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Reprod Sci ; 27(9): 1778-1790, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32124398


Progesterone therapy is a viable treatment for complex atypical hyperplasia (CAH) and endometrial adenocarcinoma, though reliable molecular determinants of response are not available. To explore if analysis of pre-therapy endometrial biopsies could yield biomarkers of response to progesterone, patients with CAH or adenocarcinoma undergoing treatment with progestins were included in this cross-sectional study. Immunohistochemistry for progesterone receptor (PR) was performed. Manual PR expression scores (PRES) were first calculated for biopsies by counting PR-positive nuclei in 12 sensitive vs 9 resistant samples. Significant differences in manual PRES were detected in the stroma (p < 0.01) and total endometrium (p < 0.01) for sensitive vs resistant patients. Manual PRES in the stroma had the highest accuracy in segregating sensitive vs resistant patients (96%). Differences in epithelial PRES were not significant. To validate these findings, a correlation between manual PRES and visual PRES was performed in the 21 patients. An additional 11 patients were analyzed to test if visual PRES would be predictive of response to progesterone. Visual PRES in epithelia and stroma in the 32 specimens was calculated. Significant differences in visual PRES were detected in the stroma for sensitive vs resistant samples (p < 0.01), while differences in epithelial and total endometrium were not significant. Whole genome bisulfite sequencing was performed on DNA isolated using pre-therapy biopsies from 6 sensitive and 6 resistant patients in this cohort. Differentially methylated regions were identified in the stroma and epithelium when evaluating sensitive vs resistant samples. Pathways involved in cell adhesion demonstrated the greatest difference in methylation in these samples.

Cell Rep ; 28(3): 759-772.e10, 2019 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-31315053


Mechanisms coordinating pancreatic ß cell metabolism with insulin secretion are essential for glucose homeostasis. One key mechanism of ß cell nutrient sensing uses the mitochondrial GTP (mtGTP) cycle. In this cycle, mtGTP synthesized by succinyl-CoA synthetase (SCS) is hydrolyzed via mitochondrial PEPCK (PEPCK-M) to make phosphoenolpyruvate, a high-energy metabolite that integrates TCA cycling and anaplerosis with glucose-stimulated insulin secretion (GSIS). Several strategies, including xenotopic overexpression of yeast mitochondrial GTP/GDP exchanger (GGC1) and human ATP and GTP-specific SCS isoforms, demonstrated the importance of the mtGTP cycle. These studies confirmed that mtGTP triggers and amplifies normal GSIS and rescues defects in GSIS both in vitro and in vivo. Increased mtGTP synthesis enhanced calcium oscillations during GSIS. mtGTP also augmented mitochondrial mass, increased insulin granule number, and membrane proximity without triggering de-differentiation or metabolic fragility. These data highlight the importance of the mtGTP signal in nutrient sensing, insulin secretion, mitochondrial maintenance, and ß cell health.

Sci Rep ; 6: 39319, 2016 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-27982116


Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.

Ensayos Analíticos de Alto Rendimiento/métodos , Imagen Óptica/métodos , Análisis de la Célula Individual/métodos , Animales , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Inmunohistoquímica , Células Secretoras de Insulina/metabolismo , Masculino , Microscopía Fluorescente , NADP/análisis , Ratas Sprague-Dawley
J Biol Chem ; 289(27): 19110-9, 2014 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-24841202


The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.

Señalización del Calcio , Citocromos c/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Animales , Señalización del Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Ayuno , Glucosa/farmacología , Glucólisis/efectos de los fármacos , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cetoácidos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley
PLoS One ; 8(7): e68793, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23894346


The suprachiasmatic nucleus (SCN) is required for the daily rhythm of plasma glucocorticoids; however, the independent contributions from oscillators within the different subregions of the SCN to the glucocorticoid rhythm remain unclear. Here, we use genetically and neurologically intact, forced desynchronized rats to test the hypothesis that the daily rhythm of the glucocorticoid, corticosterone, is regulated by both light responsive and light-dissociated circadian oscillators in the ventrolateral (vl-) and dorsomedial (dm-) SCN, respectively. We show that when the vlSCN and dmSCN are in maximum phase misalignment, the peak of the plasma corticosterone rhythm is shifted and the amplitude reduced; whereas, the peak of the plasma adrenocorticotropic hormone (ACTH) rhythm is also reduced, the phase is dissociated from that of the corticosterone rhythm. These data support previous studies suggesting an ACTH-independent pathway contributes to the corticosterone rhythm. To determine if either SCN subregion independently regulates corticosterone through the sympathetic nervous system, we compared unilateral adrenalectomized, desynchronized rats that had undergone either transection of the thoracic splanchnic nerve or sham transection to the remaining adrenal. Splanchnicectomy reduced and phase advanced the peak of both the corticosterone and ACTH rhythms. These data suggest that both the vlSCN and dmSCN contribute to the corticosterone rhythm by both reducing plasma ACTH and differentially regulating plasma corticosterone through an ACTH- and sympathetic nervous system-independent pathway.

Ritmo Circadiano , Corticosterona/sangre , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/fisiología , Glándulas Suprarrenales/metabolismo , Animales , Corticosterona/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Actividad Motora/fisiología , Neuronas/citología , Ratas , Ratas Wistar , Nervios Esplácnicos/cirugía