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ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here, we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated the ATP synthesis pathway, and analyzed intracellular ATP dynamics using an ex vivo IR-mimicking model and a cisplatin nephropathy model. Proximal tubules (PTs) were found to be strongly dependent on oxidative phosphorylation (OXPHOS) using the inhibitor oligomycin A, whereas podocytes relied on both OXPHOS and glycolysis using phloretin an active transport inhibitor of glucose. We also confirmed that an ex vivo IR-mimicking model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reoxygenation varied depending on anoxic time length, whereas ATP in distal tubules (DTs) recovered well even after long-term anoxia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor, cimetidine, suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent (Mitochonic Acid 5) delayed the cisplatin-induced ATP decrease in PTs. Thus, our novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.
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Aging generally predisposes stem cells to functional decline, impairing tissue homeostasis. Here, we report that hematopoietic stem cells (HSCs) acquire metabolic resilience that promotes cell survival. High-resolution real-time ATP analysis with glucose tracing and metabolic flux analysis revealed that old HSCs reprogram their metabolism to activate the pentose phosphate pathway (PPP), becoming more resistant to oxidative stress and less dependent on glycolytic ATP production at steady state. As a result, old HSCs can survive without glycolysis, adapting to the physiological cytokine environment in bone marrow. Mechanistically, old HSCs enhance mitochondrial complex II metabolism during stress to promote ATP production. Furthermore, increased succinate dehydrogenase assembly factor 1 (SDHAF1) in old HSCs, induced by physiological low-concentration thrombopoietin (TPO) exposure, enables rapid mitochondrial ATP production upon metabolic stress, thereby improving survival. This study provides insight into the acquisition of resilience through metabolic reprogramming in old HSCs and its molecular basis to ameliorate age-related hematopoietic abnormalities.
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Adenosina Trifosfato , Células Madre Hematopoyéticas , Mitocondrias , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Animales , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Ratones , Senescencia Celular , Ratones Endogámicos C57BL , Glucólisis , Envejecimiento/metabolismo , Estrés OxidativoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a solid-tumor malignancy. To enhance the treatment landscape of PDAC, a 3D model optimized for rigorous drug screening is essential. Within the PDAC tumor microenvironment, a dense stroma comprising a large extracellular matrix and cancer-associated fibroblasts (CAFs) is well-known for its vital role in modulating tumor growth, cellular heterogeneity, bidirectional paracrine signaling, and chemoresistance. In this study, we employed a fibroblast-populated collagen lattice (FPCL) modeling approach that has the ability to replicate fibroblast contractility in the collagenous matrix to build dense stroma. This FPCL model allows CAF differentiation by facilitating multifaceted cell-cell interactions between cancer cells and CAFs, with the differentiation further influenced by mechanical forces and hypoxia carried within the 3D structure. Our FPCL models displayed hallmark features, including ductal gland structures and differentiated CAFs with spindle shapes. Through morphological explorations alongside in-depth transcriptomic and metabolomic profiling, we identified substantial molecular shifts from the nascent to mature model stages and potential metabolic biomarkers, such as proline. The initial pharmacological assays highlighted the effectiveness of our FPCL model in screening for improved therapeutic strategies. In conclusion, our PDAC modeling platform mirrors complex tumor microenvironmental dynamics and offers an unparalleled perspective for therapeutic exploration.
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Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Microambiente Tumoral , Páncreas , Hormonas Pancreáticas , ColágenoRESUMEN
Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.
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Glucólisis , Fosfofructoquinasa-2 , Animales , Ratones , Adenosina Trifosfato/metabolismo , Anaerobiosis , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Fosforilación Oxidativa , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5'-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.
