RESUMEN
BACKGROUND: The central nucleus of the amygdala (CeA) is part of the dopaminergic reward system and controls energy balance. Recently, a cluster of neurons was identified as responsive to the orexigenic effect of ghrelin and fasting. However, the signaling pathway by which ghrelin and fasting induce feeding is unknown. AMP-activated protein kinase (AMPK) is a cellular energy sensor, and its Thr172 phosphorylation (AMPKThr172) in the mediobasal hypothalamus regulates food intake. However, whether the expression and activation of AMPK in CeA could be one of the intracellular signaling activated in response to ghrelin and fasting eliciting food intake is unknown. AIM: To evaluate the activation of AMPK into CeA in response to ghrelin, fasting, and 2-deoxy-D-glucose (2DG) and whether feeding accompanied these changes. In addition, to investigate whether the inhibition of AMPK into CeA could decrease food intake. METHODS: On a chow diet, eight-week-old Wistar male rats were stereotaxically implanted with a cannula in the CeA to inject several modulators of AMPKα1/2Thr172 phosphorylation, and we performed physiological and molecular assays. KEY FINDINGS: Fasting increased, and refeeding reduced AMPKThr172 in the CeA. Intra-CeA glucose injection decreased feeding, whereas injection of 2DG, a glucoprivation inductor, in the CeA, increased food intake and blood glucose, despite faint increases in AMPKThr172. Intra-CeA ghrelin injection increased food intake and AMPKThr172. To further confirm the role of AMPK in the CeA, chronic injection of Melanotan II (MTII) in CeA reduced body mass and food intake over seven days together with a slight decrease in AMPKThr172. SIGNIFICANCE: Our findings identified that AMPK might be part of the signaling machinery in the CeA, which responds to nutrients and hormones contributing to feeding control. The results can contribute to understanding the pathophysiological mechanisms of altered feeding behavior/consumption, such as binge eating of caloric-dense, palatable food.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Núcleo Amigdalino Central , Ingestión de Alimentos , Ayuno , Ghrelina , Ratas Wistar , Animales , Masculino , Ghrelina/metabolismo , Ghrelina/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Fosforilación/efectos de los fármacos , Núcleo Amigdalino Central/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Ratas , Transducción de Señal/efectos de los fármacos , Desoxiglucosa/farmacología , Desoxiglucosa/metabolismo , Conducta Alimentaria/efectos de los fármacos , Glucosa/metabolismoRESUMEN
BACKGROUND: Glycolytic inhibitor 2-deoxy-d-glucose (2-DG) binds to hexokinase in a non-competitive manner and phosphoglucose isomerase in a competitive manner, blocking the initial steps of the glycolytic pathway. Although 2-DG stimulates endoplasmic reticulum (ER) stress, activating the unfolded protein response to restore protein homeostasis, it is unclear which ER stress-related genes are modulated in response to 2-DG treatment in human primary cells. Here, we aimed to determine whether the treatment of monocytes and monocyte-derived macrophages (MDMs) with 2-DG leads to a transcriptional profile specific to ER stress. METHODS: We performed bioinformatics analysis to identify differentially expressed genes (DEGs) in previously reported RNA-seq datasets of 2-DG treated cells. RT-qPCR was performed to verify the sequencing data on cultured MDMs. RESULTS: A total of 95 common DEGs were found by transcriptional analysis of monocytes and MDMs treated with 2-DG. Among these, 74 were up-regulated and 21 were down-regulated. Multitranscript analysis showed that DEGs are linked to integrated stress response (GRP78/BiP, PERK, ATF4, CHOP, GADD34, IRE1α, XBP1, SESN2, ASNS, PHGDH), hexosamine biosynthetic pathway (GFAT1, GNA1, PGM3, UAP1), and mannose metabolism (GMPPA and GMPPB). CONCLUSIONS: Results reveal that 2-DG triggers a gene expression program that might be involved in restoring protein homeostasis in primary cells. GENERAL SIGNIFICANCE: 2-DG is known to inhibit glycolysis and induce ER stress; however, its effect on gene expression in primary cells is not well understood. This work shows that 2-DG is a stress inducer shifting the metabolic state of monocytes and macrophages.
