RESUMEN
AIMS: The purpose of this study was to evaluate the heterogeneities of glutamine metabolism in EGFR-TKI-resistant lung cancer cells and its potential as a therapeutic target. MAIN METHODS: Cell proliferation and cell cycle assays was performed by IncuCyte real-time analysis and flow cytometry, respectively. Tumor growth was assessed in xenografts implanted with HCC827 GR. An isotopologue analysis was conducted by LC-MS/MS using 13C-(U)-glutamine labeling to determine the amounts of metabolites. Cellular ATP and mitochondrial oxidative phosphorylation were determined by XFp analysis. KEY FINDINGS: We found that the cell growth of the two acquired EGFR-TKI-resistant lung cancer cells lines (HCC827 GR and H292 ER) depends on glutamine. In HCC827 GR, glutamine deficiency caused reduced GSH synthesis and, subsequently, enhanced ROS generation relative to their parental cells, HCC827. On the other hand, in H292 ER, glutamine mainly acted as a carbon source for TCA-cycle intermediates, and its depletion led to reduced mitochondrial ATP production. CB-839, a specific GLS inhibitor, inhibited the latter's conversion of glutamine to glutamate and exerted enhanced anti-proliferating effects on the two acquired EGFR-TKI-resistant lung cancer cell lines versus their parental cell lines. Moreover, oral administration of CB-839 significantly suppressed HCC827 GR tumor growth in the xenograft model. SIGNIFICANCE: These findings suggest that glutamine dependency in acquired EGFR-TKI-resistant lung cancer is heterogeneous and that inhibition of glutamine metabolism by CB-839 may serve as a therapeutic tool for acquired EGFR-TKI-resistant lung cancer.
Asunto(s)
Bencenoacetamidas/farmacología , Glutamina/metabolismo , Neoplasias Pulmonares/metabolismo , Tiadiazoles/farmacología , Apoptosis/efectos de los fármacos , Bencenoacetamidas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía Liquida/métodos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Glutamina/fisiología , Humanos , Mutación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Espectrometría de Masas en Tándem/métodos , Tiadiazoles/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Nutrient stress driven by glutamine deficiency activates EGFR signaling in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cells. EGFR signaling in the context of glutamine starvation is thought to be instigated by the transcriptional upregulation of EGFR ligands and functions as an adaptation mechanism to allow PDAC cells to maintain metabolic fitness. Having a clear view of the intricate signaling cascades potentiated by the metabolic induction of EGFR is important in understanding how these effector pathways influence cancer progression. In this study, we examined the complex signaling that occurs in PDAC cells when EGFR is activated by glutamine deprivation. We elucidate that the metabolic activation of EGFR is principally mediated by HB-EGF, and that other members of the ErbB receptor tyrosine kinase family are not activated by glutamine starvation. Additionally, we determine that glutamine depletion-driven EGFR signaling is associated with a specific receptor phosphorylation known to participate in a feedback loop, a process that is dependent on Erk. Lastly, we determine that K-Ras is required for glutamine depletion-induced Erk activation, as well as EGFR feedback phosphorylation, but is dispensable for Akt activation. These data provide important insights into the regulation of EGFR signaling in the context of metabolic stresses.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Carcinoma Ductal Pancreático/enzimología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Glutamina/fisiología , Factor de Crecimiento Similar a EGF de Unión a Heparina/fisiología , Humanos , Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The interplay between nutrient scarcity and signal transduction circuits is an important aspect of tumorigenesis that regulates many aspects of cancer progression. Glutamine is a critical nutrient for cancer cells, as it contributes to biosynthetic reactions that sustain cancer proliferation and growth. In tumors, because nutrient utilization can often outpace supply, glutamine levels can become limiting and oncogene-mediated metabolic rewiring triggers signaling cascades that support nutrient stress survival. Recently, we identified that in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine depletion can trigger p21-activated kinase (Pak) activation through EGFR signaling as a means to circumvent metabolic stress. Here, we elucidate that glutamine starvation, as well EGF stimulation, can enhance the presence of many different Pak phosphoforms, and that this activation only occurs in a subset of PDAC cells. Pak is a well-established effector of Rac1, and while Rac1 mutant variants can modulate the metabolic induction of Pak phosphorylation, Rac1 inhibition only partially attenuates Pak activation upon glutamine depletion. We decipher that in order to efficiently suppress metabolic activation of Pak, both EGFR and Rac1 signaling must be inhibited. These results provide a mechanistic understanding of how glutamine-regulated signal transduction can control Pak activation in PDAC cells.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Receptores ErbB/metabolismo , Neoplasias Pancreáticas/metabolismo , Transducción de Señal , Estrés Fisiológico , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Carcinoma Ductal Pancreático/enzimología , Activación Enzimática , Glutamina/fisiología , Humanos , Isoenzimas/metabolismo , Nutrientes , Neoplasias Pancreáticas/enzimología , Fosforilación , Células Tumorales CultivadasRESUMEN
Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLCVGLUT2 neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLCVGLUT2 neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLCVGLUT2 neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLCVGLUT2 neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLCVGLUT2 neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity.
