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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.
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.

3.
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
4.
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
5.
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
6.
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
7.
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
8.
J Bacteriol ; 193(21): 6032-8, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21873495

RESUMEN

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ∼50% identical to MxaF and ∼90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.


Asunto(s)
Oxidorreductasas de Alcohol/biosíntesis , Regulación Bacteriana de la Expresión Génica , Expresión Génica , Methylobacterium extorquens/enzimología , Methylobacterium extorquens/genética , Oxidorreductasas de Alcohol/genética , Medios de Cultivo/química , Formaldehído/metabolismo , Metanol/metabolismo , Methylobacterium extorquens/crecimiento & desarrollo , Methylobacterium extorquens/metabolismo , Homología de Secuencia de Aminoácido
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