RESUMEN
The retinal pigment epithelium (RPE) is omnivorous and can utilize a wide range of substrates for oxidative phosphorylation. Certain tissues with high mitochondrial metabolic load are capable of ketogenesis, a biochemical pathway that consolidates acetyl-CoA into ketone bodies. Earlier work demonstrated that the RPE expresses the rate-limiting enzyme for ketogenesis, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), and that the RPE indeed produces ketone bodies, including beta-hydroxybutyrate (ß-HB). Prior work, based on detecting ß-HB via enzymatic assays, suggested that differentiated cultures of primary RPE preferentially export ß-HB across the apical membrane. Here, we compare the accuracy of measuring ß-HB by enzymatic assay kits to mass spectrometry analysis. We found that commercial kits lack the sensitivity to accurately measure the levels of ß-HB in RPE cultures and are prone to artifact. Using mass spectrometry, we found that while RPE cultures secrete ß-HB, they do so equally to both apical and basal sides. We also find RPE is capable of consuming ß-HB as levels rise. Using isotopically labeled glucose, amino acid, and fatty acid tracers, we found that carbons from both fatty acids and ketogenic amino acids, but not from glucose, produce ß-HB. Altogether, we substantiate ß-HB secretion in RPE but find that the secretion is equal apically and basally, RPE ß-HB can derive from ketogenic amino acids or fatty acids, and accurate ß-HB assessment requires mass spectrometric analysis.
Asunto(s)
Ácido 3-Hidroxibutírico , Cuerpos Cetónicos , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/metabolismo , Cuerpos Cetónicos/metabolismo , Células Cultivadas , Ácido 3-Hidroxibutírico/metabolismo , Humanos , Pruebas de Enzimas/métodos , Hidroximetilglutaril-CoA Sintasa/metabolismo , Espectrometría de Masas , AnimalesRESUMEN
Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or rod-specific manner to better understand how glucose is utilized in the retina. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Slc2a1 from retinal neurons and Müller glia results in reduced OS growth and progressive rod but not cone photoreceptor cell death. Rhodopsin levels were severely decreased even at postnatal day 20 when OS length was relatively normal. Arrestin levels were not changed suggesting that glucose uptake is required to synthesize membrane glycoproteins. Rod-specific deletion of Slc2a1 resulted in similar changes in OS length and rod photoreceptor cell death. These studies demonstrate that glucose is an essential carbon source for rod photoreceptor cell OS maintenance and viability.
Asunto(s)
Transportador de Glucosa de Tipo 1 , Glucosa , Células Fotorreceptoras Retinianas Conos , Degeneración Retiniana , Segmento Externo de la Célula en Bastón , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Segmento Externo de la Célula en Bastón/metabolismo , Segmento Externo de la Célula en Bastón/patologíaRESUMEN
The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Simportadores/metabolismo , Animales , Bovinos , Ratones Endogámicos C57BLRESUMEN
The neural retina metabolizes glucose through aerobic glycolysis generating large amounts of lactate. Lactate flux into and out of cells is regulated by proton-coupled monocarboxylate transporters (MCTs), which are encoded by members of the Slc16a family. MCT1, MCT3, and MCT4 are expressed in the retina and require association with the accessory protein basigin, encoded by Bsg, for maturation and trafficking to the plasma membrane. Bsg-/- mice have severely reduced electroretinograms (ERGs) and progressive photoreceptor degeneration, which is presumed to be driven by metabolic dysfunction resulting from loss of MCTs. To understand the basis of the Bsg-/- phenotype, we generated mice with conditional deletion of Bsg in rods (RodΔBsg), cones (Cone∆Bsg), or retinal pigment epithelial cells (RPEΔBsg). RodΔBsg mice showed a progressive loss of photoreceptors, while ConeΔBsg mice did not display a degenerative phenotype. The RPEΔBsg mice developed a distinct phenotype characterized by severely reduced ERG responses as early as 4 weeks of age. The loss of lactate transporters from the RPE most closely resembled the phenotype of the Bsg-/- mouse, suggesting that the regulation of lactate levels in the RPE and the subretinal space is essential for the viability and function of photoreceptors.
Asunto(s)
Basigina/fisiología , Homeostasis , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Transporte Biológico , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The retina is one of the most metabolically active tissues in the body and utilizes glucose to produce energy and intermediates required for daily renewal of photoreceptor cell outer segments. Glucose transporter 1 (GLUT1) facilitates glucose transport across outer blood retinal barrier (BRB) formed by the retinal pigment epithelium (RPE) and the inner BRB formed by the endothelium. We used conditional knockout mice to study the impact of reducing glucose transport across the RPE on photoreceptor and Müller glial cells. Transgenic mice expressing Cre recombinase under control of the Bestrophin1 ( Best1) promoter were bred with Glut1flox/flox mice to generate Tg-Best1-Cre:Glut1flox/flox mice ( RPEΔGlut1). The RPEΔGlut1 mice displayed a mosaic pattern of Cre expression within the RPE that allowed us to analyze mice with ~50% ( RPEΔGlut1m) recombination and mice with >70% ( RPEΔGlut1h) recombination separately. Deletion of GLUT1 from the RPE did not affect its carrier or barrier functions, indicating that the RPE utilizes other substrates to support its metabolic needs thereby sparing glucose for the outer retina. RPEΔGlut1m mice had normal retinal morphology, function, and no cell death; however, where GLUT1 was absent from a span of RPE greater than 100 µm, there was shortening of the photoreceptor cell outer segments. RPEΔGlut1h mice showed outer segment shortening, cell death of photoreceptors, and activation of Müller glial cells. The severe phenotype seen in RPEΔGlut1h mice indicates that glucose transport via the GLUT1 transporter in the RPE is required to meet the anabolic and catabolic requirements of photoreceptors and maintain Müller glial cells in a quiescent state.