Asunto(s)
Regulación del Apetito/fisiología , Ingestión de Líquidos/fisiología , Ingestión de Alimentos/fisiología , Locus Coeruleus/citología , Red Nerviosa/fisiología , Neuronas/fisiología , Rombencéfalo/fisiología , Análisis de la Célula Individual/métodos , Animales , Apetito/fisiología , Escala de Evaluación de la Conducta , Retroalimentación , Conducta Alimentaria/fisiología , Femenino , Glutamina/metabolismo , Glutamina/fisiología , Homeostasis/fisiología , Hambre/fisiología , Masculino , Ratones , Ratones Noqueados , Motivación/fisiología , Neuronas/efectos de los fármacos , Proteínas Recombinantes , Recompensa , Rombencéfalo/citología , Rombencéfalo/diagnóstico por imagen , Gusto/fisiología , Sed/fisiologíaRESUMEN
Africa, the ancestral home of all modern humans, is the most informative continent for understanding the human genome and its contribution to complex disease. To better understand the genetics of schizophrenia, we studied the illness in the Xhosa population of South Africa, recruiting 909 cases and 917 age-, gender-, and residence-matched controls. Individuals with schizophrenia were significantly more likely than controls to harbor private, severely damaging mutations in genes that are critical to synaptic function, including neural circuitry mediated by the neurotransmitters glutamine, γ-aminobutyric acid, and dopamine. Schizophrenia is genetically highly heterogeneous, involving severe ultrarare mutations in genes that are critical to synaptic plasticity. The depth of genetic variation in Africa revealed this relationship with a moderate sample size and informed our understanding of the genetics of schizophrenia worldwide.
Asunto(s)
Esquizofrenia/etnología , Esquizofrenia/genética , Transmisión Sináptica/genética , Factores de Edad , Trastorno Autístico/genética , Trastorno Bipolar/genética , Dopamina/fisiología , Femenino , Variación Genética , Glutamina/fisiología , Humanos , Masculino , Mutación , Vías Nerviosas/fisiopatología , Esquizofrenia/fisiopatología , Factores Sexuales , Sudáfrica/etnología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Viruses may hijack glycolysis, glutaminolysis, or fatty acid ß-oxidation of host cells to provide the energy and macromolecules required for efficient viral replication. Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens and modulates metabolism of host cells. Metabolic analysis of MDV-infected chicken embryonic fibroblasts (CEFs) identified elevated levels of metabolites involved in glutamine catabolism, such as glutamic acid, alanine, glycine, pyrimidine, and creatine. In addition, our results demonstrate that glutamine uptake is elevated by MDV-infected cells in vitro Although glutamine, but not glucose, deprivation significantly reduced cell viability in MDV-infected cells, both glutamine and glucose were required for virus replication and spread. In the presence of minimum glutamine requirements based on optimal cell viability, virus replication was partially rescued by the addition of the tricarboxylic acid (TCA) cycle intermediate, α-ketoglutarate, suggesting that exogenous glutamine is an essential carbon source for the TCA cycle to generate energy and macromolecules required for virus replication. Surprisingly, the inhibition of carnitine palmitoyltransferase 1a (CPT1a), which is elevated in MDV-infected cells, by chemical (etomoxir) or physiological (malonyl-CoA) inhibitors, did not reduce MDV replication, indicating that MDV replication does not require fatty acid ß-oxidation. Taken together, our results demonstrate that MDV infection activates anaplerotic substrate from glucose to glutamine to provide energy and macromolecules required for MDV replication, and optimal MDV replication occurs when the cells do not depend on mitochondrial ß-oxidation.IMPORTANCE Viruses can manipulate host cellular metabolism to provide energy and essential biosynthetic requirements for efficient replication. Marek's disease virus (MDV), an avian alphaherpesvirus, causes a deadly lymphoma in chickens and hijacks host cell metabolism. This study provides evidence for the importance of glycolysis and glutaminolysis, but not fatty acid ß-oxidation, as an essential energy source for the replication and spread of MDV. Moreover, it suggests that in MDV infection, as in many tumor cells, glutamine is used for generation of energetic and biosynthetic requirements of the MDV infection, while glucose is used biosynthetically.
