RESUMEN
Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. SIGNIFICANCE: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription.See related commentary by Henssen, p. 293.This article is highlighted in the In This Issue feature, p. 275.
Asunto(s)
ADN/genética , Herencia Extracromosómica , Amplificación de Genes , Neoplasias/genética , Microambiente Tumoral , HumanosRESUMEN
The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.
Asunto(s)
Unión Competitiva/fisiología , Neoplasias Encefálicas/genética , Glioblastoma/genética , Factores de Transcripción SOXB1/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Dominio B30.2-SPRY , Unión Competitiva/genética , Femenino , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteómica , Factores de Transcripción SOXB1/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.
Asunto(s)
Neoplasias Encefálicas/genética , Plasticidad de la Célula/genética , Epigénesis Genética , Glioma/genética , Análisis de la Célula Individual , Estrés Fisiológico/genética , Evolución Clonal , Variaciones en el Número de Copia de ADN/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Genoma Humano , Humanos , Mutación/genética , Filogenia , Regiones Promotoras Genéticas/genética , Microambiente Tumoral/genéticaRESUMEN
Extrachromosomal, circular DNA (ecDNA) is emerging as a prevalent yet less characterized oncogenic alteration in cancer genomes. We leverage ChIA-PET and ChIA-Drop chromatin interaction assays to characterize genome-wide ecDNA-mediated chromatin contacts that impact transcriptional programs in cancers. ecDNAs in glioblastoma patient-derived neurosphere and prostate cancer cell cultures are marked by widespread intra-ecDNA and genome-wide chromosomal interactions. ecDNA-chromatin contact foci are characterized by broad and high-level H3K27ac signals converging predominantly on chromosomal genes of increased expression levels. Prostate cancer cells harboring synthetic ecDNA circles composed of characterized enhancers result in the genome-wide activation of chromosomal gene transcription. Deciphering the chromosomal targets of ecDNAs at single-molecule resolution reveals an association with actively expressed oncogenes spatially clustered within ecDNA-directed interaction networks. Our results suggest that ecDNA can function as mobile transcriptional enhancers to promote tumor progression and manifest a potential synthetic aneuploidy mechanism of transcription control in cancer.
Asunto(s)
Cromosomas/genética , ADN de Neoplasias/genética , Glioblastoma/genética , Oncogenes/genética , Carcinogénesis/genética , Cromatina/genética , HumanosRESUMEN
Extrachromosomal DNA (ecDNA) amplification promotes intratumoral genetic heterogeneity and accelerated tumor evolution1-3; however, its frequency and clinical impact are unclear. Using computational analysis of whole-genome sequencing data from 3,212 cancer patients, we show that ecDNA amplification frequently occurs in most cancer types but not in blood or normal tissue. Oncogenes were highly enriched on amplified ecDNA, and the most common recurrent oncogene amplifications arose on ecDNA. EcDNA amplifications resulted in higher levels of oncogene transcription compared to copy number-matched linear DNA, coupled with enhanced chromatin accessibility, and more frequently resulted in transcript fusions. Patients whose cancers carried ecDNA had significantly shorter survival, even when controlled for tissue type, than patients whose cancers were not driven by ecDNA-based oncogene amplification. The results presented here demonstrate that ecDNA-based oncogene amplification is common in cancer, is different from chromosomal amplification and drives poor outcome for patients across many cancer types.
Asunto(s)
Cromosomas/genética , ADN/genética , Amplificación de Genes/genética , Neoplasias/genética , Oncogenes/genética , Línea Celular Tumoral , Cromatina/genética , HumanosRESUMEN
This chapter describes a method for isolation, maintenance, and propagation of primary glioblastoma (GBM) cells in adherent monolayer cultures from patient tumor specimens. This method enables the establishment of GBM cultures with stem or progenitor-like cell characteristics, including self-renewal capacity, differentiation along restricted neural lineages, and tumor-initiating potential when orthotopically injected into immunocompromised mice. This experimentally tractable model system is therefore suitable for a wide variety of analyses in vitro as well as in vivo. Key examples of biological analyses that can be performed using these cells are also described.