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Glucosa , Monocitos , Humanos , Glucosa/metabolismo , Monocitos/metabolismo , Endorribonucleasas/metabolismo , Proteínas Serina-Treonina Quinasas , Respuesta de Proteína Desplegada/genética , Macrófagos/metabolismo , Chaperón BiP del Retículo Endoplásmico , Desoxiglucosa/farmacología , Desoxiglucosa/metabolismo , Expresión Génica , Sestrinas/metabolismoRESUMEN
Objectives: Mesenchymal Stem/Stromal Cells (MSC) are promising therapeutic tools for inflammatory diseases due to their potent immunoregulatory capacities. Their suppressive activity mainly depends on inflammatory cues that have been recently associated with changes in MSC bioenergetic status towards a glycolytic metabolism. However, the molecular mechanisms behind this metabolic reprogramming and its impact on MSC therapeutic properties have not been investigated. Methods: Human and murine-derived MSC were metabolically reprogramed using pro-inflammatory cytokines, an inhibitor of ATP synthase (oligomycin), or 2-deoxy-D-glucose (2DG). The immunosuppressive activity of these cells was tested in vitro using co-culture experiments with pro-inflammatory T cells and in vivo with the Delayed-Type Hypersensitivity (DTH) and the Graph versus Host Disease (GVHD) murine models. Results: We found that the oligomycin-mediated pro-glycolytic switch of MSC significantly enhanced their immunosuppressive properties in vitro. Conversely, glycolysis inhibition using 2DG significantly reduced MSC immunoregulatory effects. Moreover, in vivo, MSC glycolytic reprogramming significantly increased their therapeutic benefit in the DTH and GVHD mouse models. Finally, we demonstrated that the MSC glycolytic switch effect partly depends on the activation of the AMPK signaling pathway. Conclusion: Altogether, our findings show that AMPK-dependent glycolytic reprogramming of MSC using an ATP synthase inhibitor contributes to their immunosuppressive and therapeutic functions, and suggest that pro-glycolytic drugs might be used to improve MSC-based therapy.
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Proteínas Quinasas Activadas por AMP/metabolismo , Glucólisis/efectos de los fármacos , Enfermedad Injerto contra Huésped/inmunología , Hipersensibilidad Tardía/inmunología , Células Madre Mesenquimatosas/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Animales , Antimetabolitos/farmacología , Linfocitos T CD4-Positivos , Desoxiglucosa/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Humanos , Inmunoterapia , Ácido Láctico/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Ratones , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Oligomicinas/farmacología , Fosforilación Oxidativa , Consumo de OxígenoRESUMEN
Growth hormone (GH) is secreted during hypoglycemia, and GH-responsive neurons are found in brain areas containing glucose-sensing neurons that regulate the counter-regulatory response (CRR). However, whether GH modulates the CRR to hypoglycemia via specific neuronal populations is currently unknown. Mice carrying ablation of GH receptor (GHR) either in leptin receptor (LepR)- or steroidogenic factor-1 (SF1)-expressing cells were studied. We also investigated the importance of signal transducer and activator of transcription 5 (STAT5) signaling in SF1 cells for the CRR. GHR ablation in LepR cells led to impaired capacity to recover from insulin-induced hypoglycemia and to a blunted CRR caused by 2-deoxy-d-glucose (2DG) administration. GHR inactivation in SF1 cells, which include ventromedial hypothalamic neurons, also attenuated the CRR. The reduced CRR was prevented by parasympathetic blockers. Additionally, infusion of 2DG produced an abnormal hyperactivity of parasympathetic preganglionic neurons, whereas the 2DG-induced activation of anterior bed nucleus of the stria terminalis neurons was reduced in mice without GHR in SF1 cells. Mice carrying ablation of Stat5a/b genes in SF1 cells showed no defects in the CRR. In summary, GHR expression in SF1 cells is required for a normal CRR, and these effects are largely independent of STAT5 pathway.-Furigo, I. C., de Souza, G. O., Teixeira, P. D. S., Guadagnini, D., Frazão, R., List, E. O., Kopchick, J. J., Prada, P. O., Donato, J., Jr. Growth hormone enhances the recovery of hypoglycemia via ventromedial hypothalamic neurons.
