RESUMO
Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that is expressed in cancer cells. Its role in tumor metabolism is not definitively established, but investigators have suggested that regulation of PKM2 activity can cause accumulation of glycolytic intermediates and increase flux through the pentose phosphate pathway. Recent evidence suggests that PKM2 also may have non-metabolic functions, including as a transcriptional co-activator in gene regulation. We reported previously that PKM2 is abundant in photoreceptor cells in mouse retinas. In the present study, we conditionally deleted PKM2 (rod-cre PKM2-KO) in rod photoreceptors and found that the absence of PKM2 causes increased expression of PKM1 in rods. Analysis of metabolic flux from U-13C glucose shows that rod-cre PKM2-KO retinas accumulate glycolytic intermediates, consistent with an overall reduction in the amount of pyruvate kinase activity. Rod-cre PKM2-KO mice also have an increased NADPH availability could favor lipid synthesis, but we found no difference in phospholipid synthesis between rod-cre PKM2 KO and PKM2-positive controls. As rod-cre PKM2-KO mice aged, we observed a significant loss of rod function, reduced thickness of the photoreceptor outer segment layer, and reduced expression of photoreceptor proteins, including PDE6ß. The rod-cre PKM2-KO retinas showed greater TUNEL staining than wild-type retinas, indicating a slow retinal degeneration. In vitro analysis showed that PKM2 can regulate transcriptional activity from the PDE6ß promoter in vitro. Our findings indicate that both the metabolic and transcriptional regulatory functions of PKM2 may contribute to photoreceptor structure, function, and viability.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Piruvato Quinase/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/genética , Transcrição Gênica , Animais , Apoptose/genética , Isótopos de Carbono , Membrana Celular/química , Membrana Celular/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Regulação da Expressão Gênica , Humanos , Marcação In Situ das Extremidades Cortadas , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Knockout , NADP/metabolismo , Fosfolipídeos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinase/deficiência , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Transdução de Sinais , Coloração e Rotulagem/métodos , Tomografia de Coerência Óptica , Triglicerídeos/metabolismoRESUMO
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.
Assuntos
Adaptação Ocular , Metabolismo Energético , Células Ependimogliais/fisiologia , Células Fotorreceptoras/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Células Ependimogliais/metabolismo , Glucose/metabolismo , Humanos , Lactatos/metabolismo , Camundongos , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Peixe-ZebraRESUMO
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.
Assuntos
Sinalização do Cálcio/efeitos da radiação , Metabolismo Energético/efeitos da radiação , Proteínas do Olho/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Transdução de Sinal Luminoso , Retina/efeitos da radiação , Transducina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/metabolismo , Ciclo do Ácido Cítrico/efeitos da radiação , GMP Cíclico/metabolismo , Transporte de Elétrons/efeitos da radiação , Proteínas do Olho/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Glicólise/efeitos da radiação , Proteínas Heterotriméricas de Ligação ao GTP/genética , Complexo Cetoglutarato Desidrogenase/metabolismo , Luz , Metaboloma/efeitos da radiação , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Consumo de Oxigênio/efeitos da radiação , Retina/enzimologia , Retina/metabolismo , Técnicas de Cultura de Tecidos , Transducina/genéticaRESUMO
Symbiotic relationships between neurons and glia must adapt to structures, functions, and metabolic roles of the tissues they are in. We show here that Müller glia in retinas have specific enzyme deficiencies that can enhance their ability to synthesize Gln. The metabolic cost of these deficiencies is that they impair the Müller cell's ability to metabolize Glc. We show here that the cells can compensate for this deficiency by using metabolites produced by neurons. Müller glia are deficient for pyruvate kinase (PK) and for aspartate/glutamate carrier 1 (AGC1), a key component of the malate-aspartate shuttle. In contrast, photoreceptor neurons express AGC1 and the M2 isoform of pyruvate kinase, which is commonly associated with aerobic glycolysis in tumors, proliferating cells, and some other cell types. Our findings reveal a previously unidentified type of metabolic relationship between neurons and glia. Müller glia compensate for their unique metabolic adaptations by using lactate and aspartate from neurons as surrogates for their missing PK and AGC1.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiporters/metabolismo , Neuroglia/metabolismo , Piruvato Quinase/metabolismo , Neurônios Retinianos/metabolismo , Animais , Ácido Aspártico/metabolismo , Isótopos de Carbono , Células Cultivadas , Células Ependimogliais/metabolismo , Células Ependimogliais/efeitos da radiação , Glucose/metabolismo , Glutamina/metabolismo , Glicólise , Células HeLa , Humanos , Isoenzimas/metabolismo , Lactose/metabolismo , Luz , Camundongos , Modelos Biológicos , Neuroglia/efeitos da radiação , Oxirredução/efeitos da radiação , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Neurônios Retinianos/efeitos da radiaçãoRESUMO
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.
Assuntos
Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Purinonas/farmacologia , Ácido Pirúvico/metabolismo , Retina/citologia , Animais , Transporte Biológico/efeitos dos fármacos , Encéfalo/citologia , Ciclo do Ácido Cítrico/efeitos dos fármacos , Metabolômica , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Oxigênio/metabolismoRESUMO
Primary chronic subdural haematomas remains one of the commonest conditions managed by neurosurgeons. Despite this there is a relative lack of evidence regarding best management and certain treatments such as minicraniectomy, have rarely been assessed in the literature. A retrospective case note review comparing minicraniectomy and burrhole drainage of primary chronic subdural haematoma was therefore performed. We sought to determine the proportion of patients requiring repeat drainage or dandy cannula aspiration following initial surgery and to assess outcome at outpatient follow-up. The mean age of patients undergoing minicraniectomy was 73, compared to 63 in the burrhole group (p < 0.001). 130 patients underwent burrhole drainage, 23 of whom (18%) developed a symptomatic recurrence. 21 (16%) of these patients required repeat drainage. Of the 116 patients who underwent a craniectomy 23 (20%) patients suffered a symptomatic recurrence. 15 (13%) patients required the minicraniectomy to be reopened for further washout (p = 0.48). (8%) patients who underwent burrhole drainage died compared to 20 (17%) patients following craniectomy (95%CI 2 to 18%; p = 0.03). However, controlling for age using logistic logression, showed no significant difference between the two treatment groups in recurrence (p = 0.28) or death (p = 0.06). Craniectomy may be considered as a treatment option particularly in the elderly population and in patients with multiple loculated collections.
Assuntos
Craniectomia Descompressiva/métodos , Drenagem/métodos , Hematoma Subdural Crônico/cirurgia , Idoso , Drenagem/instrumentação , Medicina Baseada em Evidências , Feminino , Hematoma Subdural Crônico/mortalidade , Hematoma Subdural Crônico/fisiopatologia , Humanos , Pressão Intracraniana/fisiologia , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.