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1.
Cell Rep ; 31(5): 107606, 2020 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-32375026

RESUMEN

When O2 is plentiful, the mitochondrial electron transport chain uses it as a terminal electron acceptor. However, the mammalian retina thrives in a hypoxic niche in the eye. We find that mitochondria in retinas adapt to their hypoxic environment by reversing the succinate dehydrogenase reaction to use fumarate to accept electrons instead of O2. Reverse succinate dehydrogenase activity produces succinate and is enhanced by hypoxia-induced downregulation of cytochrome oxidase. Retinas can export the succinate they produce to the neighboring O2-rich retinal pigment epithelium-choroid complex. There, succinate enhances O2 consumption by severalfold. Malate made from succinate in the pigment epithelium can then be imported into the retina, where it is converted to fumarate to again accept electrons in the reverse succinate dehydrogenase reaction. This malate-succinate shuttle can sustain these two tissues by transferring reducing power from an O2-poor tissue (retina) to an O2-rich one (retinal pigment epithelium-choroid).

2.
J Diabetes Investig ; 2020 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-32279428

RESUMEN

AIMS/INTRODUCTION: Pancreatic islets are heterogenous. To clarify the relationship between islet heterogeneity and incretin action in the islets, we studied gene expression and metabolic profiles of non-large and enlarged islets of the Zucker fatty diabetes mellitus rat, an obese diabetes model, as well as incretin-induced insulin secretion (IIIS) in these islets. MATERIALS AND METHODS: Pancreatic islets of control (fa/+) and fatty (fa/fa) rats at 8 and 12 weeks-of-age were isolated. The islets of fa/fa rats at 12 weeks-of-age were separated into non-large islets (≤200 µm in diameter) and enlarged islets (>300 µm in diameter). Morphological analyses, insulin secretion experiments, transcriptome analysis, metabolome analysis and oxygen consumption analysis were carried out on these islets. RESULTS: The number of enlarged islets was increased with age in fatty rats, and IIIS was significantly reduced in the enlarged islets. Markers for ß-cell differentiation were markedly decreased in the enlarged islets, but those for cell proliferation were increased. Glycolysis was enhanced in the enlarged islets, whereas the tricarboxylic acid cycle was suppressed. The oxygen consumption rate under glucose stimulation was reduced in the enlarged islets. Production of glutamate, a key signal for IIIS, was decreased in the enlarged islets. CONCLUSIONS: The enlarged islets of Zucker fatty diabetes mellitus rats, which are defective for IIIS, show tumor cell-like metabolic features, including a dedifferentiated state, accelerated aerobic glycolysis and impaired mitochondrial function. The age-dependent increase in such islets could contribute to the pathophysiology of obese diabetes.

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

RESUMEN

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.

4.
Islets ; 11(3): 51-64, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31084524

RESUMEN

The aim of the study was to determine the acute contribution of fuel oxidation in mediating the increase in insulin secretion rate (ISR) in response to fatty acids. Measures of mitochondrial metabolism, as reflected by oxygen consumption rate (OCR) and cytochrome c reduction, calcium signaling, and ISR by rat islets were used to evaluate processes stimulated by acute exposure to palmitic acid (PA). The contribution of mitochondrial oxidation of PA was determined in the presence and absence of a blocker of mitochondrial transport of fatty acids (etomoxir) at different glucose concentrations. Subsequent to increasing glucose from 3 to 20 mM, PA caused small increases in OCR and cytosolic calcium (about 20% of the effect of glucose). In contrast, the effect of PA on ISR was almost 3 times that by glucose, suggesting that the metabolism of PA is not the dominant mechanism mediating PA's effect on ISR. This was further supported by lack of inhibition of PA-stimulated OCR and ISR when blocking entry of PA into mitochondria (with etomoxir), and PA's lack of stimulation of reduced cytochrome c in the presence of high glucose. Consistent with the lack of metabolic stimulation by PA, an inhibitor of calcium release from the endoplasmic reticulum, but not a blocker of L-type calcium channels, abolished the PA-induced elevation of cytosolic calcium. Notably, ISR was unaffected by thapsigargin showing the dissociation of endoplasmic reticulum calcium release and second phase insulin secretion. In conclusion, stimulation of ISR by PA was mediated by mechanisms largely independent of the oxidation of the fuel.


Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Palmitatos/metabolismo , Animales , Retículo Endoplásmico/efectos de los fármacos , Insulina/agonistas , Islotes Pancreáticos/efectos de los fármacos , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Palmitatos/farmacología , Ratas , Ratas Sprague-Dawley
5.
Elife ; 62017 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-28901286

RESUMEN

Here we report multiple lines of evidence for a comprehensive model of energy metabolism in the vertebrate eye. Metabolic flux, locations of key enzymes, and our finding that glucose enters mouse and zebrafish retinas mostly through photoreceptors support a conceptually new model for retinal metabolism. In this model, glucose from the choroidal blood passes through the retinal pigment epithelium to the retina where photoreceptors convert it to lactate. Photoreceptors then export the lactate as fuel for the retinal pigment epithelium and for neighboring Müller glial cells. We used human retinal epithelial cells to show that lactate can suppress consumption of glucose by the retinal pigment epithelium. Suppression of glucose consumption in the retinal pigment epithelium can increase the amount of glucose that reaches the retina. This framework for understanding metabolic relationships in the vertebrate retina provides new insights into the underlying causes of retinal disease and age-related vision loss.


Asunto(s)
Adaptación Ocular , Metabolismo Energético , Células Ependimogliales/fisiología , Células Fotorreceptoras/fisiología , Epitelio Pigmentado de la Retina/fisiología , Animales , Células Ependimogliales/metabolismo , Glucosa/metabolismo , Humanos , Lactatos/metabolismo , Ratones , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pez Cebra
6.
Mol Metab ; 6(6): 535-547, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28580284

RESUMEN

OBJECTIVE: The glucose stimulation of insulin secretion (GSIS) by pancreatic ß-cells critically depends on increased production of metabolic coupling factors, including NADPH. Nicotinamide nucleotide transhydrogenase (NNT) typically produces NADPH at the expense of NADH and ΔpH in energized mitochondria. Its spontaneous inactivation in C57BL/6J mice was previously shown to alter ATP production, Ca2+ influx, and GSIS, thereby leading to glucose intolerance. Here, we tested the role of NNT in the glucose regulation of mitochondrial NADPH and glutathione redox state and reinvestigated its role in GSIS coupling events in mouse pancreatic islets. METHODS: Islets were isolated from female C57BL/6J mice (J-islets), which lack functional NNT, and genetically close C57BL/6N mice (N-islets). Wild-type mouse NNT was expressed in J-islets by adenoviral infection. Mitochondrial and cytosolic glutathione oxidation was measured with glutaredoxin 1-fused roGFP2 probes targeted or not to the mitochondrial matrix. NADPH and NADH redox state was measured biochemically. Insulin secretion and upstream coupling events were measured under dynamic or static conditions by standard procedures. RESULTS: NNT is largely responsible for the acute glucose-induced rise in islet NADPH/NADP+ ratio and decrease in mitochondrial glutathione oxidation, with a small impact on cytosolic glutathione. However, contrary to current views on NNT in ß-cells, these effects resulted from a glucose-dependent reduction in NADPH consumption by NNT reverse mode of operation, rather than from a stimulation of its forward mode of operation. Accordingly, the lack of NNT in J-islets decreased their sensitivity to exogenous H2O2 at non-stimulating glucose. Surprisingly, the lack of NNT did not alter the glucose-stimulation of Ca2+ influx and upstream mitochondrial events, but it markedly reduced both phases of GSIS by altering Ca2+-induced exocytosis and its metabolic amplification. CONCLUSION: These results drastically modify current views on NNT operation and mitochondrial function in pancreatic ß-cells.


Asunto(s)
Glucosa/metabolismo , Glutatión/metabolismo , Células Secretoras de Insulina/metabolismo , NADP Transhidrogenasa AB-Específica/metabolismo , NADP/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Exocitosis , Femenino , Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , NADP Transhidrogenasa AB-Específica/genética , Oxidación-Reducción
7.
Heliyon ; 2(12): e00210, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27995203