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Hormona del Crecimiento/farmacología , Hipoglucemia/tratamiento farmacológico , Hipotálamo/efectos de los fármacos , Neuronas/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Animales , Desoxiglucosa/farmacología , Hipoglucemia/fisiopatología , Hipotálamo/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Receptores de Somatotropina/genética , Receptores de Somatotropina/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismoRESUMEN
Escherichia coli strain 15 (ATCC 9723), which forms robust biofilms, was grown under optimal biofilm conditions in NaCl-free Luria-Bertani broth (LB*) or in LB* supplemented with one of the non-metabolizable analogues 2-deoxy-d-glucose (2DG), methyl α-d-mannopyranoside (αMM), or methyl α-d-glucopyranoside (αMG). Biofilm growth was inhibited by mannose analogue 2DG even at very low concentration in unbuffered medium, and the maximal inhibition was enhanced in the presence of either 100 mM KPO4 or 100 mM MOPS, pH 7.5; in buffered medium, concentrations of 0.02 % (1.2 mM) or more inhibited growth nearly completely. In contrast, mannose analogue αMM, which should not be able to enter the cells but has been reported to inhibit biofilm growth by binding to FimH, did not exhibit strong inhibition even at concentrations up to 1.8 % (108 mM). The glucose analogue αMG inhibited biofilm growth, but much less strongly than did 2DG. None of the analogues inhibited planktonic growth or caused a change in pH of the unbuffered medium. Similar inhibitory effects of the analogues were observed in minimal medium. The effects were not strain-specific, as 2DG and αMG also inhibited the weak biofilm growth of E. coli K12.
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Antimetabolitos/farmacología , Biopelículas/crecimiento & desarrollo , Desoxiglucosa/farmacología , Escherichia coli/crecimiento & desarrollo , AMP Cíclico/farmacología , Escherichia coli/efectos de los fármacos , Glucosa-6-Fosfato/farmacología , Metilglucósidos/farmacología , Metilmanósidos/farmacologíaRESUMEN
Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca(2+) release and protein kinase activation and long-term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose and [(3) H]2-deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin-bound testosterone-induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4myc-eGFP. Subsequently, testosterone increased GLUT4myc-GFP exposure at the plasma membrane. Inhibition of Akt by the Akt-inhibitor-VIII had no effect. However, inhibition of Ca(2+) /calmodulin protein kinase (CaMKII) (KN-93 and autocamtide-2 related inhibitory peptide II) and AMP-activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4myc-eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4-dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes.
Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Glucosa/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Testosterona/farmacología , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Desoxiglucosa/análogos & derivados , Desoxiglucosa/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Androgénicos/metabolismoRESUMEN
Sucrose synthase (SUS) is a key enzyme of carbon metabolism in heterotrophic tissues of plants. The Arabidopsis genome contains six SUS genes. Two members of this family, namely AtSUS2 (At5g49190) and AtSUS3 (At4g02280) are strongly and differentially expressed in Arabidopsis seed. Expression analysis was carried out using SUS:promoter-GUS fusion lines in a wild-type genetic background or in a mutant carrying a lesion in the transcription factor LEAFY COTYLEDON 2 (LEC2; At1g28300). The accumulation patterns of mRNA, protein, and SUS activity were altered in the lec2 mutant during seed development 9-18 days after flowering. This indicates that LEC2 acts epistatically on the expression of AtSUS2 and AtSUS3. It appears that LEC2 is required for cotyledon-specific expression of both SUS genes but it is not responsible for expression in the radicle tip during embryo development. The AtSUS2 promoter was induced in planta by feeding of glucose but less so by sucrose and trehalose. Non-phosphorylable glucose analogs such as 3-O-methyl-glucose and 2-deoxyglucose also caused an induction, suggesting that sugar signaling proceeds by a hexokinase-independent pathway, possibly involving hexose sensing. Analysis of transgenic lines carrying of truncated versions of the AtSUS2:promoter fused to Beta-glucuronidase activity revealed an internal 421 bp