Asunto(s)
Glioblastoma/metabolismo , Glioblastoma/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Biomarcadores , Técnicas de Cultivo de Célula , Expresión Génica , Genes Reporteros , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Cultivo Primario de CélulasRESUMEN
Disulfiram has shown promising activity including proteasome inhibitory properties and synergy with temozolomide in preclinical glioblastoma (GBM) models. In a phase I study for newly diagnosed GBM after chemoradiotherapy, we have previously reported our initial dose-escalation results combining disulfiram with adjuvant temozolomide and established the maximum tolerated dose (MTD) as 500 mg per day. Here we report the final results of the phase I study including an additional dose-expansion cohort of disulfiram with concurrent copper. The phase I study consisted of an initial dose-escalation phase of disulfiram 500-1000 mg daily during adjuvant temozolomide, followed by a dose-expansion phase of disulfiram 500 mg daily with copper 2 mg three times daily. Proteasome inhibition was assessed using fluorometric 20S proteasome assay on peripheral blood cell. A total of 18 patients were enrolled: 7 patients received 500 mg disulfiram, 5 patients received 1000 mg disulfiram, and 6 patients received 500 mg disulfiram with copper. Two dose-limiting toxicities occurred with 1000 mg disulfiram. At disulfiram 500 mg with or without copper, only 1 patient (7%) required dose-reduction during the first month of therapy. Addition of copper to disulfiram did not increase toxicity nor proteasome inhibition. The median progression-free survival was 4.5 months (95% CI 0.8-8.2). The median overall survival (OS) was 14.0 months (95% CI 8.3-19.6), and the 2-year OS was 24%. The MTD of disulfiram at 500 mg daily in combination with adjuvant temozolomide was well tolerated by GBM patients, but 1000 mg daily was not. Toxicity and pharmacodynamic effect of disulfiram were similar with or without concurrent copper. The clinical efficacy appeared to be comparable to historical data. Additional clinical trials to combine disulfiram and copper with chemoradiotherapy or to resensitize recurrent GBM to temozolomide are ongoing.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Cobre/uso terapéutico , Disulfiram/uso terapéutico , Glioblastoma/tratamiento farmacológico , Oligoelementos/uso terapéutico , Adyuvantes Inmunológicos , Adulto , Anciano , Estudios de Cohortes , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Análisis de Supervivencia , Temozolomida/uso terapéutico , Adulto JovenRESUMEN
Accumulating evidence suggests cancer cells exhibit a dependency on metabolic pathways regulated by nicotinamide adenine dinucleotide (NAD+). Nevertheless, how the regulation of this metabolic cofactor interfaces with signal transduction networks remains poorly understood in glioblastoma. Here, we report nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting step in NAD+ synthesis, is highly expressed in glioblastoma tumors and patient-derived glioblastoma stem-like cells (GSCs). High NAMPT expression in tumors correlates with decreased patient survival. Pharmacological and genetic inhibition of NAMPT decreased NAD+ levels and GSC self-renewal capacity, and NAMPT knockdown inhibited the in vivo tumorigenicity of GSCs. Regulatory network analysis of RNA sequencing data using GSCs treated with NAMPT inhibitor identified transcription factor E2F2 as the center of a transcriptional hub in the NAD+-dependent network. Accordingly, we demonstrate E2F2 is required for GSC self-renewal. Downstream, E2F2 drives the transcription of members of the inhibitor of differentiation (ID) helix-loop-helix gene family. Finally, we find NAMPT mediates GSC radiation resistance. The identification of a NAMPT-E2F2-ID axis establishes a link between NAD+ metabolism and a self-renewal transcriptional program in glioblastoma, with therapeutic implications for this formidable cancer.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Citocinas/genética , Glioblastoma/metabolismo , NAD/biosíntesis , Nicotinamida Fosforribosiltransferasa/genética , Tolerancia a Radiación , Transcripción Genética , Animales , Antineoplásicos/farmacología , Encéfalo/patología , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Glioblastoma/radioterapia , Humanos , Ratones , Mutación , Trasplante de Neoplasias , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Células Madre/citologíaRESUMEN
Glioblastoma stem-like cells (GSCs) play a critical role in glioblastoma progression and recurrence. We discuss recent results on the role of the mitotic ubiquitin ligase cell division cycle 20-anaphase-promoting complex (CDC20-APC) in the governance of cardinal GSC functions through a mechanism involving the transcription factor sex-determining region Y-box 2 (SOX2). These findings expand the non-mitotic roles of CDC20-APC with implications for stem cell biology.