RESUMEN

OBJECTIVES: Microfluidic perfusion systems are used for assessing cell and tissue function while assuring cellular viability. Low perfusate flow rates, desired both for conserving reagents and for extending the number of channels and duration of experiments, conventionally depend on peristaltic pumps to maintain flow yet such pumps are unwieldy and scale poorly for high-throughput applications requiring 16 or more channels. The goal of the study was to develop a scalable multichannel microfluidics system capable of maintaining and assessing kinetic responses of small amounts of tissue to drugs or changes in test conditions. METHODS: Here we describe the BaroFuse, a novel, multichannel microfluidics device fabricated using 3D-printing technology that uses gas pressure to drive large numbers of parallel perfusion experiments. The system is versatile with respect to endpoints due to the translucence of the walls of the perifusion chambers, enabling optical methods for interrogating the tissue status. The system was validated by the incorporation of an oxygen detection system that enabled continuous measurement of oxygen consumption rate (OCR). RESULTS: Stable and low flow rates (1-20 µL/min/channel) were finely controlled by a single pressure regulator (0.5-2 psi). Control of flow in 0.2 µL/min increments was achieved. Low flow rates allowed for changes in OCR in response to glucose to be well resolved with very small numbers of islets (1-10 islets/channel). Effects of acetaminophen on OCR by precision-cut liver slices of were dose dependent and similar to previously published values that used more tissue and peristaltic-pump driven flow. CONCLUSIONS: The very low flow rates and simplicity of design and operation of the BaroFuse device allow for the efficient generation of large number of kinetic profiles in OCR and other endpoints lasting from hours to days. The use of flow enhances the ability to make measurements on primary tissue where some elements of native three-dimensional structure are preserved. We offer the BaroFuse as a powerful tool for physiological studies and for pharmaceutical assessment of drug effects as well as personalized medicine.

8.
Sci Rep ; 6: 39319, 2016 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-27982116

RESUMEN

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.


Asunto(s)
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
9.
Biochem J ; 473(23): 4443-4456, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27729543

RESUMEN

A real-time method to measure intracellular hydrogen peroxide (H2O2) would be very impactful in characterizing rapid changes that occur in physiologic and pathophysiologic states. Current methods do not provide the sensitivity, specificity and spatiotemporal resolution needed for such experiments on intact cells. We developed the use of HyPer, a genetic indicator for H2O2 that can be expressed in the cytosol (cyto-HyPer) or the mitochondria (mito-HyPer) of live cells. INS-1 cells or islets were permeabilized and the cytosolic HyPer signal was a linear function of extracellular H2O2, allowing fluorescent cyto-HyPer signals to be converted into H2O2 concentrations. Glucose increased cytosolic H2O2, an effect that was suppressed by overexpression of catalase. Large perturbations in pH can influence the HyPer signal, but inclusion of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] in the perfusate prevented pH changes, but did not affect glucose-induced cyto-HyPer signals, suggesting that this effect is largely pH-independent. Using the assay, two fundamental questions were addressed. Knockdown of superoxide dismutase 2 (SOD2), the mitochondrial form of SOD, completely suppressed glucose-induced H2O2 Furthermore, glucose also induced mitochondrial superoxide and H2O2 production, which preceded the appearance of cytosolic H2O2 Therefore, glucose-induced H2O2 largely originated from mitochondria. Finally, the glucose-induced HyPer signal was less than 1/20th of that induced by toxic levels of H2O2 Overall, the use of HyPer for real-time imaging allowed resolution of acute changes in intracellular levels of H2O2 and will have great utility for islet studies involving mechanisms of H2O2-mediated signaling and oxidative stress.


Asunto(s)
Peróxido de Hidrógeno/metabolismo , Islotes Pancreáticos/metabolismo , Animales , Catalasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Insulina/metabolismo , Masculino , Mitocondrias/metabolismo , Estrés Oxidativo , Consumo de Oxígeno , Propidio/metabolismo , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismo
10.
J Biol Chem ; 291(9): 4698-710, 2016 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-26677218

RESUMEN

Production of energy in a cell must keep pace with demand. Photoreceptors use ATP to maintain ion gradients in darkness, whereas in light they use it to support phototransduction. Matching production with consumption can be accomplished by coupling production directly to consumption. Alternatively, production can be set by a signal that anticipates demand. In this report we investigate the hypothesis that signaling through phototransduction controls production of energy in mouse retinas. We found that respiration in mouse retinas is not coupled tightly to ATP consumption. By analyzing metabolic flux in mouse retinas, we also found that phototransduction slows metabolic flux through glycolysis and through intermediates of the citric acid cycle. We also evaluated the relative contributions of regulation of the activities of α-ketoglutarate dehydrogenase and the aspartate-glutamate carrier 1. In addition, a comprehensive analysis of the retinal metabolome showed that phototransduction also influences steady-state concentrations of 5'-GMP, ribose-5-phosphate, ketone bodies, and purines.