region that was responsible for expression in seeds. Bioinformatic sequence analysis revealed regulatory cis-elements putatively responsible for the spatio-temporal pattern of AtSUS2 expression such as the SEF3 (aaccca) and W-box (ttgact) motifs. These findings are discussed in relation to the roles played by AtSUS2, AtSUS3 and LEC2 in the biosynthesis of seed storage products in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Glucosa/metabolismo , Semillas/crecimiento & desarrollo , Factores de Transcripción/metabolismo , 3-O-Metilglucosa/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sitios de Unión , Cotiledón/genética , Desoxiglucosa/farmacología , Flores , Regulación de la Expresión Génica de las Plantas , Glucosa/farmacología , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo , Familia de Multigenes , Mutación , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Semillas/genética , Semillas/metabolismo , Sacarosa/metabolismo , Sacarosa/farmacología , Factores de Transcripción/genética , Trehalosa/metabolismo , Trehalosa/farmacologíaRESUMEN
PURPOSE: Metformin is a widely used antidiabetic drug whose anticancer effects, mediated by the activation of AMP-activated protein kinase (AMPK) and reduction of mTOR signaling, have become noteworthy. Chemotherapy produces genotoxic stress and induces p53 activity, which can cross-talk with AMPK/mTOR pathway. Herein, we investigate whether the combination of metformin and paclitaxel has an effect in cancer cell lines. EXPERIMENTAL DESIGN: Human tumors were xenografted into severe combined immunodeficient (SCID) mice and the cancer cell lines were treated with only paclitaxel or only metformin, or a combination of both drugs. Western blotting, flow cytometry, and immunohistochemistry were then used to characterize the effects of the different treatments. RESULTS: The results presented herein show that the addition of metformin to paclitaxel leads to quantitative potentialization of molecular signaling through AMPK and a subsequent potent inhibition of the mTOR signaling pathway. Treatment with metformin and paclitaxel resulted in an increase in the number of cells arrested in the G(2)-M phase of the cell cycle, and decreased the tumor growth and increased apoptosis in tumor-bearing mice, when compared with individual drug treatments. CONCLUSION: We have provided evidence for a convergence of metformin and paclitaxel induced signaling at the level of AMPK. This mechanism shows how different drugs may cooperate to augment antigrowth signals, and suggests that target activation of AMPK by metformin may be a compelling ally in cancer treatment.
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Proteínas Quinasas Activadas por AMP/metabolismo , Antineoplásicos/farmacología , Activación Enzimática/efectos de los fármacos , Hipoglucemiantes/farmacología , Metformina/farmacología , Neoplasias/metabolismo , Animales , Antimetabolitos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxiglucosa/farmacología , Sinergismo Farmacológico , Humanos , Masculino , Ratones , Ratones SCID , Paclitaxel/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Neuroglucopenia induced by 2-deoxy-D-glucose (2DG) activates hypothalamic glucoreceptors leading to increased hepatic glucose production and insulin inhibition. This response is similar to what is observed with intravenous injection of angiotensin II (Ang II). However, the involvement of an angiotensin-converting enzyme inhibitor on neuroglucopenia has not been investigated. The aim of this study was to determine the effects of chronic enalapril treatment on plasma glucose, insulin and lipid levels in response to neuroglucopenia. Male Holtzman rats (120-170 g) were chronically treated with enalapril (10 mg/kg per day) in the drinking water for two weeks. On the day of experiment the animals received an i.v. enalapril final dose one hour before the neuroglucopenic stress by 2DG infusion (500 mg/kg), and blood samples were drawn before and 5, 10, 20, 30 and 60 minutes following infusion. The hyperglycaemic response to 2DG was not significantly changed by enalapril treatment. The enalapril-treated group exhibited a peak of plasma insulin higher than controls. Plasma triglyceride showed a significant increase only in the enalapril group after neuroglucopenic stress (p < 0.05).These data show that chronic enalapril treatment changes insulin and triglyceride responses to neuroglucopenia, suggesting an effect on glucose-induced insulin secretion and the storage of triglycerides.