RESUMEN
Disulfiram, a generic alcohol aversion drug, has promising preclinical activity against glioblastoma (GBM). This phase I study aims to evaluate its safety, maximum tolerated dose (MTD), pharmacodynamic effect, and preliminary efficacy when combined with adjuvant temozolomide in GBM patients after standard chemoradiotherapy. Patients received disulfiram 500-1000 mg once daily, in combination with 150-200 mg/m(2) temozolomide. A modified 3 + 3 dose-escalation design was used to determine the MTD. The pharmacodynamic effect of proteasome inhibition was assessed using fluorometric 20S proteasome assay on peripheral blood cells. The MTD was determined based on the dose-limiting toxicities (DLTs) within the first month of therapy. Twelve patients were enrolled to two dose levels: 500 and 1000 mg. Two DLTs of grade 3 delirium occurred after 15 days of administration at 1000 mg per day. Other possible grade 2-3 DSF-related toxicities included fatigue, ataxia, dizziness, and peripheral neuropathy. The toxicities were self-limiting or resolved after discontinuing DSF. The MTD was determined to be 500 mg per day. Limited proteasome inhibition was observed at week 4 and showed an increased trend with escalated disulfiram. Median progression-free survival with 500 mg of DSF was 5.4 months from the start of disulfiram and 8.1 months from the start of chemoradiotherapy. Disulfiram can be safely combined with temozolomide but can cause reversible neurological toxicities. The MTD of disulfiram with adjuvant temozolomide appears to produce limited proteasome inhibition on peripheral blood cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Dacarbazina/análogos & derivados , Disulfiram/uso terapéutico , Glioblastoma/terapia , Neoplasias Supratentoriales/terapia , Administración Oral , Adulto , Anciano , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Quimioradioterapia , Dacarbazina/uso terapéutico , Disulfiram/efectos adversos , Disulfiram/farmacocinética , Relación Dosis-Respuesta a Droga , Reposicionamiento de Medicamentos , Quimioterapia Combinada , Femenino , Glioblastoma/sangre , Humanos , Masculino , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/sangre , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma/efectos adversos , Inhibidores de Proteasoma/farmacocinética , Inhibidores de Proteasoma/uso terapéutico , Neoplasias Supratentoriales/sangre , Temozolomida , Resultado del Tratamiento , Adulto JovenRESUMEN
Many procedures in modern clinical medicine rely on the use of electronic implants in treating conditions that range from acute coronary events to traumatic injury. However, standard permanent electronic hardware acts as a nidus for infection: bacteria form biofilms along percutaneous wires, or seed haematogenously, with the potential to migrate within the body and to provoke immune-mediated pathological tissue reactions. The associated surgical retrieval procedures, meanwhile, subject patients to the distress associated with re-operation and expose them to additional complications. Here, we report materials, device architectures, integration strategies, and in vivo demonstrations in rats of implantable, multifunctional silicon sensors for the brain, for which all of the constituent materials naturally resorb via hydrolysis and/or metabolic action, eliminating the need for extraction. Continuous monitoring of intracranial pressure and temperature illustrates functionality essential to the treatment of traumatic brain injury; the measurement performance of our resorbable devices compares favourably with that of non-resorbable clinical standards. In our experiments, insulated percutaneous wires connect to an externally mounted, miniaturized wireless potentiostat for data transmission. In a separate set-up, we connect a sensor to an implanted (but only partially resorbable) data-communication system, proving the principle that there is no need for any percutaneous wiring. The devices can be adapted to sense fluid flow, motion, pH or thermal characteristics, in formats that are compatible with the body's abdomen and extremities, as well as the deep brain, suggesting that the sensors might meet many needs in clinical medicine.