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Adenosina Trifosfato , Proteína BRCA1 , Recombinación Homóloga , Recombinasa Rad51 , Radiación Ionizante , Humanos , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/biosíntesis , Recombinación Homóloga/efectos de la radiación , Recombinasa Rad51/metabolismo , Proteína BRCA1/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Proteína de Replicación A/metabolismo , Línea Celular Tumoral , Espacio Intracelular/metabolismo , Espacio Intracelular/efectos de la radiación , Reparación del ADN , Histonas/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a refractory tumor with a poor prognosis, and its complex microenvironment is characterized by a fibrous interstitial matrix surrounding PDAC cells. Type I collagen is a major component of this interstitial matrix. Abundant type I collagen promotes its deposition and cross-linking to form a rigid and dense physical barrier, which limits drug penetration and immune cell infiltration and provides drug resistance and metabolic adaptations. In this study, to identify the physical effect of the stroma, type I collagen was used as a 3D matrix to culture Capan-1 cells and generate a 3D PDAC model. Using transcriptome analysis, a link between type I collagen-induced physical effects and the promotion of Capan-1 cell proliferation and migration was determined. Moreover, metabolomic analysis revealed that the physical effect caused a shift in metabolism toward a glycolytic phenotype. In particular, the high expression of proline in the metabolites suggests the ability to maintain Capan-1 cell proliferation under hypoxic and nutrient-depleted conditions. In conclusion, we identified type I collagen-induced physical effects in promoting Capan-1 cells, which cause PDAC progression, providing support for the role of dense stroma in the PDAC microenvironment and identifying a fundamental method for modeling the complex PDAC microenvironment.
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Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor prognosis, largely due to its unique tumor microenvironment (TME) and dense fibrotic stroma. Cancer-associated fibroblasts (CAFs) play a crucial role in promoting tumor growth and metastasis, contributing to the metabolic adaptation of PDAC cells. However, the metabolic interactions between PDAC cells and CAFs are not well-understood. In this study, an in vitro co-culture model was used to investigate these metabolic interactions. Metabolomic analysis was performed under monoculture conditions of Capan-1 PDAC cells and CAF precursor cells, as well as co-culture conditions of PDAC cells and differentiated inflammatory CAF (iCAF). Co-cultured Capan-1 cells displayed significant metabolic changes, such as increased 2-oxoglutaric acid and lauric acid and decreased amino acids. The metabolic profiles of co-cultured Capan-1 and CAFs revealed differences in intracellular metabolites. Analysis of extracellular metabolites in the culture supernatant showed distinct differences between Capan-1 and CAF precursors, with the co-culture supernatant exhibiting the most significant changes. A comparison of the culture supernatants of Capan-1 and CAF precursors revealed different metabolic processes while co-culturing the two cell types demonstrated potential metabolic interactions. In conclusion, this study emphasizes the importance of metabolic interactions between cancer cells and CAFs in tumor progression and highlights the role of TME in metabolic reprogramming.
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Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Fibroblastos Asociados al Cáncer/metabolismo , Microambiente Tumoral , Simbiosis , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Neoplasias PancreáticasRESUMEN
In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.