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Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Glucosa/metabolismo , Neuronas/metabolismo , Neuronas/patología , Peptidil-Dipeptidasa A/metabolismo , Estrés Fisiológico/efectos de los fármacos , Animales , Glucemia/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Colesterol/sangre , Desoxiglucosa/administración & dosificación , Desoxiglucosa/farmacología , Conducta de Ingestión de Líquido/efectos de los fármacos , Enalapril/administración & dosificación , Enalapril/farmacología , Insulina/sangre , Masculino , Neuronas/efectos de los fármacos , Ratas , Factores de Tiempo , Triglicéridos/sangre , Aumento de Peso/efectos de los fármacosRESUMEN
Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.
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Represión Catabólica , Galactosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Kluyveromyces/metabolismo , Lactosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Isótopos de Carbono/análisis , Membrana Celular/enzimología , Desoxiglucosa/metabolismo , Desoxiglucosa/farmacología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Galactosa/farmacología , Glucosa/metabolismo , Glucosa/farmacología , Glucosa-6-Fosfato/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Espacio Intracelular/enzimología , Kluyveromyces/enzimología , Kluyveromyces/genética , Lactosa/farmacología , Litio/farmacología , Microscopía Fluorescente , Proteínas de Transporte de Monosacáridos/genética , Fenotipo , Fosfoglucomutasa/antagonistas & inhibidores , Fosfoglucomutasa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Brain accumulation of the amyloid-ß peptide (Aß) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aß-induced oxidative stress and HK activity. We found that Aß triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aß oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aß-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aß-induced oxidative stress and neuronal death. Results suggest that Aß-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.
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Péptidos beta-Amiloides/fisiología , Hexoquinasa/metabolismo , Mitocondrias/metabolismo , Neuronas/enzimología , Animales , Supervivencia Celular , Citosol/metabolismo , Desoxiglucosa/farmacología , Humanos , Isoenzimas/química , Neuronas/citología , Estrés Oxidativo , Ratas , Especies Reactivas de Oxígeno , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
A series of 2-deoxy-D-glucose resistant mutants was obtained from wild type Beauveria bassiana 88 (Bb 88) by UV irradiation. Five mutants were characterized on Sabouraud Dextrose Agar and Chitin Agar for both radial extension rate (V(r)) and specific growth rate (micro). These values were obtained after adjusting morphometric data to a mathematical model used for filamentous fungi. Additionally, the protease and lipase potency index, conidial size, viability, and production levels were analyzed. The highest values for those physiological measurements were obtained by mutant 882.5 which, relative to Bb 88, showed a 30% reduction in half-life (LT(50)) on Sphenarium purpurascens, 70% on Acheta domesticus, and 71% on Tenebrio molitor larvae and adults. The half lethal concentration (LC(50)) on T. molitor larvae was 2.8 x 10(5)conidia/mL (con/mL) and 1.5 x 10(6)con/mL, respectively, for mutant 882.5 and Bb 88. This demonstrates that mutant 882.5 is more virulent, with up to an 80% reduction in LC(50). This work provides a convenient method for improving strains to be used in biocontrol as a suitable alternative to transgenic constructs.