Asunto(s)
Implantes Absorbibles , Encéfalo/metabolismo , Electrónica/instrumentación , Monitoreo Fisiológico/instrumentación , Prótesis e Implantes , Silicio , Implantes Absorbibles/efectos adversos , Administración Cutánea , Animales , Temperatura Corporal , Encéfalo/cirugía , Diseño de Equipo , Hidrólisis , Masculino , Monitoreo Fisiológico/efectos adversos , Especificidad de Órganos , Presión , Prótesis e Implantes/efectos adversos , Ratas , Ratas Endogámicas Lew , Telemetría/instrumentación , Tecnología Inalámbrica/instrumentaciónRESUMEN
Glioblastoma harbors a dynamic subpopulation of glioblastoma stem-like cells (GSCs) that can propagate tumors in vivo and is resistant to standard chemoradiation. Identification of the cell-intrinsic mechanisms governing this clinically important cell state may lead to the discovery of therapeutic strategies for this challenging malignancy. Here, we demonstrate that the mitotic E3 ubiquitin ligase CDC20-anaphase-promoting complex (CDC20-APC) drives invasiveness and self-renewal in patient tumor-derived GSCs. Moreover, CDC20 knockdown inhibited and CDC20 overexpression increased the ability of human GSCs to generate brain tumors in an orthotopic xenograft model in vivo. CDC20-APC control of GSC invasion and self-renewal operates through pluripotency-related transcription factor SOX2. Our results identify a CDC20-APC/SOX2 signaling axis that controls key biological properties of GSCs, with implications for CDC20-APC-targeted strategies in the treatment of glioblastoma.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas Cdc20/metabolismo , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Neuronas/metabolismo , Transducción de Señal , Animales , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/patología , Proteínas Cdc20/genética , Células Cultivadas , Glioblastoma/patología , Humanos , Ratones , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
The ability of estrogen to shield the brain from the bioenergetic insult hypoglycemia is unclear. Estradiol (E) prevents hypoglycemic activation of the energy deficit sensor adenosine 5'-monophosphate-activated protein kinase (AMPK) in hindbrain metabolosensory A2 noradrenergic neurons. This study investigates the hypothesis that estrogen regulates A2 AMPK through control of fuel metabolism and/or upstream protein kinase/phosphatase enzyme expression. A2 cells were harvested by laser microdissection after insulin or vehicle (V) injection of E- or oil (O)-implanted ovariectomized female rats. Cell lysates were evaluated by immunoblot for glycolytic, tricarboxylic acid cycle, respiratory chain, and acetyl-CoA-malonyl-CoA pathway enzymes. A2 phosphofructokinase (PFKL), isocitrate dehydrogenase, pyruvate dehydrogenase, and ATP synthase subunit profiles were elevated in E/V vs. O/V; hypoglycemia augmented PFKL and α-ketoglutarate dehydrogenase expression in E only. Hypoglycemia increased A2 Ca(2+) /calmodulin-dependent protein kinase-ß in O and reduced protein phosphatase in both groups. A2 phospho-AMPK levels were equivalent in O/V vs. E/V but elevated during hypoglycemia in O only. These results implicate E in compensatory upregulation of substrate catabolism and corresponding maintenance of energy stability of A2 metabolosensory neurons during hypoglycemia, outcomes that support the potential viability of molecular substrates for hormone action as targets for therapies alleviating hypoglycemic brain injury.