Asunto(s)
Células Ependimogliales/metabolismo , Transportador de Glucosa de Tipo 1/biosíntesis , Glucosa/metabolismo , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Células Ependimogliales/química , Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Fotorreceptoras/química , Epitelio Pigmentado de la Retina/químicaRESUMEN
The primary energy substrate of the lens is glucose and uptake of glucose from the aqueous humor is dependent on glucose transporters. GLUT1, the facilitated glucose transporter encoded by Slc2a1 is expressed in the epithelium of bovine, human and rat lenses. In the current study, we examined the expression of GLUT1 in the mouse lens and determined its role in maintaining lens transparency by studying effects of postnatal deletion of Slc2a1. In situ hybridization and immunofluorescence labeling were used to determine the expression and subcellular distribution of GLUT1 in the lens. Slc2a1 was knocked out of the lens epithelium by crossing transgenic mice expressing Cre recombinase under control of the GFAP promoter with Slc2a1loxP/loxP mice to generate Slc2a1loxP/loxP;GFAP-Cre+/0 (LensΔGlut1) mice. LensΔGlut1 mice developed visible lens opacities by around 3 months of age, which corresponded temporally with the total loss of detectable GLUT1 expression in the lens. Spectral domain optical coherence tomography (SD-OCT) imaging was used to monitor the formation of cataracts over time. SD-OCT imaging revealed that small nuclear cataracts were first apparent in the lenses of LensΔGlut1 mice beginning at about 2.7 months of age. Longitudinal SD-OCT imaging of LensΔGlut1 mice revealed disruption of mature secondary fiber cells after 3 months of age. Histological sections of eyes from LensΔGlut1 mice confirmed the disruption of the secondary fiber cells. The structural changes were most pronounced in fiber cells that had lost their organelles. In contrast, the histology of the lens epithelium in these mice appeared normal. Lactate and ATP were measured in lenses from LensΔGlut1 and control mice at 2 and 3 months of age. At 2 months of age, when GLUT1 was still detectable in the lens epithelium, albeit at low levels, the amount of lactate and ATP were not significantly different from controls. However, in lenses isolated from 3-month-old LensΔGlut1 mice, when GLUT1 was no longer detectable, levels of lactate and ATP were 50% lower than controls. Our findings demonstrate that in vivo, the transparency of mature lens fiber cells was dependent on glycolysis for ATP and the loss of GLUT1 transporters led to cataract formation. In contrast, lens epithelium and cortical fiber cells have mitochondria and could utilize other substrates to support their anabolic and catabolic needs.
Asunto(s)
Catarata/etiología , Células Epiteliales/metabolismo , Eliminación de Gen , Transportador de Glucosa de Tipo 1/genética , Cristalino/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Humor Acuoso/metabolismo , Western Blotting , Transportador 2 de Aminoácidos Excitadores/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Glucosa/metabolismo , Glucólisis , Ratones , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Tomografía de Coherencia ÓpticaRESUMEN
The monocarboxylate transporter 1 (MCT1) is highly expressed in the outer retina, suggesting that it plays a critical role in photoreceptors. We examined MCT1 +/- heterozygotes, which express half of the normal complement of MCT1. The MCT1 +/- retina developed normally and retained normal function, indicating that MCT1 is expressed at sufficient levels to support outer retinal metabolism.
Asunto(s)
Transportadores de Ácidos Monocarboxílicos/deficiencia , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Simportadores/deficiencia , Animales , Electrorretinografía , Metabolismo Energético , Potenciales Evocados Visuales , Heterocigoto , Lactatos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neuronas Motoras/metabolismo , Oligodendroglía/metabolismo , Células Bipolares de la Retina/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.
Asunto(s)
Consumo de Oxígeno , Epitelio Pigmentado de la Retina , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Humanos , Consumo de Oxígeno/fisiología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiologíaRESUMEN
Oral squamous cell carcinoma (OSCC) is the most common subsite of head and neck cancer, with a 5-year survival rate of only 50%. There is a pressing need for animal models that recapitulate the human disease to understand the factors driving OSCC carcinogenesis. Many laboratories have used the chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) to investigate OSCC formation. The importance of the 4NQO mouse model is that it mimics the stepwise progression observed in OSCC patients. The 4NQO carcinogen model has the advantage that it can be used with transgenic mice with genetic modification in specific tissue types to investigate their role in driving cancer progression. Herein, we describe the basic approach for administering 4NQO to mice to induce OSCC and methods for assessing the tissue and disease progression.