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Beauveria/patogenicidad , Desoxiglucosa/farmacología , Animales , Beauveria/efectos de los fármacos , Beauveria/genética , Escarabajos/crecimiento & desarrollo , Escarabajos/microbiología , Farmacorresistencia Fúngica , Saltamontes/crecimiento & desarrollo , Saltamontes/microbiología , Gryllidae/crecimiento & desarrollo , Gryllidae/microbiología , Larva/microbiología , Mutación , Control Biológico de VectoresRESUMEN
Increased glucose uptake as a principal energy source is a requirement for the continued survival of tumour cells. Facilitative glucose transporter-1 (GLUT1) and -3 (GLUT3) have been previously shown to be present and regulated in breast cancer cells and are associated with poor patient prognosis. In cancer cells, the cAMP secondary messenger pathway is known to potentiate described glucose transporter activators and regulate cell fate. However, no regulation of the glucose transporters in breast cancer cells by cAMP has previously been examined. Herein, we determined in the well-characterized breast cancer cell line ZR-75, if the cAMP analogue 8-br-cAMP was capable of regulating GLUT1 and GLUT3 expression and thus glucose uptake. We demonstrated that 8-br-cAMP transiently up-regulates GLUT3 mRNA levels. The use of actinomycin-D and the cloning of 1,200 bp upstream of the human GLUT3 promoter demonstrated that this regulation was transcriptional. Immunocytochemistry and Western blotting confirmed that the increase in mRNA was reflected by an increase in protein levels. No notable regulation of GLUT1 in the presence of 8-br-cAMP was detected. Finally, we determined using the non-metabolizable glucose analogue 2-DOG if this up-regulation in GLUT3 increased glucose uptake. We observed the presence of two uptake components, one corresponding to the Km of GLUT1/4 and the other to GLUT3. A doubling in the uptake velocity was observed only at the Km corresponding to GLUT3. In conclusion, we demonstrate and characterize for the first time, an up-regulation of GLUT3 mRNA, protein and glucose uptake by the cAMP pathway in breast cancer cells.
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Neoplasias de la Mama/metabolismo , AMP Cíclico/metabolismo , Regulación Neoplásica de la Expresión Génica , Transportador de Glucosa de Tipo 3/metabolismo , Glucosa/metabolismo , Transducción de Señal , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Transporte Biológico/efectos de los fármacos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Desoxiglucosa/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Transportador de Glucosa de Tipo 3/genética , Humanos , Inmunohistoquímica , Progesterona/farmacología , ARN Mensajero/metabolismoRESUMEN
The pathogenesis of cancer anorexia is multifactorial and associated with disturbances of the central physiological mechanisms controlling food intake. However, the neurochemical mechanisms responsible for cancer-induced anorexia are unclear. Here we show that chronic infusion of 5-amino-4imidazolecarboxamide-riboside into the third cerebral ventricle and a chronic peripheral injection of 2 deoxy-d-glucose promotes hypothalamic AMP-activated protein kinase (AMPK) activation, increases food intake, and prolongs the survival of anorexic tumor-bearing (TB) rats. In parallel, the pharmacological activation of hypothalamic AMPK in TB animals markedly reduced the hypothalamic production of inducible nitric oxide synthase, IL-1beta, and TNF-alpha and modulated the expression of proopiomelanocortin, a hypothalamic neuropeptide that is involved in the control of energy homeostasis. Furthermore, the daily oral and intracerebroventricular treatment with biguanide antidiabetic drug metformin also induced AMPK phosphorylation in the central nervous system and increased food intake and life span in anorexic TB rats. Collectively, the findings of this study suggest that hypothalamic AMPK activation reverses cancer anorexia by inhibiting the production of proinflammatory molecules and controlling the neuropeptide expression in the hypothalamus, reflecting in a prolonged life span in TB rats. Thus, our data indicate that hypothalamic AMPK activation presents an attractive opportunity for the treatment of cancer-induced anorexia.