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Glucólisis , Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Glucólisis/genética , Fosforilación Oxidativa , Vía de Pentosa Fosfato/genética , Adenosina Trifosfato/metabolismoRESUMEN
Empagliflozin, a sodium-glucose co-transporter 2 inhibitor developed, has been shown to reduce cardiovascular events in patients with type 2 diabetes and established cardiovascular disease. Several studies have suggested that empagliflozin improves the cardiac energy state which is a partial cause of its potency. However, the detailed mechanism remains unclear. To address this issue, we used a mouse model that enabled direct measurement of cytosolic and mitochondrial ATP levels. Empagliflozin treatment significantly increased cytosolic and mitochondrial ATP levels in the hearts of db/db mice. Empagliflozin also enhanced cardiac robustness by maintaining intracellular ATP levels and the recovery capacity in the infarcted area during ischemic-reperfusion. Our findings suggest that empagliflozin enters cardiac mitochondria and directly causes these effects by increasing mitochondrial ATP via inhibition of NHE1 and Nav1.5 or their common downstream sites. These cardioprotective effects may be involved in the beneficial effects on heart failure seen in clinical trials.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Ratones , Animales , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Experimental/tratamiento farmacológico , Mitocondrias , Adenosina TrifosfatoRESUMEN
Wallerian degeneration (WD) occurs in the early stages of numerous neurologic disorders, and clarifying WD pathology is crucial for the advancement of neurologic therapies. ATP is acknowledged as one of the key pathologic substances in WD. The ATP-related pathologic pathways that regulate WD have been defined. The elevation of ATP levels in axon contributes to delay WD and protects axons. However, ATP is necessary for the active processes to proceed WD, given that WD is stringently managed by auto-destruction programs. But little is known about the bioenergetics during WD. In this study, we made sciatic nerve transection models for GO-ATeam2 knock-in rats and mice. We presented the spatiotemporal ATP distribution in the injured axons with in vivo ATP imaging systems, and investigated the metabolic source of ATP in the distal nerve stump. A gradual decrease in ATP levels was observed before the progression of WD. In addition, the glycolytic system and monocarboxylate transporters (MCTs) were activated in Schwann cells following axotomy. Interestingly, in axons, we found the activation of glycolytic system and the inactivation of the tricarboxylic acid (TCA) cycle. Glycolytic inhibitors, 2-deoxyglucose (2-DG) and MCT inhibitors, a-cyano-4-hydroxycinnamic acid (4-CIN) decreased ATP and enhanced WD progression, whereas mitochondrial pyruvate carrier (MPC) inhibitors (MSDC-0160) did not change. Finally, ethyl pyruvate (EP) increased ATP levels and delayed WD. Together, our findings suggest that glycolytic system, both in Schwann cells and axons, is the main source of maintaining ATP levels in the distal nerve stump.
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Axones , Degeneración Walleriana , Animales , Ratas , Ratones , Axotomía , Axones/metabolismo , Degeneración Walleriana/metabolismo , Nervio Ciático/metabolismo , Adenosina Trifosfato/metabolismo , Regeneración Nerviosa/fisiologíaRESUMEN
Intractable neuropathic pain following spinal cord injury (NP-SCI) reduces a patient's quality of life. Excessive release of ATP into the extracellular space evokes neuroinflammation via purinergic receptor. Neuroinflammation plays an important role in the initiation and maintenance of NP. However, little is known about whether or not extracellular ATP cause NP-SCI. We found in the present study that excess of intracellular ATP at the lesion site evokes at-level NP-SCI. No significant differences in the body weight, locomotor function, or motor behaviors were found in groups that were negative and positive for at-level allodynia. The intracellular ATP level at the lesion site was significantly higher in the allodynia-positive mice than in the allodynia-negative mice. A metabolome analysis revealed that there were no significant differences in the ATP production or degradation between allodynia-negative and allodynia-positive mice. Dorsal horn neurons in allodynia mice were found to be inactivated in the resting state, suggesting that decreased ATP consumption due to neural inactivity leads to a build-up of intracellular ATP. In contrast to the findings in the resting state, mechanical stimulation increased the neural activity of dorsal horn and extracellular ATP release at lesion site. The forced production of intracellular ATP at the lesion site in non-allodynia mice induced allodynia. The inhibition of P2X4 receptors in allodynia mice reduced allodynia. These results suggest that an excess buildup of intracellular ATP in the resting state causes at-level NP-SCI as a result of the extracellular release of ATP with mechanical stimulation.