Asunto(s)
Glucosa/metabolismo , Glutamina/metabolismo , Mardivirus/fisiología , Alphaherpesvirinae/metabolismo , Alphaherpesvirinae/fisiología , Animales , Embrión de Pollo , Pollos/virología , Glucosa/fisiología , Glutamina/fisiología , Glucólisis/fisiología , Herpesvirus Gallináceo 2/metabolismo , Herpesvirus Gallináceo 2/fisiología , Mardivirus/metabolismo , Enfermedad de Marek/metabolismo , Enfermedad de Marek/virología , Proteínas Virales/metabolismo , Replicación Viral/fisiologíaRESUMEN
As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.
Asunto(s)
Calcio/metabolismo , Activación del Canal Iónico/fisiología , Canales Catiónicos TRPM/fisiología , Animales , Asparagina/fisiología , Sitios de Unión , Glutamina/fisiología , Células HEK293 , Humanos , Anémonas de Mar , Canales Catiónicos TRPM/químicaRESUMEN
The green tea amino acid theanine is abundant in green tea rather than black and oolong teas, which are all made of the identical tea plant "Chanoki" (Camellia sinensis). Theanine has a molecular structure close to glutamine (GLN) compared to glutamic acid (Glu), in terms of the absence of a free carboxylic acid moiety from the gamma carbon position. Theanine efficiently inhibits [3H]GLN uptake without affecting [3H]Glu uptake in rat brain synaptosomes. In contrast to GLN, however, theanine markedly stimulates the abilities to replicate and to commit to a neuronal lineage following prolonged exposure in cultured neural progenitor cells (NPCs) prepared from embryonic and adult rodent brains. Upregulation of transcript expression is found for one of the GLN transporter isoforms, Slc38a1, besides the promotion of both proliferation and neuronal commitment along with acceleration of the phosphorylation of mechanistic target of rapamycin (mTOR) and relevant downstream proteins, in murine NPCs cultured with theanine. Stable overexpression of Slc38a1 similarly facilitates both cellular replication and neuronal commitment in pluripotent embryonic carcinoma P19â¯cells. In P19â¯cells with stable overexpression of Slc38a1, marked phosphorylation is seen for mTOR and downstream proteins in a manner insensitive to further additional phosphorylation by theanine. Taken together, theanine would exhibit a novel pharmacological property to up-regulate Slc38a1 expression for activation of the intracellular mTOR signaling pathway required for neurogenesis after sustained exposure in undifferentiated NPCs in the brain. In this review, a novel neurogenic property of the green tea amino acid theanine is summarized for embryonic and adult neurogenesis with a focus on the endogenous amino acid GLN on the basis of our accumulating evidence to date.