Asunto(s)
Anorexia/etiología , Complejos Multienzimáticos/fisiología , Neoplasias/complicaciones , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/administración & dosificación , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Anorexia/enzimología , Desoxiglucosa/administración & dosificación , Desoxiglucosa/farmacología , Vías de Administración de Medicamentos , Hipotálamo/efectos de los fármacos , Hipotálamo/enzimología , Hipotálamo/metabolismo , Masculino , Metformina/administración & dosificación , Metformina/farmacología , Complejos Multienzimáticos/metabolismo , Trasplante de Neoplasias , Neoplasias/enzimología , Neoplasias/mortalidad , Neoplasias/patología , Neuronas/enzimología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Wistar , Ribonucleótidos/administración & dosificación , Ribonucleótidos/farmacología , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
We studied the effect of different concentrations of 2-deoxy-D-glucose on the L-[U-14C]leucine, L-[1-14C]leucine and [1-14C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-D-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from L-[U-14C]leucine and from [1-14C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-D-glucose inhibited the CO2 production and lipid synthesis from L-[U-14C] leucine. This compound did not inhibit either CO2 production, or lipid synthesis from [1-14C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-D-glucose on the protein synthesis from L-[U-14C]leucine. 2-deoxy-D-glucose at 2.0 mM did not show any effect either on CO2 production, or on lipid synthesis from L-[U-14C]lactate 10 mM and glucose 5.0 mM.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Desoxiglucosa/farmacología , Glicina/metabolismo , Leucina/metabolismo , Animales , Dióxido de Carbono/metabolismo , Radioisótopos de Carbono , Corteza Cerebral/metabolismo , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Técnicas In Vitro , Ácido Láctico/metabolismo , Lípidos/biosíntesis , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
We compared growth rate, cell glucose turnover and expression of ATP-binding-cassette (ABC) transporters in Leishmania amazonensis (LTB0016; LTB) versus LTB(160) selected for resistance against the ABC transporter blocker glibenclamide. Additionally, we evaluated the influence of drug-resistance on Leishmania sensitivity against 2-mercaptoacetate and 2-deoxyglucose. Our data demonstrate that (1) LTB(160) and LTB constitutively express ABC transporters for neutral substrates, (2) glibenclamide resistance induces the expression of organic anion ABC transporters, members of the drug resistance associated transporters subfamily, (3) LTB(160) parasites use less glucose as energy substrate and exhibit a slower glucose uptake than LTB cells, and (4) LTB(160) parasites are less sensitive to 2-mercaptoacetate and 2-deoxyglucose than the glibenclamide-sensitive Leishmania LTB. Together these and previous results indicate that the metabolic adaptations expressed in drug-resistant LTB(160) differ from those described for mammalian drug resistant cells and constitute general mechanisms that underlie drug resistance in Leishmania and may be helpful for identifying alternative strategies to circumvent drug resistance in leishmaniasis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Resistencia a Medicamentos/fisiología , Gliburida/farmacología , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/metabolismo , Transportadoras de Casetes de Unión a ATP/biosíntesis , Adaptación Fisiológica , Animales , Desoxiglucosa/farmacología , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Glucosa/metabolismo , Leishmania mexicana/crecimiento & desarrollo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Tioglicolatos/farmacologíaRESUMEN
Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity.
Asunto(s)
Arabidopsis/microbiología , Glucanos/metabolismo , Nicotiana/microbiología , Polisacáridos Bacterianos/farmacología , Xanthomonas campestris/patogenicidad , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/fisiología , Desoxiglucosa/farmacología , Necrosis , Hojas de la Planta/anatomía & histología , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/microbiología , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/fisiología , Eliminación de Secuencia , Nicotiana/efectos de los fármacos , Nicotiana/fisiologíaRESUMEN
In the present study, newborn male Wistar rats were injected, subcutaneously, five times, every other day, with monosodium glutamate (MSG, 4 g/kg bw) or saline (as control, C), during the neonatal period. MSG animals developed destruction of the arcuate nuclei (ARC) with absence of NPY-immunoreactive cell bodies, which impaired both the food intake (baseline) and the 2-deoxy-D-glucose (2DG) glucoprivic feeding response. Increases in the immunoreactivity of corticotropin-releasing hormone-cell bodies in the paraventricular nuclei might have developed to compensate for the atrophy of the pituitary in MSG-treated rats. After systemic 2DG injection, neither the C nor the MSG rats increased their food intake, but they showed similar hyperglycemic responses, whereas plasma free fatty acids (FFA) increased only in the C group. In other groups, 2DG, norepinephrine (NE), neostigmine (NEO) and saline were intracerebroventricularly (i.c.v.) administered. In this condition, impairment of the hyperglycemic and food intake responses, associated to a lower increase in plasma FFA levels, were observed. As opposed to this, the MSG treatment gives support to NE effects, enhancing food intake, as well as plasma glucose and FFA levels. After NEO, plasma glucose increased only in the MSG group, while plasma FFA levels were elevated in the C rats. Taken together, the results obtained after MSG treatment point to a separate neural control of the hyperglycemic response and of the lipid mobilization when stimulated by central 2DG, NE or NEO administration. It seems likely that the excitatory neural pathway that controls lipid metabolism and is present in C rats was destroyed by the MSG treatment.