Asunto(s)
Señalización del Calcio/efectos de la radiación , Metabolismo Energético/efectos de la radiación , Proteínas del Ojo/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Fototransducción , Retina/efectos de la radiación , Transducina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animales , Antiportadores/metabolismo , Ciclo del Ácido Cítrico/efectos de la radiación , GMP Cíclico/metabolismo , Transporte de Electrón/efectos de la radiación , Proteínas del Ojo/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Glucólisis/efectos de la radiación , Proteínas de Unión al GTP Heterotriméricas/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Luz , Metaboloma/efectos de la radiación , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Consumo de Oxígeno/efectos de la radiación , Retina/enzimología , Retina/metabolismo , Técnicas de Cultivo de Tejidos , Transducina/genética
11.
Toxicol Sci ; 148(2): 594-602, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26396153

RESUMEN

There is a general need to detect toxic effects of drugs during preclinical screening. We propose that increased sensitivity of xenobiotics toxicity combined with improved in vitro physiological recapitulation will more accurately assess potentially toxic perturbations of cellular biochemistry that are near in vivo pharmacological exposure levels. Importantly, measurement of such cytopathologies avoids activating mechanisms mediating toxicity at suprapharmacologic levels not relevant to in vivo effects. We present a sensitive method to measure changes in oxygen consumption rate (OCR), a well-established parameter reflecting a potential hazard, in response to exposure to pharmacologic levels of drugs using a flow culture system and state of the art oxygen sensing system. We tested metformin and acetaminophen on rat liver slices to illustrate the method. The features of the method include continuous and very stable measurement of OCR over the course of 48 h in liver slices in a continuous flow chamber with the ability to resolve changes as small as 0.3%/h. Kinetic modeling of metformin inhibition of OCR over a wide range of concentrations revealed both a slow and fast mechanism, where the fast mechanism activated only at concentrations above 0.6 mM. For both drugs, small amounts of inhibition were reversible, but higher decrements were irreversible. Overall the study highlights the advantages of measuring low-level toxicity so as to avoid the common extrapolations made about drug toxicity based on effects of drugs tested at suprapharmacologic levels.


Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Hígado/efectos de los fármacos , Metformina/toxicidad , Modelos Biológicos , Consumo de Oxígeno/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Cinética , Hígado/metabolismo , Ratas Sprague-Dawley , Recuperación de la Función , Medición de Riesgo
12.
Stem Cells ; 33(7): 2306-19, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25919237

RESUMEN

Neural activity is tightly coupled to energy consumption, particularly sugars such as glucose. However, we find that, unlike mature neurons and astrocytes, neural stem/progenitor cells (NSPCs) do not require glucose to sustain aerobic respiration. NSPCs within the adult subventricular zone (SVZ) express enzymes required for fatty acid oxidation and show sustained increases in oxygen consumption upon treatment with a polyunsaturated fatty acid. NSPCs also demonstrate sustained decreases in oxygen consumption upon treatment with etomoxir, an inhibitor of fatty acid oxidation. In addition, etomoxir decreases the proliferation of SVZ NSPCs without affecting cellular survival. Finally, higher levels of neurogenesis can be achieved in aged mice by ectopically expressing proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), a factor that increases cellular aerobic capacity by promoting mitochondrial biogenesis and metabolic gene transcription. Regulation of metabolic fuel availability could prove a powerful tool in promoting or limiting cellular proliferation in the central nervous system. Stem Cells 2015;33:2306-2319.


Asunto(s)
Ácidos Grasos/metabolismo , Células-Madre Neurales/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Humanos , Ventrículos Laterales , Ratones
13.
Diabetes ; 64(8): 2836-46, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25845662

RESUMEN

Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance.


Asunto(s)
Polaridad Celular/fisiología , Endotelio Vascular/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Óxido Nítrico/metabolismo , Animales , Inflamación/genética , Mediadores de Inflamación/metabolismo , Resistencia a la Insulina/fisiología , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Activación de Macrófagos/fisiología , Ratones , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo III/genética , Transducción de Señal/fisiología , Triglicéridos/metabolismo
14.
J Biol Chem ; 289(27): 19110-9, 2014 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-24841202

RESUMEN

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.