Asunto(s)
Encéfalo/efectos de los fármacos , Glutamatos/farmacología , Glutamina/fisiología , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Sistema de Transporte de Aminoácidos A/fisiología , Animales , Trastornos del Conocimiento/tratamiento farmacológico , Trastornos del Conocimiento/prevención & control , Método Doble Ciego , Predicción , Glutamatos/química , Glutamatos/uso terapéutico , Glutamina/química , Hipocampo/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Transgénicos , Trastornos del Humor/tratamiento farmacológico , Células-Madre Neurales/fisiología , Neurogénesis/fisiología , Fosforilación/efectos de los fármacos , Fitoterapia , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ensayos Clínicos Controlados Aleatorios como Asunto , Ratas , Trastornos del Sueño-Vigilia/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Té/químicaRESUMEN
Certain perceptual measures have been proposed as indirect assays of brain neurochemical status in people with migraine. One such measure is binocular rivalry, however, previous studies have not measured rivalry characteristics and brain neurochemistry together in people with migraine. This study compared spectroscopy-measured levels of GABA and Glx (glutamine and glutamate complex) in visual cortex between 16 people with migraine and 16 non-headache controls, and assessed whether the concentration of these neurochemicals explains, at least partially, inter-individual variability in binocular rivalry perceptual measures. Mean Glx level was significantly reduced in migraineurs relative to controls, whereas mean occipital GABA levels were similar between groups. Neither GABA levels, nor Glx levels correlated with rivalry percept duration. Our results thus suggest that the previously suggested relationship between rivalry percept duration and GABAergic inhibitory neurotransmitter concentration in visual cortex is not strong enough to enable rivalry percept duration to be reliably assumed to be a surrogate for GABA concentration, at least in the context of healthy individuals and those that experience migraine.
Asunto(s)
Trastornos Migrañosos/fisiopatología , Neurotransmisores/fisiología , Corteza Visual/fisiopatología , Percepción Visual/fisiología , Adulto , Estudios de Casos y Controles , Femenino , Ácido Glutámico/fisiología , Glutamina/fisiología , Humanos , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana Edad , Trastornos Migrañosos/diagnóstico por imagen , Trastornos Migrañosos/psicología , Migraña con Aura/diagnóstico por imagen , Migraña con Aura/fisiopatología , Migraña con Aura/psicología , Migraña sin Aura/diagnóstico por imagen , Migraña sin Aura/fisiopatología , Migraña sin Aura/psicología , Corteza Visual/diagnóstico por imagen , Adulto Joven , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Voltage-gated Cav Ca2+ channels play crucial roles in regulating gene transcription, neuronal excitability, and synaptic transmission. Natural or pathological variations in Cav channels have yielded rich insights into the molecular determinants controlling channel function. Here, we report the consequences of a natural, putatively disease-associated mutation in the CACNA1D gene encoding the pore-forming Cav1.3 α1 subunit. The mutation causes a substitution of a glutamine residue that is highly conserved in the extracellular S1-S2 loop of domain II in all Cav channels with a histidine and was identified by whole-exome sequencing of an individual with moderate hearing impairment, developmental delay, and epilepsy. When introduced into the rat Cav1.3 cDNA, Q558H significantly decreased the density of Ca2+ currents in transfected HEK293T cells. Gating current analyses and cell-surface biotinylation experiments suggested that the smaller current amplitudes caused by Q558H were because of decreased numbers of functional Cav1.3 channels at the cell surface. The substitution also produced more sustained Ca2+ currents by weakening voltage-dependent inactivation. When inserted into the corresponding locus of Cav2.1, the substitution had similar effects as in Cav1.3. However, the substitution introduced in Cav3.1 reduced current density, but had no effects on voltage-dependent inactivation. Our results reveal a critical extracellular determinant of current density for all Cav family members and of voltage-dependent inactivation of Cav1.3 and Cav2.1 channels.
Asunto(s)
Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/fisiología , Glutamina/fisiología , Mutación , Secuencia de Aminoácidos , Canales de Calcio Tipo L/química , Señalización del Calcio/fisiología , Secuencia Conservada , Glicina/química , Pérdida Auditiva/genética , Histidina/química , Humanos , Discapacidad Intelectual/genética , Activación del Canal Iónico/fisiología , Homología de Secuencia de Aminoácido , Transmisión Sináptica/fisiología , Secuenciación del ExomaRESUMEN
Mutations in the sodium bicarbonate cotransporter NBCe1 (SLC4A4) cause proximal renal tubular acidosis (pRTA). We recently described a novel pRTA mutation p.Gln913Arg (Q913R), inherited in compound heterozygous form with p.Arg510His (R510H). Q913R causes intracellular retention of NBCe1 and a 'gain of function' Cl- leak. To learn more about the importance of glutamine at position 913, we substituted a variety of alternative amino-acid residues (Cys, Glu, Lys, Leu, Ser) at position 913. Studying cRNA-injected Xenopus oocytes by voltage clamp, we find that most de novo mutants exhibit close-to-normal NBCe1 activity; only Q913K expresses a Cl- leak. Studying transiently-transfected, polarised kidney cells by fluorescence microscopy we find that most de novo mutants (except Q913E) are intracellularly retained. A 3D homology model predicts that Gln913 is located in the gating domain of NBCe1 and neighbours the 3D space occupied by another pRTA-associated residue (Arg881), highlighting an important and conformationally-sensitive region of NBCe1. We conclude that the intracellular retention of Q913R is caused by the loss of Gln at position 913, but that the manifestation of the Cl- leak is related to the introduction of Arg at position 913. Our findings will inform future studies to elucidate the nature and the consequences of the leak.