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Neuralgia , Traumatismos de la Médula Espinal , Adenosina Trifosfato/metabolismo , Animales , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Ratones , Neuralgia/metabolismo , Calidad de Vida , Médula Espinal/metabolismo , Asta Dorsal de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismoRESUMEN
Oxygen plays an important role in diverse biological processes. However, since quantitation of the partial pressure of cellular oxygen in vivo is challenging, the extent of oxygen perturbation in situ and its cellular response remains underexplored. Using two-photon phosphorescence lifetime imaging microscopy, we determine the physiological range of oxygen tension in osteoclasts of live mice. We find that oxygen tension ranges from 17.4 to 36.4 mmHg, under hypoxic and normoxic conditions, respectively. Physiological normoxia thus corresponds to 5% and hypoxia to 2% oxygen in osteoclasts. Hypoxia in this range severely limits osteoclastogenesis, independent of energy metabolism and hypoxia-inducible factor activity. We observe that hypoxia decreases ten-eleven translocation (TET) activity. Tet2/3 cooperatively induces Prdm1 expression via oxygen-dependent DNA demethylation, which in turn activates NFATc1 required for osteoclastogenesis. Taken together, our results reveal that TET enzymes, acting as functional oxygen sensors, regulate osteoclastogenesis within the physiological range of oxygen tension, thus opening new avenues for research on in vivo response to oxygen perturbation.
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Desmetilación del ADN , Osteoclastos , Animales , Diferenciación Celular/genética , Hipoxia de la Célula , Hipoxia/metabolismo , Ratones , Osteoclastos/metabolismo , Oxígeno/metabolismoRESUMEN
In vertebrates, retinal rod and cone photoreceptor cells rely significantly on glycolysis. Lactate released from photoreceptor cells fuels neighboring retinal pigment epithelium cells and Müller glial cells through oxidative phosphorylation. To understand this highly heterogeneous metabolic environment around photoreceptor cells, single-cell analysis is needed. Here, we visualized cellular AMP-activated protein kinase (AMPK) activity and ATP levels in the retina by two-photon microscopy. Transgenic mice expressing a hyBRET-AMPK biosensor were used for measuring the AMPK activity. GO-ATeam2 transgenic mice were used for measuring the ATP level. Temporal metabolic responses were successfully detected in the live retinal explants upon drug perfusion. A glycolysis inhibitor, 2-deoxy-d-glucose (2-DG), activated AMPK and reduced ATP. These effects were clearly stronger in rods than in cones. Notably, rod AMPK and ATP started to recover at 30 min from the onset of 2-DG perfusion. Consistent with these findings, ex vivo electroretinogram recordings showed a transient slowdown in rod dim flash responses during a 60-min 2-DG perfusion, whereas cone responses were not affected. Based on these results, we propose that cones surrounded by highly glycolytic rods become less dependent on glycolysis, and rods also become less dependent on glycolysis within 60 min upon the glycolysis inhibition.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Glucólisis/fisiología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Animales , Células Ependimogliales/metabolismo , Luz , Ratones , Ratones Endogámicos C57BL , Fosforilación Oxidativa , Fotones , Retina/metabolismoRESUMEN
Spinal cord injury gradually spreads away from the epicentre of injury. The rate of degeneration on the rostral side of the injury differs from that on the caudal side. Rostral degeneration is an immediate process, while caudal degeneration is delayed. In this study, we demonstrated that the rostro-caudal differences in energy metabolism led to differences in the spread of degeneration in early thoracic cord injury using in vivo imaging. The blood flow at the rostral side of the injury showed ischaemia-reperfusion, while the caudal side presented stable perfusion. The rostral side had an ATP shortage 20 min after spinal cord injury, while the ATP levels were maintained on the caudal side. Breakdown products of purine nucleotides were accumulated at both sides of injury 18 h after spinal cord injury, but the principal metabolites in the tricarboxylic acid cycle and glycolytic pathway were elevated on the caudal side. Although the low-ATP regions expanded at the rostral side of injury until 24 h after spinal cord injury, the caudal-side ATP levels were preserved. The low-ATP regions on the rostral side showed mitochondrial reactive oxygen species production. Administration of 2-deoxy-d-glucose as a glycolysis inhibitor decreased the caudal ATP levels and expanded the low-ATP regions to the caudal side until 24 h after spinal cord injury. These results suggest that deficits in the glycolytic pathway accelerate the caudal degeneration, while immediate rostral degeneration is exacerbated by oxidative stress in early thoracic cord injury.