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Cuerpos Aórticos/patología , Estradiol/farmacología , Hipoglucemia/patología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Células Receptoras Sensoriales/enzimología , Proteínas Quinasas Activadas por AMP/genética , Animales , Glucemia , Modelos Animales de Enfermedad , Estradiol/uso terapéutico , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/genética , Receptor beta de Estrógeno/metabolismo , Femenino , Hipoglucemia/tratamiento farmacológico , Captura por Microdisección con Láser , Ovariectomía , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Nerve cell metabolic activity is monitored in multiple brain regions, including the hypothalamus and hindbrain dorsal vagal complex (DVC), but it is unclear if individual metabolosensory loci operate autonomously or interact to coordinate central nervous system (CNS) reactivity to energy imbalance. This research addressed the hypothesis that hypoglycemia-associated DVC lactoprivation stimulates hypothalamic AMPK activity and metabolic neurotransmitter expression. As DVC catecholaminergic neurons express biomarkers for metabolic monitoring, we investigated whether these cells are a source of lactate deficit signaling to the hypothalamus. Caudal fourth ventricle (CV4) infusion of the glucose metabolite l-lactate during insulin-induced hypoglycemia reversed changes in DVC A2 noradrenergic, arcuate neuropeptide Y (NPY) and pro-opiomelanocortin (POMC), and lateral hypothalamic orexin-A (ORX) neuronal AMPK activity, coincident with exacerbation of hypoglycemia. Hindbrain lactate repletion also blunted hypoglycemic upregulation of arcuate NPY mRNA and protein. This treatment did not alter hypoglycemic paraventricular oxytocin (OT) and lateral hypothalamic ORX mRNA profiles, but exacerbated or reversed adjustments in OT and ORX neuropeptide synthesis, respectively. CV4 delivery of the monocarboxylate transporter inhibitor, 4-CIN, increased A2 phosphoAMPK (pAMPK), elevated circulating glucose, and stimulated feeding, responses that were attenuated by 6-hydroxydopamine pretreatment. 4-CIN-infused rats exhibited increased (NPY, ORX neurons) or decreased (POMC neurons) pAMPK concurrent with hyperglycemia. These data show that hindbrain lactoprivic signaling regulates hypothalamic AMPK and key effector neurotransmitter responses to hypoglycemia. Evidence that A2 AMPK activity is lactate-dependent, and that DVC catecholamine cells are critical for lactoprivic control of glucose, feeding, and hypothalamic AMPK, implies A2 derivation of this metabolic regulatory stimulus.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Metabolismo Energético , Hipoglucemia/enzimología , Hipotálamo/enzimología , Ácido Láctico/metabolismo , Neuropéptidos/metabolismo , ARN Mensajero/metabolismo , Rombencéfalo/metabolismo , Neuronas Adrenérgicas/metabolismo , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Conducta Alimentaria , Regulación de la Expresión Génica , Hipoglucemia/inducido químicamente , Hipoglucemia/genética , Hipoglucemia/fisiopatología , Hipoglucemia/psicología , Hipotálamo/fisiopatología , Infusiones Intraventriculares , Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ácido Láctico/administración & dosificación , Masculino , Neuropéptido Y/genética , Neuropéptido Y/metabolismo , Neuropéptidos/genética , Orexinas , Proopiomelanocortina/genética , Proopiomelanocortina/metabolismo , Ratas , Ratas Sprague-Dawley , Rombencéfalo/fisiopatología , Transducción de SeñalRESUMEN
This study investigated the hypothesis that estrogen controls hindbrain AMP-activated protein kinase (AMPK) activity and regulation of blood glucose, counterregulatory hormone secretion, and hypothalamic nerve cell transcriptional status. Dorsal vagal complex A2 noradrenergic neurons were laser microdissected from estradiol benzoate (E)- or oil (O)-implanted ovariectomized female rats after caudal fourth ventricular (CV4) delivery of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR), for Western blot analysis. E advanced AICAR-induced increases in A2 phospho-AMPK (pAMPK) expression and in blood glucose levels and was required for augmentation of Fos, estrogen receptor-α (ERα), monocarboxylate transporter-2, and glucose transporter-3 protein in A2 neurons and enhancement of corticosterone secretion by this treatment paradigm. CV4 AICAR also resulted in site-specific modifications in Fos immunolabeling