Asunto(s)
Arginina , Mutación con Ganancia de Función/genética , Glutamina , Espacio Intracelular/metabolismo , Dominios Proteicos/genética , Simportadores de Sodio-Bicarbonato , Acidosis Tubular Renal/genética , Acidosis Tubular Renal/patología , Animales , Arginina/genética , Arginina/fisiología , Bicarbonatos/metabolismo , Membrana Celular/metabolismo , Perros , Glutamina/genética , Glutamina/fisiología , Humanos , Células de Riñón Canino Madin Darby , Oocitos , Sodio/metabolismo , Simportadores de Sodio-Bicarbonato/química , Simportadores de Sodio-Bicarbonato/genética , Xenopus laevisRESUMEN
OBJECTIVE: To investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells. METHODS: shRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. RESULTS: ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01). CONCLUSIONS: ASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/efectos de los fármacos , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/fisiología , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Glutamina/efectos de los fármacos , Antígenos de Histocompatibilidad Menor/efectos de los fármacos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/fisiología , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Línea Celular Tumoral/fisiología , Neoplasias Colorrectales/fisiopatología , Regulación hacia Abajo/efectos de los fármacos , Técnicas de Silenciamiento del Gen/métodos , Glutamina/genética , Glutamina/fisiología , Humanos , Oncogenes/efectos de los fármacos , Oncogenes/genética , ARN Mensajero/fisiología , ARN Interferente Pequeño/farmacología , TransfecciónRESUMEN
During critical periods, all cortical neural circuits are refined to optimize their functional properties. The prevailing notion is that the balance between excitation and inhibition determines the onset and closure of critical periods. In contrast, we show that maturation of silent glutamatergic synapses onto principal neurons was sufficient to govern the duration of the critical period for ocular dominance plasticity in the visual cortex of mice. Specifically, postsynaptic density protein-95 (PSD-95) was absolutely required for experience-dependent maturation of silent synapses, and its absence before the onset of critical periods resulted in lifelong juvenile ocular dominance plasticity. Loss of PSD-95 in the visual cortex after the closure of the critical period reinstated silent synapses, resulting in reopening of juvenile-like ocular dominance plasticity. Additionally, silent synapse-based ocular dominance plasticity was largely independent of the inhibitory tone, whose developmental maturation was independent of PSD-95. Moreover, glutamatergic synaptic transmission onto parvalbumin-positive interneurons was unaltered in PSD-95 KO mice. These findings reveal not only that PSD-95-dependent silent synapse maturation in visual cortical principal neurons terminates the critical period for ocular dominance plasticity but also indicate that, in general, once silent synapses are consolidated in any neural circuit, initial experience-dependent functional optimization and critical periods end.
Asunto(s)
Guanilato-Quinasas/fisiología , Proteínas de la Membrana/fisiología , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/fisiología , Sinapsis/fisiología , Corteza Visual/crecimiento & desarrollo , Corteza Visual/fisiología , Animales , Mapeo Encefálico , Homólogo 4 de la Proteína Discs Large , Predominio Ocular/fisiología , Femenino , Glutamina/fisiología , Guanilato-Quinasas/deficiencia , Guanilato-Quinasas/genética , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Plasticidad Neuronal/fisiología , Receptores AMPA/fisiologíaRESUMEN
Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid ß-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.