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Temporal and spatial colinear expression of the Hox genes determines the specification of positional identities during vertebrate development. Post-translational modifications of histones contribute to transcriptional regulation. Lysine demethylase 7A (Kdm7a) demethylates lysine 9 or 27 di-methylation of histone H3 (H3K9me2, H3K27me2) and participates in the transcriptional activation of developmental genes. However, the role of Kdm7a during mouse embryonic development remains to be elucidated. Herein, we show that Kdm7a-/- mouse exhibits an anterior homeotic transformation of the axial skeleton, including an increased number of presacral elements. Importantly, posterior Hox genes (caudally from Hox9) are specifically downregulated in the Kdm7a-/- embryo, which correlates with increased levels of H3K9me2, not H3K27me2. These observations suggest that Kdm7a controls the transcription of posterior Hox genes, likely via its demethylating activity, and thereby regulating the murine anterior-posterior development. Such epigenetic regulatory mechanisms may be harnessed for proper control of coordinate body patterning in vertebrates.
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Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Histona Demetilasas con Dominio de Jumonji , Animales , Embrión de Mamíferos/metabolismo , Femenino , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Familia de Multigenes/genéticaRESUMEN
BACKGROUND: Depletion of ATP in renal tubular cells plays the central role in the pathogenesis of kidney diseases. Nevertheless, inability to visualize spatiotemporal in vivo ATP distribution and dynamics has hindered further analysis. METHODS: A novel mouse line systemically expressing an ATP biosensor (an ATP synthase subunit and two fluorophores) revealed spatiotemporal ATP dynamics at single-cell resolution during warm and cold ischemic reperfusion (IR) with two-photon microscopy. This experimental system enabled quantification of fibrosis 2 weeks after IR and assessment of the relationship between the ATP recovery in acute phase and fibrosis in chronic phase. RESULTS: Upon ischemia induction, the ATP levels of proximal tubule (PT) cells decreased to the nadir within a few minutes, whereas those of distal tubule (DT) cells decreased gradually up to 1 hour. Upon reperfusion, the recovery rate of ATP in PTs was slower with longer ischemia. In stark contrast, ATP in DTs was quickly rebounded irrespective of ischemia duration. Morphologic changes of mitochondria in the acute phase support the observation of different ATP dynamics in the two segments. Furthermore, slow and incomplete ATP recovery of PTs in the acute phase inversely correlated with fibrosis in the chronic phase. Ischemia under conditions of hypothermia resulted in more rapid and complete ATP recovery with less fibrosis, providing a proof of concept for use of hypothermia to protect kidney tissues. CONCLUSIONS: Visualizing spatiotemporal ATP dynamics during IR injury revealed higher sensitivity of PT cells to ischemia compared with DT cells in terms of energy metabolism. The ATP dynamics of PTs in AKI might provide prognostic information.
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Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Adenosina Trifosfato/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Lesión Renal Aguda/etiología , Animales , Modelos Animales de Enfermedad , Ratones , Valor Predictivo de las Pruebas , Pronóstico , Daño por Reperfusión/etiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Factores de TiempoRESUMEN
No effective treatment is yet available to reduce infarct size and improve clinical outcomes after acute myocardial infarction by enhancing early reperfusion therapy using primary percutaneous coronary intervention. The study showed that Kyoto University Substance 121 (KUS121) reduced endoplasmic reticulum stress, maintained adenosine triphosphate levels, and ameliorated the infarct size in a murine cardiac ischemia and reperfusion injury model. The study confirmed the cardioprotective effect of KUS121 in a porcine ischemia and reperfusion injury model. These findings confirmed that KUS121 is a promising novel therapeutic agent for myocardial infarction in conjunction with primary percutaneous coronary intervention.