Asunto(s)
Glucosa/deficiencia , Glutamato de Sodio/farmacología , Animales , Animales Recién Nacidos , Núcleo Arqueado del Hipotálamo/efectos de los fármacos , Núcleo Arqueado del Hipotálamo/metabolismo , Núcleo Arqueado del Hipotálamo/patología , Glucemia/análisis , Hormona Liberadora de Corticotropina/metabolismo , Desoxiglucosa/administración & dosificación , Desoxiglucosa/farmacología , Ingestión de Alimentos/efectos de los fármacos , Ácidos Grasos no Esterificados/sangre , Hiperglucemia/fisiopatología , Inyecciones Intravenosas , Inyecciones Intraventriculares , Masculino , Neostigmina/administración & dosificación , Neostigmina/farmacología , Neuropéptido Y/metabolismo , Norepinefrina/administración & dosificación , Norepinefrina/farmacología , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas , Ratas Wistar , Glutamato de Sodio/administración & dosificaciónRESUMEN
Using an antibiotic enrichment procedure, eight mutants of Streptomyces peucetius var. caesius were isolated for their sensitivity to the glucose analogue 2-deoxyglucose (DOG), from a DOG-resistant strain (Dog(R)). These mutants (Dog(S)) and their parent strain were examined for growth sensitivity to DOG, glucose kinase (Glk) activity, glucose uptake, and sensitivity to repression by glucose and other catabolites derived from it. No correlation was found between Glk levels or glucose uptake and carbon catabolite repression (CCR) in these strains. However, the ratio of glucose uptake to Glk activity, and thus the flux through glycolysis, seemed responsible for this effect. Among several products of glucose catabolism tested, fructose-1,6-bis-phosphate and phosphoenolpyruvate showed significant repression of anthracycline formation. These compounds also reduced anthracycline formation in a Dog(R) mutant insensitive to glucose repression. Our data suggest that Glk alone is not sufficient to elicit CCR in this microorganism, and gives the first physiological evidence supporting the hypothesis that some products of glucose catabolism are involved in CCR in Streptomyces.
Asunto(s)
Antimetabolitos/farmacología , Carbono/metabolismo , Desoxiglucosa/farmacología , Streptomyces/efectos de los fármacos , Streptomyces/enzimología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Mutación , Streptomyces/genética , Streptomyces/crecimiento & desarrolloRESUMEN
Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) participates in control of expression of genes involved in adaptive thermogenesis, muscle fiber type differentiation, and fuel homeostasis. The objective of the present study was to evaluate the participation of cold-induced PGC-1alpha expression in muscle fiber type-specific activity of proteins that belong to the insulin-signaling pathway. Rats were exposed to 4 degrees C for 4 days and acutely treated with insulin in the presence or absence of an antisense oligonucleotide to PGC-1alpha. Cold exposure promoted a significant increase of PGC-1alpha and uncoupling protein-3 protein expression in type I and type II fibers of gastrocnemius muscle. In addition, cold exposure led to higher glucose uptake during a hyperinsulinemic clamp, which was accompanied by higher expression and membrane localization of GLUT4 in both muscle fiber types. Cold exposure promoted significantly lower insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and Ser473 phosphorylation of acute transforming retrovirus thymoma (Akt) and an insulin-independent increase of Thr172 phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Inhibition of PGC-1alpha expression in cold-exposed rats by antisense oligonucleotide treatment diminished glucose clearance rates during a hyperinsulinemic clamp and reduced expression and membrane localization of GLUT4. Reduction of PGC-1alpha expression resulted in no modification of insulin-induced tyrosine phosphorylation of the IR and Ser473 phosphorylation of Akt. Finally, reduction of PGC-1alpha resulted in lower Thr172 phosphorylation of AMPK. Thus cold-induced hyperexpression of PGC-1alpha participates in control of skeletal muscle glucose uptake through a mechanism that controls GLUT4 expression and subcellular localization independent of the IR and Akt activities but dependent on AMPK.