Asunto(s)
Antineoplásicos/farmacología , Daunorrubicina/farmacología , Resistencia a Antineoplásicos , Glutamina/fisiología , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Acetil-CoA C-Aciltransferasa/metabolismo , Isomerasas de Doble Vínculo Carbono-Carbono/metabolismo , Línea Celular Tumoral , Ciclosporinas/farmacología , Sinergismo Farmacológico , Enoil-CoA Hidratasa/metabolismo , Ácidos Grasos/biosíntesis , Glucólisis , Humanos , Leucemia , Metaboloma , Oxidación-Reducción , Ácido Pantoténico/metabolismo , Perhexilina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Racemasas y Epimerasas/metabolismo , TranscriptomaRESUMEN
Cellular transformation is associated with altered glutamine (Gln) metabolism. Tumor cells utilize Gln in the tricarboxylic acid (TCA) cycle to maintain sufficient pools of biosynthetic precursors to support rapid growth and proliferation. However, Gln metabolism also generates NADPH, and Gln-derived glutamate is used for synthesis of glutathione (GSH). As both NADPH and GSH are antioxidants, Gln may also contribute to redox balance in transformed cells. The Hace1 E3 ligase is a tumor suppressor inactivated in diverse human cancers. Hace1 targets the Rac1 GTPase for degradation at Rac1-dependent NADPH oxidase complexes, blocking superoxide generation by the latter. Consequently, loss of Hace1 increases reactive oxygen species (ROS) levels in vitro and in vivo. Given the link between Hace1 loss and increased ROS, we investigated whether genetic inactivation of Hace1 alters Gln metabolism. We demonstrate that mouse embryonic fibroblasts (MEFs) derived from Hace1(-/-) mice are highly sensitive to Gln withdrawal, leading to enhanced cell death compared with wild-type (wt) MEFs, and Gln depletion or chemical inhibition of Gln uptake blocks soft agar colony formation by Hace1(-/-) MEFs. Hace1(-/-) MEFs exhibit increased Gln uptake and ammonia secretion, and metabolic labeling using (13)C-Gln revealed that Hace1 loss increases incorporation of Gln carbons into the TCA cycle intermediates. Gln starvation markedly increases ROS levels in Hace1(-/-) but not in wt MEFs, and treatment with the antioxidant N-acetyl cysteine or the TCA cycle intermediate oxaloacetate efficiently rescues Gln starvation-induced ROS elevation and cell death in Hace1(-/-) MEFs. Finally, Gln starvation increases superoxide levels in Hace1(-/-) MEFs, and NADPH oxidase inhibitors block the induction of superoxide and cell death by Gln starvation. Together, these results suggest that increased ROS production due to Hace1 loss leads to Gln addiction as a mechanism to cope with increased ROS-induced oxidative stress.
Asunto(s)
Glutamina/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Apoptosis , Células Cultivadas , Ratones Noqueados , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
This review covers a broad range of topics related to the actions of zinc on the cells of the vertebrate retina. Much of this review relies on studies in which zinc was applied exogenously, and therefore the results, albeit highly suggestive, lack physiologic significance. This view stems from the fact that the concentrations of zinc used in these studies may not be encountered under the normal circumstances of life. This caveat is due to the lack of a zinc-specific probe with which to measure the concentrations of Zn(2+) that may be released from neurons or act upon them. However, a great deal of relevant information has been garnered from studies in which Zn(2+) was chelated, and the effects of its removal compared with findings obtained in its presence. For a more complete discussion of the consequences of depletion or excess in the body's trace elements, the reader is referred to a recent review by Ugarte et al. in which they provide a detailed account of the interactions, toxicity, and metabolic activity of the essential trace elements iron, zinc, and copper in retinal physiology and disease. In addition, Smart et al. have published a splendid review on the modulation by zinc of inhibitory and excitatory amino acid receptor ion channels.
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Retina/fisiología , Zinc/fisiología , Animales , Fenómenos Electrofisiológicos , Glutamina/fisiología , Humanos , Retina/citología , Retina/efectos de los fármacos , Neuronas Retinianas/efectos de los fármacos , Neuronas Retinianas/fisiología , Oligoelementos/deficiencia , Oligoelementos/metabolismo , Oligoelementos/farmacología , Zinc/deficiencia , Zinc/farmacologíaRESUMEN
Recently a large multicentre randomised controlled trial in critically ill patients reported harm to the patients given supplementary glutamine. In the original publication, no explanation was offered for why this result was obtained; a large number of studies have reported beneficial effects or no effect, but never before reported harm. These results have been commented upon in a number of communications. Now some of the authors of the multicentre randomised controlled trial present a review and meta-analysis of glutamine supplementation, and the discrepancy of results is suggested to relate to intravenous administration to patients of supplementary glutamine via parenteral nutrition or a combination of enteral and parenteral nutrition in contrast to enteral administration of supplementation or a combination of enteral and parenteral supplementation. To explain results by epidemiological means only, by combining results into a meta-analysis, is perhaps not the best way to explain mechanisms behind results. Meta-analyses are primarily hypothesis generating. Launching treatment without a solid mechanistic explanation is always risky. Glutamine supplementation of the critically ill comes into that category. Now we will all have to do our homework and try to understand whether supplementation or omission of glutamine for patients fed parenterally is a good idea or not.