Asunto(s)
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
15.
Virology ; 449: 96-103, 2014 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-24418542

RESUMEN

In this report, we further characterized the effects of silibinin (SbN), derived from milk thistle extract, and Legalon-SIL (SIL), a water-soluble derivative of SbN, on T cell metabolism and HIV infection. We assessed the effects of SbN and SIL on peripheral blood mononuclear cells (PBMC) and CEM-T4 cells in terms of cellular growth, ATP content, metabolism, and HIV infection. SIL and SbN caused a rapid and reversible (upon removal) decrease in cellular ATP levels, which was associated with suppression of mitochondrial respiration and glycolysis. SbN, but not SIL inhibited glucose uptake. Exposure of T cells to SIL (but not SbN or metabolic inhibitors) during virus adsorption blocked HIV infection. Thus, both SbN and SIL rapidly perturb T cell metabolism in vitro, which may account for its anti-inflammatory and anti-proliferative effects that arise with prolonged exposure of cells. However, the metabolic effects are not involved in SIL's unique ability to block HIV entry.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , Cardo Lechoso/química , Extractos Vegetales/farmacología , Silimarina/farmacología , Adenosina Trifosfato/metabolismo , Transporte Biológico/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Glucosa/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Silibina , Replicación Viral/efectos de los fármacos
16.
J Biol Chem ; 288(50): 36129-40, 2013 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-24187136

RESUMEN

Transport of pyruvate into mitochondria by the mitochondrial pyruvate carrier is crucial for complete oxidation of glucose and for biosynthesis of amino acids and lipids. Zaprinast is a well known phosphodiesterase inhibitor and lead compound for sildenafil. We found Zaprinast alters the metabolomic profile of mitochondrial intermediates and amino acids in retina and brain. This metabolic effect of Zaprinast does not depend on inhibition of phosphodiesterase activity. By providing (13)C-labeled glucose and glutamine as fuels, we found that the metabolic profile of the Zaprinast effect is nearly identical to that of inhibitors of the mitochondrial pyruvate carrier. Both stimulate oxidation of glutamate and massive accumulation of aspartate. Moreover, Zaprinast inhibits pyruvate-driven O2 consumption in brain mitochondria and blocks mitochondrial pyruvate carrier in liver mitochondria. Inactivation of the aspartate glutamate carrier in retina does not attenuate the metabolic effect of Zaprinast. Our results show that Zaprinast is a potent inhibitor of mitochondrial pyruvate carrier activity, and this action causes aspartate to accumulate at the expense of glutamate. Our findings show that Zaprinast is a specific mitochondrial pyruvate carrier (MPC) inhibitor and may help to elucidate the roles of MPC in amino acid metabolism and hypoglycemia.


Asunto(s)
Ácido Aspártico/metabolismo , Ácido Glutámico/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Purinonas/farmacología , Ácido Pirúvico/metabolismo , Retina/citología , Animales , Transporte Biológico/efectos de los fármacos , Encéfalo/citología , Ciclo del Ácido Cítrico/efectos de los fármacos , Metabolómica , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Oxígeno/metabolismo
17.
Cell ; 153(2): 413-25, 2013 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-23582329

RESUMEN

Here, we demonstrate that the fractalkine (FKN)/CX3CR1 system represents a regulatory mechanism for pancreatic islet ß cell function and insulin secretion. CX3CR1 knockout (KO) mice exhibited a marked defect in glucose and GLP1-stimulated insulin secretion, and this defect was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca(2+) and potentiated insulin secretion in both mouse and human islets. The KO islets exhibited reduced expression of a set of genes necessary for the fully functional, differentiated ß cell state, whereas treatment of wild-type (WT) islets with FKN led to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and high-fat diet/obesity, suggesting that decreased FKN/CX3CR1 signaling could be a mechanism underlying ß cell dysfunction in type 2 diabetes.


Asunto(s)
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores de Quimiocina/metabolismo , Transducción de Señal , Adulto , Envejecimiento , Animales , Receptor 1 de Quimiocinas CX3C , Cadáver , Quimiocina CX3CL1/administración & dosificación , Quimiocina CX3CL1/metabolismo , Dieta Alta en Grasa , Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Islotes Pancreáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Receptores de Quimiocina/genética
18.
Endocr Pract ; 19(4): 663-8, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23512385

RESUMEN

OBJECTIVE: Autoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders or type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesized that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized. METHODS: We analyzed GAD65Ab titer, GAD65 enzyme activity inhibition, and GAD65Ab epitope pattern in a cohort of type 1 diabetes patients (n = 100) and correlated our findings with neurological symptoms and diseases. RESULTS: Overall, 43% (43/100) of patients had detectable GAD65Ab titers (median = 400 U/mL, range: 142-250,000 U/mL). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90th percentile of the cohort (2,000-250,000 U/mL). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of 5 patients inhibited the enzyme activity significantly (by 34-55%). Three patients complained of muscle stiffness and pain, which was documented in 2 of these patients. CONCLUSIONS: Based on our findings, we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized.