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Cerebellar dysfunction relates to various psychiatric disorders, including autism spectrum and depressive disorders. However, the physiological aspect is less advanced. Here, we investigate the immune-triggered hyperexcitability in the cerebellum on a wider scope. Activated microglia via exposure to bacterial endotoxin lipopolysaccharide or heat-killed Gram-negative bacteria induce a potentiation of the intrinsic excitability in Purkinje neurons, which is suppressed by microglia-activity inhibitor and microglia depletion. An inflammatory cytokine, tumor necrosis factor alpha (TNF-α), released from microglia via toll-like receptor 4, triggers this plasticity. Our two-photon FRET ATP imaging shows an increase in ATP concentration following endotoxin exposure. Both TNF-α and ATP secretion facilitate synaptic transmission. Region-specific inflammation in the cerebellum in vivo shows depression- and autistic-like behaviors. Furthermore, both TNF-α inhibition and microglia depletion revert such behavioral abnormality. Resting-state functional MRI reveals overconnectivity between the inflamed cerebellum and the prefrontal neocortical regions. Thus, immune activity in the cerebellum induces neuronal hyperexcitability and disruption of psychomotor behaviors in animals.
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Cerebelo/inmunología , Depresión/metabolismo , Microglía/metabolismo , Plasticidad Neuronal/fisiología , Células de Purkinje/metabolismo , Receptor Toll-Like 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Trastorno Autístico/inmunología , Trastorno Autístico/metabolismo , Trastorno Autístico/fisiopatología , Trastorno Autístico/psicología , Cerebelo/diagnóstico por imagen , Cerebelo/metabolismo , Depresión/tratamiento farmacológico , Depresión/inmunología , Depresión/psicología , Inflamación/inmunología , Inflamación/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/toxicidad , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Plasticidad Neuronal/inmunología , Compuestos de Fenilurea/administración & dosificación , Células de Purkinje/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/inmunología , Transmisión Sináptica/fisiología , Tiazoles/administración & dosificación , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Hematopoietic stem cells (HSCs) are quiescent cells in the bone marrow niche and are relatively dependent on glycolytic ATP production. On the other hand, differentiated cells, including hematopoietic progenitor cells (HPCs), preferentially generate ATP via oxidative phosphorylation. However, it is unclear how cellular differentiation and the cell cycle status affect nutritional requirements and ATP production in HSCs and HPCs. Using a newly developed culture system, we demonstrated that survival of HPCs was strongly dependent on glucose, whereas quiescent HSCs survived for a certain duration without glucose. Among HPCs, granulocyte/monocyte progenitors (GMPs) were particularly dependent on glucose during proliferation. By monitoring the ATP concentration in live cells, we demonstrated that the ATP level was maintained for a short duration without glucose in HSCs, possibly due to their metabolic flexibility. In addition, HSCs exhibited low ATP turnover, whereas HPCs including GMPs demonstrated high ATP turnover and required efficient ATP production from glucose. These findings show that ATP turnover and nutritional requirements differ between HSCs and HPCs according to the cell cycle and differentiation status.
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Adenosina Trifosfato/metabolismo , Glucosa/metabolismo , Células Madre Hematopoyéticas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citocinas/metabolismo , Citocinas/farmacología , Femenino , Transferencia Resonante de Energía de Fluorescencia , Glucólisis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/metabolismoRESUMEN
The highly organized spatial arrangement of sensory hair cells in the organ of Corti is essential for inner ear function. Here, we report a new analytical pipeline, based on optical clearing of tissue, for the construction of a single-cell resolution map of the organ of Corti. A sorbitol-based optical clearing method enabled imaging of the entire cochlea at subcellular resolution. High-fidelity detection and analysis of all hair cell positions along the entire longitudinal axis of the organ of Corti were performed automatically by machine learning-based pattern recognition. Application of this method to samples from young, adult, and noise-exposed mice extracted essential information regarding cellular pathology, including longitudinal and radial spatial characteristics of cell loss, implying that multiple mechanisms underlie clustered cell loss. Our method of cellular mapping is effective for system-level phenotyping of the organ of Corti under both physiological and pathological conditions.