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Enfermedad Crítica/terapia , Nutrición Enteral/métodos , Glutamina/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Nutrición Parenteral/métodos , Suplementos Dietéticos/efectos adversos , Suplementos Dietéticos/normas , Glutamina/administración & dosificación , Glutamina/fisiología , Humanos , Metaanálisis como Asunto , Estudios Multicéntricos como Asunto , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Toluene/o-xylene monooxygenase (ToMO) is a bacterial multicomponent monooxygenase capable of oxidizing aromatic substrates. The carboxylate-rich diiron active site is located in the hydroxylase component of ToMO (ToMOH), buried 12 Å from the surface of the protein. A small, hydrophilic pore is the shortest pathway between the diiron active site and the protein exterior. In this study of ToMOH from Pseudomonas sp. OX1, the functions of two residues lining this pore, N202 and Q228, were investigated using site-directed mutagenesis. Steady-state characterization of WT and the three mutant enzymes demonstrates that residues N202 and Q228 are critical for turnover. Kinetic isotope effects and pH profiles reveal that these residues govern the kinetics of water egress and prevent quenching of activated oxygen intermediates formed at the diiron active site. We propose that this activity arises from movement of these residues, opening and closing the pore during catalysis, as seen in previous X-ray crystallographic studies. In addition, N202 and Q228 are important for the interactions of the reductase and regulatory components to ToMOH, suggesting that they bind competitively to the hydroxylase. The role of the pore in the hydroxylase components of other bacterial multicomponent monooxygenases within the superfamily is discussed in light of these conclusions.
Asunto(s)
Glutamina/química , Glutamina/fisiología , Oxigenasas/química , Oxigenasas/metabolismo , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Cristalografía por Rayos X , Hidroxilación , Mutagénesis Sitio-Dirigida , Oxigenasas/genética , Pseudomonas/enzimología , Pseudomonas/genética , Especificidad por Sustrato/genéticaRESUMEN
Hyperammonemia is necessary for development of the cerebral complications to liver disease including hepatic encephalopathy and cerebral edema but the mechanisms are unclear. Ammonia is taken up by the brain in proportion to its arterial concentration. The flux into the brain is most likely by both diffusion of NH3 and mediated transport of NH4 (+) . Astrocytic detoxification of ammonia involves formation of glutamine at concentrations high enough to produce cellular edema, but compensatory mechanisms reduce this effect. Glutamine can be taken up by astrocytic mitochondria and initiate the mitochondrial permeability transition but the clinical relevance is uncertain. Elevated astrocytic glutamine interferes with neurotransmission. Thus, animal studies show enhanced glutamatergic neurotransmission via the NMDA receptor which may be related to the acute cerebral complications to liver failure, while impairment of the NMDA activated glutamate-NO-cGMP pathway could relate to the behavioural changes seen in hepatic encephalopathy. Elevated glutamine also increases GABA-ergic tone, an effect which is aggravated by mitochondrial production of neurosteroids; this may relate to decreased neurotransmission and precipitation of encephalopathy by GABA targeting drugs. Hyperammonemia may compromise cerebral energy metabolism as elevated cerebral lactate is generally reported. Hypoxia is unlikely since cerebral oxygen:glucose utilisation and lactate:pyruvate ratio are both normal in clinical studies. Ammonia inhibits α-ketoglutaratedehydrogenase in isolated mitochondria, but the clinical relevance is dubious due to the observed normal cerebral oxygen:glucose utilization. Recent studies suggest that ammonia stimulates glycolysis in excess of TCA cycle activity, a hypothesis that may warrant further testing, in being in accordance with the limited clinical observations.