Asunto(s)
Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/inmunología , Glutamato Descarboxilasa/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven
19.
PLoS One ; 7(3): e33070, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22479359

RESUMEN

BACKGROUND: Oxygen consumption reflects multiple processes in pancreatic islets including mechanisms contributing to insulin secretion, oxidative stress and viability, providing an important readout in studies of islet function, islet viability and drug testing. Due to the scarcity, heterogeneity, and intrinsic kinetic properties of individual islets, it would be of great benefit to detect oxygen consumption by single islets. We present a novel method we have developed to image oxygen in single islets. METHODOLOGY/PRINCIPAL FINDINGS: Using a microfluidics system, individual islets and a fluorescent oxygen-sensitive dye were encased within a thin alginate polymer layer. Insulin secretion by the encapsulated islets was normal. Fluorescent signal from the encased dye, detected using a standard inverted fluorescence microscope and digital camera, was stable and proportional to the amount of oxygen in the media. When integrated into a perifusion system, the sensing system detected changes in response to metabolic substrates, mitochondrial poisons, and induced-oscillations. Glucose responses averaged 30.1±7.1% of the response to a metabolic inhibitor (cyanide), increases were observed in all cases (n = 6), and the system was able to resolve changes in oxygen consumption that had a period greater than 0.5 minutes. The sensing system operated similarly from 2-48 hours following encapsulation, and viability and function of the islets were not significantly affected by the encapsulation process. CONCLUSIONS/SIGNIFICANCE: An oxygen-dependent dye situated around and within a pancreatic islet encapsulated by a thin layer of alginate was sensitive to changes in oxygen consumption, and was not harmful to the function or viability of islets over the course of two days. The microcapsule-based sensing method is particularly suited to assessing the effects of compounds (dose responses and time courses) and chronic changes occurring over the course of days. The approach should be applicable to other cell types and dyes sensitive to other biologically important molecules.


Asunto(s)
Técnicas Biosensibles/métodos , Islotes Pancreáticos/metabolismo , Consumo de Oxígeno , Oxígeno/análisis , Alginatos/química , Animales , Técnicas Biosensibles/instrumentación , Membrana Celular/metabolismo , Colorantes Fluorescentes/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/química , Cinética , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados
20.
J Biol Chem ; 287(24): 19816-26, 2012 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-22528485

RESUMEN

Normal glucose-stimulated insulin secretion is dependent on interactions between neighboring ß cells. Elucidation of the reasons why this cell-to-cell contact is essential will probably yield critical insights into ß cell maturation and function. In the central nervous system, transcellular protein interactions (i.e. interactions between proteins on the surfaces of different cells) involving neuroligins are key mediators of synaptic functional development. We previously demonstrated that ß cells express neuroligin-2 and that insulin secretion is affected by changes in neuroligin-2 expression. Here we show that the effect of neuroligin-2 on insulin secretion is mediated by transcellular interactions. Neuroligin-2 binds with nanomolar affinity to a partner on the ß cell surface and contributes to the increased insulin secretion brought about by ß cell-to-ß cell contact. It does so in a manner seemingly independent of interactions with neurexin, a known binding partner. As in the synapse, transcellular neuroligin-2 interactions enhance the functioning of the submembrane exocytic machinery. Also, as in the synapse, neuroligin-2 clustering is important. Neuroligin-2 in soluble form, rather than presented on a cell surface, decreases insulin secretion by rat islets and MIN-6 cells, most likely by interfering with endogenous neuroligin interactions. Prolonged contact with neuroligin-2-expressing cells increases INS-1 ß cell proliferation and insulin content. These results extend the known parallels between the synaptic and ß cell secretory machineries to extracellular interactions. Neuroligin-2 interactions are one of the few transcellular protein interactions thus far identified that directly enhance insulin secretion. Together, these results indicate a significant role for transcellular neuroligin-2 interactions in the establishment of ß cell function.


Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Comunicación Celular/fisiología , Proliferación Celular , Regulación de la Expresión Génica/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Moléculas de Adhesión Celular Neuronal/genética , Células HEK293 , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-Dawley
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