenzimas/deficiencia , Isoenzimas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Moleculares , Estructura Molecular , Nitrilos/síntesis química , Nitrilos/química , Proteína Quinasa C/deficiencia , Proteína Quinasa C/efectos de los fármacos , Proteína Quinasa C-theta , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Estereoisomerismo , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Leptin-induced increases in insulin sensitivity are well established and may be related to the effects of leptin on lipid metabolism. However, the effects of leptin on the levels of lipid metabolites implicated in pathogenesis of insulin resistance and the effects of leptin on lipid-induced insulin resistance are unknown. The current study addressed in rats the effects of hyperleptinemia (HL) on insulin action and markers of skeletal muscle (SkM) lipid metabolism in the absence or presence of acute hyperlipidemia induced by an infusion of a lipid emulsion. Compared with controls (CONT), HL increased insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamp ( approximately 15%), and increased SkM Akt ( approximately 30%) and glycogen synthase kinase 3 alpha ( approximately 52%) phosphorylation. These improvements in insulin action were associated with decreased SkM triglycerides (TG; approximately 61%), elevated ceramides ( approximately 50%), and similar diacylglycerol (DAG) levels in HL compared with CONT. Acute hyperlipidemia in CONT decreased insulin sensitivity ( approximately 25%) and increased SkM DAG ( approximately 33%) and ceramide ( approximately 60%) levels. However, hyperlipidemia did not induce insulin resistance or SkM DAG and ceramide accumulation in HL. SkM total fatty acid transporter CD36, plasma membrane fatty acid binding protein, acetyl Co-A carboxylase phosphorylation, and fatty acid oxidation were similar in HL compared with CONT. However, HL decreased SkM protein kinase C theta (PKC theta), a kinase implicated in mediating the detrimental effects of lipids on insulin action. We conclude that increases in insulin sensitivity induced by HL are associated with decreased levels of SkM TG and PKC theta and increased SkM insulin signaling, but not with decreases in other lipid metabolites implicated in altering SkM insulin sensitivity (DAG and ceramide). Furthermore, insulin resistance induced by an acute lipid infusion is prevented by HL.