of hypothalamic metabolic structures, including the paraventricular, ventromedial, and arcuate nuclei. The current studies demonstrate that estrogen regulates AMPK activation in caudal hindbrain A2 noradrenergic neurons during pharmacological replication of energy shortage in this area of the brain, and that this sensor is involved in neural regulation of glucostasis, in part, through control of corticosterone secretion. The data provide unique evidence that A2 neurons express both ERα and -ß proteins and that AMPK upregulates cellular sensitivity to ERα-mediated signaling during simulated energy insufficiency. The results also imply that estrogen promotes glucose and lactate uptake by these cells under those conditions. Evidence for correlation between hindbrain AMPK and hypothalamic nerve cell genomic activation provides novel proof for functional connectivity between this hindbrain sensor and higher order metabolic brain loci while demonstrating a modulatory role for estrogen in this interaction.
Asunto(s)
Cuerpos Aórticos/citología , Cuarto Ventrículo/efectos de los fármacos , Células Receptoras Sensoriales/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Animales , Glucemia/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estradiol/análogos & derivados , Estradiol/farmacología , Femenino , Cuarto Ventrículo/fisiología , Hipotálamo/metabolismo , Captura por Microdisección con Láser , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas v-fos/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Adenosina A2/metabolismo , Ribonucleósidos , Factores de TiempoRESUMEN
The hypothalamic neurochemicals neuropeptide Y (NPY), orexin-A (ORX), and oxytocin (OXY) exert glucoregulatory effects upon intracerebral administration, findings that support their potential function within neural pathways that maintain glucostasis. Current understanding of how these neurotransmitter systems respond to the diabetes mellitus complication, insulin-induced hypoglycemia, is limited to knowledge of neuropeptide gene transcriptional reactivity. We investigated the hypothesis that hypoglycemia elicits hypothalamic site-specific alterations in levels of these neurochemicals, and that adjustments in local neurotransmitter availability may be regulated by catecholaminergic (CA) input from the caudal dorsomedial hindbrain. The arcuate (ARH) and paraventricular (PVH) hypothalamic nuclei and lateral hypothalamic area (LHA) were each microdissected from adult male rats pretreated by caudal fourth ventricular administration of the selective CA neurotoxin, 6-hydroxydopamine (6-OHDA), or vehicle prior to insulin (INS)-induced hypoglycemia. Hypoglycemia stimulated ARH NPY gene expression and NPY accumulation in the ARH and LHA, but not PVH. 6-OHDA pretreatment did not modify the positive NPY mRNA response to INS, but blunted hypoglycemic augmentation of ARH and LHA NPY content while increasing PVH NPY levels in response to hypoglycemia. INS-treated rats exhibited diminished LHA ORX gene expression and increased [ARH; LHA] or decreased [PVH] tissue ORX protein levels. 6-OHDA+INS animals showed a comparable decline in ORX transcripts, but attenuated augmentation of ARH and LHA ORX content and elevated PVH ORX levels. OT mRNA and protein were respectively decreased or unchanged during hypoglycemia, responses that were uninfluenced by hindbrain CA nerve cell destruction. These results illustrate divergent adjustments in glucoregulatory neurotransmitter gene expression and site-specific protein accumulation in the hypothalamus during hypoglycemia. Evidence that 6-OHDA pretreatment does not modify NPY or ORX transcriptional reactivity to hypoglycemia, but alters hypoglycemic patterns of NPY and ORX accretion implicates dorsomedial hindbrain CA neurons in regulation of translation/post-translational processing and site-specific availability of these neurotransmitters in the hypothalamus during hypoglycemia.